PROCESS FOR OBTAINING AQUEOUS EXTRACT ENRICHED WITH SMALL RNA FROM PLANT MATERIAL AND EXTRACTS FROM

专利摘要:
The invention relates to a process for obtaining an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides from a plant material. The invention is characterized in that it comprises the following steps according to which: a) the plant material is brought into contact with water; b) tetrasodium ethylenediamine tetraacetic acid (EDTA) is added to the mixture obtained in a) at a pH of between 10.5 and 11; c) the pH of the mixture obtained in b) is then adjusted to a value of between 6 and 8; d) purifying the mixture obtained in c) so as to remove the plant material and recover an aqueous crude extract; and e) controlling the pH and adjusting it if necessary to a value between 6 and 8. The invention also relates to an aqueous extract of plant material, enriched in small RNAs of a length of at most 150 nucleotides, capable of to be obtained by such a method, as well as compositions comprising such an extract and their cosmetic uses to fight against the signs of skin aging, and improve skin hydration.
公开号:FR3043554A1
申请号:FR1670672
申请日:2016-11-10
公开日:2017-05-19
发明作者:Valerie Lequoy；Elodie OGER；Frederique Portolan
申请人:ISP Investments LLC；
IPC主号:

专利说明:

The invention relates to a process for obtaining an aqueous extract enriched in small RNA (ribonucleic acids) of a small amount of RNA. length of at most 150 nucleotides (nt) from a plant material, extracts from such a process, as well as compositions comprising such extracts and their cosmetic uses.
The standard extraction protocols for ribonucleic acids (RNA, small molecular weight RNA) carried out in the laboratory, and therefore on a small scale, must be carried out under a chemical hood because these processes involve the use of solvents such as phenol and chloroform. which are toxic and are not considered cosmetic solvents (Zumbo, P. 2014 "Phenol-chloroform Extraction", 2014). These solvents are added to an aqueous phase of lysed cells or plant or animal tissues ground very finely, or directly on the ground material made in the presence of liquid nitrogen. The solution thus obtained is centrifuged at a high speed of the order of 10 000 g at 4 ° C., in order to create two distinct phases, an organic phase containing the proteins and an aqueous phase containing the total RNAs, the intermediate phase containing the DNA. The aqueous phase must then be carefully recovered so as not to inadvertently take the intermediate and organic phase. A step of precipitation of the RNA is carried out with isopropanol. The total RNA pellet is then washed with ethanol. Then, the total RNAs are resolubilized in water and must be stored at a very low temperature (-20 ° C and better still -80 ° C).
In order to obtain only the small molecular weight RNAs, it is possible to use kits containing purification columns such as the Sigma kit, mirPremier ™ microRNA Isolation Kit. These kits can only be used on a laboratory test scale because the volumes considered are of the order of one mL and therefore can not be adapted to large-scale product development.
In addition, all these extraction and purification protocols will only make it possible to obtain the purified nucleic acid fraction. This RNA or DNA or small RNA nucleic acid fraction will be devoid of any other molecule of interest such as secondary metabolites, vitamins, sugars, peptides, etc. which can have beneficial effects for the skin and are therefore of cosmetic interest.
Furthermore, for example, document WO8403835 describes a process for obtaining an aqueous extract of plant embryos enriched with pure DNA. Such a method uses in particular milled wheat and / or soy embryos in a buffered extraction solution at pH 9.5 containing sucrose, EDTA (0.05 M) and sodium chloride. After filtration, the residue is resuspended in a buffered saline solution at pH 7.4 with anionic detergent with stirring at room temperature, then adding sodium perchlorate with stirring at 0 ° C, and addition of chloroform and octanol before centrifugation at low temperature (4 ° C) and very high speed (25000 g). An aqueous phase containing essentially DNA with a small amount of RNA and amino acids is recovered, on which RNAse can advantageously be carried out.
This document therefore discloses the obtaining of an aqueous extract of plant embryos specifically enriched in DNA using a large number of treatments, including treatments with an anionic detergent and various solvents including chloroform and octanol which are likely to leave toxic traces in the products obtained and can not be used in cosmetics.
Also known is FR2831168 which describes a process for obtaining a nucleic acid-rich extract (DNA and / or RNA) from a plant material, in particular plant embryos or seeds rich in DNA or RNA. The process consists in extracting the plant material in an aqueous medium in the presence of cellulolytic enzymes at an initial pH of 9 to 13, the pH evolving towards neutrality (optimum pH for the action of these enzymes) in a few minutes, then separating the plant material to recover an aqueous extract, and finally treat the extract with a protease and separate the insolubles to recover a purified aqueous extract. The freeze-dried product thus obtained may contain, in particular, 0.1 to 1% by weight of DNA, 0.2 to 1.5% by weight of RNA as well as carbohydrates, proteins, minerals, vitamin B and lipids. According to the data described in this document, the freeze-dried product obtained thus seems to contain, in particular, from 1 to 10 mg / l of DNA and from 10 to 75 mg / l of RNA.
Considering the foregoing, a problem to be solved by the invention is to develop a process for obtaining an aqueous extract enriched specifically in small RNAs and not containing DNA, from a plant material, which is easy and inexpensive to implement on an industrial scale, not necessarily requiring the use of cellulolytic enzymes (cellulase, hemicellulase) or DNAse, offering good yields of small RNA, and not having the disadvantages of methods of the prior art cited, such as the use of detergent and potentially toxic solvents. The extract thus obtained can then be used directly in cosmetics. The invention therefore firstly relates to a process for obtaining an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides from a plant material comprising the following steps: vegetable matter with water; b) tetrasodium ethylenediamine tetraacetic acid (EDTA) is added to the mixture obtained in a), the pH of the mixture being between 10.5 and 11; c) the pH of the mixture obtained in b) is then adjusted to a value of between 6 and 8; d) purifying the mixture obtained in c) so as to remove the plant material and recover an aqueous crude extract; and e) at least one filtration is performed of the aqueous crude extract to obtain the aqueous extract enriched in small RNAs of a length of at most 150 nucleotides whose pH is controlled and if necessary readjusted to a value between 6 and 8, preferably between 6 and 6.5.
In addition, the subject of the invention is an aqueous extract of plant material enriched in small RNAs having a length of at most 150 nucleotides obtained by the process according to the invention, characterized in that it comprises, by weight, total weight of the extract, from 5 to 60 g / kg of solids and from 10 to 1000 mg / kg of small RNAs of a maximum length of 150 nucleotides and does not include DNA. The third subject of the invention is a composition comprising, as an anti-aging active agent, an effective amount of an extract according to the invention, and a physiologically acceptable medium. The fourth subject of the invention is the cosmetic use of a composition according to the invention for combating the signs of skin aging.
Finally, the invention has as a fifth object the cosmetic use of a composition according to the invention for improving the hydration of the skin. 1 / invention and the advantages thereof will be better understood on reading the description and the non-limiting embodiments which follow, written with reference to the appended figures in which: FIG. 1A represents a 2-agarose gel electrophoresis; showing the effect of pH on the extraction of low molecular weight RNA in extracts according to Example 2 (rice seeds) of the invention; Figure 1B shows 2% agarose gel electrophoresis demonstrating the effect of enzymatic hydrolysis on the extraction of low molecular weight RNA in extracts according to Example 2 (rice germs) of The invention; FIG. 2 represents the Bioanalyseur® quantification of the small molecular weight RNAs of an extract according to Example 3 (lenses) of the invention; 3 represents the Bioanalyseur® quantification of the small molecular weight RNAs of the extract according to Example 4 (lenses) of the invention before and after treatment with DNAse; FIG. 4A represents the effect of various baobab extracts (Adansiona digitata) according to Examples 7, 8 and 9 on the expression of hyaluronan synthase 2 (HAS2) on human passage fibroblasts 8 (P8); and FIG. 4B represents the effect of various baobab extracts (Adansiona digitata) according to Examples 7, 8 and 9 on the expression of hyaluronan synthase 2 (HAS2) on human fibroblasts of passage 32 (P32) .
In this description, unless otherwise indicated, it is understood that, when an interval is given, it includes the upper and lower bounds of said interval. 1 / invention relates to a method implemented for obtaining an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides from a plant material. It should be noted that the process of the invention may be preceded by any type of preliminary extraction step known to those skilled in the art (chemical, enzymatic hydrolysis, etc.) allowing, for example, the prior elimination of certain fractions of plant that could hinder the smooth running of the process of the invention.
"Small RNA" or "low molecular weight RNA" means non-coding ribonucleic acid (RNA) of low molecular weight, of a maximum length of 150 nucleotides, such as all types of small non-messenger RNAs, single and / or double strands, for example micro-RNAs, interfering RNAs, introns, small nuclear RNAs or any RNA fragment.
A plant material is generally a plant, living organism forming part of the vegetable kingdom characterized by a very low motility, which feeds on mineral substances and absorbs carbon dioxide, and includes plants whose life cycle generally takes place in the middle aquatic such as algae.
Advantageously, for the implementation of the method, the plant material according to the invention is an entire plant or preferably a plant part (fruit, leaves, roots, bulbs, seeds, seeds, etc.), which can be used in the form of a plant. fresh, dried, sprouted, whole, powdered, frozen, but also in the form of a plant residue obtained after processing, such as cakes, or curds. To be done all parts of the plant species mentioned can be used to obtain an aqueous extract extract enriched in small RNAs of a length of at most 150 nucleotides. Thus the examples relating to certain parts of the plant are given for illustrative and not limiting.
May be advantageously considered as starting vegetable material to obtain an aqueous extract enriched in small molecular weight RNA any fruit or part of fruit. For example, whole fruits may be mentioned in pieces, in the form of powder, fresh or frozen, in the form of a grain. The fruits of the family Passifloraceae, but also fruits of other families, such as those of the family Malvaceae as Hibiscus esculentus (okra). There may also be fruits of the family Punicaceae such as Punica granatum (pomegranate), those of the family Actinidiaceae Actinidia chinensis (kiwi), family Myrtaceae as Psidum guajava (guava), fruits of the family Clusiaceae like Garcinia cambodgia (the tamarind of Malabar), family Caricaceae as Carica papaya (papaya). But still fruits belonging to the Malphigiaceae family such as Malphigia glabra (acerola), fruits in the form of berries such as those of the Ericaceae family such as Vaccinium myrtillus (blueberry) and the family Grossulariaceae as Ribes nigrum (the Cassis). Or any fruit of the family Solanaceae, such as Lycium barbarum (berries goj i).
An aqueous extract enriched in small molecular weight RNA can also be obtained from any flower in whole, powdered, fresh or frozen form, for example from the Rosaceae family such as roses or from the Rutaceae family as well. Citrus aurantium (bitter orange blossom). We can also mention the use of flowers from other families such as flowers of the Oleaceae family such as Jasminum officinalis (jasmine) or flowers of the Asteraceae family such as Centaurea cyanus (blueberry).
The seeds can also be used as starting vegetable material to obtain an aqueous extract enriched with low molecular weight RNA. Any seed, whole in powder form, sprouted or ungerminated. As an indication and not restrictive, the seeds may be from plants of the Chenopodiaceae family such as Quinoa chenopodium, from the family of leguminous plants or Fabaceae such as Lens esculenta (the lens) or from the family of Poaceae such as Oryza sativa (rice ). Or from the family Cucurbitaceae such as Cucurbita pepo (squash), or Lamiaceae. Also useful for the invention are the small seeds or seeds of the family Rutaceae, or Rosaceae or any other species producing fruits containing seeds or nuclei. Underground parts of a plant such as roots, or rhizomes, bulbs may also be considered as starting plant material to obtain an aqueous extract enriched with low molecular weight RNA. These subterranean parts may be in whole form, powder, fresh, dried or frozen. As a guide and not restricted to the roots or bulbs or rhizomes, come from the family of Liliaceae such as Lilium candidum or Lilium tigrinum, but may also be considered for 1'invention all plant species of the family Iridaceae, Amaryllidaceae, Alliaceae .
Can also be considered as starting plant material to obtain an aqueous extract enriched in small molecular weight RNA of any type of leaf in whole form, powder, fresh or frozen, include plants of the family Araliaceae or Dioscoreaceae or any other type of plant family with a developed leaf system.
The plants used in the examples are preferably chosen from the family of Poaceae (formerly grasses), Fabaceae commonly called legumes, Malvaceae, Bombacaceae, Cucurbitaceae, Chenopodiaceae or pseudo cereals, Rosaceae, Rutaceae, Liliaceae, Passifloraceae. As a nonlimiting illustrative example, the plant material is chosen from the species Hibiscus esculentus (okra), Adansonia digitata (baobab), Chenopodium quinoa (quinoa), Lens esculenta (lentil), Oryza sativa (rice), Cucurbita pepo ( squash), Rosa centifolia (pink) r Citrus aurantium (bitter orange), Lilium candidum (lily), Lilium tigrinum (tiger lily), Passiflora alata (grenadilla).
