Phytocomplexes and extracts of meristem cell lines selected from plants belonging to the genus Rosa

Abstract

The present invention relates to meristematic cell lines selected from tissues, preferably callus tissues, of plants belonging to the genus Rosa. The present invention also relates to derivatives of the cell lines, i.e., phytocomplexes or extracts of the cell lines. The meristematic cell lines are characterized by a high polysaccharide content. Furthermore, the present invention relates to cosmetic, functional food, and medical uses of the selected meristematic cell lines or their derivatives.

Description

Technical Field

[0001] The present invention relates to a meristem cell line derived from a plant belonging to the genus Rosa (Rosa) characterized by a high polysaccharide content, and to the cosmetic, functional food, and medical uses of said meristem cell line or its derivatives.

Background Art

[0002] Medium molecular weight polysaccharides such as dextran exert a moisturizing and anti-inflammatory activity and contribute to the improvement of the mechanical properties of the skin and tissues by retaining water by an osmotic mechanism. Furthermore, dextran, in association with other active ingredients and a suitable vehicle, can reduce edema and erythema even in individuals with sensitive skin, and can further reduce skin aging and irritation due to the action of weak carboxylic acids. In the pharmaceutical field, dextran sulfate has antithrombotic and anticoagulant activities and can also be used topically to regulate skin blood flow.

[0003] There are numerous plant sources containing medium molecular weight polysaccharides and many of their uses are already known. The most well-known among them is Aloe vera, and its leaves rich in polysaccharides are widely used in cosmetic formulations to increase skin moisturization.

[0004] Polysaccharides present in Camellia sinensis have also been demonstrated to be active in skin moisturization by increasing the expression of aquaporin. Aquaporin is a family of membrane proteins that allow the passage of water and small solutes across cell membranes. In particular, aquaporin 3 plays an important role in regulating skin moisturization, skin elasticity, and the barrier function of the epidermis.

[0005] The preparation of standardized plant derivatives (i.e., extracts containing metabolites of reproducible content) causes many problems related to the variation in metabolite content in different plant tissues, seasonal variation in metabolite content and type, contamination by plant parasites, differences related to cultivation areas, and loss of biological activity of molecules during harvesting, storage, and extraction. Extreme variations in the content of plant components in plant preparations obtained directly from plants or parts thereof by extraction negatively affect their effectiveness.

[0006] Another method for obtaining contaminant-free standardized plant phytocomplexes in industrial quantities is to use in vitro cell cultures. This technique makes it possible to solve the problems related to the variability of plant extracts, since it provides preparations containing active substances of reproducible content that can be reproduced in a standardized manner. The present invention relates to this technical basis and provides selected meristematic cell lines capable of obtaining phytocomplexes (and also extracts) containing standardized and reproducible contents of active substances.

[0007] The present invention provides selected meristematic cell lines and their derivatives derived from plants belonging to the genus Rosa, having a high polysaccharide content.

Summary of the Invention

Means for Solving the Problems

[0008] The first aspect of the present invention relates to a meristematic cell line derived from a plant belonging to the genus Rosa, preferably the species Rosa canina, or the species Rosa chinensis, preferably a cell line derived from callus tissue obtained from the plant itself.

[0009] The second aspect of the present invention relates to derivatives of the meristematic cell line, i.e., phytocomplexes or extracts of the cell line.

[0010] The meristematic cell line and its derivatives are preferably characterized by a high content of polysaccharides having an intermediate molecular weight.

[0011] A third aspect of the present invention relates to a composition comprising a meristematic cell line or a derivative thereof, mixed with an excipient acceptable from the viewpoint of cosmetics and / or pharmaceuticals.

[0012] The present applicant has demonstrated that the cell line or its derivative has antioxidant activity and can increase the biosynthesis of aquaporin, particularly aquaporin 3 (both for topical and systemic application to skin models). Aquaporin is a substance involved in maintaining skin hydration and wound healing.

[0013] Therefore, the present invention also relates to the cosmetic use of a meristematic cell line or a derivative thereof for protecting the skin from signs of aging and maintaining skin hydration (hydrating activity). The cell line or its derivative also has anti-wrinkle and antioxidant activities.

[0014] Furthermore, the meristematic cell line or a derivative thereof can be used for the treatment or prevention of skin-affecting pathologies such as erythema, irritation, local inflammation or cracking, or to promote the wound healing process (i.e., to accelerate wound healing).

[0015] Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Rosa having a high polysaccharide content.

[0016] The present invention will be described in detail below, with reference to the accompanying drawings by way of example.

Brief Description of the Drawings

[0017]

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Embodiments for Carrying Out the Invention

[0018] Definitions In connection with the present invention, "meristematic tissue line" or "meristematic tissue cell" means a plant line or cell capable of maintaining the ability to divide by mitosis to produce new cells. All meristematic tissue cells are derived from another meristematic tissue cell. The function of plant meristematic tissue cells is equivalent to that of animal stem cells.

[0019] In connection with the present invention, "callus tissue" refers to an unorganized mass of undifferentiated cells or cells that are hardly specialized, having a thin cell wall and large vacuoles in which secondary metabolites are accumulated.

[0020] In connection with the present invention, "medium molecular weight polysaccharide" means a polysaccharide having a molecular weight of 1000 to 5000 Da.

[0021] In connection with the present invention, "low molecular weight polysaccharide" means a polysaccharide having a molecular weight of less than 1000 Da.

[0022] In connection with the present invention, unless otherwise specified, "w / w" means a weight / weight quantity with respect to the dry mass of the cell line.

[0023] The first aspect of the present invention relates to a meristematic tissue cell line derived from a plant belonging to the genus Rosa, preferably the species Rosa canina, or the species Rosa chinensis.

[0024] In one embodiment, the meristematic tissue cell line is as follows: 1) planting a tissue obtained from a plant of the genus Rosa on a solid medium; 2) isolating a plurality of cell clones; 3) inoculating each of the isolated clones into a liquid medium; 4) quantifying the polysaccharide content for each clone; 5) selecting the cell clone having the highest polysaccharide content and is obtained by a process comprising:

[0025] In step 1), tissue obtained from a plant of the genus Rosa (Rosa) is placed in a solid medium to obtain undifferentiated callus tissue. The Rosa tissue is preferably at least one shoot of Rosa or a plurality of shoots of Rosa.

[0026] In a preferred embodiment of the present invention, the solid and liquid media contain salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), and 6-benzylaminopurine (BAP).

[0027] The solid medium further contains agar, while the liquid medium does not contain agar.

[0028] The solid and liquid media preferably each contain sucrose at a concentration of 10 - 45 g / L, preferably 15 - 40 g / L; NAA at a concentration of 0.5 - 2.5 mg / L, preferably 0.8 - 2 mg / L, and BAP at a concentration of 0.1 - 0.5 mg / L, preferably 0.15 - 0.3 g / L.

[0029] In both the solid and liquid media, the salts suitable for the growth of plant cells are selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4, and combinations thereof.

[0030] In both the solid and liquid media, the salts suitable for the growth of plant cells are preferably selected from the following: CoCl2·6H2O, CuSO4·5H2O, NaEDTA·2H2O, FeSO4·7H2O, H3BO3, Kl, MnSO4·H2O, Na2MoO4·2H2O, ZnSO4·7H2O, and combinations thereof.

