阅读说明：本技术 获得莳萝的小rna富集水性提取物的方法 (Method for obtaining small RNA-enriched aqueous extract of dill ) 是由 E·奥热 J-M·博托 于 2018-04-27 设计创作，主要内容包括：本发明涉及一种从植物材料获得富集最大长度为150个核苷酸的小RNA的莳萝(Anethum graveolens)水性提取物的方法,所述方法包含以下步骤：a)将莳萝地上部分与水接触；b)在pH为10.5至11之间时加入乙二胺四乙酸(EDTA)四钠；c)然后将b)中获得的混合物的pH调节至6至8之间的值；d)将c)中获得的混合物纯化；并且e)检查pH并且如果需要,将其重新调节为6至8之间的值。本发明还涉及通过该方法获得的富集最大长度为150个核苷酸的小RNA的莳萝水性提取物。(The present invention relates to a method for obtaining an aqueous extract of dill (Anethum graveolens) enriched in small RNAs of maximum length 150 nucleotides from plant material, the method comprising the steps of: a) contacting the aerial parts of dill with water; b) adding tetrasodium ethylenediaminetetraacetic acid (EDTA) at a pH of between 10.5 and 11; c) then adjusting the pH of the mixture obtained in b) to a value between 6 and 8; d) purifying the mixture obtained in c); and e) checking the pH and, if necessary, readjusting it to a value between 6 and 8. The invention also relates to an aqueous dill extract enriched in small RNAs of maximum length 150 nucleotides obtained by this process.)

1. A process for obtaining an aqueous extract of the aerial parts of dill (Anethum graveolens) from plant material, the extract being enriched in small RNAs of maximum length 150 nucleotides, the process comprising the steps of:

a) contacting the aerial parts of dill with water;

b) adding tetrasodium ethylenediaminetetraacetic acid (EDTA) to the mixture obtained in a), the pH of the mixture being between 10.5 and 11;

c) then adjusting the pH of the mixture obtained in b) to a value between 6 and 8;

d) purifying the mixture obtained in c) to eliminate residual solid plant material and obtain a purified crude aqueous extract; and is

e) The pH is checked and readjusted if necessary to a value between 6 and 8, preferably between 6 and 6.5.

2. The method according to claim 1, characterized in that in step a) the plant material, previously dried and then ground, is contacted with water in a plant/water ratio of 4 to 20% (weight/weight).

3. The process according to any one of claims 1 or 2, characterized in that step b) by treatment with tetrasodium ethylenediaminetetraacetic acid (EDTA) is carried out with stirring for at least 1 hour and at a temperature between 20 and 80 ℃.

4. The method according to any one of claims 1 to 3, characterized in that in step d) the mixture obtained in c) is centrifuged.

5. The process of any one of claims 1 to 4, characterized in that the crude aqueous extract obtained in d) is subjected to at least one filtration before step e), and the crude aqueous extract is preferably continuously filtered by lowering the filtration threshold from 20-50 μm to 0.1-0.30 μm.

6. The method according to any of the preceding claims, characterized in that the concentration of tetrasodium EDTA is between 2 and 15mM, preferably 10 mM.

7. An aqueous extract of dill aerial parts enriched in small RNAs of maximum length 150 nucleotides and comprising no DNA obtained by the method of any one of claims 1 to 6, characterized in that it comprises: 0 to 30g/kg of dry weight extract, 2 to 10g/kg of protein fragments, 2 to 10g/kg of sugar, 0.2 to 3g/kg of amino acids, 100 to 2000mg/kg of phenol compounds, and 10 to 100mg/kg of low molecular weight RNA having a maximum length of 150 nucleotides, based on the weight relative to the total weight of the extract, and does not contain DNA.

8. Extract as claimed in claim 7, characterized in that it is diluted in a solvent and comprises, by weight relative to the total weight of the extract, 5 to 15g/kg of dry extract, 50 to 1000mg/kg of polyphenols, 0.5 to 10g/kg of protein fragments, 0.5 to 10g/kg of sugars, 0.1 to 1g/kg of amino acids, and 10 to 100mg/kg of small RNAs having a maximum length of 150 nucleotides.

Detailed Description

The present invention relates to a process for obtaining an aqueous extract enriched in small RNAs of maximum length 150 nucleotides from dried or fresh aerial parts of dill.

The term "aerial parts" refers to the leaves, stems and flowers of dill.

In the sense of the present invention, seeds are not included in the "aboveground parts".

In the course of the description, the terms "dill" and "Anethum grandis" are used synonymously and have the same meaning.

The term "small RNA" or "low molecular weight RNA" refers to non-coding RNA (ribonucleic acid) of low molecular weight and a maximum length of 150 nucleotides, such as all types of non-messenger small RNA (single and/or double stranded), e.g. micro RNA, interfering RNA, introns, small nuclear RNA or any RNA fragment.

In the first step a) of the process according to the invention, the dill plant material or ground, dried above-ground portion is contacted with water, preferably at a plant material/water ratio of from 4 to 20% w/w, more preferably at a ratio of from 5 to 10% w/w, for example at a ratio of 5.7 or 10 w/w.

