Process for the preparation of an extract obtained from undifferentiated cell suspension cultures of clary sage

Abstract

The invention relates to a process for the preparation of an extract obtained from undifferentiated cell suspension cultures of clary sage, to the extract obtained by this process, to the use of this extract as an active ingredient in the prevention and / or treatment of inflammation or microbial infections, to a pharmaceutical composition comprising said extract, to a sanitizing composition comprising said extract, to the use of said sanitizing composition for sanitizing a surface or increasing the surface's resistance to microbe transmission, to the cosmetic use of said extract as an anti-aging active ingredient and to a cosmetic composition comprising said extract

Description

[0001] PROCESS FOR THE PREPARATION OF AN EXTRACT OBTAINED FROM UNDIFFERENTIATED CELL SUSPENSION CULTURES OF CLARY SAGE

[0002] FIELD OF THE INVENTION

[0003] The invention belongs to the technical field of production of bioactive compounds from plant origin.

[0004] In particular, the invention concerns a process for the preparation of an extract obtained from undifferentiated cell suspension cultures of clary sage, the extract obtained by this process, the use of this extract as an active ingredient in the prevention and / or treatment of inflammation or microbial infections, a pharmaceutical composition comprising said extract, a sanitizing composition comprising said extract, the use of said sanitizing composition for sanitizing a surface or increasing the surface's resistance to microbe transmission, the cosmetic use of said extract as an anti-aging active ingredient and a cosmetic composition comprising said extract.

[0005] TECHNICAL BACKGROUND

[0006] Plants materials are of increasing interest for their applications in pharmaceutical, nutritional and cosmetic application. They are the source of useful bioactive ingredients known since long time by traditional uses for medical purposes. Plants contain many active compounds such as alkaloids, steroids, tannins, glycosides, volatile oils, fixed oils, resins, phenols and flavonoids which are accumulated in their specific tissues / organs, such as leaves, flowers, bark, seeds, fruits, and root.

[0007] Sage is an aromatic plant in the Lamiaceae family, scientifically known as Salvia. It is native to the Mediterranean region, but is cultivated in many parts of the world for its culinary, medicinal and ornamental properties.

[0008] Sage is rich in antioxidant compounds such as flavonoids, phenols and terpenes, which help to neutralise free radicals in the body, reducing oxidative damage to cells and helping to prevent chronic oxidative diseases such as cardiovascular disease, neurodegenerative diseases and certain types of cancer. Sage has anti-inflammatory properties thanks to its bioactive compounds, such as rosmarinic acid, which can help reduce inflammation in the body. This can be beneficial in relieving the symptoms of inflammatory conditions such as arthritis, respiratory ailments and digestive disorders. Sage also has antimicrobial properties, thanks to its active compounds, such as flavonoids and terpenes, which can help fight bacterial, viral and fungal infections. It can be used to relieve respiratory ailments, skin and mouth infections.

[0009] There are several varieties of Sage, each with unique characteristics that set it apart from the others, whether for culinary, medicinal, aromatic or decorative uses. Sage Salvia officinalis') is used mainly for its medicinal properties and in cooking. Clary sage (Salvia sclarea) is known for its relaxing properties and its use in aromatherapy. White Sage (Salvia apiana) is renowned for its purifying properties, often used in purification rituals. Purple Sage (Salvia officinalis 'Purpurascens') is appreciated for its decorative leaves and culinary uses. Pineapple Sage (Salvia elegans) is recognizable by its fruity aroma, mainly used for its aromatic and decorative qualities.

[0010] Several methods exist to extract active compounds from Sage and more generally from plants. These techniques can be called conventional (or traditional methods) and use organic solvents or water that penetrate into the solid plant materials and solubilize the compounds of similar polarity.

[0011] However, these traditional methods of producing plant extracts face several significant issues such as the overharvesting of plants, leading to environmental degradation and loss of biodiversity. Additionally, the availability and quality of plant materials collected from the wild are subject to seasonal variability, while many plants require long growth periods to reach maturity. This creates difficulties, especially when working with rarer species, and contributes to inconsistent phytochemical composition in the extracts, as growing conditions can vary substantially.

[0012] These challenges create a non-negligible gap in the market for consistent, sustainable, and high-quality plant extracts, particularly in industries like cosmetics and pharma. With the increasing demand for plant-based bioactive ingredients, there is a need for more efficient, scalable, and consistent methods of production.

[0013] The inventors set out themselves to solve this technical problem and developed an alternative process to access to the active compounds contained in the plant Salvia sclarea. OBJECTS AND SUMMARY

[0014] A first object of the present invention is a process for the preparation of an extract derived from a Salvia sclarea undifferentiated cell culture, said process comprising at least the steps of: a) providing a suspension-cultured cell line cultured in a liquid basal medium from a callus of a plant of the species Salvia sclarea, b) culturing said suspension-cultured cell line of step a) in a liquid basal medium free from any elicitor and in the dark, to obtain Salvia sclarea undifferentiated cells, c) recovering said cells from the liquid basal medium, d) converting said cells into cell fragments, e) homogenizing said cells fragments in a liquid polar solvent, f) removing said solvent, partially or in totality, to obtain said extract.