[0031] Both the solid and liquid media preferably further contain vitamins suitable for the growth of plant cells, selected from the following: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl, and combinations thereof.

[0032] In one embodiment, salts suitable for the growth of plant cells in both solid and liquid media are as follows: selected from CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4, CoCl2·6H2O, CuSO4·5H2O, NaEDTA·2H2O, FeSO4·7H2O, H3BO3, Kl, MnSO4·H2O, Na2MoO4·2H2O, ZnSO4·7H2O and combinations thereof. This combination of salts is Gamborg B5 medium.

[0033] In one embodiment, both solid and liquid media further contain vitamins suitable for the growth of plant cells, selected from the following: inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof, in addition to the salts listed above.

[0034] The solid and liquid media preferably each contain CaCl2 at a concentration of 120 - 170 mg / L, preferably 130 - 160 mg / L; KNO3 at a concentration of 800 - 3700 mg / L, preferably 1000 - 3100 mg / L; MgSO4 at a concentration of 220 - 270 mg / L, preferably 230 - 260 mg / L; NaH2PO4 at a concentration of 100 - 180 mg / L, preferably 110 - 150 mg / L; and (NH4)2SO4 at a concentration of 100 - 180 mg / L, preferably 110 - 150 mg / L.

[0035] The solid and liquid media preferably each contain CoCl2·6H2O at a concentration of 0.01 - 0.05 mg / L, preferably 0.015 - 0.03 mg / L; CuSO4·5H2O at a concentration of 0.01 - 0.05 mg / L, preferably 0.015 - 0.03 mg / L; NaEDTA·2H2O at a concentration of 20 - 60 mg / L, preferably 30 - 45 mg / L; FeSO4·7H2O at a concentration of 15 - 45 mg / L, preferably 20 - 35 mg / L; H3BO3 at a concentration of 1 - 7 mg / L, preferably 2 - 5 mg / L; Kl at a concentration of 0.1 - 2 mg / L, preferably 0.4 - 1 mg / L; MnSO4·H2O at a concentration of 5 - 20 mg / L, preferably 7 - 15 mg / L; Na2MoO4·2H2O at a concentration of 0.1 - 0.5 mg / L, preferably 0.15 - 0.3 mg / L, and ZnSO4·7H2O at a concentration of 0.5 - 5 mg / L, preferably 1 - 3 mg / L.

[0036] Both the solid and liquid media preferably each contain myo-inositol at a concentration of 70 - 130 mg, preferably 90 - 110 mg; pyridoxine-HCl at a concentration of 70 - 130 mg, preferably 90 - 110 mg; and thiamine-HCl at a concentration of 5 - 20 mg / L, preferably 7 - 15 mg / L.

[0037] After step 1), the callus tissue is preferably divided into a plurality of parts and stabilized by continuously transferring them to a solid medium (step 1a)) to obtain stabilized cells. This step is called the stabilization step.

[0038] After the stabilization step 1a), the stabilized cells preferably undergo the first "clone selection". Clone selection involves culturing the stabilized cells for an appropriate period, preferably 5 - 20 days of culture, more preferably 10 - 15 days (step 1b). These cells are incubated in the dark at a temperature of 15°C - 35°C, preferably 24°C - 26°C.

[0039] In step 2), a plurality of cell clones are isolated by removing aggregates of stabilized cells from the solid medium.

[0040] In step 3), each cell clone is inoculated into the above-described liquid medium.

[0041] According to one embodiment, in step 4), after a time sufficient to achieve proper growth of the cell clones, preferably after a growth phase of 10 to 15 days, the polysaccharide content of each clone is quantified.

[0042] In step 5) of the selection of cell clones, it is preferable to perform a second clone selection according to step 1b) until a Rosa plant cell line with optimal production of polysaccharides having a preferably medium molecular weight is obtained.

[0043] In a preferred embodiment, the clone selection of step 5) is repeated until a Rosa cell line containing polysaccharides in an amount of more than 25% w / w, preferably 25% - 70% w / w, more preferably 30% - 65% w / w is obtained.

[0044] The polysaccharides present in the selected cell line contain 50% - 90%, preferably 55% - 85% of medium molecular weight polysaccharides.

[0045] The polysaccharides present in the selected cell line contain 20% - 30%, preferably 25% - 35% of low molecular weight polysaccharides.

[0046] In other words, the selected cell line contains 15% - 60% w / w, preferably 18% - 50% w / w of medium molecular weight polysaccharides and 5% - 20% w / w, preferably 8% - 15% w / w of low molecular weight polysaccharides, based on the dry weight of the cell line.

[0047] In one embodiment, the selected cell line contains more than 0.2% w / w, preferably 0.3% - 20% w / w, more preferably 0.4% - 18% w / w of polyphenols.

[0048] Cell lines derived from the Rosa canina species preferably contain polyphenols in an amount of 3% to 25% w / w, preferably 4% to 20% w / w.

[0049] Cell lines derived from the Rosa chinensis species preferably contain polyphenols in an amount of 0.3% to 10% w / w, preferably 0.4% to 8% w / w.

[0050] In one embodiment, the polyphenols are selected from flavonoids and non-flavonoids, where the flavonoids are preferably selected from catechin, epicatechin, procyanidin, and proanthocyanidin. The polyphenols are preferably catechin and proanthocyanidin, preferably selected from type P2, type P3, and type P4 (P represents a polymer, and the numbers 2, 3, and 4 indicate the number of catechin monomers contained in the molecule).

[0051] The non-flavonoids are selected from hydroxybenzoic acid, preferably gallic acid, and hydroxycinnamic acid, preferably selected from coumaric acid and caffeic acid.

[0052] In a preferred embodiment, the cell line contains protein in an amount of 10% to 40% w / w, preferably 12% to 38% w / w, more preferably 15% to 35% w / w.

[0053] In a preferred embodiment, the cell line contains hydroxyproline in an amount of 0.1% to 1.3% w / w, preferably 0.2% to 1.2% w / w, more preferably 0.3% to 1% w / w.

[0054] In a preferred embodiment, the cell line contains lipids in an amount of 1% to 10% w / w, preferably 2% to 8% w / w, more preferably 3% to 6% w / w.

[0055] The cell line derived from the Rosa canina species is preferably the Rc-F2P strain containing 30 - 55% w / w of polysaccharides, 65 - 80% of which are medium molecular weight polysaccharides, and preferably 5 - 15% w / w of total polyphenols, 15 - 20% w / w of proteins, and 0.2 - 0.6% w / w of hydroxyproline.

[0056] The cell line derived from the Rosa Chinensis species is preferably the Rch-PsMW strain containing 30 - 60% w / w of polysaccharides, 65 - 80% of which are medium molecular weight polysaccharides, and preferably 0.5 - 5% w / w of total polyphenols, 16 - 35% w / w of proteins, and 0.7 - 1.2% w / w of hydroxyproline.

[0057] The second aspect of the present invention relates to a derivative of a cell line which is a phytocomplex or extract of the selected meristematic cell line described above.

