Phytocomplexes and extracts of meristematic cell lines selected from Echinacea purpurea

Abstract

The present invention relates to meristematic cell lines selected from plant tissues, preferably callus tissue, of Echinacea purpurea. 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 polyphenol content, particularly a high chicoric acid 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 selected meristem cell lines derived from plants of Echinacea purpurea, which are characterized by a high polyphenol content, and to the cosmetic, functional food and medical uses of said meristem cell lines or their derivatives.

Background Art

[0002] Plants of the genus Echinacea are widely used, inter alia, because of their well-known immunostimulatory activity.

[0003] Polyphenols, in particular chicoric acid, chlorogenic acid, cichoric acid and caftaric acid, are typical components of Echinacea purpurea. Said compounds can inhibit the production of free radicals and lipid peroxidation (Georgieva S.et al.,2014,J.Exp.Integr.Med.). The content of chicoric acid and other polyphenol components of Echinacea purpurea is associated with high variability related to several factors that are difficult to control: season, plant age, cultivation area and the tissue used for the preparation of the product.

[0004] Furthermore, there are also many problems related to the cultivation of Echinacea: fungal infections, seed dormancy, low germination rate, long maturation time and difficulty in standardizing extracts.

[0005] For the above reasons, products based on E. purpurea available on the market today are often contaminated or, in many cases, substituted with another plant species that is not specified.

[0006] The preparation of standardized plant derivatives (i.e., containing metabolites of reproducible content) causes many problems: variations in metabolite content in different plant tissues, seasonal variations, 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.

[0007] 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 containing standardized and reproducible contents of active substances.

[0008] European Patent No. 2319914 describes the preparation of meristematic cells derived from plants of the genus Echinacea, preferably Echinacea angustifolia, having a high content of caffeic acid.

[0009] The present invention provides selected meristematic cell lines and their derivatives from plants belonging to the Echinacea purpurea species, which contain high contents of polyphenols other than caffeic acid.

Summary of the Invention

Means for Solving the Problems

[0010] A first aspect of the present invention relates to a selected meristematic cell line derived from a plant belonging to the Echinacea purpurea species, preferably a cell line derived from callus tissue obtained from the plant itself.

[0011] The second aspect of the present invention relates to a derivative of a selected meristematic cell line, namely, the phytocomplex or extract of the cell line.

[0012] The selected meristematic cell line and its derivatives are characterized by a high content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, caffeic acid, ferulic acid, phthalic acid and mixtures thereof.

[0013] The third aspect of the present invention relates to a composition comprising a selected meristematic cell line or a derivative thereof, mixed with excipients acceptable from the viewpoint of cosmetics and / or pharmaceuticals.

[0014] The applicant has demonstrated that the selected cell line or its derivative is active in inducing the expression of the genes of collagen III and IV, and has significant anti-inflammatory activity mediated by the inhibition of the expression of iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase 2), IL-1β (interleukin 1β) and TNFα (tumor necrosis factor α), as well as antioxidant activity mediated by the inhibition of ROS (reactive oxygen species), MDA (malondialdehyde) and the release of NO2-.

[0015] Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Echinacea purpurea, which contain a high content of polyphenols other than caffeic acid.

[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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Mode for Carrying Out the Invention

[0018] Definitions In the context of 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 meristematic tissue cells is equivalent to that of animal stem cells.

[0019] In the context of the present invention, "callus tissue" refers to an unorganized mass of undifferentiated cells or almost non-specialized cells with thin cell walls and large vacuoles in which secondary metabolites have accumulated.

[0020] In the context of the present invention, unless otherwise stated, "w / w" means a weight / weight quantity with respect to the dry mass of the cell line.

[0021] The first aspect of the present invention relates to a meristematic tissue cell line derived from a plant belonging to the Echinacea purpurea species.

[0022] In one embodiment, the meristematic tissue cell line is as follows: 1) The step of planting the tissue obtained from a plant belonging to the genus Echinacea purpurea on a solid medium; 2) The step of isolating a plurality of cell clones; 3) The step of inoculating each of the isolated clones into a liquid medium; 4) For each clone, the step of quantifying the content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caffeic acid, and ferulic acid; 5) Selecting a cell clone containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, caffeic acid, ferulic acid, phthalic acid, and mixtures thereof in an amount of 0.05% w / w, preferably 0.5% - 25% w / w.

[0023] In step 1), tissues obtained from plants of the genus Echinacea purpurea are placed in a solid medium to obtain undifferentiated callus tissue. The tissues of Echinacea purpurea are preferably at least one shoot of Echinacea purpurea or a plurality of shoots of Echinacea purpurea.