[0015] By cultivating Salvia sclarea undifferentiated cells in a controlled environment such as stirred-tank or wave-tank reactors, the need for large-scale plant harvesting is eliminated, thereby preserving natural ecosystems and reducing environmental impact. The process of the invention also ensures consistent production of bioactive compounds, regardless of seasonal or geographic factors, leading to high-quality extracts. The use of this Salvia sclarea undifferentiated cell culture is scalable, making it possible to meet growing market demands for plant extracts while maintaining a smaller environmental footprint. Moreover, because the production process occurs in a controlled environment (in bioreactors), the risk of contamination is minimized, resulting in purer extracts with no contaminants nor pathogens. This leads to safer and more sustainable products. Additionally, the extracts obtained according to this process have a higher content of rosmarinic acid compared to cell cultures of other Salvia species growing in similar conditions reported in the prior art, for example in Chinese patent application CN105613285; the bioprocess of this invention is independent of the use of light and elicitors. With the technology described here, a production of rosmarinic acid equivalent to 5.68 ± 0.60 pg / mg of dry weight cells is obtained with the hydrophilic extract. A second object of the present invention is an extract derived from a Salvia sclarea undifferentiated cell culture, wherein said extract is obtained by the process as defined according to the first object of the invention.

[0016] A third object of the present invention is an extract derived from a Salvia sclarea undifferentiated cell culture for its use as an active ingredient in the prevention and / or treatment of inflammation or microbial infections, wherein said extract is obtained by the process as defined according to the first object of the invention.

[0017] A fourth object of the present invention is a pharmaceutical composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention, and a pharmaceutically acceptable vehicle.

[0018] A fifth object of the present invention is a sanitizing composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention, and a sanitizing acceptable vehicle.

[0019] A sixth object of the present invention is the use of said sanitizing composition, for sanitizing a surface or increasing the surface's resistance to microbe transmission.

[0020] A seventh object of the present invention is a cosmetic use of an extract derived from a Salvia sclarea undifferentiated cell culture, wherein said extract is obtained by a process as defined according to the first object of the invention or as defined according to the second object of the invention, as an anti-aging active ingredient.

[0021] An eighth object of the present invention is a cosmetic composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention or as defined according to the second object of the invention, and a cosmetically acceptable vehicle.

[0022] DEFINITIONS

[0023] In the following disclosure the expression "a liquid basal medium free from any elicitor » means that said medium is free from any (a)biotic elicitor, i.e. those belonging to the class of physical and chemical substances, as well as those derived from living organisms.

[0024] In particular, auxins (as for example 2,4-dichlorophenoxyacetic acid) and cytokinins (as for example kinetin) that can be added in the liquid basal medium are not elicitors but phytohormones.

[0025] The term "sanitizing" refers to the fact that due to the presence of the extract according to the invention in the composition, the application of such a composition makes it possible to avoid and / or reduce the proliferation of pathogens on a surface and eliminate pathogens present on a surface, in particular the following pathogenic microorganisms: Staphylococcus aureus, multiresistant strains of Staphylococcus aureus (including Methicillin-resistant S. aureus - MR.SA), Staphylococcus epidermidis, Escherichia coll or Candida albicans.

[0026] DETAILED DESCRIPTION

[0027] First object of the invention

[0028] According to a preferred embodiment, the liquid basal medium used in steps a) and b) of the process is a liquid Murashige and Skoog (MS) medium, i.e. a MS medium without agar.

[0029] The culture of said of step b) can be carried out in flasks or in bioreactors of different capacities ranging for example from 2-4 L to up to 30 L or more.

[0030] According to preferred embodiment, said liquid basal medium is supplemented with at least one phytohormone.

[0031] The phytohormone is preferably chosen in the group comprising auxins and cytokinins.

[0032] As examples of auxins that can be present in the liquid basal medium, one can mention 2,4-dichlorophenoxyacetic.

[0033] When present, the amount of auxins may be from about 0.01 to 10 mg / mL, preferably from 0.1 to 2 mg / mL of liquid basal medium.

[0034] As examples of cytokinins that can be present in the liquid basal medium, one can mention kinetin, benzylaminopurine, zeatin, and mixtures thereof.

[0035] When present, the amount of cytokinins may be from about 0.1 to 10 mg / mL, preferably fromO.l to 1 mg / mL of liquid basal medium. According to a particular and preferred embodiment of the present invention, said liquid medium is supplemented with 2,4-dichlorophenoxyacetic acid and / or kinetin, and even more preferably with 2,4-dichlorophenoxyacetic acid and kinetin.

[0036] The callus of the plant of the species Salvia sclarea can be established from leaf explants of S. sclarea in v / tro-grown plants, preferably on MS solid medium. This MS solid medium can also be supplemented with at least one phytohormone, preferably with at least one auxin, such as for example 2,4-dichlorophenoxyacetic acid and with at least one cytokinin, such as for example kinetin.

[0037] According to a preferred embodiment, step b) is carried out at a temperature of 22 to 32°C, more preferably at a temperature of 22 to 28°C and even more preferably at a temperature of 25±1°C.

[0038] The duration of step b) may be from about 10 to 21 days.

[0039] Step b) is preferably carried out under agitation for example in a wave-tank reactors or in a stirred-tank bioreactor equipped with one or several impellers to help keeping the cell culture evenly distributed and also to ensure a good oxygenation of the liquid basal medium.

[0040] The speed of stirring during step b) is preferably from about 100 to 300 rpm and more preferably from about 100 to 200 rpm.

[0041] According to a preferred embodiment of the invention, step b) is carried out under oxygenation, i.e. with an external input of air into the liquid basal medium. In that case, airflow can be set between about 0.025 and 0.5 volume of air per volume of liquid basal medium per minute (VVM) and preferably from 0.1 to 0.25 VVM.

[0042] Step b) can be carried at a pressure of 0 to 0.5 bar, preferably at a pressure of 0 to 0.3 bar.

[0043] At step c), the recovering of the cells from the liquid basal medium can for example be performed by vacuum filtration or via the use of a filter press, according to the techniques well-known from the skilled person.