In a first step a) of the process according to the invention, the plant material is brought into contact with water, preferably in a vegetable matter / water ratio of 4 to 20% w / w, more preferably in a ratio of 5 to 15%, for example in a ratio of 5, 10 or 15% w / w. The water used is a distilled or demineralised water or a water rich in mineral salts and / or trace elements, preferably distilled water.
Preferably, the plant material is milled before being placed in the presence of water in step a). Grinding is a mechanical action that allows better extraction. Mechanical grinding followed by alkaline lysis in the presence of EDTA promotes the complete destructuration of the cell membrane and in particular of the nuclear membrane.
Tetrasodium EDTA is then added in a step b) to the mixture obtained in a). The pH at this stage is basic and should be adjusted, if necessary, to a value between 10.5 and 11 by the addition of sodium hydroxide (NaOH). During step b) it is essential to maintain the basic pH between 10.5 and 11. In fact, this pH level, associated with the action of EDTA, causes the destructuring of the cell membrane, including nuclear membrane, lysis of cells and denaturation of DNA (the two strands of the double helix are separated). The pH control in step b) shows that it remains basic and stabilizes between 9 and 11.
The concentration of tetrasodium EDTA is preferably between 2 and 15 mM, and more preferably 10 mM.
This concentration is chosen to optimize the extraction yield of the small molecular weight RNAs in the final extract. Tetrasodium EDTA will weaken, destructure the pectocellulosic membranes of plant cells by sequestering by complexation divalent ions such as calcium ions that form ionic bridges between the pectin molecules surrounding the cellulose microfibrils. This has the effect of promoting the release of cellular content during extraction. The EDTA treatment step is essential to enrich the small molecular weight RNA extract. The EDTA treatment step preferably lasts at least 1 h at a temperature of between 20 and 80 ° C. During this step, the mixture obtained in a) is advantageously stirred.
In a step c), the pH of the mixture obtained in b) is then adjusted to a value of between 6 and 8.
For example, the pH is adjusted by adding a solution of hydrochloric acid (HCl) or any acid that can regulate the pH that is compatible with a cosmetic use such as citric or lactic acid.
This acidification step causes the sudden renaturation of the DNA (reapparriage of duplex strands). Nevertheless, the very long chromosomal DNA fails to reappear completely and forms insoluble entanglements. On the contrary, the small RNAs, much shorter, remain in solution. DNA and small RNAs are thus separated into two distinct phases; a solid phase containing inter alia chromosomal DNA, and a liquid phase containing, inter alia, small RNAs.
In a step d) the mixture obtained in c) is purified so as to remove the plant material and recover an aqueous crude extract. Any method known to those skilled in the art may be used. Preferably the mixture obtained in c) is centrifuged at low speed, for example for at least 10 min at 4000 g, so as to sediment the residual plant material in the pellet and recover an aqueous crude extract in the supernatant.
Advantageously, diatomaceous earth or silica at a concentration of 10 to 20 g / kg are added to the mixture before step d) in order to obtain a compact pellet during centrifugation, which allows obtain a supernatant free of unwanted solids.
In a step e) the pH is controlled and readjusted to a value between 6 and 8. Preferably the pH is readjusted to a value between 6 and 6.5, even more preferably to 6.5. The pH is readjusted by adding a solution of hydrochloric acid (HCl) or sodium hydroxide (NaOH).
Indeed, a pH below 6 can cause the precipitation of nucleic acids in general, so that of small molecular weight RNAs of a length of at most 150 nucleotides. The step of adjusting the pH in step e) of the process according to the invention is an essential step for the optimal extraction of small molecular weight RNAs.
Advantageously, the readjustment of the pH of step e) is preceded by at least one filtration of the aqueous crude extract obtained in d). Preferentially, successive filtrations will be carried out by lowering the filtration threshold from 20 to 50 μm and then from 0.1 to 0.3 μm.
According to an advantageous embodiment, the process according to the invention comprises an additional step of hydrolysis carried out before step c), either directly on the mixture obtained in b), or on the supernatant after centrifugation of the mixture obtained in b) and removing the plant material, by action of at least one enzyme selected from a carbohydrase, a cellulase and / or a protease, preferably a protease, for at least 1 h, at a temperature between 45 and 65 ° C and at a pH adjusted according to the enzyme or enzymes used, generally between 6 and 8.5.
When an additional step of hydrolysis is carried out, the pH is then readjusted if necessary between 6 and 8 and preferably between 6 and 6.5 to preserve the small molecular weight RNAs extracted during the previous steps and to avoid that they precipitate because of too acid pH.
In this advantageous embodiment, the method also necessarily comprises a step of deactivating the enzyme at a temperature of between 65 and 80 ° C. for at least 1 h, this deactivation step being carried out directly after the hydrolysis step. or between 2 successive filtration steps in e). The second object of the invention is that an aqueous extract of plant material, enriched in small RNAs with a length of at most 150 nucleotides of the invention, can be obtained by the method described above.
In a particular embodiment, the aqueous extract enriched in small RNAs having a length of at most 150 nucleotides of the invention is obtained by the method described above.
This extract does not contain DNA (deoxyribonucleic acid).
Such an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides comprises, before dilution, by weight of the total weight of the extract, from 5 to 60 g / kg of dry extract, and 10 to 1000 mg / kg of small RNAs up to 150 nucleotides in length. The extract obtainable by the process according to the invention further comprises from 0.5 to 30 g / kg of protein fragments and from 0.5 to 50 g / kg of sugars. Such an extract may further comprise 0.01 to 5 g / kg of amino acids and 0.01 to 4 g / kg of phenolic compounds. The extract thus obtained is considered concentrated. It can then be diluted in a physiologically acceptable solvent for cosmetic use, so that the concentration of the extract is then adjusted to a particular dry weight of interest. As illustrative and nonlimiting examples of physiologically acceptable solvents, mention may be made of water, glycerol, ethanol, propanediol and its natural version called Zemea® from corn, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols or any mixture of these solvents.
Preferably, the extract obtainable by the process according to the invention is diluted in a solvent such as 30% glycerol and water, and comprises, by weight, the total weight of the extract, from 5 to 35% by weight. g / kg of dry extract, and 10 to 500 mg / kg of small RNAs with a length of at most 150 nucleotides. This diluted extract further comprises from 0.5 to 20 g / kg of protein fragments and from 0.5 to 30 g / kg of sugars. Such a diluted extract may further comprise 0.01 to 3 g / kg of amino acids, 0.01 to 2 g / kg of phenolic compounds. By way of illustration, preferred examples of embodiment of the method according to the invention are described below.
Example 1 Preparation of a rice seed extract (Oryza sativa) of the family Poaceae, enriched in small RNA
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from rice (Oryza sativa) of the family Poaceae (formerly grasses).
In a first step, 5% of rice seeds in powder form are placed in distilled water and 10 mM of tetrasodium EDTA are added, ie 50 g of rice seed powder in 1 kg of distilled water and 3.8 g of tetrasodium EDTA. The pH at this stage should be basic and between 10.5 and 11 for optimal enrichment of the low molecular weight RNA extract.
The mixture is stirred for 2 hours at room temperature.
Enzymatic hydrolysis is then carried out with proteases (of alkalase® which is a serine endopeptidase and bromelain) added to 2% each relative to the plant material involved, ie 1 g of each enzyme is added to the mixture. The pH is adjusted between 7.5 and 8.
The mixture is then heated for 2 hours at 55 ° C. and then for 2 hours at 80 ° C. to deactivate these same enzymes.
The mixture is then centrifuged with diatomaceous earth (10 g per 1 kg of mixture), 10 min at 4000 g, to remove the solid matter. At the end of this step, the pH is controlled, before possible dilution, to place it if necessary between 6 and 6.5 and preserve the small RNA extract.
Sequential filtrations on decreasing porosity filters are then carried out in order to clarify the plant extract until sterilizing filtration at 0.2 μm. In general, an aqueous extract of light yellow-colored rice germ containing 20 to 50 g / kg of dry weight extract, 3 to 15 g / kg of protein fragments, 5 to 30 g / kg is obtained. of sugars, 1.5 to 3 g / kg of amino acids, 300 to 750 mg / kg of phenolic compounds and 50 to 400 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. However, for rice sprouts of the species Oryza sativa, the extracts obtained may present a significant variability depending on factors such as the place of harvest, the year of harvest, the season, climatic conditions, etc.
In this example, an aqueous extract containing 23 g / kg of dry weight extract is more particularly obtained. The physicochemical analysis shows that this extract has a concentration of 4.8 g / kg of protein fragments, 8.2 g / kg of sugars, 2.3 g / kg of amino acids, 328 mg / kg of compounds phenolics and 132 mg / kg of small molecular weight RNA of up to 150 nucleotides in length. This extract can be subsequently diluted in water and preserved by the addition of a cosmetic solvent such as glycerine and a preservative such as 1.5% of phenoxyethanol.
Example 2 Study of the Influence of pH and Enzymatic Hydrolysis in the Implementation of the Process
In order to study the influence of pH in the implementation of the process according to the invention, extraction tests were carried out at different pH and for different types of plants.
These extractions were carried out in particular on the rice seed (as described in Example 1). Comparable results can be obtained generally for all types of plants considered and more particularly described according to the invention. 5% of rice seed powder is placed in water, ie 50 g of rice seed powder in 1 kg of distilled water, and 10 mM of tetrasodium EDTA are added (ie 3.8 g). The pH was adjusted to either 7 or 11 by adding 1 to 3 mL of concentrated sodium hydroxide, and the mixture was stirred for 2h at room temperature.
Samples are then taken during the extraction process to observe the enrichment of small molecular weight RNA for each extract after this key step.
2% agarose gel electrophoresis is thus performed in order to visualize the presence of the small molecular weight RNAs (Fig IA).
As illustrated by Figure 1A, it can be seen that the optimum pH for enriching the low molecular weight RNA extract is a basic pH of 11; indeed, at pH 7, the extract contains no RNA of small molecular weight (absence of characteristic band). The use of a basic pH during the EDTA treatment step is therefore an essential condition for the implementation of the method according to the invention.
In a second step, an enzymatic hydrolysis is carried out with proteases (alkalase® and bromelain) at 2% each relative to the plant material involved, ie 1 g of each enzyme, in the mixture. The pH is adjusted to between 7.5 and 8, optimal pH of enzyme activity, and optimal for maintaining soluble low molecular weight RNAs in the mixture with a pH below 6 precipitating them.
The mixture is then heated for 2 hours at 55 ° C., optimal enzyme activity temperature, then 2 hours at 80 ° C. to deactivate these same enzymes.
The mixture is then centrifuged with diatomaceous earth (10 g per 1 kg of mixture), 10 min at 4000 g, to remove the solid matter. At the end of this step, the pH is controlled, before possible dilution, to place it between 6 and 6.5 and preserve the small RNA extract.
Sequential filtrations on decreasing porosity filters are then carried out in order to clarify the plant extract until a sterilizing filtration with a porosity of 0.2 μm. The final extract is visualized by 2% agarose gel electrophoresis (Fig IB).
As illustrated in Figure 1B, advantageously, it is found that the use of enzyme (s) improves the extraction efficiency of the small molecular weight RNAs in the final extract.
This result has been observed on different types of plants extracted. The fact that enzymes potentiate the extraction of low molecular weight RNA may be due to the fact that proteins are often linked to nucleic acids; the degradation of proteins by enzymes that break the peptide bonds of proteins thus creating protein fragments of small sizes (less than 10 kDa), would dissociate the RNA bound to proteins and release them increasing thus their final yield in the extract .
Example 3 Preparation of a lens extract (Lens esculenta) of the Fabaceae family enriched in small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from lentils (Lens esculenta).
In a first step, the lenses are germinated the day before the extraction process, namely 40 g of lenses coated with distilled water.
The next day, distilled water is added to obtain the equivalent of 4% of lentils engaged in the extraction process.
The lenses (40 g qs 1 kg distilled water) are then milled and 10 mM tetrasodium EDTA (ie 3.8 g) is added. The pH is adjusted to 11 by adding a solution of NaOH and the mixture is stirred for 2 hours at room temperature.
Filtration with a large porosity is advantageously carried out to remove solid debris.
Proteases (2% bromelain and 2% alkalase® with respect to the plant material involved) are then added to the filtrate to perform enzymatic hydrolysis, taking care to allow the enzymes to solubilize before adjusting the pH to 7. , 5 and 8 by adding a solution of HCl.
The filtrate is stirred for 2 hours at 55 ° C, the time that takes place hydrolysis.
Sequential filtrations are then carried out on decreasing porosity filters of sizes of between 20 and 50 μm and then 7 and 20 μm in order to clarify the aqueous crude plant extract. The extract is then heated at 80 ° C. for 1 hour to deactivate the enzymes.