[0024] In a preferred embodiment of the present invention, the solid and liquid media contain salts, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA), and kinetin suitable for the growth of plant cells.

[0025] The solid medium further contains agar, while the liquid medium does not contain agar.

[0026] The solid and liquid media each contain sucrose at a concentration of 15 - 50 g / L, preferably 18 - 45 g / L, naphthylacetic acid (NAA) at a concentration of 0.1 - 2.5 mg / L, preferably 0.5 - 2 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2.5 mg / L, preferably 0.3 - 1 mg / L, and kinetin at a concentration of 0.1 - 2 mg / L, preferably 0.2 - 1 mg / L.

[0027] In a preferred embodiment, the solid medium contains sucrose at a concentration of 10 - 30 g / L, preferably 15 - 25 g / L, naphthaleneacetic acid (NAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, and kinetin at a concentration of 0.2 - 1 mg / L, preferably 0.3 - 0.8 mg / L.

[0028] In a preferred embodiment, the liquid medium contains sucrose at a concentration of 25 - 50 g / L, preferably 30 - 45 g / L, naphthaleneacetic acid (NAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, and kinetin at a concentration of 0.2 - 1 mg / L, preferably 0.3 - 0.8 mg / L.

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

[0030] For both solid and liquid media, 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 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, for both solid and liquid media, 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 compounds is Gamborg B5 medium.

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

[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 - 3000 mg / L, preferably 1000 - 2600 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 these are 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 part of the stabilized cells preferably undergoes a first "clone selection". The clone selection is to culture the stabilized cells for an appropriate period, preferably for a period of 5 to 20 days of culture, more preferably for a period of 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.

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

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

[0041] 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 polyphenol content of each clone is quantified.

[0042] In the step 5) of selection of cell clones, it is preferable to perform a second clone selection according to step 1b) until a plant cell line of Echinacea purpurea containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caffeic acid, ferulic acid and mixtures thereof in an amount of more than 0.5% w / w, preferably 0.5% to 25% w / w is obtained.

[0043] The meristem cell line selected by the process described above preferably contains polyphenols in an amount of 3 to 15% w / w, more preferably 3.5 to 10% w / w, as listed above.

[0044] The polyphenol mixture preferably contains chicoric acid, preferably chicoric acid and caffeylquinic acid, more preferably chicoric acid, caffeylquinic acid and chlorogenic acid, and even more preferably chicoric acid, caffeylquinic acid, chlorogenic acid, caftaric acid and ferulic acid.

[0045] In a preferred embodiment, the polyphenol contains at least 40%, at least 35% or at least 30% w / w, preferably 38% - 50% w / w of chicoric acid.

[0046] The selected cell line preferably contains polyphenol in an amount of 3.5 - 10% w / w, and the polyphenol contains at least 40%, or at least 35% or at least 30% w / w, preferably 38% - 50% w / w of chicoric acid.

[0047] The selected meristem cell line of the present invention further contains polysaccharide in an amount of 30 - 70% w / w, preferably 40% - 65% w / w.

[0048] The selected meristem cell line of the present invention further contains protein in an amount of 5 - 40% w / w, preferably 10 - 30% w / w.

[0049] The selected meristem cell line of the present invention further contains lipid in an amount of 3 - 30% w / w, preferably 5 - 10% w / w.

[0050] In a preferred embodiment, the selected meristem cell line is the Ep - 2P strain containing 0.5 - 20% of polyphenol, of which 35 - 40% is chicoric acid.

[0051] A second aspect of the present invention relates to a derivative of a cell line which is a phytocomplex or extract of a selected meristem cell line.

[0052] Phytocomplex means dried or lyophilized cells, cell homogenates, or cell walls and their components. The phytocomplex is preferably a cell homogenate.

[0053] The phytocomplex contains polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, caffeic acid, ferulic acid, coumaric acid and mixtures thereof, in an amount of more than 0.5% w / w, preferably 0.5% - 25% w / w, based on the dry mass of the phytocomplex. More preferably, the phytocomplex contains the aforementioned polyphenols in an amount of 3 - 15% w / w, even more preferably 3.5 - 10% w / w, based on the dry mass of the phytocomplex.

[0054] The mixture of polyphenols preferably contains chicoric acid, preferably chicoric acid and caffeic acid, even more preferably chicoric acid, caffeic acid and chlorogenic acid, and more preferably chicoric acid, caffeic acid, chlorogenic acid, ferulic acid and coumaric acid.

[0055] In a preferred embodiment, the polyphenols contain at least 40%, or at least 35% or at least 30% w / w, preferably 32 - 40% w / w, of chicoric acid, based on the dry mass of the phytocomplex.