[0058] Phytocomplex means dried or lyophilized cells, cell homogenates, or cell walls and their components. The phytocomplex is preferably a cell homogenate.

[0059] The phytocomplex contains polysaccharides in an amount of more than 25% w / w, preferably 25% - 70% w / w, more preferably 30% - 65% w / w.

[0060] The polysaccharides present in the phytocomplex contain 50% - 90%, preferably 55% - 85% of medium molecular weight polysaccharides.

[0061] The polysaccharides present in the phytocomplex contain 20% - 30%, preferably 25% - 35% of low molecular weight polysaccharides.

[0062] In other words, the phytocomplex contains intermediate molecular weight polysaccharides in an amount of 15% - 60% w / w, preferably 18% - 50% w / w, and low molecular weight polysaccharides in an amount of 5% - 20% w / w, preferably 8% - 15% w / w, based on the dry mass of the phytocomplex.

[0063] The phytocomplex also contains proteins in an amount of 10% - 40% w / w, preferably 12% - 38% w / w, more preferably 15% - 35% w / w, based on the dry mass of the phytocomplex.

[0064] The phytocomplex contains hydroxyproline in an amount of 0.1 - 1.3% w / w, preferably 0.2% - 1.2% w / w, more preferably 0.3% - 1% w / w, based on the dry mass of the phytocomplex.

[0065] The phytocomplex contains lipids in an amount of 1 - 10% w / w, preferably 2 - 8% w / w, preferably 3 - 6% w / w, based on the dry mass of the phytocomplex.

[0066] In a preferred embodiment, the phytocomplex is derived from the selected meristematic cell line Rc - F2P or the selected meristematic cell line Rch - PsMW. The phytocomplex is preferably a cell homogenate of the selected meristematic cell line Rc - F2P or a cell homogenate of the selected meristematic cell line Rch - PsMW.

[0067] The extract means the extract of the cell line itself or the phytocomplex of the cell line in an alcohol solvent, such as methanol or ethanol, or in a water / ethanol mixture in various ratios: 50:50 or 60:40 or 70:30. The extract is preferably an extract of the cell homogenate of the cell line. The content of the extract coincides with the content of the phytocomplex or the cell line from which it is obtained and can vary depending on the extraction technique.

[0068] The third aspect of the present invention relates to a composition comprising a split tissue cell line and / or its derivatives (phyto complex and / or extract) combined with at least one excipient acceptable from a cosmetic, functional food and / or pharmaceutical perspective.

[0069] In one embodiment, the composition comprises the cell line and / or its derivatives at a concentration of 0.01% - 30% w / w, preferably 0.03% - 15% w / w, more preferably 0.05% - 10% w / w, based on the weight of the composition.

[0070] In one embodiment, the cell line and / or its derivatives are dispersed before being mixed with the excipient to prepare the composition of the present invention. By way of example, suitable dispersants are glycerin, propylene or butylene glycol.

[0071] The composition of the present invention comprises at least one excipient acceptable for pharmaceutical, functional food and / or cosmetic use, which is useful in the preparation of the composition and is generally biologically safe and non-toxic.

[0072] The excipient is at least one conditioning agent, humectant, or occlusive agent for the skin, surfactant, stabilizer, preservative or emollient.

[0073] The composition of the present invention is formulated for topical use as a cream, gel cream, gel, serum, oil, emulsion, emulsion-gel (emagel) ointment, eye drops, mouthwash, spray, preferably nasal spray or stick (such as lip cream). For example, formulation of the composition as a face serum or cream having moisturizing, anti-wrinkle, antioxidant and / or scar-forming activity, preferably hand cream or face cream, is particularly preferred.

[0074] The composition may also be formulated for oral administration, preferably as pills, capsules, tablets, granular powder, hard shell capsules, orally disintegrating granules, sachets or lozenges.

[0075] In one embodiment, the composition is formulated to rapidly release the active ingredient contained therein or to release it in a delayed and / or controlled manner after administration, preferably formulated as liposomes.

[0076] The experimental data included herein indicate that the aforementioned cell lines or their derivatives can exert antioxidant (reduction of free radicals), moisturizing, and scar-forming effects.

[0077] In particular, the Applicant has demonstrated that derivatives of the cell line have the ability to reduce the level of free radicals and increase the expression of aquaporins, especially aquaporin 3, which is involved in maintaining skin hydration and the wound healing process.

[0078] Furthermore, the cell lines obtained by the Applicant exhibit a high content of hydroxyproline, which accounts for approximately 20% of the amino acids of glycoproteins present in the plant cell wall. These glycoproteins rich in hydroxyproline contain extensin, which is structurally similar to animal collagen. Therefore, many aspects of extensin are targeted for cosmetics.

[0079] Another aspect of the present invention relates to the cosmetic use of the cell line or its derivatives.

[0080] Cosmetic use means the prevention, reduction, and / or treatment of signs of skin aging, moisturization of the skin or cutin, anti-wrinkle activity, and antioxidant activity.

[0081] Another aspect of the present invention relates to the use as a drug, particularly for the treatment or prevention of skin-invasive conditions, preferably irritation, local inflammation, cracks or redness, or for promoting the wound healing process (scar-forming effect), of the cell line or its derivatives.

[0082] Another aspect of the present invention relates to the use of a cell line or a derivative thereof as a dietary supplement for the purpose of preventing, reducing or treating signs of skin aging and / or an increase in free radicals and / or systemic or skin dehydration symptoms. In this case, the cell line or a derivative thereof is formulated into a composition for oral use such as pills, capsules, tablets, granular powder, hard shell capsules, orally disintegrating granules, sachets or lozenges.

[0083] Another aspect of the present invention relates to the use of a cell line or a derivative thereof for body care and hygiene; in this case, the cell line or a derivative thereof is formulated, together with a suitable excipient, as a bath foam, shower gel, soap, shampoo or hair conditioner.

[0084] Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells having a high polysaccharide content, preferably a polysaccharide content of more than 25% w / w based on the dry weight of the cell line. The method comprises the following: 1) planting tissue obtained from a plant of the genus Rosa on a solid medium; 2) isolating cell clones; 3) inoculating each of the isolated clones into a liquid medium; 4) quantifying the polysaccharide content for each clone; 5) selecting the cell clone having the highest polysaccharide content including.

[0085] In one embodiment, the preparation of meristematic cells preferably involves collecting shoot tissue from a plant selected from the Rosa canina species and / or the Rosa chinensis species, for example washing it with water, cutting it into small pieces, and sterilizing it on a plate by successive treatments using, for example, ethanol, sodium hypochlorite and mercurous salts.

[0086] In a preferred embodiment of the present invention, the solid and liquid media contain salts, sucrose, naphthylacetic acid (NAA), and 6-benzylaminopurine (BAP) suitable for the growth of plant cells.

[0087] The solid medium further contains agar, while the liquid medium does not contain agar.

[0088] The solid and liquid media preferably each contain sucrose at a concentration of 10 - 45 g / L, preferably 15 - 40 g / L; NAA at a concentration of 0.5 - 2.5 mg / L, preferably 0.8 - 2 mg / L, and BAP at a concentration of 0.1 - 0.5 mg / L, preferably 0.15 - 0.3 g / L.