The filtrations are continued to a porosity of 0.3 to 0.4 μm. At this stage of the extraction process, it is important to check the pH, before any dilution, in order to place it advantageously between 6 and 6.5 and to preserve the small RNAs of the extract. In general, an aqueous extract of amber-red colored lenses containing 15 to 25 g / kg of dry weight extract, 3 to 8 g / kg of protein fragments, 2 to 8 g / kg of sugars, 1 to 3 g / kg of amino acids, 300 to 750 mg / kg of phenolic compounds and 50 to 150 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for lentils of the same species (Lens esculenta), the extracts obtained can present a significant variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 15.4 g / kg of dry weight extract, 6.9 g / kg of protein fragments, 2.1 g / kg of sugars, 1.4 g / kg is more particularly obtained. of amino acids, 400 mg / kg of phenolic compounds and 96 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. The extract is then diluted in water or in a physiologically acceptable solvent comprising, for example, water and 30% of glycerol, so that the final extract is adjusted to 10 g / kg of weight extract. dry. The physicochemical analysis shows that after dilution this extract has a protein fragment concentration of 5.1 g / kg, in sugars of 1.5 g / kg, in amino acids of 0.8 g / kg, in compounds phenolics of 280 mg / kg and RNA of small molecular weight (up to 150 nucleotides in length) of 70 mg / kg as illustrated more particularly in Figure 2 by the quantification of low molecular weight RNAs at Bioanalyzer® (Agilent).
The Bioanalyseur® is a device that allows miniaturized electrophoresis to be carried out using electronic chips specific to nucleic acid analysis such as small molecular weight RNAs. It allows to determine the size and concentration contained in an extract from a few microliters. The result is in the form of a graph with an arbitrary fluorescence unit on the ordinate (FU) and on the abscissa the number of nucleotides (nt). An internal marker is added to each analysis (peak 4 nt in Figure 2), and serves as internal control to validate the smooth running of the analysis.
Example 4 Test on the Absence of DNA in the Extract According to Example 3 (Lenses)
In order to verify that the nucleic acid obtained in the extracts according to the invention is indeed RNA, and more particularly RNA of small molecular weight (of a length of at most 150 nucleotides), and not of DNA, a test using DNAse (OPTIZYME ™ Fisher Bioreagent) that specifically degrades the DNA was performed following the protocol recommended by the supplier.
A control solution containing 500 μg / mL of salmon DNA (Sigma, 31149-10g-F) and another control solution containing 500 μg / mL of small molecular weight RNA from Torula yeast (Sigma R6625-25G) have been executed. The reaction volume contains 1 μg of DNA or RNA, 1 μl of RNAse, 1 μl of 10X buffer and supplemented to 10 μl with 0.1% v / v DEPC (diethylpyrocarbonate) water. The reaction mixture is then incubated for 30 min at 37 ° C., an optimal reaction condition for the DNAse. The enzyme is then quenched by adding 50 mM tetrasodium EDTA and heating for 10 min at 65 ° C.
To visualize the profile of the control solutions and of the extract according to Example 3 after action of the DNAse, the small RNAs were quantified by Bioanalyseur®, before and after treatment with DNAse. The profiles obtained with or without DNAse treatment of the plant extract are identical, the amount remaining the same is about 30 mg / kg, as shown in Figure 3. An internal marker is added to each analysis (peak to 4 nt in Figure 3), and serves as internal control to validate the smooth running of the analysis.
The DNAse test, an enzyme which specifically degrades the DNA and not the RNA, demonstrates that the nucleic acid of the extract according to Example 3 is still present after the DNAse treatment. It is thus well RNA, in particular RNA of small molecular weight, a length of at most 150 nucleotides, and not of DNA.
Example 5 Preparation of a Okra Extract (Hibiscus esculentus) from the Malvaceae Family, Enriched in Small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from okra fruits of the species Hibiscus esculentus.
In a first step, after thawing, 10% of Hibiscus esculentus fruits are mixed in distilled water, for example 100 g of fruit in 1 kg of distilled water, then ground for 10 minutes with the addition of EDTA. tetrasodium at a final concentration of 10 mM for 1kg, 3.8 g. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 2 hours at 45 ° C. Even if the temperature at this stage can vary from 20 ° C to 80 ° C, for this species a temperature of 45 ° C is the temperature that allows to obtain the best results in terms of enrichment of the extract aqueous solution of small molecular weight RNA. At the end of the 2 hours, diatomaceous earth (or silica) is added at a concentration of 10 g / kg and the mixture is stirred for another 10 min, followed by centrifugation at 4000 g for 10 min.
The supernatant is then harvested. This crude extract contains especially protein fragments, sugars, and small molecular weight RNAs.
Proteases (2% bromelain and 2% alkalase® relative to the amount of plant material involved) are added to perform enzymatic hydrolysis, taking care to allow the enzymes to solubilize before adjusting the pH to 7, 5 and 8.
The crude solution is stirred for 2h at 55 ° C, the time that takes place hydrolysis. The enzymatic hydrolysis will make it possible to obtain protein fragments of small molecular weight (less than 10 kDa, the high molecular weight proteins being allergenic). Such protein fragments thus obtained may also have a biological activity of interest in the skin.
In order to begin the clarification of the crude extract, sequential filtrations are then carried out on filters of decreasing porosity of sizes of between 20 and 50 μm and then 7 and 20 μm, followed by a step of heating the mixture at high temperature. extracted at 80 ° C on the night. This step makes it possible to deactivate the proteases which under the action of a strong heat denature and become inactive then.
The filtrations are then continued until a sterilizing filtration of 0.1 to 0.3 μm. The plant extract should have a final pH between 6 and 8 to prevent small molecular weight RNA from precipitating. At this stage of the extraction process, it is necessary to check the pH, before any dilution, in order to place it even more preferentially between 6 and 6.5.
In general, a pale yellow aqueous extract with a content of 10 to 20 g / kg of dry weight extract, 2 to 5 g / kg of protein fragments, 2.5 to 5 g / kg of sugars, is obtained. , 1 to 2 g / kg of amino acids, 0.2 to 3 g / kg of phenolic compounds, and 10 to 100 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for okra fruits of the same species (Hibiscus esculentus), the extracts obtained can present a large variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc. .
In this example, an aqueous extract containing 13.3 g / kg of dry weight extract, 3.2 g / kg of protein fragments, 3.9 g / kg of sugars, 790 mg / kg of amino acids, 490 mg / kg of phenolics, and 60 mg / kg of small molecular weight RNA of up to 150 nucleotides in length. The extract is then diluted in a physiologically acceptable solvent comprising, for example, water and 30% glycerol, so that the final extract is adjusted to 10 g / kg of dry weight extract. The physicochemical analysis shows that, after dilution, the extract has a concentration of protein fragments of 2.5 g / kg, sugars of 2.7 g / kg, amino acids of 520 mg / kg, and phenolic compounds. of 320 mg / kg and 35 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides.
Example 6 Demonstration of the Role of an EDTA Treatment Step in the Implementation of a Small RNA Extraction Process from Okra (Hibiscus esculentus)
In order to demonstrate the role of an EDTA treatment step in the extraction of small molecular weight RNAs, a fruit extract of Hibiscus esculentus was obtained by modifying certain essential steps of the process according to invention, not allowing to enrich the RNA extract of small molecular weight. 15% of thawed fruits of Hibiscus esculentus are mixed with distilled water and then crushed, ie 150 g of fruit in 1 kg of distilled water.
Proteases are then added sequentially: 2% alkalase® with respect to the plant material involved (ie 3 g) at pH 8 for 2 h at 55 ° C (optimal condition for this enzyme), then 2% bromelain ( or 3 g) at a pH adjusted to between 4 and 4.5 for 2 h at 55 ° C.
This mixture is then centrifuged to remove solid debris.
In order to begin the clarification of the crude extract, sequential filtrations are then carried out on filters of decreasing porosity of sizes of between 20 and 50 μm and then 7 and 20 μm, followed by a step of heating the mixture at high temperature. extracted at 80 ° C on the night.
The filtrations are continued until a sterilizing filtration of 0.1 to 0.3 μm. The aqueous extract obtained is then diluted in a physiologically acceptable solvent comprising, for example, water and 30% glycerol, to reach 9.6 g / kg of dry weight extract. The pH of the final extract is between 4 and 4.5. The physicochemical analysis shows that the final vegetable extract, after dilution, has a protein fragment concentration of 2.2 g / kg, sugars of 3.8 g / kg, 550 mg / kg of amino acids and 243 mg / kg of phenolic compounds. Bioanalyzer analysis indicates that the concentration of low molecular weight RNA is zero for this extract. This result demonstrates that an extract obtained from an extraction process not having an EDTA treatment step does not contain low molecular weight RNA. The EDTA treatment step is therefore essential to obtain a low molecular weight RNA rich extract according to the invention.
Example 7 Preparation of a baobab extract (Adansonia diqitata) of the Bombacaceae family enriched in small RNAs
An aqueous extract enriched in RNA of small molecular weight (of a length of at most 150 nucleotides) is obtained from baobab of the species Adansonia digitata.
In a first step, 5% baobab seed cake (Adansonia digitata) is dry milled or directly in water containing tetrasodium EDTA at a final concentration of 10 mM, ie 50 g of baobab cake. in 1 kg of distilled water to which 3.8 g of tetrasodium EDTA is added. The pH at this stage is basic and more particularly between 10.5 and 11, the optimum pH for enriching the extract with low molecular weight RNA.
The mixture is stirred for 2 h at 58 ° C.
For this species, advantageously a hydrolysis step with a proteolytic enzyme is carried out: 2% of papain are added relative to the amount of plant material involved (ie 1 g). The pH of the mixture is adjusted between 7 and 8, if necessary, and stirred for 2 hours at 58 ° C., an optimum condition for this enzyme.
Then, the pH is adjusted to 8 and then the extract is centrifuged for 10 min at 4000 g to remove the solid matter.
Sequential filtrations are then carried out on decreasing porosity filters of size between 20 and 50 pm and then 7 and 20 pm in order to clarify the plant extract. The extract is then heated to 80 ° C between 8 and 12 hours to thermally deactivate the enzyme.
The filtrations are continued until a porosity of 0.3 to 0.4 μιη. At this stage of the extraction process, it is necessary to check the pH, before any dilution, in order to place it between 6 and 6.5 and preserve the small RNA of the extract. An acidic pH can cause the precipitation of nucleic acids in general therefore also that of the RNAs of small molecular weight.
In general, an aqueous amber-red baobab extract containing 15 to 25 g / kg of dry weight extract, 3 to 8 g / kg of protein fragments, 2 to 8 g / kg of sugars, is obtained. 0.05 to 1 g / kg of amino acids, 0.05 to 1 g / kg of phenolic compounds and 10 to 80 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, extracts obtained from the species Adansonia digitata can present a significant variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 17 g / kg of dry weight extract, 5.3 g / kg of protein fragments, 5.4 g / kg of sugars and 650 mg / kg of amino acids is more particularly obtained. , 441 mg / kg of phenolic compounds, and 57 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. The extract is then diluted in a mixture of water and 30% of glycerol and 1.5% of phenoxyethanol are added, which makes it possible to obtain a final extract at 12 g / kg of dry weight extract. The physicochemical analysis shows that after dilution this extract has a concentration of protein fragments of 2.9 g / kg, sugars of 4 g / kg, amino acids of 400 mg / kg, phenolic compounds of 310 mg / kg, and 41 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides.
Example 8 Demonstration of the Role of an EDTA Treatment Step for the Implementation of a Process for Extracting Small RNAs from Baobab (Adansonia digitata)
In order to highlight the role of an EDTA treatment step in the extraction of small molecular weight RNAs, an extract of baobab (Adansonia digitata) was also obtained, by modifying certain essential steps of the process according to the invention does not allow to enrich the RNA extract of small molecular weight.
In a first step, 10% baobab seed cake (Adansonia digitata) is crushed and water is added, ie 100 g of baobab cake in 1 kg of distilled water.
For this species, a hydrolysis with a proteolytic enzyme is carried out: 2% of papain are added in relation to the amount of plant material involved, ie 2 g. The pH is adjusted between 7 and 8, and the mixture is stirred for 2 hours at 58 ° C, optimal conditions for the activity of the enzyme. After this period, the pH was lowered to 4.5, the pH commonly used for cosmetic ingredients.
Then, the extract is centrifuged for 10 min at 4000 g to remove the solid matter. The extract is then heated to 80 ° C between 8 and 12h in order to deactivate the enzyme by high temperature.
Sequential filtrations are then carried out on filters of decreasing porosity with a size of between 20 and 50 μm and then up to a porosity of 0.3 to 0.4 μm.
A light yellow-colored aqueous extract is then obtained, yielding 12.5 g / kg of dry weight extract, 5.8 g / kg of protein fragments, 7.6 g / kg of sugars, 540 mg / kg of acids. amines, and 440 mg / kg of phenolic compounds. The extract is then diluted in water and glycerol so as to obtain a 30% final extract of glycerol and adjusted to 10 g / kg of dry weight extract. The physicochemical analysis shows that, after dilution, the plant extract has a concentration of protein fragments of 3.25 g / kg, sugars of 5.1 g / kg, amino acids of 310 mg / kg, and phenolic compounds of 250 mg / kg. Under these extraction conditions (no treatment with EDTA), the Bioanalyseur® analysis indicates that the concentration of low molecular weight RNA is zero for this extract. This result confirms that an extract obtained from an extraction process in the absence of EDTA treatment (at basic pH) does not contain low molecular weight RNA. The EDTA treatment step is essential to obtain an extract rich in low molecular weight RNA according to the invention.
Example 9: Study of the influence of the final pH in the preparation of extracts of qombo (Hibiscus esculentus) and baobab (Adansonia diqitata)
The extraction process is carried out under the same operating conditions as Examples 5 and 7, in order to enrich a small molecular weight RNA extract, except for the final step of adjusting the pH.
