WO2019034614A1

WO2019034614A1 - Pseudotsuga menziesii extract, method for obtaining same and use thereof

Info

Publication number: WO2019034614A1
Application number: PCT/EP2018/071935
Authority: WO
Inventors: Oriane BEAUDUC; Stéphane TARRADE; Vincent SOL; Philippe Bressollier; Christian RIBES
Original assignee: Centre De Valorisation Agro Ressources; Universite De Limoges; Xylex
Priority date: 2017-08-18
Filing date: 2018-08-13
Publication date: 2019-02-21
Also published as: FR3070126A1; FR3070126B1

Abstract

The invention concerns a method for simultaneously extracting at least one lipophilic molecule and at least one polyphenolic compound from Pseudotsuga menziesii, comprising at least one step of extraction by percolation diffusion of a pressurised solvent in a fixed bed reactor carried out from a raw material derived from Pseudotsuga menziesii. The invention also concerns the concentrate obtained during the implementation of the method, and the extracts obtained and the uses of same.

Description

PSEUDOTSUGA MENZIESII EXTRACT, PROCESS FOR OBTAINING AND USES

The present invention relates to a method for extracting molecules from a raw material derived from Pseudotsuga menziesii which makes it possible to extract at least one lipophilic molecule and at least one polyphenolic compound. The method makes it possible to obtain, in a first phase, a concentrate which contains at least one lipophilic molecule and at least one polyphenolic compound which is also the subject of the invention, and then the separation of the molecules of interest leads to obtaining at least one Pseudotsuga menziesii extract comprising at least one lipophilic molecule and at least one Pseudotsuga menziesii extract comprising at least one polyphenolic compound. These extracts are also the subject of the present invention, as well as their uses.

Plant extracts are increasingly used in the industry, especially in the medical, cosmetic and dietary supplements. Among the raw materials used, plants and flowers have long been used, but more recently research has focused on tree-derived products, including pine extracts that contain molecules of interest such as polyphenols. This is particularly the case of Pseudotsuga menziesii.

Pseudotsuga menziesii also known as Douglas fir or Oregon pine or Pseudotsuga douglasii Carr or Pseudotsuga taxifolia or British Columbia Pine, is a tree native to the western part of the North American continent. Discovered in 1792 by A. MENZIES, it was imported to Europe in 1827, thanks to Sir D. DOUGLAS Scottish botanist. His first appearance in France dates from 1842 when he was first installed in parks. Since 1960, as part of the reforestation policy initiated by the State with the support of the National Forestry Fund (created in 1946), the Douglas has gradually supplanted the Spruce, to the point of becoming today the first essence of reforestation of the national territory. As a result, France is the European country with the most important Douglas resource. It occupies today, a surface of 420 000 ha. Five territories alone account for more than 70% of Douglas stands: New Aquitaine (Limousin), Burgundy- Franche-Comté (Burgundy), Auvergne-Rhône-Alpes (Auvergne), Brittany, Rhône-Alpes and Occitanie (Midi-Pyrenees).

Wood residues, such as bark, are mainly exploited for energy purposes. Nevertheless, Douglas fir and in particular its bark, is relatively rich in waxes, fibers, polyphenolic compounds including lignans, flavonoids including flavonols such as Quercetine / Dihydroquercetine (Taxifoline), condensed tannins (JF Hall, 1971; Kiehlmann and Li 1995, Trivaleto et al 2016). This makes it an economically attractive raw material because of the bioactive compounds it contains.

More recently, a more comprehensive study of the composition of Douglas-fir bark has been completed (Ferreira et al., 2015, Ferreira et al., 2016). These publications reveal for the barks, an ash content of 5.9% with a majority of Calcium and Potassium; a high proportion of lignin (29.5%) and suberin (22%) as well as many extractibles (various polyphenols including lignans, flavonoids (flavonols, condensed tannins ...), triterpenes, phytosterols, derivatives of ferrulic acid, fatty acids and esterified fatty alcohols ...). The polysaccharides of the Douglas bark are predominantly composed of glucose.

Currently no suitable method exists for the simultaneous extraction of these molecules. Indeed, the few processes described in the prior art are not efficient, they are difficult to industrialize and only make it possible to value only a part of the molecules contained in the raw material, either polyphenols or lipophilic molecules. This is the case, for example, with the process described in the application US3131198, which describes a process for the aqueous extraction of Taxifolin from Douglas-fir bark and a purification process using pentyl acetate, and process described in the application US3789058 which describes a process for extracting waxes from Douglas-fir with a mixture of alkanes derived from petrochemistry. These processes do not meet the criteria of eco-extraction.

An object of the invention is therefore to overcome the disadvantages of the prior art and to provide an efficient process in terms of efficiency, feasible on an industrial scale and respectful of the environment, which allows the simultaneous extraction of a majority of the molecules of interest contained in the products derived from Pseudotsuga menziesii, so as to maximize the value of the co-products of the sector.