[0089] In both the solid and liquid media, the salts suitable for the growth of plant cells are selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4, and combinations thereof.

[0090] In both the solid and liquid media, the salts suitable for the growth of plant cells are preferably selected from the following: CoCl2·6H2O, CuSO4·5H2O, NaEDTA·2H2O, FeSO4·7H2O, H3BO3, Kl, MnSO4·H2O, Na2MoO4·2H2O, ZnSO4·7H2O, and combinations thereof.

[0091] Both the solid and liquid media further contain vitamins suitable for the growth of plant cells, preferably selected from the following: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl, and combinations thereof.

[0092] In one embodiment, in both the solid and liquid media, the salts suitable for the growth of plant cells are selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4, CoCl2·6H2O, CuSO4·5H2O, NaEDTA·2H2O, FeSO4·7H2O, H3BO3, Kl, MnSO4·H2O, Na2MoO4·2H2O, ZnSO4·7H2O, and combinations thereof. This combination of salts is Gamborg B5 medium.

[0093] In one embodiment, both the solid and liquid media further contain vitamins suitable for the growth of plant cells selected from, in addition to the salts listed above, myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl, and combinations thereof.

[0094] The solid and liquid media preferably each contain CaCl2 at a concentration of 120 - 170 mg / L, preferably 130 - 160 mg / L; KNO3 at a concentration of 800 - 3700 mg / L, preferably 1000 - 3100 mg / L; MgSO4 at a concentration of 220 - 270 mg / L, preferably 230 - 260 mg / L, NaH2PO4 at a concentration of 100 - 180 mg / L, preferably 110 - 150 mg / L; and (NH4)2SO4 at a concentration of 100 - 180 mg / L, preferably 110 - 150 mg / L.

[0095] The solid and liquid media preferably each contain CoCl2·6H2O at a concentration of 0.01 - 0.05 mg / L, preferably 0.015 - 0.03 mg / L; CuSO4·5H2O at a concentration of 0.01 - 0.05 mg / L, preferably 0.015 - 0.03 mg / L; NaEDTA·2H2O at a concentration of 20 - 60 mg / L, preferably 30 - 45 mg / L; FeSO4·7H2O at a concentration of 15 - 45 mg / L, preferably 20 - 35 mg / L; H3BO3 at a concentration of 1 - 7 mg / L, preferably 2 - 5 mg / L; Kl at a concentration of 0.1 - 2 mg / L, preferably 0.4 - 1 mg / L; MnSO4·H2O at a concentration of 5 - 20 mg / L, preferably 7 - 15 mg / L; Na2MoO4·2H2O at a concentration of 0.1 - 0.5 mg / L, preferably 0.15 - 0.3 mg / L, and ZnSO4·7H2O at a concentration of 0.5 - 5 mg / L, preferably 1 - 3 mg / L.

[0096] Both the solid and liquid media preferably each contain myo-inositol at a concentration of 70 - 130 mg / L, preferably 90 - 110 mg / L; pyridoxine-HCl at a concentration of 70 - 130 mg / L, preferably 90 - 110 mg / L; and thiamine-HCl at a concentration of 5 - 20 mg / L, preferably 7 - 15 mg / L.

[0097] After step 1), the callus tissue is preferably divided into a plurality of parts, and these are stabilized by continuously transferring them to a solid medium (step 1a)) to obtain stabilized cells. This step is called the stabilization step.

[0098] After the stabilization step 1a), the stabilized cells preferably undergo an initial "clone selection". The clone selection is to culture the stabilized cells for a suitable period, preferably 5 to 20 days of culture, more preferably 10 to 15 days (step 1b). These cells are incubated in the dark at a temperature of 15°C to 35°C, preferably 24°C to 26°C.

[0099] In step 2), a plurality of cell clones are isolated by removing aggregates of stabilized cells from the solid medium.

[0100] In step 3), each cell clone is inoculated into the liquid medium described above.

[0101] According to one embodiment, in step 4), after a time to achieve appropriate growth of the cell clones, preferably after a growth phase of 10 to 15 days, the polysaccharide content of each clone is quantified.

[0102] In the step 5) of cell clone selection, it is preferable to perform a second clone selection according to step 1b) until a plant cell line of Rosa with optimal production of polysaccharides having a preferably medium molecular weight is obtained.

[0103] Next, the selected cell line is grown in a flask or a bioreactor or a fermenter to achieve an increase in biomass.

[0104] The increase in biomass occurs in the first stage in the liquid growth medium. The liquid growth medium is a medium containing the Gamborg salts described above, the vitamins listed above, sucrose, NAA, and BAP.

[0105] Liquid growth media, namely RP for Rosa canina and RPS for Rosa chinensis, contain, among Gamborg salts, KNO3 in an amount of 2 g / L to 4 g / L, preferably 2 g / L to 3 g / L. Sucrose is preferably contained at 20 g / L to 30 g / L. NAA is preferably contained at 0.5 mg / L to 2 mg / L, and BAP is contained at 0.1 to 0.5.

[0106] Cells grown in the liquid growth media are transferred, for the final growth phase, to a final liquid medium containing the Gamborg salts listed above, the vitamins listed above, and sucrose, namely RP-F for Rosa canina and RPS-F for Rosa chinensis, thereby inducing an increase in polysaccharide content and biomass.

[0107] The final liquid medium contains, among Gamborg salts, KNO3 in an amount of 2 g / L to 5 g / L, preferably 2 g / L to 4 g / L. Sucrose is preferably contained at 25 g / L to 45 g / L. NAA is preferably contained at 0.5 mg / L to 2 mg / L, and BAP is contained at 0.1 mg / L to 0.5 mg / L.

[0108] According to a preferred embodiment, in both the liquid growth medium and the final liquid medium, the growth of cell lines in flasks, bioreactors or fermenters is carried out under dark conditions at a temperature of 15 °C to 35 °C, typically 25 °C, for 7 to 30 days, preferably 14 to 21 days.

[0109] At the end of the growth in the final liquid medium, after filtering the cell line, the cells are recovered and used in the next step in the form of a phytocomplex, or alternatively, they can be subjected to an extraction phase in a subsequent alcohol solvent to produce a cell extract characterized by a high polysaccharide content.

[0110] The phytocomplex can be obtained by freeze-drying or drying of living cells; in this case, the phytocomplex is a freeze-dried product of dead cells.

[0111] In one embodiment, at the end of growth in a flask, bioreactor or fermenter, the cells are preferably homogenized, for example by mechanical grinding, in an acidifying solution (e.g., containing ascorbic acid or citric acid or acetic acid), and then lyophilized or dried. In the latter case, the phytocomplex is a cell homogenate in which the cells and their internal structures are disrupted. All of these different types of phytocomplexes are characterized by having a high polysaccharide content as described above.

[0112] Alternatively, the phytocomplex, preferably in the form of a cell homogenate, is subjected to extraction in an alcohol solvent (e.g., methanol or ethanol) using conventional techniques. The extract thus obtained is characterized by a high polysaccharide content as detailed above and can be used for the preparation of cosmetic or pharmaceutical compositions as described above.