This extraction process is carried out with tetrasodium EDTA treatment step, followed by the enzymatic hydrolysis step, but with a final adjustment of the extract at an acidic pH of between 4 and 4.5 instead of one. pH between 6 and 8.
This results in the precipitation of small molecular weight RNAs, results confirmed by the Bioanalyseur® analysis which gives a zero concentration of low molecular weight RNA for each of the extracts of Okra (Hibiscus esculentus) and Baobab (Adansonia digitata) thus obtained. .
Example 10 Preparation of a pumpkin extract (Cucurbita pepo) of the family Cucurbitaceae enriched in small RNA
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from squash (Cucurbita pepo) of the cucurbitaceae family.
In a first step, 10% of squash seed meal is mixed with water and tetrasodium EDTA is added to obtain a final concentration of 10 mM, ie 100 g of squash cake in 1 kg of water. distilled and 3.8 g of tetrasodium EDTA.
The pH at this stage must be basic and more particularly between 10.5 and 11 for optimum enrichment of the small molecular weight RNA extract.
The mixture is stirred for 2 h at 45 ° C.
An enzymatic hydrolysis is then carried out with proteases: 2% alkalase® and 4% papain relative to the amount of plant material involved, ie 2 g of alkalase® and 4 g of papain, are thus added to the mixture . The pH is adjusted between 7.5 and 8, optimal pH of activity for these two enzymes, this pH remaining optimal to maintain the small molecular weight RNAs soluble in the mixture, a pH below 6 that can precipitate them.
The mixture is then heated for 2 hours at 50 ° C., optimal temperature of enzyme activity and then 2 hours at 80 ° C. to deactivate these same enzymes.
The mixture is then centrifuged with diatomaceous earth (10 g per 1 kg of mixture), 10 min at 4000 g, to remove the solid matter. At the end of this step, the pH is controlled, before possible dilution, to place it if necessary between 6 and 6.5 and preserve the small RNA extract.
Sequential filtrations are then carried out on decreasing porosity filters of size between 20 and 50 pm and then 7 and 20 pm in order to clarify the plant extract. Then, the filtrations continue until the 0.2 micron sterilizing filtration.
In general, a light yellow-colored aqueous extract is obtained containing 20 to 50 g / kg of dry weight extract, 3 to 25 g / kg of protein fragments, 1 to 10 g / kg of sugars, and 50 to 250 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for squash of the same species (Cucurbita pepo), the extracts obtained can present a considerable variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 37.3 g / kg of dry weight extract, 18.4 g / kg of protein fragments, 3.8 g / kg of sugars and 168 mg / kg of alcohol is more particularly obtained. RNA of small molecular weight of up to 150 nucleotides in length. The extract can then be diluted in a physiologically acceptable solvent comprising, for example, water and 30% glycerol.
EXAMPLE 11 Preparation of Quinoa Extract (Chenopodium Quinoa) from the Chenopodiaceae or Pseudo Cereal Family, fortified with Small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from quinoa Chenopodium quinoa, from the family chenopodiaceae or pseudo cereal.
In a first step, the sprouted quinoa seeds (10% final weight, ie 100 g) are engaged in the extraction process and mixed in 1 kg of distilled water and then crushed, and tetrasodium EDTA is added. to obtain a final concentration of 10 mM or 3.8 g. The pH at this stage must be basic and between 10.5 and 11, the pH being thus adjusted with NaOH, for optimum enrichment of the extract according to the invention in low molecular weight RNA with a length of at most 150 nucleotides. The mixture is stirred for 2h at 55 ° C.
Enzymatic hydrolysis is then carried out with proteases: 2% alkalase® and 2% bromelain are added to the mixture (relative to the material involved). The pH is adjusted between 7.5 and 8, the optimum pH of activity for these two enzymes, this pH remaining optimal for keeping the small molecular weight RNAs soluble in the mixture, a pH below 6 making them precipitate.
The mixture is heated for 2 hours at 45 ° C., the optimum temperature for the activity of the enzymes.
The mixture is then centrifuged with diatomaceous earth (10 g per 1 kg of mixture), 10 min at 4000 g, to remove the solid matter.
Then, the filtrate is heated for 2 hours at 80 ° C. to deactivate these enzymes.
Sequential filtrations are then carried out on decreasing porosity filters of size between 20 and 50 pm and then 7 and 20 pm in order to clarify the plant extract.
After this filtration step, it is possible to dilute the extract in preferentially water and glycerol so as to obtain a final extract containing 30% glycerol.
Then, the filtrations continue until the 0.2 micron sterilizing filtration.
In general, a light yellow-colored extract is obtained containing 20 to 50 g / kg of dry weight extract, 3 to 15 g / kg of protein fragments, 10 to 30 g / kg of sugars, 0.5 to 5 g / kg of amino acids, 100 to 700 mg / kg of phenolic compounds and 50 to 250 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides.
Similar results can be obtained from non-sprouted quinoa seeds or quinoa flour. Nevertheless, for quinoa of the same species (Chenopodium quinoa), the extracts obtained can present a considerable variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 33 g / kg of dry weight extract, 9.5 g / kg of protein fragments, 21.6 g / kg of sugars, 1.8 g / kg of amino acids, 364 mg / kg of phenolic compounds, and 173 mg / kg of small molecular weight RNA of up to 150 nucleotides in length. The extract can then be diluted in a physiologically acceptable solvent comprising for example water and 30% glycerol.
Example 12 Preparation of a Rose Extract (Rosa centifolia) of the Rosaceae Family Enriched in Small RNAs
An aqueous extract enriched in RNA of small molecular weight (of a length of at most 150 nucleotides) is obtained from rose (Rosa centifolia) of the Rosaceae family. In this example, the whole fresh flower is used.
In a first step, 10% of roses are mixed in distilled water, for example 100 g of flowers supplemented with 1 kg of distilled water, and are ground for 10 minutes with addition of tetrasodium EDTA to a final concentration. 10 mM for 1kg, 3.8 g. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 1 h at 80 ° C. Even if the temperature at this stage can vary from 50 ° C to 80 ° C, for this species a temperature of 80 ° C turns out to be the temperature which allows to obtain the best results in terms of enrichment of the extract aqueous solution of small molecular weight RNA. At the end of this step, sequential filtrations are then carried out on decreasing porosity filters with a size of between 20 and 50 μm and then 7 and 20 μm in order to remove the solid matter and then to clarify the plant extract. At this stage, the pH is controlled, to place it if necessary between 6 and 6.5 and preserve the small RNA of the extract.
Then, the filtrations continue until the 0.2 micron sterilizing filtration.
In general, an amber-colored aqueous extract containing 5 to 20 g / kg of dry weight extract, 1 to 10 g / kg of protein fragments, 1 to 10 g / kg of sugars, 0.5 to 2 g / kg of phenolic compounds and 20 to 200 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for roses of the same species (Rosa centifolia), the extracts obtained can present a significant variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 11.3 g / kg of dry weight extract, 5.1 g / kg of protein fragments, 2.6 g / kg of sugars, 1 g / kg of phenolic compounds and 96 mg / kg is more particularly obtained. small molecular weight RNA of up to 150 nucleotides in length. The extract may then be diluted with, for example, 30% glycerol, which makes it possible to obtain a final extract at 8 g / kg of dry weight extract. The physicochemical analysis shows that, after dilution, this extract has a concentration of protein fragments of 4.6 g / kg, sugars of 2 g / kg, amino acids of 280 mg / kg, and phenolic compounds of 800 mg / kg. and 83 mg / kg of small molecular weight RNA of up to 150 nucleotides in length.
EXAMPLE 13 Preparation of a Bitter Orange Flower Extract (Citrus Aurantium) of the Rutaceae Family Enriched in Small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from bitter orange (Citrus aurantium) flowers of the family Rutaceae. In this example, the fresh and whole flower is used.
In a first step, 5% of orange blossom flowers are mixed in distilled water, for example 50 g of flowers supplemented with 1 kg of distilled water, and are ground for 5 minutes with addition of tetrasodium EDTA to a final concentration of 10 mM for 1kg, 3.8 g. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 1 h at 45 ° C. Even if the temperature at this stage can vary from 25 ° C to 50 ° C, for this species a temperature of 45 ° C turns out to be the temperature that allows to obtain the best results in terms of enrichment of the extract aqueous solution of small molecular weight RNA. At the end of this step, sequential filtrations are then performed on decreasing porosity filters with a size of between 20 and 50 μm and then 7 and 20 μm, in order to remove the solid matter and then to clarify the plant extract. At this stage, the pH is controlled, to place it if necessary between 6 and 6.5 and preserve the small RNA of the extract.
The filtrations are then continued to a porosity of 0.3 to 0.5 μm.
In general, an amber-colored aqueous extract containing 5 to 20 g / kg of dry weight extract, 1 to 10 g / kg of protein fragments, 1 to 10 g / kg of sugars, 200 to 1000 mg is obtained. / kg of phenolic compounds and 10 to 100 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for flowers of the same species (Citrus aurantium), the extracts obtained can present a considerable variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 11.8 g / kg of dry weight extract, 4.5 g / kg of protein fragments, 3.8 g / kg of sugars, 560 mg / kg of phenolic compounds and 20 mg / kg is more particularly obtained. small molecular weight RNA of up to 150 nucleotides in length. The extract can then be diluted in a physiologically acceptable solvent such as water or glycerol.
Example 14 Preparation of a lily extract (Lilium candidum) enriched in small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from white lily bulb (Lilium candidum) of the family Liliaceae.
In a first step, 15% of lily bulbs are placed in distilled water, for example 150 g of bulbs in 1 kg of distilled water containing tetrasodium EDTA at a final concentration of 10 mM or 1 kg. 3.8 g, then grinding the soltuion for 5 minutes. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 30 minutes at 65 ° C. Even if the temperature at this stage can vary from 50 ° C to 80 ° C and the stirring time from 30 minutes to 1 hour, for this species a temperature of 65 ° C for 30 minutes proves to be the conditions that allow obtain the best results in terms of enrichment of the final aqueous extract in low molecular weight RNA. At the end of this step, sequential filtrations are then performed on decreasing porosity filters with a size of between 20 and 50 μm and then 7 and 20 μm, in order to remove the solid matter and then to clarify the plant extract. At this stage, the pH is controlled, to place it if necessary between 6 and 6.5 and preserve the small RNA of the extract.
The filtrations are continued to a porosity of 2 to 4 μm. The extract can then be preserved by adding 30% glycerol and 1.5% phenoxyethanol. The filtrations are continued to a porosity of 0.2 to 0.3 μm.
In general, a yellow-colored aqueous extract with a content of 10 to 25 g / kg of dry weight extract, 0.5 to 5 g / kg of protein fragments, 2 to 15 g / kg of sugars, 100 to 500 mg / kg of phenolic compounds and 10 to 100 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for lilies of the same species (Lilium candidum), the extracts obtained can present a considerable variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 16.3 g / kg of dry weight extract, 1.5 g / kg of protein fragments, 5.3 g / kg of sugars, 200 mg / kg of phenolic compounds and 20 mg is more particularly obtained. / kg of small molecular weight RNA with a length of at most 150 nucleotides. The extract can then be diluted in a physiologically acceptable solvent such as water or glycerol.
Example 15 Preparation of a lily extract (Lilium tigrinum) of the family Liliaceae enriched in small RNA
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from the tiger lily bulb (Lilium tigrinum) of the family Liliaceae.
In a first step, after washing and thawing, 10% of lily bulbs are mixed in distilled water, for example 100 g of bulbs in 1 kg of distilled water containing tetrasodium EDTA at a final concentration of 10 mM for 1kg, 3.8 g then the mixture is ground for 5 minutes. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 1 hour at 80 ° C. Even if the temperature at this stage can vary from 50 ° C. to 80 ° C. and the stirring time from 30 minutes to 1 hour, for this species a temperature of 80 ° C. for 1 hour proves to be the conditions which make it possible to obtain the best results in terms of enrichment of the final aqueous extract in low molecular weight RNA. At the end of this step, sequential filtrations are then performed on decreasing porosity filters with a size of between 20 and 50 μm and then 7 and 20 μm, in order to remove the solid matter and then to clarify the plant extract. At this stage, the pH is controlled, to place it if necessary between 6 and 6.5 and preserve the small RNA of the extract. The filtrations are continued to a porosity of 0.2 to 0.3 μm.
In general, a yellow-colored aqueous extract with a content of 10 to 25 g / kg of dry weight extract, 0.5 to 5 g / kg of protein fragments, 5 to 20 g / kg of sugar, 100 to 500 mg / kg of phenolic compounds and 10 to 100 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. Nevertheless, for lilies of the same species (Lilium tigrinum), the extracts obtained can present a considerable variability according to factors such as the place of harvest, the year of harvest, the season, the climatic conditions, etc.
In this example, an aqueous extract containing 17.9 g / kg of dry weight extract, 2.1 g / kg of protein fragments, 11.4 g / kg of sugars, 200 mg / kg of phenol compounds and 54 mg / kg is more particularly obtained. small molecular weight RNA of up to 150 nucleotides in length. The extract is then diluted in a mixture of water, 30% of glycerol and 1.5% of phenoxyethanol, which makes it possible to obtain a final extract at 10 g / kg of dry matter. The physicochemical analysis shows that after dilution this extract has a protein fragment concentration of 1.0 g / kg, sugars of 5.8 g / kg, phenolic compounds of 100 mg / kg, and 30 mg / kg of RNA of small molecular weight of up to 150 nucleotides in length.
EXAMPLE 16 Preparation of a passion fruit extract (Passiflora alata) from the Passifloraceae family enriched in small RNAs