[0056] The phytocomplex preferably contains polyphenols in an amount of 3.5 - 10% w / w, based on the dry mass of the phytocomplex, and the polyphenols are composed of 38 - 50% w / w of chicoric acid.

[0057] The phytocomplex also further contains polysaccharides in an amount of 30 - 70% w / w, preferably 40% - 65% w / w, based on the dry mass of the phytocomplex.

[0058] The phytocomplex also further contains proteins in an amount of 5 - 40% w / w, preferably 10 - 30% w / w, based on the dry mass of the phytocomplex.

[0059] The phytocomplex also further contains lipids in an amount of 3 - 30% w / w, preferably 5 - 10% w / w, based on the dry mass of the phytocomplex.

[0060] In a preferred embodiment, the phytocomplex is derived from the selected meristematic cell line Ep-2P, contains 0.5-20% polyphenols, 35-40% of which are chicoric acid.

[0061] The phytocomplex is preferably a cell homogenate of the selected meristematic cell line Ep-2P.

[0062] The extract means the extract of the cell line itself or the phytocomplex of the cell line in an alcohol solvent, for example, methanol or ethanol, or in water / ethanol mixtures in various ratios: 50:50 or 60:40 or 70:30. The extract is preferably an extract of a 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 may vary depending on the extraction technique.

[0063] A third aspect of the present invention relates to a composition comprising a meristematic cell line and / or its derivatives (phytocomplex and / or extract) combined with at least one excipient acceptable from a cosmetic, functional food and / or pharmaceutical point of view.

[0064] In one embodiment, the composition contains 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. The composition preferably contains a phytocomplex which is a cell homogenate.

[0065] 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.

[0066] The composition of the present invention contains at least one excipient acceptable for pharmaceutical and / or cosmetic use, which is useful for the preparation of the composition and is generally biologically safe and non-toxic.

[0067] The excipient is at least one conditioning agent, moisturizing agent, or occluding agent, surfactant, stabilizer, preservative, or emollient agent for skin.

[0068] The composition of the present invention is formulated for topical use as a cream, gel cream, gel, serum, oil, emulsion, emulsion-gel (emulgel), ointment, eye drops, mouthwash, spray, preferably a nasal spray, or stick (such as lip cream).

[0069] The composition can also be formulated for oral administration, preferably as pills, capsules, tablets, granular powder, hard shell capsules, orally disintegrating granules, sachets, or lozenges.

[0070] 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.

[0071] The experimental data included herein indicate that the aforementioned cell line or its derivatives can stimulate the synthesis of collagen III and IV, resulting in improved skin elasticity and density. Furthermore, it has been demonstrated that the cell line or its derivatives have anti-inflammatory activity related to the reduction of the synthesis of inflammatory enzymes iNOS and COX-2 and cytokines TNFα and IL-1β stimulated by LPS, and further antioxidant activity related to the reduction of ROS production after oxidative stress.

[0072] Another aspect of the present invention relates to the cosmetic use of the cell line or its derivatives.

[0073] Cosmetic use means the prevention, alleviation, and / or treatment of signs of skin aging, loss of skin elasticity and density, and redness, moisturization of the skin, anti-wrinkle activity, and antioxidant activity.

[0074] 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, a shower gel, a soap, a shampoo or a hair conditioner.

[0075] Another aspect of the present invention relates to a cell line or a derivative thereof for use as a medicament or a dietary supplement, in particular for the treatment or prevention of inflammations and infections affecting the upper respiratory tract.

[0076] Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of plants belonging to the genus Echinacea purpurea, which comprises the following: 1) planting tissue obtained from a plant of the genus Echinacea purpurea on a solid medium; 2) isolating a plurality of cell clones; 3) inoculating each of the isolated clones into a liquid medium; 4) for each clone, quantifying the content of polyphenols selected from the group comprising chicoric acid, chlorogenic acid, cichoric acid, caftaric acid and ferulic acid; 5) selecting cell clones containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caftaric acid, ferulic acid and mixtures thereof in an amount of more than 0.05% w / w, preferably from 0.5% to 25% w / w and includes.

[0077] In one embodiment, the preparation of the meristematic cell line comprises collecting tissue, preferably a shoot, from a plant of Echinacea purpurea, washing it, for example, with water, cutting it into small pieces, and sterilizing it on a plate, for example, by successive treatment with ethanol, sodium hypochlorite and a mercury salt.

[0078] In step 1), the collected tissue is placed in a solid medium to obtain undifferentiated callus tissue.

[0079] In a preferred embodiment of the present invention, the solid and liquid media contain salts, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA), and kinetin suitable for the growth of plant cells.

[0080] The solid medium further contains agar, while the liquid medium does not contain agar.