For this purpose the invention is directed to a process for simultaneous extraction of at least one lipophilic molecule and at least one polyphenolic compound of Pseudotsuga menziesii, comprising at least one step of extraction by percolation diffusion of a solvent under pressure in fixed bed reactor made from a raw material derived from Pseudotsuga menziesii. Advantageously, the invention makes it possible to obtain, with a single process, the extraction of lipophilic molecules and at least one polyphenolic compound.

The implementation of the method according to the invention makes it possible to obtain an intermediate product, a concentrate, which comprises at least one lipophilic molecule and at least one polyphenolic compound. The separation of the different phases of this concentrate makes it possible to obtain on the one hand an insoluble extract between 2 and 8 ° C comprising at least one lipophilic molecule and on the other hand a soluble extract between 2 and 8 ° C comprising at least one compound polyphenol. The concentrate and the extracts are also the subject of the invention.

Advantageously, the process according to the invention makes it possible to use all of the raw material, without generating non-valuable co-products or waste.

Finally, the extracts obtained according to the invention have properties, in particular:

The insoluble extract has physical properties (hydrophobic, texturizing, melting temperature between 63 and 65 ° C, co-emulsifier) and biological and can therefore be used in cosmetic compositions, or cleaning products and / or cleaning .

The soluble extract has antioxidant, anti-inflammatory and / or antimicrobial properties, and can therefore be used in cosmetic compositions, food supplements, food compositions or cleaning products.

Therefore, the invention also relates to these uses.

Other features and advantages of the invention will emerge from the description in detail which follows with reference to the appended figures in which:

FIG. 1 is a histogram representing the cytotoxicity measured as a function of the dilution of a soluble extract according to the invention in the cell culture medium,

FIG. 2 is a histogram representing the anti-inflammatory activity of a soluble extract according to the invention, measured in the presence of LPS for the dilutions considered as being non-cytotoxic. CHALLENGE NITIONS

By "raw material derived from Pseudotsuga menziesii" within the meaning of the invention is meant any raw material derived from all or part of the Pseudotsuga menziesii tree, in particular bark, knots, platelets, strains, roots and / or sawdust.

For the purposes of the invention, the term "agro-solvent" is intended to mean a solvent produced from biomasses originating from agriculture.

By "insoluble extract" or "insoluble extract between 2 and 8 ° C" within the meaning of the invention means an extract obtained from a raw material derived from Pseudotsuga menziesii, insoluble in the extraction solvent between + 2 and + 8 ° C.

By "soluble extract" or "soluble extract between 2 and 8 ° C" within the meaning of the invention is meant an extract obtained from a raw material derived from Pseudotsuga menziesii, soluble in the extraction solvent between +2 and + 8 ° C.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the invention is therefore a process for the simultaneous extraction of at least one lipophilic molecule and at least one polyphenolic compound of Pseudotsuga menziesii, comprising at least one step of extraction by percolation of diffusion of a solvent under pressure. Fixed bed reactor, made from a raw material derived from Pseudotsuga menziesii.

The raw material derived from Pseudotsuga menziesii is preferably chosen from the barks, knots, platelets, strains, roots and / or sawdust of Pseudotsuga menziesii. It is collected especially at sawmills, usually after treatment of felled trees, so that it can be assured of its quality and traceability.

Preferably, the raw material comes from forests and sawmills certified PEFC.

Similarly, the raw material can optionally be selected according to particular characteristics of the trees (such as the age of the plantations, the altitude of the plantations, the period of slaughter, etc.) identical or similar to ensure better reproducibility of the extracts obtained.

Preferably, the raw material derived from Pseudotsuga menziesii harvested is dried and / or milled. Drying limits the degradation of the raw material and facilitates grinding. Grinding is performed to obtain particles of size preferably between 0.05 and 10 mm. According to one embodiment, between 20 and 40% by weight of particles have a size of between 2 and 10 mm and between 60 and 80% by weight of the particles have a size between 0.05 and 2 mm.

The raw material, if not used immediately after harvesting and / or drying and / or grinding, is preferably stored in a dry place.

Pressure percolation in a fixed-bed reactor with a solvent is an essential process step according to the invention since it makes it possible to extract both the lipophilic molecules and polyphenolic compounds contained in the raw material. Preferably, the extraction by percolation under pressure in a fixed-bed reactor is carried out with a solvent chosen from solvents produced from biomasses of plant origin, namely agro-solvents, bio-solvents or green solvents. This solvent may especially be chosen from 2-methyltetrahydrofuran, ethyl acetate, bioethanol, water and mixtures thereof. According to a particularly suitable embodiment, it is 2-methyltetrahydrofuran.

Percolation diffusion of the solvent under pressure in fixed bed reactor can be carried out in dynamic mode or in static mode. Preferably it is carried out at a pressure of between 1 and 100 bar, more preferably between 1 and 10 bar. The duration of an extraction process is 3 to 7 hours to treat 500 kg of raw material, this time can vary depending on the diameter of the tank, the flow rate, the solvent and the raw material.

The solvent diffusion percolation step is preferably followed by a step of evaporation of the solvent to recover a concentrate comprising at least one lipophilic molecule and at least one polyphenolic compound. This evaporation is preferably carried out by a system of evaporation under reduced pressure and / or controlled temperature.