An aqueous extract enriched in small molecular weight RNA (up to 150 nucleotides in length) is obtained from passion fruit (Passiflora alata) of the passifloraceae family.
In a first step, 5% of the powder is mixed in distilled water, for example 50 g of fruit powder supplemented with 1 kg of distilled water, then the solution is stirred for 5 minutes and then tetrasodium EDTA at a final concentration of 10 mM for 1kg, 3.8 g is added. The pH at this stage is between 10.5 and 11, optimal pH to enrich the extract in small RNA.
This mixture is then stirred for 1 hour at 50 ° C. Even if the temperature at this stage can vary from 25 ° C to 80 ° C and the stirring time from 30 minutes to 1 hour, for this species a temperature of 50 ° C for 60 minutes proves to be the conditions that allow obtain the best results in terms of enrichment of the final aqueous extract in low molecular weight RNA. At the end of this step, the mixture is centrifuged for 10 min at 4000 g to remove the solid matter.
Then sequential filtrations are then carried out on decreasing porosity filters of size between 20 and 50 pm and then 7 and 20 pm up to 2-4 pm, in order to clarify the plant extract. At this stage, the pH is controlled, in order to place it if necessary between 6 and 6.5 and preserve the small RNAs of the extract which are sensitive to an acidic pH.
The filtrations are continued to a porosity of 0.3 to 0.5 μm.
In general, an amber-colored aqueous extract with a content of 10 to 30 g / kg of dry weight extract, 0.5 to 5 g / kg of protein fragments, 2 to 15 g / kg of sugars, 100 to 100 g of 1500 mg / kg of phenolic compounds and 10 to 100 mg / kg of small molecular weight RNA with a length of at most 150 nucleotides. The extract can then be diluted or preserved by adding a physiologically acceptable solvent such as 30% glycerol.
In this example, an aqueous extract containing 15 g / kg of dry weight extract, 2.3 g / kg of protein fragments, 3.0 g / kg of sugars, 200 mg / kg of compounds is obtained after addition of 30% of solvent. phenolics and 35 mg / kg of small molecular weight RNA of up to 150 nucleotides in length.
According to a third aspect of the invention, the aqueous extracts enriched in small RNAs obtained according to the invention are advantageously used in the preparation of cosmetic compositions comprising, as an anti-aging active agent, an effective amount of such an extract. small RNA according to the invention, and a physiologically acceptable medium.
By effective amount is meant the minimum amount of extract according to the invention which is necessary to obtain the activity of the extract, in particular cosmetic and more particularly against the signs of skin aging or for the improvement of the hydration of the skin, without this quantity being toxic.
Advantageously, the small RNA extract according to the invention is used in its diluted form, with a dry weight of between 5 and 35 g / kg.
Advantageously, the small RNA extract according to the invention is present in the composition at a concentration of 0.1 to 5%, preferably at a concentration of 1 to 5% by weight relative to the total weight of the composition.
A physiologically acceptable medium means a vehicle adapted for contact with the outer layers of the skin or mucous membranes, without toxicity, irritation, undue and similar allergic response or intolerance reaction, and proportionate to a reasonable benefit / risk ratio.
The composition that can be used according to the invention can be applied by any appropriate route, in particular oral or external topical, and the formulation of the compositions will be adapted by those skilled in the art.
Preferably, the compositions according to the invention are in a form suitable for topical application. These compositions must therefore contain a physiologically acceptable medium, that is to say, compatible with the skin and integuments, without risk of discomfort during their application and cover all suitable cosmetic forms.
By topical application, it is meant to apply or spread the aqueous extract enriched in small RNA according to the invention, and more particularly a composition containing it, on the surface of the skin or mucosa.
The skin refers more particularly to the skin of the face, in particular the outline of the eyes and the mouth, the nose, the forehead, the neck, the hands, as well as the skin of the rest of the body.
The compositions for the implementation of the invention may especially be in the form of an aqueous solution, aqueous-alcoholic or oily, an oil-in-water emulsion, water-in-oil or multiple emulsions; they may also be in the form of suspensions, or powders, suitable for application to the skin, mucous membranes, lips and / or hair.
These compositions may be more or less fluid and may also have the appearance of a cream, lotion, milk, serum, ointment, gel, paste or paste. a foam. They can also be in solid form, as a stick or be applied to the skin in aerosol form. As a physiologically acceptable medium commonly used in the intended field of application, mention may be made, for example, of adjuvants necessary for the formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.
In all cases, those skilled in the art will ensure that these adjuvants and their proportions are chosen so as not to adversely affect the desirable properties of the composition according to the invention. These adjuvants may, for example, correspond to 0.01 to 20% of the total weight of the composition. When the composition according to the invention is an emulsion, the fatty phase may represent from 5 to 80% by weight and preferably from 5 to 50% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition are chosen from those conventionally used in the field under consideration. For example, they may be used in a proportion ranging from 0.3 to 30% by weight relative to the total weight of the composition.
According to another advantageous embodiment of the invention, the aqueous extract enriched in small RNA according to the invention may be encapsulated or included in a cosmetic vector such as liposomes or any other nanocapsule or microcapsule used in the field of cosmetics or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites.
Advantageously, the composition according to the invention may comprise, in addition to the active agent according to the invention, at least one other active agent having cosmetic effects similar to and / or complementary to those of the invention. According to the invention, this active agent is defined as an "additional active agent".
For example, the additional active agent (s) may be chosen from: anti-aging, firming, lightening, moisturizing, draining, microcirculatory promoting agents, exfoliants, desquamating agents, stimulating the extracellular matrix, activating energy metabolism, antibacterials, antifungal agents, soothing, anti-radical, anti-UV, anti-acne, anti-inflammatory, anesthetic, providing a feeling of warmth, providing a feeling of freshness, slimming.
Such additional active agents may be chosen from the groups comprising: vitamin A and in particular retinoic acid, retinol, retinol propionate, retinol palmitate; vitamin B3 and more particularly niacinamide, tocopherol nicotinate; - vitamin B5, vitamin B6, vitamin B12, panthenol; vitamin C, especially ascorbic acid, ascorbyl glucoside, ascorbyl tetrapalmitate, magnesium and sodium ascorbyl phosphate; vitamins E, F, H, K, PP, coenzyme Q10; metalloproteinase inhibitors, or a TIMP activator; - DHEA, its precursors and derivatives; amino acids such as arginine, ornithine, hydroxyproline, hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine, methionine and its derivatives, N-acylated amino acid compounds; the natural or synthetic peptides, including di-, tri-, tetra-, penta- and hexapeptides and their lipophilic derivatives, isomers and complexed with other species such as a metal ion (for example copper, zinc, manganese) , magnesium, and others). By way of examples, mention may be made of the peptides commercially known under the name MATRIXYL®, ARGIRELINE®, CHRONOGEN ™, LAMINIXYL IS ™, PEPTIDE Q10 ™, COLLAXYL ™ (patent FR2827170, ASHLAND®), PEPTIDE VINCI 01 ™ (patent FR2837098, ASHLAND®), PEPTIDE VINCI 02 ™ (patent FR2841781, ASHLAND®), ATPeptide ™ (patent FR2846883, ASHLAND®) or the synthetic peptide of Arg-Gly-Ser-NH2 sequence, sold under the name ATPeptide ™ by ASHLAND®; extract of Artemia salina, marketed under the name GP4G ™ (FR2817748, ASHLAND®); plant peptide extracts such as flax extracts (Lipigenin ™, patent FR2956818, ASHLAND®), extracts of soya, spelled, grapevine, rapeseed, flax, rice, maize, pea; yeast extracts, for example Dynagen ™ (patent FR2951946, ASHLAND®) or Actopontine ™ (patent FR2944526, ASHLAND®); - dehydroacetic acid (DHA); phystosterols of synthetic or natural origin; salicylic acid and its derivatives, alpha- and beta-hydroxy acids, silanols; amino sugars, glucosamine, D-glucosamine, N-acetyl glucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl mannosamine, galactosamine, N-acetyl galactosamine; extracts of polyphenols, isoflavones, flavonoids, such as grape extracts, pine extracts, olive extracts; lipids such as ceramides or phospholipids, oils of animal origin, such as squalene or squalane; vegetable oils, such as sweet almond oil, coconut oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soya bean oil, of cotton, alfalfa, poppy, pumpkin, evening primrose, millet, barley, rye, safflower, passionflower, hazelnut, palm, apricot kernel, avocado, calendula ; ethoxylated vegetable oils, shea butter; - all UV screens and sunscreens; cyclic AMP and its derivatives, the activating agents of the adenylate cyclase enzyme and the phosphodiesterase enzyme inhibitory agents, the Centella asiatica extract, the asatic acid and the Asian acid, the methyl xanthines, the theine , caffeine and its derivatives, theophylline, theobromine, forskolin, esculin and esculoside, ACE inhibitors, Val-Trp peptide, neuropeptide Y inhibitors, enkephalin, extract of Ginkgo biloba, dioscorea extract, rutin, yerba mate extract, guarana extract, oligosaccharides, polysaccharides, carnitine, ivy extract, fucus extract, extract hydrolyzed Prunella vulgaris, hydrolysed extract of Celosia cristata, Anogeissus leiocarpus extract, Manihot utilisissima leaf extract, palmitoylcarnitine, carnosine, taurine, elderberry extract, seaweed extracts such as Palmaria Palmata extract. By way of illustration, mention is made hereinafter of examples of formulations of a cosmetic composition containing an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides obtained according to the invention:
Example 17: Balm for the eye contour
Ingredients (Brand Name) INCI% w / w
Preparation process: 1. Homogenize phase A in the main container until it is clear; 2. At 25 ° C, sprinkle in Phase B and homogenize for 10 minutes until homogeneous; 3. At 25 ° C, prepare phase C in a separate beaker, mix until homogeneous. Sprinkle in phase D and mix well until well blended; 4. At 25 ° C, add the C + D phase to the main vessel and mix until homogeneous; 5. At 25 ° C, add phase E to the main container and mix until homogeneous; 6. At 25 ° C, premix phase F, add it to the main container and mix until homogeneous; 7. Stop at 25 ° C.
The composition is thus in the form of a violet pearly cream gel, with a pH of between 5.70 and 6.20 and a viscosity (OD) of 80000 - 130000 cps (Brookfield RVT / Spindle C / 5 RPM / 1 minute / 25 ° C).
Example 18: Rich Cream
Ingredients (Brand Name) INCI% w / w
Preparation process: 1. Homogenize phase A in the main container and start heating at 75-80 ° C; 2. At 30 ° C, sprinkle in Phase B and homogenize while warming;
3. In a separate beaker, prepare phase C, heat to 75-80 ° C until homogeneous; 4. At 75 ° C, add phase C to the main vessel and homogenize for 10 minutes; 5. Allow the temperature to cool down and add phase D at 65 ° C. Mix well to homogenize for 10 minutes; 6. Premix E phase before adding it to the main container; 7. Add phase E at 60 ° C. Mix well to homogenize for 10 minutes; 8. At 35 ° C, premix F phase before adding and mixing thoroughly; 9. Premix phase G before adding it to the main container; 10. Add phase G at 35 ° C. Mix well to homogenize; 11. In a separate beaker, prepare phase H: sprinkle Natrosol ™ in water at room temperature and homogenize while heating at 60 ° C; 12. Add phase H at 30 ° C. Mix well to homogenize; 13. Stop at 25 ° C.
The composition is thus in the form of a cream butter rose, with a pH between 4.90 and 5.40 and a viscosity (D0) of 160000 - 210000 cps (Brookfield RVT / Spindle D / 5 RPM / 1 minute / 25 ° C).
Example 19: Face Serum
Ingredients (Brand Name) _INCI_% w / w
Process of preparation: 1. In a beaker at room temperature, weigh the ingredients of phase A and mix. Sprinkle phase B and homogenize; 2. At room temperature, sprinkle in phase C and continue homogenizing the assembly; 3. At room temperature, add phase D to phase ABC and continue homogenizing; 4. At room temperature, add phase E and homogenise; 5. At room temperature, add phase F and homogenise the assembly; 6. At room temperature, add phase G and mix until homogeneous; 7. Stop at 25 ° C.
The composition is thus in the form of a smooth gel, translucent, creamy yellow, with a pH between 6.30 and 7.10 and a viscosity (OD) of 10,000-15,000 cps (Brookfield RVT / Spindle B / 5 RPM / 1 minute / 25 ° C).
Example 20: Anti-aging Mask
Ingredients (Brand Name)
INCI% w / w
Preparation process: 1. At 25 ° C, homogenize phase A in the main container; 2. At 25 ° C sprinkle in phase B and mix well until homogeneous; 3. At 25 ° C add phase C and mix well until homogeneous; 4. Premix phase D in a separate beaker and add to the main container at 25 ° C; 5. At 25 ° C, add phase E to the main container and mix well; 6. Premix phase F and add slowly. Mix well until homogeneous;
7. Premix phase G into a separate beaker and add to the main container until homogeneous; 8. Stop at 25 ° C.
The composition is thus in the form of a cream gel with scintillating green effects, with a pH between 5.30 and 5.80 and a viscosity (OD) of 70000 - 100000 cps (Brookfield RVT / Spindle C / 5 RPM / 1 minute / 25 ° C).
Example 21 Serum
Ingredients (Brand Name) INCI% w / w
Preparation process: 1. Add water to the main container and begin mixing with a hi-lo propeller blade; 2. Add the remaining ingredients, one after the other while mixing between each addition.
The composition is thus in the form of a smooth serum, semi-opaque, with a pH between 5.75 and 6.25 and a viscosity (D0) of 1,100 - 1,400 cps (Brookfield RVT / spindle 3/20 rpm / 25 ° C / 1 minute).
According to a fourth aspect, the invention relates to the cosmetic use of the composition according to the invention for combating the signs of cutaneous aging.