[0081] The solid and liquid media preferably each contain sucrose at a concentration of 15 - 50 g / L, more preferably 18 - 45 g / L, naphthylacetic acid (NAA) at a concentration of 0.1 - 2.5 mg / L, more preferably 0.5 - 2 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2.5 mg / L, more preferably 0.3 - 1 mg / L, and kinetin at a concentration of 0.1 mg / L - 2 mg / L, more preferably 0.2 mg / L - 1 mg / L.

[0082] In a preferred embodiment, the solid medium contains sucrose at a concentration of 10 - 30 g / L, preferably 15 - 25 g / L, naphthaleneacetic acid (NAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, and kinetin at a concentration of 0.2 mg / L - 1 mg / L, preferably 0.3 mg / L - 0.8 mg / L.

[0083] In a preferred embodiment, the liquid medium contains sucrose at a concentration of 25 - 50 g / L, preferably 30 - 45 g / L, naphthaleneacetic acid (NAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, indoleacetic acid (IAA) at a concentration of 0.1 - 2 mg / L, preferably 0.5 - 1.5 mg / L, and kinetin at a concentration of 0.2 mg / L - 1 mg / L, preferably 0.3 mg / L - 0.8 mg / L.

[0084] For both solid and liquid media, salts suitable for the growth of plant cells are selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4, and combinations thereof.

[0085] For both solid and liquid media, 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, and ZnSO4·7H2O, and combinations thereof.

[0086] Both 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.

[0087] In one embodiment, for both solid and liquid media, 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. This combination of salts is Gamborg B5 medium.

[0088] In one embodiment, both solid and liquid media 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, in addition to the salts listed above.

[0089] The solid and liquid media preferably each contain CaCl2 at a concentration of 120-170 mg / L, more preferably 130-160 mg / L; KNO3 at a concentration of 800-3000 mg / L, more preferably 1000-2600 mg / L; MgSO4 at a concentration of 220-270 mg / L, more preferably 230-260 mg / L; NaH2PO4 at a concentration of 100-180 mg / L, more preferably 110-150 mg / L; and (NH4)2SO4 at a concentration of 100-180 mg / L, more preferably 110-150 mg / L.

[0090] 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, more preferably 0.015-0.03 mg / L; NaEDTA·2H2O at a concentration of 20-60 mg / L, more preferably 30-45 mg / L; FeSO4·7H2O at a concentration of 15-45 mg / L, more preferably 20-35 mg / L; H3BO3 at a concentration of 1-7 mg / L, more preferably 2-5 mg / L; Kl at a concentration of 0.1-2 mg / L, more preferably 0.4-1 mg / L; MnSO4·H2O at a concentration of 5-20 mg / L, more preferably 7-15 mg / L; Na2MoO4·2H2O at a concentration of 0.1-0.5 mg / L, more preferably 0.15-0.3 mg / L; and ZnSO4·7H2O at a concentration of 0.5-5 mg / L, more preferably 1-3 mg / L.

[0091] Both the solid and liquid media preferably each contain inositol at a concentration of 70-130 mg, more preferably 90-110 mg; pyridoxine-HCl at a concentration of 70-130 mg, more preferably 90-110 mg; and thiamine-HCl at a concentration of 5-20 mg / L, preferably 7-15 mg / L.

[0092] After step 1), the callus tissue is preferably divided into a plurality of parts and these are transferred successively to the solid medium for stabilization (step 1a) to obtain stabilized cells. This step is called the stabilization step.

[0093] After the stabilization step 1a), the stabilized cells preferably undergo an initial "clone selection". The clone selection involves culturing the stabilized cells for a suitable period, preferably 5 to 20 days of culture, more preferably for a period of 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.

[0094] In step 2), a plurality of cell clones are isolated by removing aggregates of stabilized cells from the solid medium.

[0095] In step 3), each cell clone is inoculated into the above-described liquid medium.

[0096] According to one embodiment, in step 4), after a growth phase of a time sufficient to achieve appropriate growth of the cell clones, preferably 10 to 15 days, the polyphenol content of each clone is quantified.

[0097] In step 5) of the selection of cell clones, it is preferable to perform a second clone selection according to step 1b) until an Echinacea purpurea plant cell line that optimally produces polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, caffeic acid, ferulic acid, phthalic acid, and mixtures thereof is obtained.

[0098] Next, the selected cell line is grown in a flask or a bioreactor or a fermenter to achieve an increase in biomass. The increase in biomass occurs in the first stage in a liquid growth medium called EP. The liquid growth medium EP is a medium containing the above-mentioned Gamborg salts, the vitamins listed above, sucrose, NAA, and IAA.