The concentrate can then be winterized, especially at temperatures between 2 and 8 ° C. This step makes insoluble fatty alcohols, fatty acids whose lengths of hydrogen carbon chains are> 22 carbon atoms.

The constituents of the concentrate can then be separated in particular on the basis of their difference in solubility at temperatures between 2 and 8 ° C. This separation step can be carried out by centrifugation. It leads preferentially to obtaining:

a liquid fraction, soluble between 2 and 8 ° C., comprising polyphenolic compounds and lipophilic molecules with molecular masses of less than 600 Daltons, such as phytosterols; this liquid fraction can optionally be dried, for example by lyophilization or atomization; and

a solid fraction, insoluble between 2 and 8 ° C, comprising lipophilic compounds including fatty acids and fatty alcohols whose carbon-carbon chain lengths are> 22 carbon atoms.

According to a particular embodiment of the invention, the method comprises the implementation of the following steps:

drying and / or grinding of the raw material derived from Pseudotsuga menziesii, extraction by percolation diffusion of a solvent under pressure in a fixed bed reactor, evaporation of the solvent to recover a concentrate comprising at least one lipophilic molecule and at least one polyphenolic compound,

winterization of the concentrate at temperatures between 2 and 8 ° C,

separating the constituents of the concentrate on the basis of their solubility difference at temperatures between 2 and 8 ° C.

The process according to the invention also preferably comprises a step of recycling the extraction solvent. In the same way the raw material exhausted in extractables of interest can be used like fuel. Thus, the process according to the invention limits the production of waste.

The concentrate obtained during the implementation of the process is also an object of the invention. In particular the invention relates to a concentrate obtained from raw material derived from Pseudotsuga menziesii, consisting of:

at least one molecule chosen from polyphenols and / or at least one lipophilic compound having a molecular mass of less than 600 Da

at least one lipophilic molecule selected from fatty alcohols and long-chain fatty acids and their esters.

Preferably the concentrate is obtained during the implementation of a method according to the invention.

The invention also relates to the extracts obtained by the implementation of the method according to the invention. These extracts are undenatured, biodegradable and contaminant free.

A first extract targeted by the invention is an extract of Pseudotsuga menziesii soluble between 2 and 8 ° C. I l preferably comprises at least one molecule chosen from polyphenols and at least one lipophilic compound having a molecular weight of less than 600 Da, chosen in particular from phytosterols, even more preferentially, it comprises polyphenols and phytosterols. Among the polyphenols, the extract preferably comprises at least polyphenols and preferably at least flavonoids, in particular dihydrofavanol and in particular Taxifolin (or dihydroquercetin).

Said extract may comprise in particular between 5 and 15 grams of total polyphenols per 100 grams of dry initial raw material. According to a particularly suitable embodiment, it comprises between 1 and 6 grams of dihydroflavonol per 100 grams of dry initial raw material and / or between 4 to 150 mg of phytosterols per 100 grams of dry initial raw material. Preferably, it comprises at least lg of taxifolin per 100 grams of dry initial raw material.

The soluble extract according to the invention preferably comprises at least one of the molecules mentioned in Table 1 below, or a mixture of several of these molecules, and may possibly contain all of them:

Identified compounds CAS number

Taxifolin (dihydroquercetine) 480-18-2

Matairésinol or isomer * 580-72-3

Pinoresinol * 487-36-5

Phloroglucinol 108-73-6

Phenol, 3,4-dimethoxy 2033-89-8 1,3-benzenediol, 4-propyl 18979-60-7

Benzenepropanol, 4-hydroxy-3-methoxy 2305-13-7

Vanillin 121-33-5

Vanilic acid 121-34-6

Isovanilic acid 645-08-9

Alpha-pinene 80-56-8

Alpha-terpineol 98-55-5

Dehydroabietic acid 1740-19-8

Abietic acid 514-10-3

Stigmast-5-en-3-ol, oleate Not found

Ergost-5-en-3-ol, (3 beta) - 474-62-4

Beta sitosterol * 83-46-5

4-campestene-3-one 51014-22-3

Stigmast-4-in-3-one 1058-61-3

Gamma-sitostenone 84924-96-9

Azelaic acid 123-99-9

N-hexadecanoic acid 57-10-3

Linoleic acid 60-33-3

Oleic acid 112-80-1

Stearic acid 57-11-4

Docosanoic acid 112-85-6

Tetracosanoic acid 557-59-5

Cis-vaccenic acid 506-17-2

Tetracosanol 506-51-4

Behenic alcohol 661-19-8

Beta-D-glucopyranose, 1,6-anhydro-498-07-7

Ethyl ester of docosanoic acid 5908-87-2

Ethyl ester of tetracosanoic acid 24634-95-5

Ferrulate-C22 ^; Not found

Ferrulate -C24 'Not found

Bicyclo [3.1.1] hept-2-ene-2-carboxylic acid 19250-17-0

Table 1

*: Majority compound in the extracts

Δ: Quantified compound by HPLC The soluble extract can be used as is or enriched. By way of example, an enrichment in Taxifolin can be carried out by delipidating filtration (for example using the Zeta Plus - 3M filter) and / or by adsorption on macroporous resin (for example using FPX 66 or XAD 16 HP resin). .