[0044] Step d) of converting the cells recovered from the liquid basal medium into cell fragments can be carried either by lyophilization followed by a step of grinding the lyophilized cells or by lysing the cell, for example with an osmotic chock. Step e) of homogenizing the cells fragments in a liquid polar solvent is an extraction step. Depending on the nature of the liquid polar solvent used during step e) a hydrophilic extract or a lipophilic extract will be obtained at the end of step f).

[0045] According to a first embodiment of step e), said liquid polar solvent is a polar protic solvent selected in the group comprising water, methanol, ethanol, acetic acid, isopropanol, 1,3-propanediol, glycerol, and mixtures thereof. Among these solvents, ethanol is particularly preferred.

[0046] According to this first embodiment, the extract obtained at the end of step f), i.e. after the total evaporation of the polar protic solvent, is a dry hydrophilic extract.

[0047] According to this first embodiment of step e), the solid S / liquid L ratio (S / L) of the cells fragments (in g) / volume of solvent (in L) can vary from about 1 / 5 to 1 / 50, and preferably from about 1 / 10 to 1 / 20.

[0048] According to a second embodiment of step e), said liquid polar solvent is a polar aprotic solvent selected in the group comprising ethyl acetate, acetone, dimethyl sulfoxide, acetonitrile, tetra hydrofuran, dichloromethane, and mixtures thereof. Among these solvents, ethyl acetate is particularly preferred.

[0049] According to this second embodiment, the evaporation of the solvent cannot be carried out in totality and the extract obtained at the end of step f), is concentrated lipophilic extract.

[0050] According to this second embodiment of step e), the solid S / liquid L ratio (S / L) of the cells fragments (in g) / volume of polar aprotic solvent (in L) can vary from about 1 / 5 to 1 / 50, and preferably from about 1 / 10 to 1 / 20.

[0051] At the end of step f), the extracts can be kept in a dry place, preferably in dark and in a sealed container.

[0052] Second object of the invention

[0053] The extracts obtained according to the above detailed process constitute the second object of the present invention.

[0054] Said extracts comprise different families of known compounds such as for example polyphenols (e.g. rosmarinic acid), pentacyclic triterpenoids, cinnamides, and hydroxycinnamic acid esters. The dry hydrophilic extract is characterized in that it comprises rosmarinic acid. The content of rosmarinic acid in the dry hydrophilic extract of the invention may range from about 2 to 10 pg / g of dry weight of cells...

[0055] Third object of the invention

[0056] The third object of the present invention is an extract derived from a Salvia sclarea undifferentiated cell culture for its use as an active ingredient in the prevention and / or treatment of inflammation or microbial infections, wherein said extract is obtained by the process as defined according to the first object of the invention.

[0057] The dry hydrophilic extract may in particular be used as an active ingredient in the prevention and / or treatment of microbial infections caused by Staphylococcus aureus, multiresistant strains of Staphylococcus aureus, Staphylococcus epidermidis or Escherichia coli.

[0058] The lipophilic extract may in particular be used as an active ingredient in the prevention and / or treatment of microbial infections caused by Staphylococcus epidermidis or Candida albicans.

[0059] Fourth object of the invention

[0060] The fourth object of the present invention is a pharmaceutical composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention, and a pharmaceutically acceptable vehicle.

[0061] The pharmaceutical compositions according to the invention may be formulated in solid forms, such as granulates, tablets, capsules, semisolid forms, such as ointments and the like or liquid forms, such as solutions, emulsions or suspensions. Preferably gels, ointments, dusting powders for wounds, solutions and suspensions as well as the combinations of powder and solvent ampoules are prepared.

[0062] The pharmaceutical compositions according to the invention may in particular be in the form of a topical composition to be administered to the skin, such as a cream, a lotion, or a spray. Depending on the formulation, commonly used carriers, as well as other additives such as vehicles, disintegrating, sliding and emulsifying agents may be used.

[0063] Fifth object of the invention

[0064] The fifth object of the present invention is a sanitizing composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention, and a sanitizing acceptable vehicle.

[0065] Sixth object of the invention

[0066] The sanitizing composition can be used for sanitizing a surface or increasing the surface's resistance to microbe transmission.

[0067] According to a particular embodiment, the surface is a community surface, for example hospitals, nurseries or even accommodation establishments for the elderly, or a surface of a public or private establishment, for example kitchens.

[0068] According to this use, the sanitizing composition can be applied according to the usual procedures for sanitizing or increasing the surface's resistance to microbe transmission of a surface in addition to or replacing disinfection procedures known to those skilled in the art.

[0069] Seventh object of the invention

[0070] The seventh object of the present invention is a cosmetic use of an extract derived from a Salvia sclarea undifferentiated cell culture, wherein said extract is obtained by a process as defined according to the first object of the invention or as defined according to the second object of the invention, as an anti-aging active ingredient.

[0071] According to a preferred embodiment, said extract is a dry hydrophilic extract.

[0072] Eighth object of the invention

[0073] The eighth object of the present invention is a cosmetic composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined according to the first object of the invention or as defined according to the second object of the invention, and a cosmetically acceptable vehicle.

[0074] According to a preferred embodiment, said extract is a dry hydrophilic extract.

[0075] According to an embodiment, the extract of the invention is present in the cosmetic composition in an amount ranging from about 0.01 to 5 wt. % relative to the total weight of said cosmetic product, preferably in amount ranging from about 0.1 to 2 wt.%

[0076] The cosmetic composition according to the invention may also further comprise one or several additional active ingredients capable of reinforcing and / or completing the advantageous properties of the extract of the invention. At the occasion, those skilled in the art will take care that this(these) additional active ingredient(s) does(do) not interfere or decrease advantageous properties of the extract of the invention.