[0099] The liquid growth medium EP contains, among Gamborg salts, KNO3 in an amount of 1.5 g / L to 3.5 g / L, preferably 2 g / L to 3 g / L. Sucrose is preferably contained at 15 g / L to 25 g / L. NAA is preferably contained at 0.5 mg / L to 1.5 mg / L. IAA is preferably contained at 0.2 mg / L to 1 mg / L. Kinetin is preferably contained at 0.2 mg / L to 1 mg / L.

[0100] Cells grown in the liquid medium EP are transferred, for the final growth phase, into the final medium EP-F containing Gamborg salts, vitamins, sucrose, NAA, and IAA, thereby inducing an increase in polyphenol content and biomass.

[0101] The final medium EP-F contains, among Gamborg salts, KNO3 in an amount of 0.5 g / L to 2 g / L. Sucrose is preferably contained at 30 g / L to 50 g / L. NAA is preferably contained at 0.2 mg / L to 1 mg / L. IAA is preferably contained at 0.2 mg / L to 1 mg / L, and kinetin is preferably contained at 0.2 mg / L to 1 mg / L.

[0102] According to a preferred embodiment, in both the EP growth medium and the final EP-F medium, the growth of the cell line in a flask, bioreactor or fermenter 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.

[0103] At the end of the growth in the final EP-F medium, after filtering the cell line, the cells are recovered and used in the next step in the form of phytocomplexes, or alternatively, they can be subjected to an extraction phase in a subsequent alcohol solvent to produce a cell extract characterized by a high polyphenol content.

[0104] The phytocomplexes can be obtained by freeze-drying or drying of living cells; in this case, the phytocomplexes are freeze-dried products of dead cells.

[0105] 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 (for example, containing ascorbic acid and / or citric acid and / 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 have disintegrated. All of these different types of phytocomplexes are characterized by having a high polyphenol content as described above.

[0106] Alternatively, the phytocomplex, preferably in the form of a cell homogenate, is subjected to extraction in an alcohol solvent (for example, methanol and / or ethanol and / or water) using conventional techniques. The extract thus obtained is characterized by a high polyphenol content as detailed above and can be used, as described above, for the preparation of cosmetic, functional food or pharmaceutical compositions.

[0107] Alternatively, after purification, the live cells can be used directly as such for the preparation of the compositions of the present invention.

[0108] The biological data obtained in the subsequent experiments confirm the effectiveness of the cell lines and / or derivatives containing, in surprisingly high amounts, polyphenols selected from the group consisting of chicoric acid, caffeylquinic acid, chlorogenic acid, caffeic acid, ferulic acid and mixtures thereof, for the uses described herein.

[0109] The unexpected effectiveness of the derivatives of the selected cell lines is likewise demonstrated, in this case the derivatives being homogenates containing all cell components, or phytocomplexes, without exposing the cells to solvents or other extraction procedures that could impair the integrity of the active ingredients.

Example

[0110] Preparation and selection of a meristematic cell line of Echinacea purpurea Callus tissue induction was achieved using standard procedures described in the literature. This procedure involves the collection of young tissues (shoots) from plants of Echinacea purpurea, for example, their washing with tap water, cutting into sections of 2 - 5 cm, and sterilization on plates by treatment, in order, with an aqueous solution of 70% ethanol for about 15 seconds, 2% sodium hypochlorite and 0.1% Tween 20 for about 5 minutes, and finally at least 4 washes with sterile distilled water. It is solidified by the addition of agar, and all fragments of the crushed plant tissue (explants) are placed in Petri dishes containing a nutrient medium supplemented with growth hormones without antibiotics. After incubation for a suitable period in the dark at 25 °C, undifferentiated callus tissue is formed; this is then transferred to a larger surface containing fresh medium and grown.

[0111] Meristematic cell lines obtained from undifferentiated callus tissue are stabilized by transferring them several times (subculturing) to a solid medium. This medium consists of a solid Gamborg B5 type (containing 2.5 g / L of KNO3) with a final pH of 6.5, supplemented with 20 g / L of sucrose, 1 mg / L of NAA, 0.5 mg / L of IAA, 0.5 mg / L of kinetin, and 0.8 - 1% plant agar (EP medium). The cell line obtained in this specific medium after clone selection was designated Ep - 2P.

[0112] It was confirmed by DNA fingerprint analysis that the obtained meristematic cells belong to the plant species Echinacea purpurea.

[0113] Cell line Ep - 2P is grown to obtain a sufficient amount of biomass to transfer the cells to a liquid medium (EP medium without agar). After growth in the liquid medium, the cell suspension can be transferred to a bioreactor, which may contain a final production medium or an EP growth medium for a further growth phase.