[0113] Alternatively, after purification, the live cells can be used directly as such for the preparation of the compositions of the present invention.

Examples

[0114] Preparation and selection of meristem cell lines of Rosa canina and Rosa chinensis Callus tissue induction was achieved using the standard procedures described in the literature. By this procedure, collection of young tissues (shoots) from plants of Rosa canina and Rosa Chinensis, for example, their washing with tap water, cutting into sections of 2 - 5 cm, and disinfection by treatment, for example, successively with an aqueous solution of 70% ethanol for about 15 minutes, 2% sodium hypochlorite and 0.1% Tween 20 for about 5 minutes, and finally washing at least 4 times with sterile distilled water are carried out. All fragments (explants) of the crushed plant tissue are further placed in a Petri dish containing a nutrient medium solidified by the addition of agar and supplemented with growth hormones. After incubation for a suitable period in the dark at 25°C, undifferentiated callus tissue is formed; this is then transferred to a wider surface containing fresh medium and grown.

[0115] The obtained meristematic cells are stabilized by transferring them several times to a solid medium (subculture).

[0116] In the case of Rosa canina, the medium is Gamborg B5 (Gamborg O.L. et al, 1968, Exp. Cell Res., 50, 151) (RP medium) with a final pH of 6.5 containing 2.5 g / L of KNO3, and supplemented with 20 g / L of sucrose, 1 mg / L of NAA and 0.2 mg / L of BAP, and 0.7 - 0.9% plant agar. After clone selection carried out in solid RP medium (containing agar) and liquid RP medium (without agar), the cell line obtained in this specific medium was designated Rc - F2P. It was confirmed by DNA fingerprint analysis that the obtained meristematic cells belong to the plant species Rosa canina.

[0117] The medium for Rosa chinensis contains 2.5 g / L of KNO3, and 25 g / L of sucrose, 1.5 mg / L of NAA and 0.25 mg / L of BAP, and 0.7 - 0.9% of phytagel are added, which is Gamborg's B5 (RPS medium) with a final pH of 6.5. After clone selection carried out in solid RPS medium (containing phytagel) and liquid RPS medium (not containing phytagel), the cell line obtained in this specific medium was designated as RCh-PsMW. It was confirmed by DNA fingerprint analysis that the obtained meristematic cells belong to the plant species Rosa chinensis.

[0118] Grow the selected plant cell line to obtain an amount sufficient to transfer the biomass into a liquid medium (RP and RPS media without phytagel).

[0119] After growth in RP and RPS liquid media, transfer the cell suspension into a bioreactor, which contains a final production medium (RP-F or RPS-F) for a further growth phase or a liquid growth medium (RP or RPS without phytagel).

[0120] The production liquid medium for Rosa canina is Gamborg's B5 (RP-F medium) with a final pH of 6.5, to which 30 g / L of sucrose, a total of 3 g / L of KNO3, 1 mg / L of NAA and 0.2 mg / L of BAP are added.

[0121] The production liquid medium for Rosa chinensis is Gamborg's B5 (RPS-F medium) with a final pH of 6.5, to which 35 g / L of sucrose, a total of 3 g / L of KNO3, 1.5 mg / L of NAA and 0.25 mg / L of BAP are added.

[0122] At the end of growth in the production medium, filter the cell suspension and then recover the biomass, which is used in the steps after preparing the phytocomplex.

[0123] The characteristics of cell lines of Rosa canina and Rosa chinensis are described as non-limiting examples.

[0124] Morphological characteristics of cell lines A selected cell line of Rosa canina designated Rc-F2P is maintained on solid RP medium. This strain is beige-mixed hazelnut color and has a brittle texture (Figs. 1 - 3).

[0125] A selected cell line of Rosa chinensis designated Rch-PsMW is maintained on solid RPS medium. This strain is light yellow with brown spots and has a brittle texture (Figs. 4 - 6).

[0126] Homogenization procedure The procedure for homogenizing the biomass of cells selected and grown in a bioreactor is as follows: a) Obtaining only the cells by filtration of the biomass obtained from the growth of the Rc-F2P or Rch-PsMW cell culture in production liquid medium RP-F for the Rc-F2P strain or RPS-F for the Rch-PsMW strain, and discarding the medium; b) Washing the cells with twice the volume of saline (0.9% W / V NaCl in sterile water); c) Adding 1.5% w / w (0.5 - 2% w / w) ascorbic acid (or citric acid or a mixture of citric acid and ascorbic acid) to the filtered and washed biomass; d) Homogenizing the mixture using, for example, an Ultra-Turrax or other suitable instrument for disrupting cells and their internal structures; e) Drying the biomass by freeze-drying or air-circulation drying or rotary cylinder drying or fluidized bed drying or spraying is included.

[0127] Using the procedures described for the Rc-F2P strain and the Rch-PsMW strain, homogenates A) or B) are obtained respectively: A) After growing for 14 days in the dark at 25 °C (±2), a homogenate of the cell line Rc-F2P containing a high content of medium molecular weight polysaccharides in RP-F medium.

[0128] Details of homogenate A), the content of Rc-F2P: 30 - 55% polysaccharides, of which 65 - 80% are medium molecular weight polysaccharides; 5 - 15% total polyphenols; 15 - 20% proteins; 0.2 - 0.6% hydroxyproline; 3 - 5% lipids; 2 - 4% moisture; 2 - 4% ash; 5 - 30% citric acid. B) After growing for 14 days in the dark at 25 °C (±2), a homogenate of the cell line Rch-PsMW containing a high content of medium molecular weight polysaccharides in RPS-F medium.

[0129] Details of homogenate B), the content of Rch-PsMW: 30 - 60% polysaccharides, of which 65 - 80% are medium molecular weight polysaccharides; 0.5 - 5% total polyphenols; 16 - 35% proteins; 0.7 - 1.2% hydroxyproline; 3 - 6% lipids; 2 - 5% moisture; 2 - 6% ash; 5 - 30% citric acid.

[0130] Examples of the preparation of the cell line Rc-F2P in RP-F medium and the cell line Rch-PsMW in RPS-F medium are provided as non-limiting examples.

[0131] Preparation and analysis of Rc-F2P phytocomplex Meristematic cells (designated as Rc-F2P), which were stabilized and selected as described above, derived from a strain of Rosa canina cultured in solid RP medium (Gamborg's B5 containing 20 g / L sucrose, 1 mg / L NAA, 0.2 mg / L BAP, final pH 6.5, and 0.8% (w / v) agar), were inoculated into five 1-liter flasks containing 200 ml of RP-F liquid medium (Gamborg's B5 with 30 g / L sucrose, 3 g / L KNO3 in total, 1 mg / L NAA, and 0.2 mg / L BAP added, final pH 6.5). The amount of meristematic cells inoculated into the liquid medium was equal to 6% w / v. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 14 days of incubation, the plant biomass (1 liter of cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm, and washed with 900 ml of sterile saline (0.9% w / v). 8 g of citric acid was added to the washed cells (fresh weight 450 g) and homogenized with an Ultra-Turrax.