[0077] The additional active ingredients may for example be chosen among plant extracts (different from the extract of the invention and such as green teas and grape extracts), moisturizers, humectants, antioxidants, skin-protecting agents, anti-ageing active agents, agents for protecting against rays, and mixtures thereof.

[0078] The cosmetic composition according to the invention may further comprise one or several formulation ingredients or additives such as, as a non-limiting example, penetrating agents, thickening agents such as natural gums and synthetic polymers, surfactants, emulsifying agents such as polyglycerol derivatives, preservatives agents such as phenoxyethanol and dehydroacetic acid (DHA), oils, pigments such as titanium dioxide and zinc dioxide, dyes, film-forming agents, mineral charges, perfumes, etc...

[0079] The cosmetic composition according to the invention may be presented into various galenic formulations adapted to a topical application, in particular in the form of a gel, an emulsion (e.g. cream or milk), in particular an oil-in-water or water-in-oil bi-phasic emulsion, a microemulsion, a serum, an oil, a mask, a salve, an ointment, a lotion, a concentrated solution, a suspension, a foam, solid sticks or aerosols. The cosmetic composition according to the invention is preferably in the form of a cream, a serum, a lotion or a gel. The manner in which the invention can be carried out and the advantages which result from it will become clearer from the examples of implementation which follow, given for informational and non-limiting purposes, in support of the appended figures.

[0080] BRIEF DESCRIPTION OF THE FIGURES

[0081] - Figure 1 gives graphs showing the antimicrobial properties of the hydrophilic extract of example 1 against two particular strains of S. aureus. The graph of Figure la gives the results obtained on the strain ATCC 6538. Curve 1 corresponds to the positive control, curve 2 to the assay with the hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract at 5 mg / mL and curve 4 to a negative control with chloramphenicol at 50 pg / mL. The graph of Figure lb represents the results obtained on strain DSM 11822. Curve 1 corresponds to a positive control, curve 2 to the negative control with chloramphenicol at 50 pg / mL, curve 3 to the assay with the hydrophilic extract a 1 mg / mL, curve 4 to the assay with the hydrophilic extract at 3 mg / mL, and curve 5 to the assay with the hydrophilic extract at 5 mg / mL.

[0082] - Figure 2 is a graph presenting the antimicrobial properties of the hydrophilic extract of example 1 against an E. coli strain. Curve 1 corresponds to a positive control, curve 2 corresponds to the assay with the hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract at 3 mg / mL and curve 4 to the assay with the hydrophilic extract at 5 mg / mL.

[0083] - Figure 3 is a graph presenting the antimicrobial properties of the hydrophilic extract of example 1 against a strain of Staphylococcus epidermidis. On this figure, curve 1 corresponds to the positive control, On figure 2, curve 1 corresponds to the positive control, curve 2 corresponds to the assay with the hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract at 3 mg / mL and curve 4 to the assay with the hydrophilic extract at 5 mg / mL.

[0084] - Figure 4 is a graph presenting the antimicrobial properties of the lipophilic extract of example 1 against C. albicans. On this figure, curve 1 corresponds to the positive control + 15 pL of ethanol and curve 2 to the assay with the lipophilic extract at 5 mg / mL + 15 pl of ethanol.

[0085] - Figure 5 is a graph presenting the antimicrobial properties of the lipophilic extract of example 1 against S. epidermidis. On this figure, curve 1 corresponds to the positive control + 15 pL of ethanol and curve 2 to the assay with the lipophilic extract at 5 mg / mL + 15 pl of ethanol.

[0086] - Figure 6 gives the chromatograms obtained from a S. sclarea cell extract according to the process of the invention (upper curve) comparatively to an extract obtained fromm S. sclarea leaves (lower curve).

[0087] EXAMPLES

[0088] EXAMPLE 1: Preparation of extracts of Salvia sclarea according to the process of the invention

[0089] 1.1 Preparation of calli

[0090] Calli were established from leaf explants of S. sclarea in v / tro-grown plants on Murashige and Skoog (MS) (Duchefa Biochemie, NL) solid medium containing 2,4-dichlorophenoxyacetic acid 1 mg / L + kinetin 0.2 mg / L.

[0091] 1.2. Preparation of an undifferentiated cells suspension

[0092] Cell suspensions were established from the calli obtained in step 1.1, using the same culture MS medium, but without agar. The cell suspensions were maintained in darkness at 26°C.

[0093] 1.3. Cultivation specifications

[0094] The cell suspension obtained in step 1.2 were then introduced in a 30-litre bioreactor (INFOR.S HT, Switzerland) equipped with 2 marine impellers and 1 Rushton impeller. The cell cultivation has been carried out in the conditions detailed in Table 1 below:

[0095] TABLE 1

[0096] In table 1, the acronym VVM stands for "1 Volume of Air per Volume of Medium per Minute." It is a unit used to describe the rate of aeration in bioreactors, particularly in the context of cell culture. This parameter was automatically controlled by cascade based on pO2 set point which corresponds to the amount of oxygen (in %) required in the bioreactor. The range indicates the lower and higher limit reached during the run.

[0097] Biomass yield is expressed as grams of dried weight cells per liter (g DW cells / L).

[0098] At the end of the cultivation time, the cells were recovered from the bioreactor and lyophilized.

[0099] 1.4. Preparation of the extracts

[0100] The extracts (lipophilic and hydrophilic) were prepared according to the following protocol.