[0114] The production liquid medium (optimized to increase the polyphenol content) is Gamborg's B5 (containing 1 g / L of KNO3) with a final pH of 6.5, supplemented with 40 g / L of sucrose, 1 mg / L of NAA, 0.5 mg / L of IAA, and 0.5 mg / L of kinetin (EP-F medium).

[0115] The characteristics of the cell line Ep-2P of Echinacea purpurea are described as an example.

[0116] Morphological characteristics of the cell line A selected cell line of Echinacea purpurea designated Ep-2P is maintained on solid EP medium and is beige in color with a whitish tint and has a brittle texture (Figs. 1 - 3).

[0117] Homogenization procedure The procedure for homogenizing the biomass of cells selected and grown for 14 days in a bioreactor at 25°C (±2) is as follows: a) Obtaining only the cells by filtration of the biomass obtained from the growth of Ep-2P cell cultures in EP-F medium and discarding the medium; b) Washing the cells with twice the volume of saline (0.9% W / V NaCl in sterile water); c) Adding 1.0% W / W (0.5 - 2% W / W) of citric acid (or ascorbic 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.

[0118] Using the described procedure, a phytocomplex designated EchiPure-PC® is obtained. Details of the content of EchiPure-PC (registered trademark) homogenate: 40 - 65% polysaccharide 0.5 - 20% total polyphenols (about 38% of which is chicoric acid) 10 - 30% protein 5 - 10% lipid 2 - 6% moisture 2 - 17% ash 5 - 30% citric acid Examples of the preparation of the standard EchiPure-PC (registered trademark) A phytocomplex in EP-F medium are provided as non-limiting examples.

[0119] The phytocomplex thus obtained is used as it is or dispersed in a suitable dispersion medium at a concentration in the range of 0.5 - 5% w / w. The suspension thus obtained is designated as EchiPure CRO (registered trademark)-G.

[0120] Preparation and analysis of EchiPure-PC (registered trademark) phytocomplex Meristematic cells (designated as Ep-2P), which were stabilized and selected as described above, derived from a strain of Echinacea purpurea cultured in solid EP medium (Gamborg's B5 supplemented with 20 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA, 0.5 mg / L kinetin and 0.8% phytagel for plants), were inoculated into five 1-liter flasks containing 200 ml of liquid EP-F medium (Gamborg's B5 with a final pH of 6.5 supplemented with 40 g / L sucrose, 1 g / L KNO3 instead of 2.5 g / L, 1 mg / L NAA, 0.5 mg / L IAA and 0.5 mg / L kinetin). The amount of meristematic cells inoculated into the liquid medium was 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, 5 mg / L methyl jasmonate was added to the cell suspension (95%, Sigma-Aldrich, CAS Number: 39924-52-2). After 7 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 700 ml of sterile saline (0.9% W / V). 3.5 g of citric acid was added to the washed cells (fresh weight 350 g) and homogenized with an Ultra-Turrax.

[0121] The homogenized cells were freeze-dried. From 1 liter of cell suspension, 25 g of freeze-dried product (EchiPure-PC® phytocomplex) containing the following was obtained: 1.05 g of polyphenols, 13 g of polysaccharides, 3.75 g of proteins, 2.2 g of lipids, 0.75 g of ash and 3.5 g of citric acid.

[0122] Table 1 shows the characterization of the phytocomplex.

Table 1

[0123] The characterization of the phytocomplex was carried out using the method described below: a) Quantification of polyphenols in EchiPure-PC® phytocomplex by UPLC-DAD 100 mg of powder of EchiPure-PC® phytocomplex was weighed and introduced into a 15 mL test tube, and 30 volumes of ethanol and water 60:40 (V / V) were added. After the suspension was mixed for 30 seconds with a vortex mixer, it was sonicated in an ice bath for 15 minutes; finally, it was centrifuged at 4000 rpm for 15 minutes at 6 °C. At the end of centrifugation, the supernatant was recovered. 15 mL of the supernatant was transferred to a new test tube and stored in ice until loaded onto the UPLC system. The sample was diluted 1:10 (first 1:5 with solvent and then 1:2 with water). The diluted sample was filtered through a 0.22 μm filter and then loaded onto the UPLC system. The chromatography system used for the quantification of polyphenols was an Acquity UPLC BEH C18 1.7 μm column, size 2.1×100 mm, connected to an Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 3 / Pk, size 2.1×5 mm. The platform used for UPLC-DAD analysis included a UPLC system (Waters) composed of an eluent management module, Binary Solvent Manager model I Class, and an autosampler, Sample Manager-FTN model I Class, connected to a PDA eλ diode array detector. Data were acquired and analyzed using Empower 3 (Waters) software. The chromatography method used was as follows: Solvent A: water, 0.1% formic acid; Solvent B: 100% acetonitrile. The initial condition was 99% Solvent A; furthermore, the flow rate remained constant at 0.350 ml / min throughout the analysis period. The chromatography column was temperature-controlled at 30 °C. As shown in Table 2, elution of the molecules was carried out by alternating gradient and isocratic phases.