Another extract targeted by the invention is an extract of Pseudotsuga menziesii insoluble between 2 and 8 ° C. It preferably comprises at least one molecule chosen from fatty alcohols, fatty acids whose carbon-carbon chain lengths are greater than or equal to 22 carbon atoms and their esters (in particular waxes having a number of carbon atoms greater than or equal to 40). .

Said insoluble extract may comprise in particular between 0.1 and 7 grams of fatty alcohols and / or long-chain fatty acids and / or their esters per 100 grams of dry initial raw material.

The insoluble extract according to the invention preferably comprises at least one of the molecules mentioned in Table 2 below, or a mixture of several of these molecules, and may possibly contain all of them:

Table 2

*: Majority compound in the extracts

The insoluble extract may comprise a wax fraction which consists of esters of acids and fatty alcohols whose sizes are between 40 and 50 carbon atoms. Of these different esters, moles per 100 moles have a carbon chain less than or equal to 42 carbon atoms, 74 moles per 100 moles have a carbon chain composed of 44 to 46 carbon atoms and 11 moles per 100 moles have a higher carbon chain. or equal to 48 carbon atoms (proportion made from a single extract). The extracts according to the invention have numerous efficiencies that allow their use in different fields. In particular : The insoluble extract has physical properties (hydrophobic, texturizing, melting temperature between 63 and 65 ° C, co-emulsifier) and biological and can therefore be used in cosmetic compositions, or cleaning products and / or cleaning .

The invention therefore also relates to an extract according to the invention for its use as a medicament, as well as the use of an extract according to the invention in cosmetic compositions, food supplements or cleaning products. Advantageously, the method according to the invention makes it possible to valorize co-products with low added value, by developing effective and usable products for different sectors. In fact, the Douglas fir derivatives produced by sawmill logging initially have a low added value, and are mainly used as fuel energy sources. In addition, the process according to the invention is an eco-extraction process, that is to say an environmentally friendly green process, since it minimizes the number of stages and the energy consumptions and preferably uses solvents. green. In addition, the process according to the invention can make it possible to favor the creation of co-products instead of waste to integrate the path of the bio- or agro-refinery.

In addition, the simultaneous extraction of the molecules of interest makes it possible to reduce the unit operations to be implemented. The process according to the invention is a safe, robust and controlled process.

The invention is now illustrated by examples of the process according to the invention, and by test results demonstrating the effectiveness of the extracts obtained as well as comparative test results.

EXAMPLES

For the analysis of the extracts according to the invention, the following analytical methods can be used. These are the methods that were used in the examples: Dosage Analytical Methods

Total polyphenols Colorimetric determination by Folin-Ciocalteu method

(Singleton and Rossi, 1965)

FLUOstar OMEGA BMG LABTECH Microplate Reader Condensed Tannins Vanillin / HCI Colorimetric Assay

(Broadhurst and Jones 1978).

PerkinElmer Lambda 25 UV / VIS Spectrophotometer

Taxifoline Identification: HPLC-MS-MS system [Shimadzu LC pump

10 ADvp with SIL-Ht auto-injector, CTO-10ASvp furnace, Atlantis T3 column (150 x 2.1mm id, 5μιη) (Waters). The detection is carried out by a quadrupole triple-quadrupole hybrid mass spectrometer / 4000 Q-TRAP ^® (Applied Biosystems) linear ion spectrometer equipped with a source

Ion Spray Electrospray Ionization (ESI) and Controlled by Analyst 1.5 Software (Applied Biosystems)

Quantification: by LC-DAD (Schimadzu LC-20AD / T LPGE KIT coupled SPD-M20A IVDD with a Luna column 5μ C18 100A

CPG / MS identification:

compounds / soluble fraction Shimadzu GCMS-Q.P2010 SE

at 4 ° C Phenomenex ZB5 HT column (25m x 0.25mm). Injection in SPLIT mode, division report

340 ° C to 8 ° C / min

Composition of waxes Gas Chromatography - FID

Table 3 Examples of processes and extracts obtained

The examples were made from Douglas-fir bark.

The process implemented is as follows:

a method: static fixed-bed reactor under pressure (100 bar) in static mode (ASE 350 - Accelerated Solvent Extractor System, Thermo Scientific Dionex), a solvent: 2-methyltetrahydrofuran (the volumes by extracts are controlled automatically by the ASE 350 and vary according to the number of cycles, the percentage of rinsing per cycle ...),

Quantity of raw material per 22ml ASE cells: 5 to 12 grams.

An experimental plan has been put in place. The principle of experimental design is to simultaneously vary the levels (modalities) of one or more factors at each test. The number of factors to be tested and their modalities make it possible to define the number and the nature of the experiments to be carried out and thus to generate an experimental field which should be explored both uniformly and economically.