[0101] Solvents used:

[0102] - Hydrophilic extraction with 70% ethanol (solid S / liquid L ratio (w / v): 1 / 20)

[0103] - Lipophilic extraction with 100% Ethyl acetate (S / L ratio (w / v): 1 / 20)

[0104] Method:

[0105] 50 g of lyophilized cells were ground with 500 pm sieve (Retch ZM200) in 2L-Schott bottle with IL solvent (ethanol or ethyl acetate). The mixture was agitated for 10 min at 140 rpm to ensure a complete mixing and then sonicated for 20 min in an ultrasonic bath. After sonication, the mixture was further agitated for 2 hours at 140 rpm at room temperature. The resulting mixture was filtered into a new flask using a filter paper and a Buchner funnel or separated through centrifugation.

[0106] 5 mL of extract were collected in a pre-weighed glass tube and the solvent was evaporated under vacuum a 40°C with a rotary evaporator.

[0107] For the hydrophilic extract, it was possible to completely evaporate ethanol to obtain a dry extract. For the lipophilic extract, the evaporation of ethyl acetate was not complete resulting in a viscous extract.

[0108] The tubes were weighed again to calculate the extraction yield (DW extract / DW cells).

[0109] After solvent removal, the hydrophilic dry extract has been homogenized in a mortar with pestle.

[0110] The dry and homogeneous hydrophilic extract powder was then stored at a temperature below 25 °C in light protection tube / bottle e.g. Amber Eppendorf Tubes® (VWR: EPPE0030122.330). The viscous lipophilic extract was stored in the same conditions.

[0111] The amount of rosmarinic acid present in the hydrophilic extract was quantified by HPLC chromatography using dedicated standard (Extrasynthese). The amount of rosmarinic acid in the hydrophilic extract was 270.22 ± 4.08 pg / mL.

[0112] EXAMPLE 2: Determination of the antioxidant power of the extracts

[0113] A spectrophotometric assays has been performed to determine the antioxidant power of the hydrophilic and lipophilic extracts obtained according to the process described in example 1, before the step of removing the solvents used for the extraction.

[0114] 2.1 Method

[0115] The antioxidant activities were determined using the Ferric Reducing Antioxidant Power (FRAP) assay. In a 96-well plate, 10 pL of extract, and 190 pL of FRAP reagent were pipetted. The reagent was composed of acetate buffer, ferric 2,4,6-tripyridyl-s-triazine (TPTZ) solution and FeCh at a ratio 10: 1 : 1 v / v / v. The plate was then incubated in darkness for 20 minutes and the absorbance was read with a spectrophotometer (Spark20M ®, TECAN) at 593 nm.

[0116] 2.2 Results

[0117] The results are reported in Table 2 below, the antioxidant power being expressed in acid ascorbic equivalent:

[0118] TABLE 2

[0119] Stability tests conducted by incubating the extracts for 4 weeks at 40°C revealed a decrease of 15% and 21% in the antioxidant activity in the hydrophilic and lipophilic extracts, respectively. The loss of activity in the hydrophilic extract coincided with a decrease in rosmarinic acid concentration from 270.22 ± 4.08 pg / ml to 175.4 ± 2.31 pg / ml. EXAMPLE 3: Antimicrobial of the extracts

[0120] The hydrophilic and lipophilic extracts prepared above in example 1 were used to assess their antimicrobial properties.

[0121] 3.1 Method

[0122] Antimicrobial analyses were performed by a liquid culture method.

[0123] Following microbial strains were screened:

[0124] - Staphylococcus aureus VTT E-70045 (ATCC 6538),

[0125] - Pseudomonas aeruginosa VTT E-84219 (ATCC 15692),

[0126] - S. aureus MR.SA VTT E-183582 (DSM 11822),

[0127] - E. coll VTT E-94564T (ATCC 11775),

[0128] - Staphylococcus epidermidis VTT E-97768T (ATCC 14990), and

[0129] - Candida albicans VTT C-85161 (ATCC 10231).

[0130] Bacterial strains were cultivated aerobically in BD Difco ® Nutrient Broth (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C, shaking at 150 rpm. C. albicans was cultivated accordingly in Yeast Mannitol Broth. Microbial stock cultures were grown on solid media for 1-2 days as described above for each strain. Single colonies were transferred to liquid media, incubated for 20-24 h, and used as the source of inoculum for antimicrobial activity tests. Assessed extracts (at 1 mg / mL or 3 mg / mL or 5 mg / mL depending on the assay) were weighed in a sterile Eppendorf tube of 2 mL and suspended with a microbial inoculum of 1 mL in microbial culture medium (Nutrient Broth / Yeast Mannitol Broth). Microbial cultures without extract were included as positive controls, and antibiotics (chloramphenicol 50 pg / mL for bacterial cultures and hygromycin 150 pg / mL for yeast culture) as negative controls. The cultures with the extracts in Eppendorf tubes were incubated at +37 °C, shaking at 150 rpm for 48 h. Samples were taken during cultivation on occasions of 0, 3, 6, 24, and 48 h for antimicrobial activity evaluation. Plate counts (cfu / mL) of the samples were measured by serial dilutions on plates (plate count method), and the antimicrobial activity of the extracts was evaluated by comparison of control growth curves of the microbial liquid cultures with the ones with plant cell extracts.

[0131] 3.2 Results

[0132] The results of the assays are shown on Figures 1-5 annexed. The results obtained with the hydrophilic extracts on the two strains of S. aureus are reported on Figure 1. Figure la is a graph representing the results obtained on strain S. aureus (strain ATCC 6538) on which CFU / mL are expressed as a function of time (in hours) for each assay. Curve 1 corresponds to the positive control, curve 2 to the assay with hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract a 5 mg / mL and curve 4 to the negative control with chloramphenicol at 50 pg / mL.