"Signs of skin aging" means any changes in the appearance of the skin due to aging such as, for example, fine lines and wrinkles, cracks, bags under the eyes, dark circles, wilting, loss of skin. elasticity, firmness and / or tone of the skin, but also any internal changes in the skin that do not systematically result in a modified external appearance such as, for example, thinning of the skin, or any internal damage to the skin. skin resulting from environmental stresses such as pollution and UV radiation. The study of the expression of collagens in the skin is a means of judging the anti-aging effect of the invention. Indeed, collagen synthesized by skin fibroblasts has an important biological role. It is responsible for the cohesion of tissues such as the skin and imparts resistance and suppleness properties to the skin. The invention also relates to the cosmetic use of the composition according to the invention for improving the hydration of the skin.
The term "skin hydration" is understood to mean any improvement in the appearance of the skin due to dehydration, such as drought, tightness and discomfort. The study of the expression of hyaluronic acid as well as the enzyme involved in the synthesis of hyaluronic acid is a means of judging the moisturizing effect of the invention. Indeed, hyaluronic acid is a major component of the extracellular matrix of the dermis, also present in the epidermis, and is involved in cutaneous hydration. As such, the invention is illustrated below, by the different test results. For this purpose, similar results (not shown) to those shown in Examples 22 to 25 below were obtained with other aqueous extracts enriched in small RNAs of a length of at most 150 nucleotides according to the invention. obtained from vegetable matter, and more particularly from Chenopodium quinoa (quinoa), Lens esculenta (lentil), Oryza sativa (rice sprouts), or Cucurbita pepo (squash).
EXAMPLE 22 Evaluation of the Effects of Okra Extracts (Hibiscus esculentus) According to Examples 5, 6 and 9 on the Extracellular Matrix of the Derm by the Study of Collagens I and III
The aim of this study is to compare the effects on the extracellular matrix of the dermis of three extracts of okra (Hibiscus esculentus). The first extract enriched in RNA of small molecular weight, obtained according to Example 5, the second extract obtained according to Example 6 (no treatment with EDTA) and the last extract obtained after precipitation according to Example 9.
The aim of this study is to evaluate the effects of these three extracts of Hibiscus esculentus on the expression of collagen I and III proteins involved in the structure of the extracellular matrix. Collagen is very important for maintaining the elasticity and firmness of the skin.
Protocol:
Human skin biopsies 6 mm in diameter are maintained in culture ex vivo in the presence of a specific medium (DMEM 1 g / L, HAMF12, SVF and antibiotics) on inserts deposited in 6-well plates. The biopsies are cultured for 48 hours and receive 2 applications per day of a Okra extract (Hibiscus esculentus) according to Examples 5, 6 and 9 diluted 1/100 or 3 / 100th in PBS, respectively at the final concentration. 1% and 3% volume / volume. The control condition is carried out using PBS IX. The applications are carried out in the form of a drop of approximately 20 μl deposited on the surface of the biopsy. The biopsies are then fixed in formaldehyde and then included in the paraffin. Skin sections of 4 μm thick are then made. The labels of collagens I and III are carried out after unmasking of the specific sites by incubation in the microwave and then trypsin treatment. The immunolabelings are carried out using a polyclonal rabbit antibody specific for collagen I (Rockland, 600-401-103-0.5), a rabbit polyclonal antibody specific for collagen III (Rockland, Ref. 401-105-0.5), followed by a fluorochrome-coupled anti-rabbit secondary antibody (Invitrogen, Ref A21206). The biopsies are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantitation of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) was performed from the photographs obtained. Results:
The treatments with okra extract (Hibiscus esculentus) enriched in small RNAs, obtained according to Example 5, at 1% and 3% make it possible to observe a significant increase in the expression of collagens I and III compared with the condition. control treated with IX PBS and compared with treatments with extracts of okra (Hibiscus esculentus) obtained according to Examples 6 (no treatment with EDTA) and 9 (obtained after precipitation), for the ex vivo study.
CONCLUSIONS: Okra extract (Hibiscus esculentus) enriched in small RNA (example 5) at 1 and 3% further stimulates the expression of collagens I and III in ex vivo human skin compared to two extracts of Hibiscus esculentus no. enriched in small RNA (Examples 6 and 9) at 1% and 3%.
EXAMPLE 23 Evaluation of the Effects of Baobab Extracts (Adansiona digitata) According to Examples 7, 8 and 9 on the Extracellular Matrix of the Derm by the Study of Collagens I and III
The aim of this study is to compare the effects on the extracellular matrix of the dermis of three baobab extracts (Adansiona digitata). The first extract enriched in RNA of small molecular weight, obtained according to Example 7, the second extract obtained according to Example 8 (no treatment with EDTA) and the last extract obtained after precipitation according to Example 9.
The aim of this study is to evaluate the effects of these three baobab extracts (Adansiona digitata) on the expression of collagen I and III proteins involved in the structure of the extracellular matrix. Collagen is very important for maintaining the elasticity and firmness of the skin.
Protocol:
Human skin biopsies 6 mm in diameter are maintained in culture ex vivo in the presence of a specific medium (DMEM 1 g / L, HAMF12, SVF and antibiotics) on inserts deposited in 6-well plates. Biopsies are grown for 48 hours and
receive 2 applications per day of a baobab extract (Adansiona digitata) according to Examples 7, 8 and 9 diluted 1/100 or 3 / 100th in PBS, respectively at the final concentration of 1% and 3% volume / volume. The control condition is carried out using PBS IX. The applications are carried out in the form of a drop of approximately 20 μl deposited on the surface of the biopsy. The biopsies are then fixed in formaldehyde and then included in the paraffin. Skin sections of 4 μm thick are then made. The labels of collagens I and III are carried out after unmasking of the specific sites by incubation in the microwave and then trypsin treatment. The immunolabelings are carried out using a polyclonal rabbit antibody specific for collagen I (Rockland, 600-401-103-0.5), a rabbit polyclonal antibody specific for collagen III (Rockland, Ref. -401-105-0.5), followed by a fluorochrome-coupled anti-rabbit secondary antibody (Invitrogen, Ref A21206). The biopsies are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantitation of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) was performed from the photographs obtained. Results:
Treatment with Baobab extract (Adansiona digitata) enriched in small RNAs, obtained according to Example 7, at 1% makes it possible to observe a significant increase in the expression of collagen I compared with the control condition treated with PBS. IX and compared to the 1% treatments with the Adansiona digitata extracts obtained according to Examples 8 (no treatment with EDTA) and 9 (obtained after precipitation), for the ex vivo study.
Treatment with Baobab extract (Adansiona digitata) enriched in small RNAs, obtained according to Example 7, at 3% makes it possible to observe a significant increase in the expression of collagens I and III compared to the condition control treated with of PBS IX and compared with 3% treatments with baobab extracts (Adansiona digitata) obtained according to Examples 8 (no treatment with EDTA) and 9 (obtained after precipitation), for the ex vivo study.
Conclusions; Baobab extract (Adansiona digitata) enriched in small RNA (example 7) at 1% further stimulates the expression of collagen I in ex vivo human skin compared to extracts of Adansiona digitata not enriched in small RNA (Examples 8). and 9) at 1%. Baobab extract (Adansiona digitata) enriched in small RNA (example 7) at 3% further stimulates the expression of collagens I and III in ex vivo human skin compared to extracts of Adansiona digitata not enriched in small RNA ( Examples 8 and 9) at 3%.
EXAMPLE 24 Evaluation of the Effects of Baobab Extracts (Adansiona digitata) According to Examples 7, 8 and 9 on the Synthesis of Hyaluronic Acid in the Derm by the Study of Hyaluronan Synthase 2 (HAS2)
The purpose of this study is to compare the effects of three baobab extracts (Adansiona digitata) on the expression of
HAS2, an enzyme involved in the synthesis of hyaluronic acid. The first extract enriched in low molecular weight RNA is obtained according to Example 7, the second extract obtained according to Example 8 (no treatment with EDTA) and the last extract obtained after precipitation according to Example 9. Hyaluronic acid is a major component of the extracellular matrix of the dermis, involved in the hydration of the skin. During aging, its renewal is disrupted, as well as the expression of its synthetic enzyme HAS2 (Rock et al., 2014).
Protocol:
Human fibroblasts are cultured in a specific medium and maintained in a long treatment culture (for more than 32 passages). At each passage, part of the cells is frozen. Then, two opposite passages are chosen: passage 8 (passage young) and passage 32 (passage senescent). After thawing, the senescent fibroblasts or not are treated with baobab extracts (Adansiona digitata) according to Examples 7, 8 and 9 diluted 1/100 in culture medium, or at the final concentration of 1% volume / volume, 48 hours (2 applications per day). The evaluation of the expression of HAS2 on senescent or non-senescent fibroblasts treated or not treated with the three extracts of Adansiona digitata according to Examples 7, 8 and 9 at 1% is observed by immunostaining.
To do this, the cells are rinsed and fixed with cold methanol for 5 minutes. After saturation of non-specific sites with 1% bovine serum albumin for 15 min, the cells are incubated with a mouse monoclonal antibody solution specific for HAS2 (Thermo Fisher, Ref MAS-17087), then a fluorochrome-coupled anti-mouse secondary antibody solution (Invitrogen, Ref A21202). The cells are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantitation of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) was performed from the photographs obtained. Results:
As illustrated by FIG. 4A, treatment with baobab extract (Adansiona digitata) enriched in small RNAs of a length of at most 150 nucleotides, obtained according to Example 7 at 1% on passage fibroblasts 8 (passages considered young) allows to observe a significant increase in the expression of HAS2 compared to the untreated condition and also compared to the 1% treatments with the extracts of Adansiona digitata obtained according to Examples 8 (no treatment with EDTA) and 9 (obtained after precipitation).
In addition, in agreement with the literature, as shown in Figure 4B, a significant decrease in HAS2 expression is observed between the passage (P8) and passage (P32) fibroblasts (senescent).
Treatment with Baobab extract (Adansiona digitata) enriched in small RNA according to the invention, obtained according to Example 7 at 1% (volume / volume) on senescent fibroblasts makes it possible to observe a maintenance of HAS2 expression. This maintenance is not observed on the cells treated with the extracts of Adansiona digitata obtained respectively according to Examples 8 (no treatment with EDTA) and 9 (obtained after precipitation).
Conclusion: Baobab extract (Adansiona digitata) enriched in small RNAs up to a maximum of 150 nucleotides (Example 7) at 1% further stimulates the expression of HAS2 in fibroblasts compared to the non-essential condition. treated and in comparison with the extracts of Adansiona digitata obtained according to Examples 8 (no treatment with EDTA) and 9 (obtained after precipitation).
In addition, on senescent fibroblasts, Baobab extract (Adansiona digitata) enriched in small RNA according to the invention (Example 7) at 1% makes it possible to maintain the level of expression of HAS2 observed on the non-senescent fibroblasts. The other extracts of Adansiona digitata not enriched in small RNA (Examples 8 and 9) at 1% do not allow this maintenance.
EXAMPLE 25 Evaluation of the Effects of the Okra Extract (Hibiscus esculentus) According to Example 5 and of the Baobab Extract (Adansiona digitata) According to Example 7 on the Expression Level of Hyaluronic Acid
The purpose of this study is to visualize the effect of the extracts of Okra (Hibiscus esculentus) and Baobab (Adansiona digitata) respectively obtained according to Examples 5 and 7, extracts enriched in RNA of small molecular weight of a length of maximum 150 nucleotides, on the expression of hyaluronic acid by labeling. Hyaluronic acid is a major component of the extracellular matrix of the dermis and involved in the hydration of the skin.
Protocol:
Human skin biopsies 6 mm in diameter are maintained in culture ex vivo in the presence of a specific medium (DMEM 1 g / L, HAMF12, SVF and antibiotics) on inserts deposited in 6-well plates. The biopsies are cultured for 48 hours and receive 2 applications per day of the extract of okra (Hibiscus esculentus) or of the baobab extract (Adansiona digitata) according to Examples 5 and 7 (respectively) diluted to 1% (volume / volume). ) in PBS or IX PBS for the control condition. The applications are carried out in the form of a drop of approximately 20 μl deposited on the surface of the biopsy. The biopsies are then fixed in formaldehyde and then included in the paraffin. Skin sections of 4 μm thick are then made. The sections are incubated in the presence of a biotinylated protein specific for hyaluronic acid (Coger, ref: 400-763-1A), then in the presence of streptavidin coupled to a fluorochrome (Invitrogen, Ref .: S32354). The biopsies are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantitation of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) was performed from the photographs obtained. Results:
Treatment with the okra extract (Hibiscus esculentus) or the baobab extract (Adansiona digitata) enriched in small RNAs, obtained respectively according to Examples 5 and 7, at 1% makes it possible to observe a significant increase in the expression hyaluronic acid in the epidermis and in the dermis compared to the control condition treated with PBS IX.
Conclusion: Okra extract (Hibiscus esculentus) and baobab extract (Adansiona digitata) enriched in small RNAs with a maximum length of 150 nucleotides (according to Examples 5 and 7) at 1% stimulate expression. hyaluronic acid in ex vivo human skin compared to control condition treated with IX PBS.
Of course, the invention is not limited to the embodiments and to the examples presented above, and the person skilled in the art, by virtue of routine operations, may be led to make other embodiments that are not explicitly described. which fall within the broad scope of the invention.