The experimental design that has been used is of hypergreco-Latin square type with 5 factors and 4

Modalities by factor (Table 4):

% Rinse

Granulometry Number of

Temperature Time / solvent cycle

FACTORS of matter cycles

Extraction extraction per cycle

First extraction extraction

Method 1 0,5-lmm 70 ° C 5min 10% -, Modality 2 l-2mm 50 ° C 7min 20%

Modality 3 2-5mm 80 ° C 9min 40%

Modality 4 Gross (<10mm) 90 ° C 3min 60%

Table 4

The total number of possible tests to be carried out with 5 factors and 4 modalities would be 4 1024 tests. The experimental design established with these same factors and modalities allows to minimize the number of tests to 16, which are defined below (Table 5):

Table 5

Each of these tests was characterized by the measurement of different responses such as insoluble fraction content between 2 and 8 ° C (Winterization), total polyphenols (Folin), Taxifolin (HPLC), β-sitosterol (CPG) , in antioxidant activity (DPPH test - test described in the EFFECT OF EXTRACTS OBTAINED section). Reference Fraction Polyphenols Taxifolin β-sitosterol Activity

insoluble total test in g in g / 100g in mg / 100 antioxidant

4 ° C in g eq. Ac. MP g of MP (DPPH) in g eq. Ac.

/ 100g of Ascorbic Gallic M / 100g

/ 100g MP of MP

Test 1 0,226 2,783 1,182 14,830 2,728

Test 2 0,942 3,249 1,140 12,730 4,016

Test 3 0,363 3,242 1,176 14,210 3,400

Test 4 0,246 2,989 1,598 16,900 2,827

Test 5 0,286 3,135 2,018 20,480 2,385

Test 6 0,273 2,358 1,433 14,710 1,953

Test 7 2,280 3,077 2,104 18,600 3,965

Test 8 0,729 3,744 2,368 22,250 3,640

Trial 9,363 4,488 5,270 26,120 3,749

Test 10 0,306 2,890 2,236 15,600 2,343

Test 11 0.393 3.36 2.965 17.800 2.711

Test 12 0,200 3,060 2,470 15,470 2,435

Test 13,573 3,359 1,933 12,890 3,315

Test 14 0.552 3.330 2.104 14.660 3.075

Test 1,571 4,923 4,202 26,620 4,479

Test 16 0.682 4,603 3,687 23,940 4,061

Min 0.200 2.388 1.140 12.730 1.953

Max 2,280 4,923 5,270 26,620 4,479

Table 6

From the 16 combinations tested and the model "average effects plot" of the various factors, it is possible, after statistical treatment of the results, to predict the values of each of the measurable responses for the 1024 possible combinations. By applying a filter that corresponds to the threshold values of the different responses to be obtained (example: [Taxifolin]> 3.25g / 100g, [beta-sitosterol]> 21 mg / 100g ...), a subset of 6 is obtained. combinations of the experimental domain that are supposed to meet the criteria of optimization of the process.

The 6 combinations tested to validate the experimental design are presented below:

Table 7 Each of these 6 validation tests is carried out and the performances are evaluated by the measurement of the responses such as: content of insoluble fraction between 2 and 8 ° C (Winterization); total polyphenol content (Folin); condensed tannin content (vanillin test); Taxifoline content (HPLC); β-sitosterol content (GPC), antioxidant activity (DPPH test - test described in the EFFECT OF EXTRACTS OBTAINED).

The results are presented below:

Polyphenol Tannins Activity

Fraction β-

Total references in Condensed Taxifolin antioxidant insoluble to sitosterol

eq. Ac. in g eq. e in (DPPH) in g eq.

4 ° C (Wax ...) in

validatio Gallic Catechin g / 100g Ac.

in g / 100g mg / 100g

n / 100g of / 100g MP Ascorbic MP / 100 MP MP

MP MP g of MP

E0558 1,590 5,698 0,592 4,086 41,220 4,892

E0798 1,410 5,730 1,157 3,431 51,140 5,262

E0814 1,967 6,361 1,402 3,539 29,890 5,630

E0926 1,610 6,184 1,133 4,040 27,590 5,425

E0942 1,780 6,927 1,099 3,655 66,440 5,715

E1006 2,247 6,566 1,081 4,173 28,490 5,438

Table 8

A characterization of the soluble fraction between 2 and 8 ° C was carried out by HPLC and CPG on the sample E0814, the molecules listed below were identified therein: Taxifolin, Matairésinol, Pinoresinol, Phloroglucinol, Phenol, 3,4- dimethoxy, 1,3-benzenediol, 4-propyl, vanilic acid, alpha-pinene, dehydroabietic acid, Stigmast-5-en-3-ol, oleate; Ergost-5-en-3-ol, (3 beta) -; Beta sitosterol; 4-campestene-3-one, Stigmast-4-en-3-one, Azelaic acid, N-hexadecanoic acid, Linoleic acid, Stearic acid, Tetracosanoic acid, Cis-vaccenic acid, Tetracosanol, Behen alcohol, Ethyl ester of the docosanoic acid, Ferrulate-C22, Ferrulate -C24. A characterization of the insoluble fraction between 2 and 8 ° C was carried out by GC on sample E0814, the molecules listed below were identified therein: hexadecanoic acid, hexadecanoic acid-14 methyl, octadecanoic acid (C18), acid Octadecadienoic acid (C18-2), Octadecenoic acid (C18-1), Nonadecanoic acid, Methyl-18-methylnonadecanoic acid, Docosanoic acid, Tricosanoic acid, Tetracosanoic acid, Pentacosanoic acid, Hexacosanoic acid, Octacosanoic acid, Hexadecanedioic acid, Docosanol, Tetracosanol, Heneicosane, Beta sitosterol, Stigmastanol, Stigmast-4-en-3one A characterization of the length of the carbon chains of the waxes of the insoluble fraction between 2 and 8 ° C was carried out on the sample E0814, the results are presented in Table 9 below:

Length of the chain Proportion in moles / 100 moles

carbonaceous

C 40 ₆

C 42 9

WBÊÊÊ

C 46

Efii

C 50 2

Table 9 In addition, an analysis of the content of certain molecules and the biological activity in several extracts obtained according to the process of the invention with different solvents was carried out.

The minimum concentrations that can be found in these extracts are presented in the table below:

Table 10 EFFECTS OF EXTRACTS OBTAINED

Tests have been conducted to demonstrate the effectiveness of the extracts obtained according to the invention. These tests focused on the following measures:

Measurement of antioxidant activity

- Cytotoxicity measurement

Measurement of anti-inflammatory activity

Measurement of antibacterial activity

Antioxidant Activity Evaluation Test: DPPH Test, 2,2-DIPHENYL-1 -PICRYLHYDRAZYL The antioxidant activity of the extracts is evaluated by measuring its ability to trap the free radical DPPH. The reduction in DPPH results in a color change of the solution from purple to yellow, resulting in a decrease in its absorbance at 517 nm. Therefore, the greater the decrease in absorbance, the higher the antioxidant activity of the solution involved. The antioxidant activity can be expressed in equivalent concentration of a reference antioxidant compound (vitamin C, chlorogenic acid, etc.).

The reaction involved is as follows: Z ^* + AH -> ZH + A ^*

With: AH = polyphenolic compounds and Z- = DPPH (free radical form)

Evaluation of Cytotoxicity: MTT Test (In vitro)

Evaluation of the cytotoxicity of the extracts was performed by measuring cell viability through determination of mitochondrial dehydrogenase activity.

The cytotoxicity of active ingredients is evaluated by an MTT test (3- [4,5-dimethylthiazol-2yl] -2,5-diphenyltetrazolium bromide) allowing a rapid and sensitive quantification of cell viability HACAT (human keratinocytes). The tetrazolium salts of the MTT reagent are reduced, by mitochondrial dehydrogenase type enzymes, to formazan violet insoluble crystals. The formazan crystals are then solubilized with a solvent, DMSO, and the absorbance is measured spectrophotometrically at 595 nm. The cell viability is proportional to the mitochondrial enzymatic activity and therefore to the amount of purple formazan crystals formed. Evaluation of anti-inflammatory activity: Determination of NO (in vitro)

The estimation of the anti-inflammatory activity of an extract is based on its ability to reduce the production of nitric oxide, NO, by RAW264.7 cells (murine macrophages) undergoing inflammation due to LPS (lipopolysaccharide) . The nitric oxide product is dissolved in the cell medium in the form of nitrite N0 ₂ ^~ . This ion is quantified by an absorbance measurement at 535 nm, after adding the Griess reagent which causes the formation of a pink color in the presence of N0 ₂ ^" (formation of a diazonium salt with sulphanilic acid, which gives a pink coloring with α-naphthylamine.) The amount of N0 ₂ ^" is determined by a range of sodium nitrite NaNO ₂ , it is compared to the positive control consisting of L-nitroargininemethyl ester (anti-inflammatory) and the negative control where there is no anti-inflammatory agent.

Evaluation of the anti-bacterial activity:

The estimation of the antimicrobial activity of an extract is based on its ability to inhibit the growth of a microbial population (Staphylococcus aureus). The extract is placed in the presence of a suspension of Staphylococcus aureus and incubated at 37 ° C. for 24 h, a follow-up of the bacterial growth is carried out throughout these 24 hours by measurement of absorbance at 620 nm. If the absorbance increases during 24 hours, there is therefore growth of the bacterial suspension not revealing antimicrobial activity, on the contrary if the absorbance does not evolve, the extract reveals an antimicrobial activity. In parallel with the analysis, a growth control of the strain is made to ensure the viability of the strain.

Results obtained

The results obtained for the extracts, were collected.

The content of total polyphenols was determined by a Folin-Ciocalteu assay and expressed in gallic acid equivalent. The measured activities are related to an equivalent amount of total polyphenols present in the extract. In Table 11 below, it is indicated the amount of total polyphenols extracted from 100 g of raw material as well as the antioxidant activity (expressed in vitamin C equivalent) and the antimicrobial activity measured against Staphylococcus aureus which are associated. Antibacterial (Staph Aureus)

Total Polyphenols Antioxidant Activity

(I n growth inhibition over 24h to

Test (Gallic Acetate Equivalent) (DPPH) (mg eq Vitamin C / g

from Xμg of / 100g of MP) of polyphenols)

polyphenols / ml / 500000 bacteria)

Extract

according to

the invention

Table 11

In addition, among the polyphenols extracted, tests of anti-inflammatory activities in vitro extracts were carried out while controlling their cytotoxicity. The results are shown in Figures 1 and 2.