Table 2

[0124] For the quantification of polyphenols, the chromatogram related to the wavelength of 330 nm was used. The polyphenols were quantified as the equivalent amount of chicoric acid, and chicoric acid is the most representative molecule in the above group. The polyphenols were quantified by a calibration curve of a reliable commercial standard of chicoric acid (CAS 6537-80-0; purity ≥ 95%; Sigma Aldrich). Data analysis was carried out using Empower 3 software. The chromatographic profile of EchiPure-PC® phytocomplex is shown in Figure 4. From the UPLC analysis, it was found that the EchiPure-PC® phytocomplex contains 4.2% of polyphenols (expressed as the equivalent amount of chicoric acid). The percentage of chicoric acid in the phytocomplex was equal to 1.6. b) HPLC-ESI-MS analysis of EchiPure-PC® phytocomplex The powder of EchiPure-PC® phytocomplex was stirred for 30 seconds with a vortex mixer and then extracted under ice in a sonicator at 40 Hz for 15 minutes using 20 volumes of methanol: water 90:10; after centrifugation at 18000 g for 10 minutes at 4 °C, the extract was recovered. Before analysis, the sample was diluted with a suitable amount of the same solvent (methanol: water 90:10) to obtain a spectrophotometric absorbance of 0.4 - 0.6 at 320 nm. The sample was further diluted with water and analyzed by HPLC-ESI-MS.

[0125] For mass spectrometry involving chromatographic separation, an HPLC system (Beckman Coulter System Gold 1, Solvent Module equipped with an autosampler) "online" connected to an Esquire 6000 mass spectrometer (Bruker Daltonik GmbH, Germany) equipped with an ESI source was used. Chromatographic and mass data were collected using the Bruker Daltonics Esquire 5.2 - EsquireControl 5.2 program, the following parameters were applied, and the data were processed using the Bruker Daltonics Esquire 5.2 - Data Analysis 3.2 program (Bruker Daltonik GmbH, Germany). Flow rate: 200 μl / min, 25 °C; Injection volume: 10 μl. Column used: Alltima HP C18 3 μm 150×2.1 mm (Alltech Associates, Inc, Derfield, IL) connected to a 7.5×2.1 mm guard column. ESI: Nebulizer gas N2, pressure 50 psi, temperature 350 °C, drying gas 10 l / min Mass acquisition: Full scan alternating in the range of 50 - 1500 m / z; Vacuum pressure: 1.4×10~5 mbar.

[0126] For tandem mass spectrometry: Fragmentation amplitude: 1 V; Collision gas: Helium.

[0127] The chromatographic separation method used was as follows: Eluent: - Solution A: 5% ACN and 0.5% formic acid aqueous solution, Solution B: 100% ACN Total elution time: 42 minutes Gradient: - Start of analysis: 100% eluent A; - Gradient 1: 10% eluent B for 2 minutes; - Gradient 2: At 2 minutes, 20% B for 10 minutes; - Gradient 3: At 12 minutes, 25% B for 2 minutes; - At the gradient 4:14 time point, 70% of B for 7 minutes; - Isocratic 1: 70% of B for 6 minutes; - Gradient 5: At the 27-minute time point, 90% of B for 5 minutes; - Isocratic 2: 90% of B for 10 minutes; - Final re-equilibration of the column: At the 42-minute time point, 0% of B for 1 minute, and the end of analysis at the 60-minute time point.

[0128] Using the Bruker Daltonics Esquire 5.2 - Data Analysis 3.2 software program (Bruker Daltonik GmbH, Germany), data regarding chromatograms and mass spectrometry were converted from the proprietary Bruker format to the.netcdf format.

[0129] Information was extracted from chromatographic analysis using the MZmine - version 2.21 software (http: / / mzmine.sourceforge.net / ).

[0130] Figure 5 shows the chromatogram of the EchiPure - PC (registered trademark) phytocomplex of meristematic cells of Echinacea purpurea.

[0131] This profile shows a dominant peak at a retention time of 25 minutes. This peak has an m / z value and fragmentation profile (MS / MS) equal to that of chicoric acid (commercial standard, Sigma Aldrich).