[0132] The homogenized cells were freeze-dried. From 1 liter of cell suspension, 42.71 g of freeze-dried product (a homogenate of meristematic cells of Rosa canina designated as Rc-F2P) was obtained, containing the following: 5.21 g of total polyphenols, 19.2 g of total polysaccharides (of which 15.3 g were medium molecular weight polysaccharides), 6.7 g of proteins, 2 g of lipids, 0.9 g of ash, and 7.5 g of citric acid (Table 1).

Table 1

[0133] The characterization of the homogenate was carried out using the methods described below. a) Quantification of total polyphenols in cell line Rc-F2P using Folin-Ciocalteau

[0134] The Folin-Ciocalteu reagent is a bright yellow aqueous solution of phosphotungstic acid and phosphomolybdic acid. This method utilizes the redox reaction between a reducing compound and the Folin-Ciocalteu reagent in a basic environment, from which a blue-colored complex is formed, and its intensity increases with the increase in the amount of the reducing compound present, which can be detected at 725 nm. Analysis using the Folin-Ciocalteu reagent enables the quantitative spectrophotometric determination of phenolic compounds and reducing substances present in the sample, which are expressed as gallic acid.

[0135] 20 mg of phytocomplex Rc-F2P was weighed and extracted in 10 ml of methanol / water 50:50 in an ultrasonic bath for 20 minutes. As a reference, standard solutions of gallic acid at concentrations of 200 - 20 μg / mL were used for the preparation of the calibration curve. Next, 0.5 mL of the solution containing the extract was taken out, and 0.5 mL of a 20% w / v sodium carbonate solution and 0.5 mL of Folin-Ciocalteu were added. Then, the mixture was made up to 10 mL with deionized water, and the resulting solution was allowed to stand in the dark away from the heat source for 30 minutes. Five standard gallic acid solutions were treated in the same manner as the extract.

[0136] The solution was read at 725 nm using a UV-Vis spectrophotometer. The % of total polyphenols is expressed as % of gallic acid, which is shown in Table 2.

Table 2

[0137] b) UPLC-qTOF analysis of cell line RcF2P The cell homogenate powder was stirred with a shaker for 30 seconds and then extracted with 6 volumes of methanol in a sonicator at 40 Hz for 15 minutes under ice cooling; the extract was recovered after centrifugation at 18,000 g for 10 minutes at 4 °C. The extract was diluted 1:5 with methanol and then 1:2 with twice-distilled water. After filtering the extract, it was analyzed by UPLC-qTOF. The platform used was an Acquity UPLC I-class (Waters) that was connected online with a cooled autosampler, a diode array type UV / VIS detector, and a high-resolution mass spectrometer of the Xevo G2-XS QTof 4k type (Waters).

[0138] Prior to the guard column, a C18 "reverse phase" chromatography column, Acquity UPLC BEH C18 100×2.1 mm, 1.7 μm particles (Waters) was used.

[0139] The elution conditions were as follows: Solution A: 0.1% aqueous formic acid; Solution B: 100% acetonitrile.

Table 3

[0140] Figure 7 shows the UV / VIS chromatogram obtained using a diode array detector, and the UPLC-MS chromatograms (qTOF) obtained in positive and negative ionization modes. Table 4 shows the id, m / z values, and, if possible, the identification of the main peaks.

[0141] Figure 8 shows the id, m / z values, and, if possible, the identification of the main peaks.

[0142] Identification was carried out based on the m / z values in the negative mode, comparison with the literature, and fragmentation patterns.

[0143] Furthermore, targeted analysis of putative fabane-3-ol and proanthocyanidins was performed. From this additional analysis conducted, it was found that the phytocomplex of the meristematic cells of Rosa canina contains significant amounts of catechins, as well as proanthocyanidins of types P2, P3 and P4 (where P represents the polymer and the numbers 2, 3 and 4 indicate the number of catechin monomers contained in the molecule). c) Qualitative - quantitative analysis of the polysaccharide content of cell line Rc - F2P This analysis was carried out by HPLC - ELSD - SEC (size exclusion chromatography).

[0144] 50 mg of the powder of the homogenate of cell line Rc - F2P was weighed and dissolved in 25 ml of water. The sample was sonicated for 30 minutes, centrifuged and then placed in a vial.

[0145] By HPLC - SEC analysis, compounds can be separated based on their molecular size, which represents their molecular weight: Compounds of low to intermediate molecular weight (glucose - dextran 1000) will have a longer retention time as they remain in the column for a longer time than high - molecular - weight polysaccharides. As reference standards, glucose as a monosaccharide (molecular weight 180 Da) and dextrans with different molecular weights, especially 1000 - 5000 Da, were used.

[0146] A 300×7.8 mm PolySep - GFC - P 3000 column equipped with a 5×7.8 mm guard column was used as the stationary phase, and 0.1 M ammonium acetate and acetonitrile (99:1) were used as the mobile phase. As the detector, a Sedex 60 LT evaporative light scattering detector (ELSD) was used. The gain was set to 9 au, the pressure to 2.3 bar, and the evaporation temperature to 60 degrees.

[0147] According to the analysis performed, the polysaccharides of the homogenate of Rosa canina are distinguished by a pattern of substances showing a low molecular weight peak (21 - 22 minutes, such compounds can be monosaccharides and disaccharides) compared to that of glucose, and a peak (19 minutes) falling in the region attributable to 1000 Da. Figure 9 shows the chromatogram of the homogenate of the phytocomplex of Rosa canina analyzed.

[0148] The results obtained are shown in Table 4.

Table 4

[0149] d) Analysis of the protein content of cell line Rc - F2P As described in Lynch, J M. et al, “Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products” Journal of AOAC International (1999), 82(6), 1389 - 1398, the total content of protein nitrogen in cell line Rc - F2P was measured using the Kjeldahl method.

[0150] The protein content in cell line Rc - F2P was found to be equal to 15.68% W / W. e) Analysis of the hydroxyproline content of cell line Rc - F2P The hydroxyproline content of cell line Rc - F2P was quantified by hydrolysis with 6N HCl and HPLC analysis after derivatization of the amino acids with o - phthalaldehyde (OPA). This analytical quantification was repeated three times. The content of hydroxyproline was equal to 447 mg in 100 g of the dry cell line (0.44% W / W Hyp in the cell line). f) Analysis of the lipid content of cell line Rc - F2P The extraction of the total lipid fraction from cell line Rc-F2P was carried out by Soxhlet extraction with dichloromethane for at least 12 h according to the method described in Martinez M. et al., “Soxhlet lipids extraction from cotton from different producing areas. Comparison of dichloromethane or successive dichloromethane-methanol extractions”. Grasas y Aceites (1997), 48(4), 226-230.

[0151] The lipid content in cell line Rc-F2P was equal to 4.6% w / w. g) Analysis of moisture and ash in cell line Rc-F2P The measurement of moisture was carried out on the cell line homogenate by leaving the material on a 40 °C stove for 12 h. The measurement of ash was obtained by treating the material in a muffle furnace at 300 °C until a constant weight was reached.