Figure 1 is a histogram showing the cytotoxicity measured as a function of the dilution of the extract in the culture medium of the cells, and Figure 2 is a histogram representing the anti-inflammatory activity measured in the presence of LPS for the dilutions. considered non-cytotoxic.

A significant cytotoxic effect (<50% viability) is observed until the dilution l / ^160th (0.625%). The soluble extract according to the invention in amounts of 0.313% and 0.156%, 10 present a cytotoxic effect less important since the cell viability was 70% and 95%, respectively for dilutions l / 320 ^th (0.313%) and 1/640 ^th (0.156%).

In addition, the extract (0.313% and 0.156%) has an anti-inflammatory effect from a dilution of l / 320 ^th (= no production of nitrogen monoxide (NO) in the condition "+ LPS" ).

From these results, cytotoxicity and anti-inflammatory activity as a function of the amount of polyphenols could be determined for the extract E0814. These values are shown in Table 12: Anti-inflammatory cytotoxicity

Total polyphenols

(Absence or negligible Xμg (Activity observed for Xμg

Test (Gallic Acq)

polyphenols / ml / 10,000 polyphenols / ml / 10,000 / 100g of MP)

cells)

Extract

according to

the invention

Table 12

It is important to note that the soluble extract according to the invention has an anti-inflammatory activity in vitro for an equivalence of polyphenols (equivalent μg Gallic Ac) well below the threshold of appearance of cytotoxicity. COMPARATIVE TRIALS

Comparison of extraction processes

This test relates to the comparison of the performance of the extraction process according to the method used (3 methods tested). The example developed concerns:

a Douglas fir derivative: the bark

- a raw material granulometry (Brute)

an agro-solvent: 2-methyltetrahydrofuran

3 different processes: a) stirred batch reactor; (b) Static pressure type fixed bed reactor in static mode; c) pressurized fixed bed type reactor in dynamic mode.

The conditions for implementing the 3 processes are described in Table 13 below:

Table 13

The extractions are carried out with each of the 3 processes. The characterization of the extracts was carried out by measuring the following responses: contents of insoluble fraction at 4 ° C (winterization); total polyphenols (Folin); condensed tannins (vanillin test); in Taxifolin (HPLC); β-sitosterol (CPG), with antioxidant activity (DPPH test).

Table 14

It can be seen that the fixed-bed reactor makes it possible to obtain better results than in stirred batch reactor.

Examples with different extraction solvents

The examples relate to:

a derivative of Douglas fir: bark

a raw material granulometry (Brute)

a method of implementation: fixed-bed type reactor under pressure in dynamic mode.

- 4 solvents: 2-methyltetrahydrofuran; ethyl acetate ; bio ethanol; hydroalcoholic solution (50/50 v / v)

The conditions of extraction of the fixed bed reactor method in dynamic mode are described in the table below:

Table 15

The performances of the four solvents used with the same process (fixed-bed type reactor in dynamic mode) are quantified by measuring different responses such as insoluble fraction contents at 4 ° C (Winterization); total polyphenols (Folin); in condensed tannins (vanillin test); in taxifolin (HPLC); in β-sitosterol, in antioxidant activity DPPH test). The results are shown in Table 14 below:

Table 16

2-Methyltetrahydrofuran and ethyl acetate allow a good simultaneous extraction of waxes and derivatives, taxifolin and β-sitosterol. Ethanol and the mixture hydroalcoholic also; ethanol allows better extraction of waxes while the hydroalcoholic mixture (50/50) allows to extract more polyphenols.

Influence of Extraction Solvent Recycling on Process Performance

This test relates to the recycling of the extraction solvent, the process according to the invention may comprise a recovery step of the extraction solvent, in order to limit the consumption and consequently the process costs.

In order to validate this possibility, it was necessary to check the quality of the extraction after each cycle of use of the solvent. The example developed concerns:

a derivative of Douglas fir: bark

- a raw material granulometry (Brute)

a method: fixed type fixed bed reactor under static mode.

an agro-solvent: 2-methyltetrahydrofuran

The conditions for extraction in fixed-bed reactor under static pressure are described in Table 17 below:

Table 17 The results presented are obtained after 6 successive extractions, 5 of which are carried out with the recycled solvent.

Solvent Extraction: 2-Methyltetrahydrofuran

Extraction 1 2-Methyltetrahydrofuran

Extraction 2 2-Methyltetrahydrofuran recycled after extraction 1

Extraction 3 2-Methyltetrahydrofuran recycled after extraction 2

Extraction 4 2-Methyltetrahydrofuran recycled after extraction 3

Extraction 5 2-Methyltetrahydrofuran recycled after extraction 4

Extraction 6 2-Methyltetrahydrofuran recycled after extraction 5

Table 18

The extracts obtained at the end of each of the 6 cycles were characterized to determine the impact of solvent recycling on the performance of the process. The measured responses relate to insoluble fraction contents at 4 ° C (Winterization); total polyphenols (Folin); condensed tannins (vanillin test); in Taxifolin (HPLC); β-sitosterol (CPG), with antioxidant activity (DPPH test). In addition, the evolution of the purity of the solvent (2-methyltetrahydrofuran) after each cycle was analyzed by GC-MS.