[0133] Figure lb is a graph representing the results obtained on strain S. aureus (strain DSM 11822) on which CFU / mL are expressed as a function of time (in hours) for each assay. Curve 1 corresponds to the positive control, curve 2 to the negative control with chloramphenicol at 50 pg / mL, curve 3 to the assay with the hydrophilic extract a 1 mg / mL, curve 4 to the assay with the hydrophilic extract at 3 mg / mL, and curve 5 to the assay with the hydrophilic extract at 5 mg / mL.

[0134] Results presented on figure 1 demonstrate that the hydrophilic extracts of S. sclarea cell cultures show strong antimicrobial activity at 5 mg / mL level against S. aureus (strain ATCC 6538) and also against S. aureus (strain DSM 11822) at levels 1 - 5 mg / mL. The results obtained on S. aureus (strain DSM 11822) are particularly interesting since the antimicrobial activity of the hydrophilic extract of S sclarea obtained according to the process of the invention is higher than the antimicrobial activity of the antibiotic solution of chloramphenicol.

[0135] The results obtained with the hydrophilic extract on the E. coll strain are reported on Figure 2 annexed on which CFU / mL are expressed as a function of time (in hours) for each assay. On figure 2, curve 1 corresponds to the positive control, curve 2 corresponds to the assay with the hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract at 3 mg / mL and curve 4 to the assay with the hydrophilic extract at 5 mg / mL. These results show that the hydrophilic extract of S. sclarea cell cultures according to the invention exhibits an antimicrobial activity against E. coll at 3 and 5 mg / mL.

[0136] The results obtained with the hydrophilic extracts on the Staphylococcus epidermidis strain are reported on Figure 3 annexed on which CFU / mL are expressed as a function of time (in hours) for each assay. On this figure 3, curve 1 corresponds to the positive control, On figure 2, curve 1 corresponds to the positive control, curve 2 corresponds to the assay with the hydrophilic extract at 1 mg / mL, curve 3 to the assay with the hydrophilic extract at 3 mg / mL and curve 4 to the assay with the hydrophilic extract at 5 mg / mL. These results show that the hydrophilic extract of S. sclarea cell cultures according to the invention exhibits a weak / m ode rate antimicrobial activity against E. coli at 5 mg / mL.

[0137] The results obtained with the lipophilic extracts on C. albicans are reported on Figure 4 annexed on which CFU / mL are expressed as a function of time (in hours) for each assay. On this figure, curve 1 corresponds to the positive control + 15 pL of ethanol and curve 2 to the assay with the lipophilic extract at 5 mg / mL + 15 pl of ethanol. These results show that the lipophilic extract of S. sclarea cell cultures according to the invention exhibits a weak antimicrobial activity against C. albicans at 5 mg / mL.

[0138] The results obtained with the lipophilic extracts on S. epidermidis are reported on Figure 5 annexed on which CFU / mL are expressed as a function of time (in hours) for each assay. On this figure, curve 1 corresponds to the positive control + 15 pL of ethanol and curve 2 to the assay with the lipophilic extract at 5 mg / mL + 15 pl of ethanol. These results show that the lipophilic extract of S. sclarea cell cultures according to the invention exhibits a weak antimicrobial activity against S. epidermidis at 5 mg / mL.

[0139] EXAMPLE 4: Antioxidant properties of the extracts

[0140] The hydrophilic and lipophilic extracts prepared above in example 1 were used to assess their anti-inflammatory and antioxidant properties.

[0141] 4.1 Assessment of the anti-inflammatory properties of the extracts of the invention in normal human epidermal keratinocvtes

[0142] 4, 1_ M_a_teri a ls_ a _nd jrethods

[0143] NHEK-KBF-luc cells have been stably transfected with the plasmid KBF-Luc plasmid, which contains three copies of N F-KB binding site (from major histocompatibility complex promoter), fused to a minimal simian virus 40 promoter driving the luciferase gene. Cells (15xl04cells / ml) were seeded the day before the assay. Then the cells were treated with the test substances at 300pg / mL (n=4) for 15 min and then stimulated with 30 ng / ml phorbol 12-myristate 13-acetate (PMA) or TNFo. After 6 h, the cells were washed twice with PBS and lysed in lOOpL lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgC , 1 mM dithiothreitol (DTT), 1% Triton X-100, and 7% glycerol during 15 min at room temperature in a horizontal shaker. After centrifugation, the supernatants were used to measure luciferase activity using an Autolumat LB 9510 (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The protein concentration in the cell extracts was measured by the Bradford method (BioRad). The RLU / pg was calculated and the results were expressed in % of inhibition compared to the control (80% threshold).

[0144] 4,k2_ Results

[0145] The NF-kappaB inhibitions in normal Human epidermal fibroblast (NHEK) (in % compared to the control) are expressed for each extract in the following Table 3:

[0146] TABLE 3

[0147] These results show that the extracts of the invention exhibits antiinflammatory properties at the tested concentration.

[0148] 4.2 Assessment of the antioxidant properties of the extracts of the invention in normal human dermal fibroblasts

[0149] 4, 2 1_ M_a_teri a ls_ a _nd jrethods

[0150] Normal Human Dermal Fibroblasts (NHDF) cells were seeded at a density of 2xl04cells / well in 96-well plates and incubated overnight in DMEM medium at 37°C in a humidified atmosphere of 5% CO2. Then, cell cultures were stimulated with the test extract at 300pg / ml for 48h (4 doses, n=4). Cells were then collected and treated according the 8-isoprostane ELISA kit protocol (Abeam). Cells were first collected and homogenized using a solution containing a final concentration of 0.1 mM triphenylphosphine (TPP). The lysate was stabilized at pH=4 with acetic acid and an extraction with an equal volume of ethyl acetate was performed. After evaporation, 10 pL ethanol was added to dissolve the residue and further diluted with the solution provided by the manufacturer. lOOpL of proceeded sample were further using ELISA in 96-well plate according to the manufacturer guidelines.