权利要求:
Claims (20)
[1" id="c-fr-0001]
1. A process for obtaining an aqueous extract enriched in small RNAs having a length of at most 150 nucleotides from a plant material comprising the following steps: a) the plant material is brought into contact with the plant material; water; b) tetrasodium ethylenediamine tetraacetic acid (EDTA) is added to the mixture obtained in a) at a pH of between 10.5 and 11; c) the pH of the mixture obtained in b) is then adjusted to a value of between 6 and 8; d) purifying the mixture obtained in c) so as to remove the residual solid plant material and obtain a purified aqueous crude extract; and e) the pH is controlled and readjusted if necessary to a value between 6 and 8, preferably between 6 and 6.5.
[2" id="c-fr-0002]
2. Method according to claim 1 characterized in that in step a) the plant material is brought into contact with water in a vegetable matter / water ratio of 4 to 20% (w / w).
[3" id="c-fr-0003]
3. Method according to one of claims 1 or 2 characterized in that the b) treatment step with tetrasodium ethylenediamine tetraacetic acid (EDTA) is carried out with stirring for a time of at least 1 h and at a temperature of between 20 and 80 ° C.
[4" id="c-fr-0004]
4. Method according to one of claims 1 to 3 characterized in that in step d) centrifuging the mixture obtained in c).
[5" id="c-fr-0005]
5. Method according to one of claims 1 to 4 characterized in that step e) is preceded by at least one filtration of the aqueous crude extract obtained in d) and preferably by successive filtrations of the aqueous crude extract by lowering the filtration threshold from 20-50 μιη to 0.1-0.30 μπι.
[6" id="c-fr-0006]
6. Method according to one of claims 1 to 5, characterized in that it comprises an additional step of hydrolysis carried out before step c), either directly on the mixture obtained in b), or on the supernatant after centrifugation of the mixture obtained in b) and elimination of the residual plant material by the action of at least one enzyme chosen from a carbohydrase, a cellulase and / or a protease, for at least 1 h, at a temperature of between 45 and 65 ° C. and at a pH of between 6 and 8.5.
[7" id="c-fr-0007]
7. Method according to claim 6, characterized in that it comprises a step of deactivating the enzyme at a temperature between 65 and 80 ° C for at least 1 h, this deactivation step being carried out directly after step d hydrolysis or between 2 successive filtration steps preceding step e).
[8" id="c-fr-0008]
8. Method according to one of the preceding claims, characterized in that diatomaceous earth or silica at a concentration of 10 to 20 g / kg are added to the mixture before step d).
[9" id="c-fr-0009]
9. Method according to one of the preceding claims, characterized in that the concentration of tetrasodium EDTA is between 2 and 15 mM, preferably 10 mM.
[10" id="c-fr-0010]
10. Method according to one of the preceding claims, characterized in that the plant material is milled before being placed in the presence of water in step a).
[11" id="c-fr-0011]
11. Method according to one of the preceding claims, characterized in that the plant material is a plant part selected from seeds, fruits, bulbs, flowers, leaves, roots, germ or cakes and the dreches.
[12" id="c-fr-0012]
12. Method according to one of the preceding claims, characterized in that the plant material is selected from the families of Passifloraceae, Malvaceae, Punicaceae, Actinidiaceae, Myrtaceae, Clusiaceae, Caricaceae, Malphigiaceaer Ericaceaer Grossulariaceae, Solanaceae, Poaceae, Fabaceae, Malvaceae, Bombacaceae, Cucurbitaceae, Chenopodiaceae, Rosaceae, Rutaceae, Liliaceae, Iridaceae, Amaryllidaceae, Alliaceae.
[13" id="c-fr-0013]
13. The method of claim 12, characterized in that the plant material is selected from the species Hibiscus esculentus (okra), Adansonia digitata (baobab), Chenopodium quinoa (quinoa), Lens esculenta (lens), Oryza sativa (rice germ ), Cucurbita pepo (squash), Rosa centifolia (pink), Citrus aurantium (bitter orange), Lilium candidum (lily), Lilium tigrinum (tiger lily) r Passiflora alata (grenadilla).
[14" id="c-fr-0014]
14. An aqueous extract of vegetable matter, enriched in small RNAs of a length of at most 150 nucleotides obtainable by the method according to one of claims 1 to 13, characterized in that it comprises by weight of total weight of the extract, 5-60 g / kg of solids and 10-1000 mg / kg of small RNAs up to 150 nucleotides in length, 0.5 to 30 g / kg of protein fragments and 0.5 to 50 g / kg of sugars and and does not include DNA.
[15" id="c-fr-0015]
15. Extract according to claim 14, characterized in that it is diluted in a solvent and comprises by weight of the total weight of the extract, 5-35 g / kg of dry extract, 0.5-20 g / kg. protein fragments, 0.5-30 g / kg of sugars, and 10-500 mg / kg of small RNAs up to 150 nucleotides in length.
[16" id="c-fr-0016]
16. A composition comprising, as an antiaging active agent, an effective amount of the extract of one of claims 13 to 15, and a physiologically acceptable medium.
[17" id="c-fr-0017]
17. Composition according to claim 16, characterized in that the extract is present at a concentration of between 1 and 5% by weight of the total weight of the composition.
[18" id="c-fr-0018]
18. Composition according to one of claims 16 to 18, characterized in that it is formulated to be applied topically to the skin.
[19" id="c-fr-0019]
19. Cosmetic use of the composition of one of claims 16 to 18 for combating the signs of skin aging.
[20" id="c-fr-0020]
20. Cosmetic use of the composition of one of claims 16 to 18 for improving the hydration of the skin.