[0152] The moisture of the phytocomplex was equal to 2.8% and the ash was equal to 2.1%. Preparation and analysis of the Rch-PsMW phytocomplex Meristematic cells (designated Rch-PsMW) stabilized and selected as described above, derived from a strain of Rosa chinensis cultured in solid RPS medium (Gamborg's B5 with a final pH of 6.5 supplemented with 25 g / L sucrose, 1.5 mg / L NAA, 0.25 mg / L BAP, and 0.8% phytagel), were inoculated into five 1-liter flasks containing 200 ml of RPS-F liquid medium (Gamborg's B5 with a final pH of 6.5 supplemented with 35 g / L sucrose, 3 g / L KNO3, 1.5 mg / L NAA, and 0.25 mg / L BAP). The amount of meristematic cells inoculated into the liquid medium was equal to 5% W / V. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 14 days of incubation, the plant biomass (1 liter of cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm, and washed with 900 ml of sterile saline (0.9% W / V). 6 g of ascorbic acid was added to the washed cells (fresh weight 480 g) and homogenized with an Ultra-Turrax.

[0153] The homogenized cells were freeze-dried. From 1 liter of cell suspension, 42.25 g of freeze-dried product (homogenate of meristematic cells of Rosa chinensis designated Rch-PsMW) containing 2.15 g of total polyphenols, 15.38 g of total polysaccharides (of which 9.9 g are medium molecular weight polysaccharides), 16 g of protein, 2.8 g of lipids, 0.9 g of ash, and 5.5 g of citric acid was obtained (Table 5).

Table 5

[0154] The phytocomplex characterization was performed using the method described below: a) Quantification of total polyphenols in cell line Rch-PsMW using Folin-Ciocalteau The method used was described above. The results obtained are shown in Table 6.

Table 6

[0155] b) UPLC-qTOF analysis of cell line Rch-PsMW Analysis was performed as described for the Rc-F2P strain.

[0156] Figure 10 shows the UV / VIS chromatogram obtained by a diode array detector and the UPLC-MS chromatogram (qTOF) obtained in the negative ionization mode. c) Qualitative-quantitative analysis of the polysaccharide content of cell line Rch-PsMW Analysis was performed as described for the Rc-F2P strain.

[0157] Figure 11 shows the chromatogram of the analyzed Rch-PsMW strain.

[0158] The results obtained are shown in Table 7. d) Analysis of the hydroxyproline content of cell line Rch-PsMW The hydroxyproline content of cell line Rch-PsMW was quantified by HPLC analysis after hydrolysis with 6N HCl and derivatization of the amino acids with o-phthalaldehyde (OPA). This analytical quantification was repeated three times. The content of hydroxyproline was equal to 996 mg in 100 g of the dry cell line (0.99% W / W Hyp in the phytocomplex cell line).

Table 7

[0159] Preparation of cell line Rc-F2P on an industrial scale Meristematic cells (designated Rc-F2P) that were stabilized and selected as described above, derived from a strain of Rosa canina cultured in solid RP medium (Gamborg's B5 with a final pH of 6.5 supplemented with 20 g / L sucrose, 1 mg / L NAA, 0.2 mg / L BAP, and 0.8% w / v phytagel), were inoculated into ten 1-liter flasks containing 200 ml of RP liquid medium. The amount of meristematic cells inoculated into the liquid medium was equal to 6% w / v. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 7 days of incubation, the cell suspension was used to inoculate ten 3-liter flasks containing 800 ml of RP liquid medium. 200 ml of the cell suspension was transferred into 800 ml of RP medium in the 3-liter flasks. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 7 days of incubation, the cell suspension was used to inoculate a bioreactor containing 90 liters of RP-F medium (Gamborg's B5 with a final pH of 6.5 supplemented with 30 g / L sucrose, 3 g / L total KNO3, 1 mg / L NAA, and 0.2 mg / L BAP).

[0160] After 14 days of growth in the bioreactor, the plant biomass (100 liters of cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm, and washed with 64 L of sterile saline (0.9% w / v). 320 g of citric acid was added to the washed cells (fresh weight 32 kg) and homogenized with an Ultra-Turrax.

[0161] The homogenized cells were dried. 4090 g of homogenate Rc-F2P was obtained from 100 liters of cell suspension. Preparation of the cell line Rch-PsMW on an industrial scale Meristematic cells (designated Rch-PsMW) that were stabilized and selected as described above, derived from a strain of Rosa chinensis cultured in solid RPS medium (Gamborg's B5 with a final pH of 6.5 supplemented with 25 g / L sucrose, 1.5 mg / L NAA, 0.25 mg / L BAP, and 0.8% w / v phytagel), were inoculated into ten 1-liter flasks containing 200 ml of RPS liquid medium. The amount of meristematic cells inoculated into the liquid medium was equal to 6% w / v. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 7 days of incubation, the cell suspension was used to inoculate ten 3-liter flasks containing 800 ml of RPS liquid medium. 200 ml of the cell suspension was transferred into the 800 ml of RPS medium contained in the 3-liter flasks. The suspension thus obtained was incubated in the dark at 25 °C and placed on an orbital shaker set at 120 RPM. After 7 days of incubation, the cell suspension was used to inoculate a bioreactor containing 90 liters of RPS-F medium (Gamborg's B5 with a final pH of 6.5 supplemented with 35 g / L sucrose, 3 g / L KNO3, 1.5 mg / L NAA, and 0.25 mg / L BAP).

[0162] After 14 days of growth in the bioreactor, the plant biomass (100 liters of cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm, and washed with 70 L of sterile saline (0.9% w / v). 350 g of citric acid was added to the washed cells (fresh weight 35 kg) and homogenized with an Ultra-Turrax.

[0163] The homogenized cells were dried. 4450 g of Rch-PsMW homogenate was obtained from 100 liters of cell suspension. Biological test of antioxidant activity using cell line Rc-F2P The antioxidant activity was assayed using the DPPH method. This assay using DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical aims to evaluate in vitro the antioxidant activity of the Rosa canina Rc-F2P strain against the stained stable radical and measure its disappearance by spectrophotometry.

[0164] The methanol solution of DPPH is purple and shows a maximum absorbance at a wavelength of 517 nm. When DPPH is reacted with an antioxidant compound capable of generating hydrogen radicals, the disappearance of the DPPH radical causes decolorization of the solution from purple to yellow, which can be monitored by spectrophotometry over time at the wavelength of the maximum absorbance, 517 nm.

[0165] The Rc-F2P homogenate of the meristematic cells of Rosa canina was extracted in methanol and placed in an ultrasonic bath for 20 minutes. Various aliquots of the solution were taken and added to the same amount of DPPH solution, and then made to an appropriate amount using methanol to evaluate the antioxidant capacity of samples at various concentrations. The results are expressed as the EC50, i.e., the concentration of the Rc-F2P phytocomplex capable of reducing the initial absorbance of the radical at 517 nm by 50%. Subsequently, the results are compared with those obtained from a methanol solution of ascorbic acid used as a reference. The lower the EC50, the higher the antioxidant capacity.

[0166] Table 8 shows the antioxidant activity of the homogenate of the Rc-F2P strain.