Table 19 Solvent purity in%

Solvent

(2-methyltetrahydrofuran)

Solvent not recycled 99,88

Solvent after extraction 1 99,86

Solvent after extraction 2 99,87

Solvent after extraction 3 99,85

Solvent after extraction 4 99,87

Solvent after extraction 5 99,87

Solvent after extraction 6 99.86

Table 20

This solvent recycling study did not show a significant decrease in its extraction capacity or a change in purity after 6 consecutive cycles. This confirms the possibility of recycling for economic and ecological reasons.

Claims

1. Process for the simultaneous extraction of at least one lipophilic molecule and at least one polyphenolic compound from Pseudotsuga menziesii, comprising at least one step of extraction by percolation diffusion of a solvent under pressure in a fixed bed reactor, carried out from a raw material derived from Pseudotsuga menziesii.

2. Method according to claim 1, characterized in that the extraction by percolation under pressure in a fixed bed reactor is carried out with a solvent selected from solvents produced from biomasses of plant origin.

3. Method according to one of the preceding claims, characterized in that the extraction percolation diffusion under pressure in a fixed bed reactor is carried out with a solvent selected from 2-methyltetrahydrofuran, ethyl acetate, ethanol , water and their mixtures.

4. Method according to one of the preceding claims, characterized in that the raw material derived from Pseudotsuga menziesii is constituted by barks, knots, platelets, strains, roots and / or sawdust of Pseudotsuga menziesii.

5. Method according to one of the preceding claims, characterized in that the raw material derived from Pseudotsuga menziesii is previously dried and / or milled.

6. Method according to claim 5, characterized in that the grinding is performed to obtain particles of size between 0.05 and 10mm.

7. Method according to one of the preceding claims, characterized in that the diffusion percolation of the solvent under pressure in fixed bed reactor is performed in dynamic mode or in static mode.

8. Method according to one of the preceding claims, characterized in that the diffusion percolation of the solvent under pressure in fixed bed reactor is carried out at a pressure between 1 and 100 bar.

9. Method according to one of the preceding claims, characterized in that it comprises the implementation of the following steps:

- winterization of the concentrate at temperatures between 2 and 8 ° C, separating the constituents of the concentrate on the basis of their solubility difference at temperatures between 2 and 8 ° C.

10. Process according to the preceding claim, characterized in that the separation is carried out by centrifugation.

11. Method according to one of claims 9 or 10, characterized in that the separation is performed so as to obtain firstly a liquid fraction, soluble between 2 and 8 ° C, comprising compounds with molecular weights less than 600 Da and on the other hand a solid fraction, insoluble between 2 and 8 ° C, comprising lipophilic compounds mainly consisting of fatty alcohols and fatty acids having hydrocarbon chains of length> 22 carbon atoms and their esters.

12. Process according to the preceding claim, characterized in that the liquid fraction is dried.

13. Concentrate obtained from raw material derived from Pseudotsuga menziesii, consisting of:

at least one molecule chosen from polyphenols and / or a lipophilic compound having a molecular mass of less than 600 Da

14. Concentrate according to the preceding claim, characterized in that it is obtained during the implementation of a method according to one of claims 1 to 12.

15. Extract of Pseudotsuga menziesii soluble between 2 and 8 ° C, obtained by the implementation of a method according to one of claims 1 to 12.

16. Extract according to claim 15, characterized in that it comprises at least one molecule chosen from polyphenols and phytosterols.

17. Extract according to claim 15 or 16, characterized in that it comprises between 5 and 15 grams of total polyphenols per 100 grams of dry initial raw material.

18. Extract according to claim 15 or 16, characterized in that it comprises between 1 and 6 grams of dihydroflavonol per 100 grams of dry initial raw material and / or between 4 to 150 mg of phytosterols per 100 grams of dry initial raw material.

19. Extract according to one of claims 15 to 18, characterized in that it comprises at least 1 g of taxifolin per 100 grams of dry initial raw material.

20. Pseudotsuga menziesii extract insoluble between 2 and 8 ° C obtained by the implementation of a method according to one of claims 1 to 12.

21. Extract according to claim 20, characterized in that it comprises at least one molecule chosen from fatty alcohols, fatty acids having hydrocarbon chains of lengths between 22 and 28 carbon atoms and their esters.

22. Extract according to claim 21, characterized in that it comprises between 0.1 and 7 grams of fatty alcohols and / or long-chain fatty acids and / or their esters per 100 grams of dry initial raw material.

23. Extract according to one of claims 15 to 22, for use as a medicament.

24. Use of an extract according to one of claims 15 to 22, in cosmetic compositions or food supplements or food compositions or cleaning products.