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US11021505B2|2021-06-01|

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KR20180080319A|2018-07-11|

---

EP3377623A1|2018-09-26|

---

EP3377623B1|2021-10-20|

---

WO2017084958A1|2017-05-26|

---

JP2018537455A|2018-12-20|

---

CN108473980A|2018-08-31|

---

CA3005605A1|2017-05-26|

---

HK1256880A1|2019-10-04|

---

FR3043554B1|2019-07-12|

---

US20180371000A1|2018-12-27|

---

HK1251015A1|2019-01-18|

---

CN108291222A|2018-07-17|

---

EP3377624A1|2018-09-26|

---

US20200246252A1|2020-08-06|

---

JP2018533615A|2018-11-15|

---

BR112018009930A2|2018-11-13|

---

MX2018005944A|2018-08-14|

---

JP6826597B2|2021-02-03|

---

FR3043695B1|2019-10-25|

---

WO2017087245A1|2017-05-26|

---

EP3377624B1|2021-07-21|

---

KR20180081800A|2018-07-17|

---

CA3005605C|2020-10-13|

---

FR3043695A1|2017-05-19|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

DE19820629A1|1998-05-08|1999-11-11|Forschungsanstalt Geisenheim|An extraction process for RNA and DNA from wooden, non-wooden and herbal plant materials|

---

WO2002057289A1|2001-01-16|2002-07-25|Invitrogen Corporation|Reagent for the isolation of rna|

---

FR2831168A1|2001-10-22|2003-04-25|Rocher Yves Biolog Vegetale|PROCESS FOR OBTAINING A NUCLEIC ACID-RICH EXTRACT FROM PLANT MATERIAL|

---

WO2003101376A2|2002-06-03|2003-12-11|L'oreal|Topical use of at least one double-stranded rna oligonucleotide |

---

WO2004046305A2|2002-11-18|2004-06-03|Agency For Science, Technology And Research|Method and system for cell and/or nucleic acid molecules isolation|

---

WO2005034648A2|2003-10-01|2005-04-21|Sensient Flavors Inc.|Method for the production of pending natural botanical extracts|

---

EP1723958A2|2005-05-19|2006-11-22|L'oreal|dsRNA vectorisation with cationic particles and their topical use|

---

FR2912313A1|2007-02-13|2008-08-15|Biolog Vegetale Yves Rocher Sa|Combination, useful for the preparation of cosmetic or dermatological composition to prevent and/or treat skin aging, comprises wheat germ extract and esculin|

---

CN101601635A|2008-06-13|2009-12-16|殷勤伟|The prescription of little RNA/DNA whitening product and manufacture method|

---

FR2940112A1|2008-12-22|2010-06-25|Rocher Yves Biolog Vegetale|Extraction of compounds and/or active ingredients present in plant material, comprises grinding the plant material at temperature lower than the freezing temperature of plant material, and adding enzyme to the obtained plant homogenate|CN111346142A|2018-12-22|2020-06-30|大江生医股份有限公司|Application of seville orange flower extract in preparation of composition for improving anti-saccharification activity, inhibiting fat accumulation and promoting lipolysis|DE1540597C3|1965-11-29|1975-01-16|Siemens Ag, 1000 Berlin Und 8000 Muenchen|High voltage bushing|

---

FR2543438B1|1983-03-30|1987-07-31|Duraffourd Alain|COMPOSITION FOR REGENERATION OF CELLS AND TISSUES OF THE SKIN AND PROCESS FOR OBTAINING SAME|

---

FR2817748B1|2000-12-13|2003-01-17|Seporga|COSMETIC AND / OR DERMATOLOGICAL COMPOSITION CONTAINING ARTEMIA SALINA EXTRACT|

---

FR2827170B1|2001-07-13|2004-07-16|Soc Extraction Principes Actif|USE OF PEPTIDES TO INCREASE CELL ADHESION|

---

FR2837098B1|2002-03-18|2004-05-28|Vincience|COSMETIC OR PHARMACEUTICAL COMPOSITION COMPRISING PEPTIDES, METHODS OF TREATMENT AND USES|

---

FR2840217B1|2002-06-03|2005-06-24|Oreal|COSMETIC COMPOSITIONS COMPRISING AT LEAST ONE DOUBLE-STRANDED RNA OLIGONUCLEOTIDE AND USES THEREOF|

---

FR2841781B1|2002-07-03|2005-12-16|USE OF PEPTIDES TO PROMOTE SKIN REGENERATION|

---

FR2846883B1|2002-11-08|2004-12-24|Vincience|COSMETIC COMPOSITION COMPRISING, AS ACTIVE INGREDIENT, AT LEAST ONE PEPTIDE AND USE OF THIS PEPTIDE|

---

US9139850B2|2005-05-19|2015-09-22|L'oreal|Vectorization of dsRNA by cationic particles and topical use|

---

JP5025253B2|2006-03-31|2012-09-12|日生バイオ株式会社|Formulation for basic cosmetics and basic cosmetics|

---

FR2944526B1|2009-04-15|2013-05-10|Isp Investments Inc|COSMETIC AND / OR PHARMACEUTICAL COMPOSITION COMPRISING A PEPTIDE HYDROLYZATE CAPABLE OF STRENGTHENING BARRIER FUNCTION|

---

JP2011016760A|2009-07-09|2011-01-27|Noevir Co Ltd|Skin care external preparation, oral agent, moisturizing agent, anti-aging agent, bleaching agent, and anti-oxidizing agent containing bulb of lilium plant and/or callus extract as effective ingredient|

---

FR2951946B1|2009-11-03|2012-05-11|Isp Investments Inc|USE OF PEPTIDE HYDROLYSAT OF YEAST AS AN ACTIVE AGENT FOR STRENGTHENING THE HAIR|

---

FR2956818B1|2010-02-26|2012-07-20|Isp Investments Inc|USE OF PEPTIDE LINK HYDROLYSAT IN A COMPOSITION FOR SOOTHING SKIN|

---

CN102146374B|2011-01-27|2012-10-31|四川农业大学|Cell lysis solution for extracting animal DNA, kit and method|

---

CN103211738B|2011-09-27|2015-08-26|浙江欧诗漫生物股份有限公司|A kind of shining face containing peony root extractive and Margarita powder repaiies the preparation method holding cream composition|

---

CN102776174A|2012-08-21|2012-11-14|昆明理工大学|Method for simultaneously extracting DNA and RNA from lily tissue|

---

CN103705392B|2012-09-29|2015-02-18|黄兵|Small nucleic acid whitening and freckle-removing cream, and preparation method thereof|

---

CN104138346B|2014-08-07|2016-09-21|广州雅纯化妆品制造有限公司|A kind of anti-wrinkle cosmetic composition and preparation method thereof|KR102041867B1|2018-02-23|2019-11-06|주식회사 위노바|Skin cosmetic composition comprising the extract of Hibiscus Esculentus, Chenopodium quinoa, PunicagranatumL., Perilla frutescens, Chamaecyparis obtusa, Propolis and synthetic peptide|

---

KR102237543B1|2019-02-22|2021-04-07|모아캠|Method of Producing DNA Derived from Aloe Genus Plant, and Composition Containing DNA Derived from Aloe Genus Plant for Anti-Aging and Anti-Inflammation|

---

WO2020179823A1|2019-03-04|2020-09-10|三井化学株式会社|Method for determining whether organism having cell wall exists and method for identifying organism having cell wall|

---

FR3106754B1|2020-02-04|2022-01-07|Isp Investments Llc|METHOD FOR OBTAINING AN AQUEOUS LAVENDER EXTRACT, COMPOSITIONS COMPRISING SUCH AN EXTRACT AND THEIR COSMETIC USES|

法律状态:
2017-08-11| PLSC| Publication of the preliminary search report|Effective date: 20170811 |

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2017-11-27| PLFP| Fee payment|Year of fee payment: 2 |

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2019-11-25| PLFP| Fee payment|Year of fee payment: 4 |

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2020-11-25| PLFP| Fee payment|Year of fee payment: 5 |

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2021-11-24| PLFP| Fee payment|Year of fee payment: 6 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1502361A|FR3043695B1|2015-11-17|2015-11-17|PROCESS FOR OBTAINING AQUEOUS EXTRACT ENRICHED IN SMALL RNA FROM PLANT MATERIAL AND EXTRACTS FROM THE PROCESS|

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FR1502361|2015-11-17|

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