[0151] The results are expressed in % of inhibition compared to the control (80% threshold). 4.2^ 2_ Results

[0152] The Isoprostane inhibitions in NHDF (in % compared to the control) are expressed for each extract in the following Table 4:

[0153] TABLE 4 These results show that the extracts of the invention exhibits antioxidant properties at the tested concentration.

[0154] EXAMPLE 5: Antiaaing cosmetic composition

[0155] The hydrophilic extract obtained according to example 1 has been used to prepare a antiaging day cream in the form of an oil-in-water emulsion comprising the following ingredients (in wt. %):

[0156] Phase A

[0157] - Glycerin (moisturizing agent) 3

[0158] - Demineralized water 69.8

[0159] Phase B - Glyceryl stearate (thickener) 12

[0160] - Cetyl alcohol (texturizing agent) 2

[0161] - Ceteareth 20 (emulsifier) 2

[0162] - Caprylic / capric triglyceride (emollient) 8

[0163] - Argania Spinosa oil (oil base) 1 - Prunus dulcis oil (oil base) 1

[0164] Phase C

[0165] - Perfume (fruit essence) 0.1

[0166] - Phenoxyethanol (conservative) 0.1

[0167] Extract of the invention - Hydrophilic cell culture extract as obtained according to example 1 (active ingredient) 1 Glycerin has been hydrated in water at a temperature of 80°C. Phase B, previously homogenized under a cooled turbine, has been added to phase A at a temperature of 80°C. The mixture of Phase A and Phase B has been cooled down until 40°C then Phase C and the hydrophilic extract of the invention have been added successively at 40°C, the whole is mixed and gradually cooled.

[0168] This antiaging cream can be applied to the face once or twice a day, for example during the morning and evening routines, for a period of 1 to 3 months.

[0169] EXAMPLE 6: COMPARATIVE ASSESSMENT OF SOME METABOLITES OF CLASSICAL EXTRACTS FROM LEAVES AND CELLS OBTAINED ACCORDING TO THE PROCESS OF THE INVENITON

[0170] In this example, a comparative qualitative and semi-quantitative study has been conducted to assess some metabolites in an alcoholic extract obtained from dried leaves of S. sclarea and in cells obtained according to the in vitro process of the invention.

[0171] 6.1. Material and methods

[0172] In this example, the cells have been prepared according to steps 1.1 to 1.3 of Example 1.

[0173] As a comparison purpose, dried leaves of S. sclarea were also used.

[0174] S. sclarea dried leaf and dried cell samples were first ground using a Retsch ZM200 in a 50mL tube filled with two 4.5 mm beads (25Hz, 3min). About 50 mg sample powder was extracted using a 70% ethanol / water mixture at a solid liquid ratio of 1 :30 w:v. The mixture was sonicated in a bath for 10 minutes (37Hz, 100% power, sweep mode), followed by an incubation for lh at 25°C with 150 rpm agitation. A centrifugation was carried out to recover the extracts (5000rpm for lOmin at RT). Samples were recovered (ImL) in a fresh 2 mL-tube and further dried. Dried extracts were resuspended in 0.6 mL 5% (v / v) methanol before analysis. Extracts were filtered through a 0.22 pm PTFE syringe filter (Millex-LG, Merck KGaA, Darmstadt, Germany) and analyzed using an Acquity UPLC I-Class ultra-high-pressure liquid chromatography (UHPLC) system equipped with a diode array detector (DAD) (Waters, Milford, MA, USA) coupled to a hybrid quadrupoletime of flight mass spectrometer (TripleTOF 6600+, SCIEX, Framingham, MA, USA) in negative ionization mode. 10 microliters of the sample were injected and separated on a reverse-phase Acquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 |jm particle size) (Waters, Milford, MA, USA) at a flow rate of 0.5 mL / min and a column temperature of 50°C. The mobile phase consisted of 0.1% (v / v) formic acid in water (A) and 0.1% (v / v) formic acid in acetonitrile (B), with the following gradient: 0 min, 1% B; 4 min, 1% B; 16 min, 5% B; 35 min, 40% B; 45 min, 100% B; 50 min, 100% B; 54 min, 1% B; and 60 min, 1% B. UV-visible spectra were acquired between 190 and 800 nm at a rate of 10 points / sec.

[0175] Electrospray ionization (ESI) was performed on analytes using the following parameter values for positive and negative modes: source temperature 650°C; ion spray voltage of 4.5 and -4.5 kV, respectively, curtain gas (nitrogen) of 30 psi, nebulizer gas (air) of 55 psi and turbine gas (air) of 50 psi. The declustering potential was set up at 60 V in positive and -60 V in negative mode. Survey scans of 175 ms were acquired for information-dependent acquisition. The ten highest MS ions were selected for fragmentation if they were singly charged and had an intensity exceeding a threshold of 100 counts / sec. Product ion scans were collected with an accumulation time of 200 ms in high sensitivity mode, thus leading to a total cycle time of 2.225 s. A sweeping collision energy of 15 V below and above 15 and -15 V, for the positive and negative modes, respectively, was applied to all precursor ions. The dynamic exclusion was set for 2 s after three occurrences before the precursor could be fragmented again. Data for all varieties were acquired in negative mode, while positive mode analysis was run on one variety of each species to aid in the identification of the compounds.