Table 8

[0167] Biological test on the increased biosynthesis of aquaporin 3 in reconstructed skin using the cell line Rch-PsMW The evaluation of the increased expression of aquaporin 3 was carried out at VitroScreen laboratories (Milan) using the reconstituted human epidermis (RHE) model for homeostasis reconstruction. The evaluation of the increased biosynthesis of aquaporin 3 by the Rch-PsMW strain of Rosa chinensis was carried out using both topical application (15 μl of saline containing 0.1% w / w cell homogenate) and systemic application (0.1% w / w homogenate in the maintenance medium of the explants) to the RHE.

[0168] After topical application, the RHE was incubated for 24 hours (at 37 °C, 5% CO2). Systemic application was carried out by adding 0.1% of the extract to the maintenance medium of the epidermis and incubating the RHE for 24 hours.

[0169] After incubation, the explants were collected and immunostaining analysis and PCR were carried out. To evaluate the expression of aquaporin 3, an immunohistochemical technique considering the use of an anti-aquaporin 3 antibody (Abcam) was used, and then the evaluation of antigen-antibody binding was carried out using a fluorescence microscope. Figure 12 shows the results obtained regarding the increased biosynthesis of aquaporin 3.

[0170] Aquaporin 3 is involved in maintaining skin hydration and wound healing. The Rch-PsMW homogenate acts favorably on the biosynthesis of the transmembrane aquaporin 3, thereby promoting water transport. Formulation of the Rch-PsMW and Rc-F2P strains into two-phase and multiple emulsions, gels, and skin and hair cleansing systems The homogenate of the Rch-PsMW strain or the Rc-F2P strain was dispersed in glycerin at a concentration of 3% w / w (INCI name: Glycerin (and) Rosa Chinensis Callus Lysate (and) Citric Acid). The dispersion was added at 3% to the following formulations.

Table 9

Table 10

Table 11

Claims

A process for preparing and selecting a plant meristem cell line derived from a plant belonging to the Rosa canina species or Rosa chinensis species, wherein the cell line has a polysaccharide content of more than 25% w / w based on the dry mass of the cell line, and the process comprises the following: 1) Planting tissue obtained from a plant of the Rosa canina species or Rosa chinensis species on a solid medium; 2) Isolating a plurality of cell clones; 3) Inoculating each of the isolated clones into a liquid medium; 4) Quantifying the polysaccharide content for each clone; 5) Selecting the cell clone having the highest polysaccharide content comprising, wherein the solid and liquid media contain salts, sucrose, naphthylacetic acid (NAA) and 6-benzylaminopurine (BAP) suitable for the growth of plant cells, when the plant is of the Rosa canina species, the solid medium is Gamborg's B5 medium with a final pH of 6.5 containing 2.5 g / L of KNO₃, and 20 g / L of sucrose, 1 mg / L of NAA and 0.2 mg / L of BAP, and 0.7 - 0.9% plant agar added; when the plant is of the Rosa chinensis species, the solid medium is Gamborg's B5 medium with a final pH of 6.5 containing 2.5 g / L of KNO₃, and 25 g / L of sucrose, 1.5 mg / L of NAA and 0.25 mg / L of BAP, and 0.7 - 0.9% plant agar added; when the plant is of the Rosa canina species, the liquid medium is Gamborg's B5 medium with a final pH of 6.5 containing 30 g / L of sucrose, a total of 3 g / L of KNO₃, 1 mg / L of NAA and 0.2 mg / L of BAP added; when the plant is of the Rosa chinensis species, the liquid medium is Gamborg's B5 medium with a final pH of 6.5 containing 35 g / L of sucrose, a total of 3 g / L of KNO₃, 1.5 mg / L of NAA and 0.25 mg / L of BAP added, process. The process according to claim 1, wherein the cell line contains intermediate molecular weight polysaccharides in an amount of 50% to 90% based on the total amount of the polysaccharides. **Claim 3**: The process according to claim 1 or 2, wherein the cell line contains low molecular weight polysaccharides in an amount of 20% to 30% based on the total amount of the polysaccharides. **Claim 4**: The process according to any one of claims 1 to 3, wherein the cell line contains polyphenols in an amount exceeding 0.2% based on the dry mass of the cell line. **Claim 5**: The process according to claim 4, wherein the cell line is derived from the Rosa canina species and contains polyphenols in an amount of 3% to 25% based on the dry mass of the cell line. **Claim 6**: The process according to claim 4, wherein the cell line is derived from the Rosa chinensis species and contains polyphenols in an amount of 0.3% to 10% based on the dry mass of the cell line. **Claim 7**: The process according to any one of claims 1 to 6, wherein the cell line contains proteins in an amount of 10% to 40% w / w, lipids in an amount of 1% to 10% w / w based on the dry mass of the cell line, and hydroxyproline in an amount of 0.1% to 1.3% w / w based on the dry mass of the cell line. **Claim 8** A process for preparing a phyto complex and / or extract of the meristematic tissue cell line using the meristematic tissue cell line prepared by the process according to any one of claims 1 to 7, wherein the phyto complex and / or extract is dried or lyophilized cells, or cell homogenates, or cell walls and their components, or homogenates of the meristematic tissue cell line. **Claim 9** A process for preparing a composition comprising the meristematic tissue cell line prepared by the process according to any one of claims 1 to 7 and / or the phyto complex and / or extract prepared by the process according to claim 8. **Claim 10**: The process according to claim 9, wherein the composition contains the meristematic tissue cell line and / or the phyto complex and / or the extract at a concentration of 0.01% to 1% by weight based on the total composition. **Claim 11**: The process according to claim 9 or 10, wherein the composition is formulated as a cream, gel cream, gel, serum, oil, emulsion, emulsion gel (emagel), ointment, eye drops, mouthwash, spray, stick, pill, capsule, tablet, granular powder, hard shell capsule, orally disintegrating granule, sachet, lozenge or liposome.

12. A split tissue cell line adjusted by the process according to any one of Claims 1 to 7, a phytocomplex and / or extract adjusted by the process according to Claim 8, or a composition adjusted by the process according to any one of Claims 9 to 11, for use as a cosmetic for the purpose of preventing, reducing or treating signs of skin aging and / or an increase in free radicals in the skin, for the purpose of preventing or reducing wrinkles, or for the purpose of preventing, reducing or treating signs of dehydration symptoms of the skin and / or the cutin, the process according to any one of Claims 1 to 11.

13. A split tissue cell line adjusted by the process according to any one of Claims 1 to 7, a phytocomplex and / or extract adjusted by the process according to Claim 8, or a composition adjusted by the process according to any one of Claims 9 to 11, for use as a dietary supplement, the process according to any one of Claims 1 to 11.

14. A split tissue cell line adjusted by the process according to any one of Claims 1 to 7, a phytocomplex and / or extract adjusted by the process according to Claim 8, or a composition adjusted by the process according to any one of Claims 9 to 11, for use as a medicament for the treatment or prevention of skin-invading pathologies such as redness, irritation, local inflammation or cracking, or for the purpose of promoting the wound healing process, the process according to any one of Claims 1 to 11.

15. Salts, vitamins, sucrose, NAA and 6-benzylaminopurine (BAP) suitable for the growth of plant cells, and KNO ３ The process according to any one of claims 1 to 7, further comprising the step of transferring said cells to a final liquid medium containing

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