The water used is distilled demineralized water or water enriched with mineral salts and/or oligoelements, preferably distilled water.

The aerial parts of dill are preferably in dry form.

Preferably, the plant material is ground in step a) before it is contacted with water. Grinding is a mechanical action that improves the extraction. Mechanical grinding followed by alkaline lysis in the presence of EDTA promotes complete deconstruction of cell membranes, particularly nuclear membranes.

Tetrasodium ethylenediaminetetraacetate is then added to the mixture obtained in a) in step b). The pH is basic in this step and must be adjusted to a value between 10.5 and 11 by adding sodium (NaOH) if necessary. During step b), the basic pH must be maintained between 10.5 and 11. In fact, this pH, in combination with the action of EDTA, causes the deconstruction of the cell membrane (including the nuclear membrane), the lysis of the cells and the denaturation of the DNA (2-strand separation of the double helix). pH check in step b) showed that it remained basic and stabilized between 9 and 11.

The concentration of tetrasodium ethylenediaminetetraacetate is preferably between 2 and 15mM, more preferably 10 mM.

This concentration was chosen to optimize the extraction yield of low molecular weight RNA in the final extract. Tetrasodium ethylenediaminetetraacetate will weaken and destroy the pectin cellulose membrane of plant cells by chelation of divalent ions, such as calcium ions, which form ionic bridges between pectin molecules surrounding the cellulose microfibrils. The result is enhanced release of cell contents during extraction. The step of EDTA treatment is critical for the enrichment of the extract with low molecular weight RNA.

The step of EDTA treatment is preferably carried out at a temperature between 20 and 80 ℃ for at least 1 hour. During this step, the mixture obtained in a) is advantageously stirred.

In step c), the pH of the mixture obtained in b) is adjusted to a value between 6 and 8.

For example, the pH is adjusted by adding a solution of hydrochloric acid (HCl) or any acid capable of changing the pH and compatible with cosmetic use, such as citric acid or lactic acid.

This acidification step results in sudden renaturation of the DNA (strand re-pairing of the double strand). However, very long chromosomal DNA cannot perfectly re-pair and form insoluble tangles. In contrast, much shorter small RNAs remain in solution. DNA and small RNA were then separated into two separate phases: a solid phase comprising, inter alia, chromosomal DNA, and a liquid phase comprising, inter alia, small RNA.

In step d), the mixture obtained in c) is purified to remove plant material and to recover a crude aqueous 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 at 4000g for at least 10 minutes, to precipitate the residual plant material in the precipitate and to recover the crude aqueous extract in the supernatant.

In step d) the pH is checked and readjusted to a value between 6 and 8. The pH is preferably readjusted to a value between 6 and 6.5, even more preferably 6.5. The pH is readjusted by adding hydrochloric acid (HCl) or sodium (NaOH) solution.

In fact, a pH below 6 can generally cause precipitation of nucleic acids and thus low molecular weight RNA with a maximum length of 150 nucleotides.

The step of adjusting the pH in step d) of the method according to the invention is an essential step for optimal extraction of low molecular weight RNA.

Advantageously, the crude aqueous extract obtained in d) is subjected to at least one filtration before readjusting the pH in step d). Continuous filtration is preferably performed by lowering the filtration threshold from 50 to 20 μm to 0.4, 0.3, 0.2 or 0.1 μm.

The second subject of the present invention is the aqueous dill extract of the present invention, obtained by the above process, which is enriched in small RNAs with a maximum length of 150 nucleotides.

The extract contains no DNA (deoxyribonucleic acid).

This aqueous extract enriched for small RNAs with a maximum length of 150 nucleotides contained, before dilution: 10 to 30g/kg of dry extract, 2 to 10g/kg of protein fragments, 2 to 10g/kg of sugar, 0.2 to 3g/kg of amino acids, 100 to 2000mg/kg of phenol compounds, and 10 to 100mg/kg of low molecular weight RNA having a maximum length of 150 nucleotides, by weight relative to the total weight of the extract. The extract thus obtained is considered to be concentrated. It can then be diluted in a physiologically acceptable solvent for cosmetic use, in order to adjust the concentration of the extract to the particular dry extract weight required.

Illustrative and non-limiting examples of physiologically acceptable solvents include water, glycerol, ethanol, propylene glycol (natural forms derived from corn are known as) Butylene glycol, dipropylene glycol, ethoxylated or propoxylated diethylene glycol, cyclic polyols, or any mixture of these solvents.

The extract obtained by the process of the present invention is preferably diluted in a solvent such as 30% glycerol and water and contained by weight relative to the total weight of the extract. The extract obtained by the method of the invention also comprises 5-15g/kg of dry extract, 50 to 1000mg/kg of polyphenols, 0.5-10g/kg of protein fragments, 0.5-10g/kg of sugars, 0.1 to 1g/kg of amino acids, and 10-100mg/kg of small RNAs with a maximum length of 150 nucleotides.

Preferred embodiments of the process according to the invention will be described hereinafter by way of example.