[0176] For the identification and relative quantification, raw data files were processed using Progenesis QI (v2.3, Nonlinear Dynamics, Newcastle upon Tyne, UK) to align all runs, normalize the data, and perform relative quantitative analysis based on sample groups (species). Only features with MS / MS data were retained for the identification stage. The output data were manually reviewed using PeakView software (vl.2, SCIEX, Framingham, MA). Initial identification relied on the use of an in-house database exported in msp-format. All database hits were manually validated. For compounds which were not present in this in-house database, identification was achieved through a literature search and MS / MS comparison using external databases such as GNPS (https: / / gnps.ucsd.edu / ProteoSAFe / libraries.jsp), MZCIoud™

[0177] (https: / / beta.mzcloud.org / ), LipidMaps (https: / / www.lipidmaps.org / ), and PubChem (https: / / pubchem.ncbi.nlm.nih.gov). Accepted identifications were subsequently incorporated into the in-house database for future dereplication. All identifications reported in this study adhere to level 2 standards as defined by the Metabolomics Standards Initiative (MSI).

[0178] 6.2 Results Figure 6 gives the chromatograms obtained from S. sc / area cell extract

[0179] (upper curve) and from the S. sclarea leaf extract (lower curve).

[0180] It emerges from these chromatograms that the two extracts display strong differences in chemical patterns.

[0181] Identifications relative to the peaks of major peaks displayed in figure 6 (leaf and cell extracts) are given in the following table 5. Abundance was calculated from the ionisation intensity. DL means that the compound was below the Detection Limits. RT: retention time, m / z: mass-to-charge ratio.

[0182] TABLE 5 Conclusions:

[0183] S. sclarea leaf and cell extracts display totally different chemical profiles (R2=0.24, figure 6, table 5).

[0184] Rosmarinic acid and derivatives (rosmarinic acid-glucoside, rosmarinic acid dimer, salvianolic acid, etc...) are more abundant in the extract produced using S. sclarea plant cell suspension culture process according to the invention (Figure 6, Table 5).

[0185] These results demonstrate that the process according to the present invention is not a simple alternative to classical extraction methods from plant leaves but leads to qualitatively and quantitatively different extracts. In particular, the process of the invention is particularly suitable for obtaining extracts having a very hight content in rosmarinic acid and its derivatives.

Claims

CLAIMS1. A process for the preparation of an extract derived from a Salvia sclarea undifferentiated cell culture, said process comprising at least the steps of: a) providing a suspension-cultured cell line cultured in a liquid basal medium from a callus of a plant of the species Salvia sclarea, b) culturing said suspension-cultured cell line of step a) in a liquid basal medium free from any elicitor and in the dark, to obtain Salvia sclarea undifferentiated cells, c) recovering said cells from the liquid basal medium, d) converting said cells into cell fragments, e) homogenizing said cells fragments in a liquid polar solvent, f) removing said solvent, partially or in totality, to obtain said extract.

2. The process according to claim 1, wherein said liquid basal medium is supplemented with at least one phytohormone.

3. The process according to claim 2, wherein said phytohormone is selected from the group comprising auxins and cytokinins.

4. The process according to claim 2 or 3, wherein said liquid medium is supplemented with 2,4-dichlorophenoxyacetic acid and / or kinetin.

5. The process according to any one of claims 1 to 4, wherein said callus is obtained from leaf explants of Salvia sclarea in v7tro-grown plants.

6. The process according to any one of the preceding claims, wherein step b) is carried out at a temperature ranging from 22 to 32°C.

7. The process according to any one of the preceding claims, wherein step b) is carried out at a temperature of 25±1°C for 10 to 21 days under agitation.

8. The process according to any one of the preceding claims, wherein said polar solvent is a polar protic solvent selected in the group comprising water, methanol, ethanol, acetic acid, isopropanol, 1,3-propanediol, glycerol, and mixtures thereof, and further wherein during step f) said solvent is totally removed and said extract is a dry hydrophilic extract.

9. The process according to any one of claims 1 to 7, wherein said polar solvent is a polar aprotic solvent selected in the group comprising ethyl acetate, acetone, dimethyl sulfoxide, acetonitrile, tetra hydrofuran, dichloromethane, and mixtures thereof, and further wherein during step f) said solvent is only partially removed and said extract is a concentrated lipophilic extract.

10. An extract derived from a Salvia sclarea undifferentiated cell culture, wherein said extract is obtained by the process as defined in any one of claims 1- 9.

11. The extract according to claim 10, wherein said extract is a dry hydrophilic extract and further wherein it comprises rosmarinic acid.

12. An extract derived from a Salvia sclarea undifferentiated cell culture for its use as an active ingredient in the prevention and / or treatment of inflammation or microbial infections, wherein said extract is obtained by the process as defined in any one of claims 1-9.

13. The extract for its use according to claim 12, wherein said extract is a dry hydrophilic extract and further wherein said microbial infections are infections caused by Staphylococcus aureus, multiresistant strains of Staphylococcus aureus, Staphylococcus epidermidis or Escherichia coli.

14. The extract for its use according to claim 12, wherein said extract is a concentrated lipophilic extract and further wherein said microbial infections are infections caused Staphylococcus epidermidis or Candida albicans.

15. A pharmaceutical composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined in any one of claims 1-9, and a pharmaceutically acceptable vehicle.

16. A sanitizing composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined in any one of claims 1-9, and a sanitizing acceptable vehicle.

17. Use of a composition according to claim 16, for sanitizing a surface or increasing the surface's resistance to microbe transmission.

18. A cosmetic use of an extract derived from a Salvia sclarea undifferentiated cell culture, wherein said extract is obtained by a process asdefined in any one of claims 1 to 9 or is as defined in claim 10 or 11, as an antiaging active ingredient.

19. A cosmetic composition comprising an extract derived from a Salvia sclarea undifferentiated cell culture obtained by the process as defined in any one of claims 1 to 9 or is as defined in claim 10 or 11 and a cosmetically acceptable vehicle.

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