[0132] Figure 6 shows in three dimensions the chromatogram of the analysis performed on the EchiPure - PC (registered trademark) phytocomplex using the negative ionization mode. c) Quantitative analysis of the polysaccharide content in the EchiPure - PC (registered trademark) phytocomplex The analysis was carried out by modifying the phenol-sulfuric acid method (Segarra et al., 1995, Am J Enol Vitic.). This method involves the acid hydrolysis of polysaccharides, which releases monosaccharides. The monosaccharides react with phenol to develop a yellow color, which can be measured using a spectrophotometer at 490 nm. The results obtained showed an amount of polysaccharide equivalent to 520 mg / g of EchiPure-PC® phytocomplex, which corresponds to 52%. d) Analysis of the protein content of EchiPure-PC® phytocomplex 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 Kjeldahl method was used to measure the total content of proteinaceous nitrogen in the EchiPure-PC® phytocomplex.

[0133] The protein content in the EchiPure-PC® phytocomplex was 15% w / w. e) Analysis of the lipid content of EchiPure-PC® phytocomplex 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, the extraction of the total lipid fraction was carried out on the EchiPure-PC® phytocomplex by Soxhlet extraction with dichloromethane for at least 12 hours.

[0134] The lipid content in EchiPure-PC (registered trademark) phytocomplex was equal to 8.8% w / w. f) Analysis of moisture and ash in EchiPure-PC (registered trademark) phytocomplex The moisture of the phytocomplex was measured by leaving the material on a 40 °C stove for 12 hours. The ash measurement was obtained by treating the material in a muffle furnace at 300 °C until a constant weight was reached.

[0135] The moisture of the phytocomplex was equal to 3%, and the ash was equal to 3%.

[0136] Comparison of polyphenols before and after cell line selection Calli of meristematic cells of Echinacea purpurea were analyzed to quantify their polyphenol content before selection, particularly their chicoric acid content. The analytical method used was the UPLC analysis described above. As is evident from Figure 4, the selected strain Ep-2P (A) shows a total polyphenol content (expressed as equivalent chicoric acid) 39 times higher than that of the initial cells (B).

[0137] Preparation of EchiPure-PC (registered trademark) phytocomplex on an industrial scale The meristematic cells, which were stabilized and selected as described above and derived from a strain of Echinacea purpurea designated Ep-2P, cultured in solid EP medium, were inoculated into a 1-liter capacity flask containing 200 ml of liquid EP 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 a 3-liter capacity flask containing 800 ml of liquid EP medium. 200 ml of the cell suspension was transferred into 800 ml of EP medium in a 3-liter capacity flask. 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 80 liters of EP-F medium. After 7 days of growth in the bioreactor in the dark at 25°C, 5 mg / L of methyl jasmonate was added to the cell suspension.

[0138] After a further 7 days of incubation, the plant biomass (90 liters of cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm, and washed with 56 L of sterile saline (0.9% W / V). 280 g of citric acid was added to the washed cells (fresh weight 28 Kg) and homogenized with an Ultra-Turrax.

[0139] The homogenized cells were dried. 2950 g of EchiPure-PC® phytocomplex was obtained from 90 liters of cell suspension. Biological test: Increase in gene expression of collagen III and IV Using the microtissues of human fibroblasts from elderly donors, the evaluation of the stimulation of collagen III and IV biosynthesis was carried out. The assays were set up using EchiPure-PC® phytocomplex at concentrations of 0.03% and 0.1%. The concentrations used in the assays were equivalent to the final concentrations of the phytocomplex in cosmetic formulations that would be used by consumers.

[0140] EchiPure-PC® phytocomplex at 0.03% and 0.1% was allowed to contact the tissue explants for 72 hours. The skin tissue explants were prepared from primary dermal fibroblasts collected from 60-year-old donors. At the end of the 72-hour incubation in a specific medium (Fibroblast Culture Medium) filtered through a 0.22 μm filter, the tissue explants were collected to perform PCR and histological analysis. The PCR analysis was carried out by extracting RNA using a kit. Using the extracted RNA, cDNA was synthesized and the expression of biomarker genes was tested using PCR technology. The variation in gene expression was evaluated by comparing the samples treated with the extract with the untreated ones. Figure 7 shows the results regarding the gene expression of collagen III and collagen IV.

[0141] The results show that the EchiPure-PC® phytocomplex induces an increase in the gene expression of collagen IV, while in the case of collagen III, the effect is not so significant. Cryosections of the tissue explants prepared 72 hours after treatment with the phytocomplex were treated with an anti-collagen III antibody (Abcam) and the nuclei were stained with DAPI. The signal was observed using a Leica DM 2500 fluorescence microscope. In Figure 8, an increase in the deposition of collagen III in the tissue explants treated with the EchiPure-PC® phytocomplex is observed compared to the untreated tissue explants. Anti-inflammatory activity of the EchiPure-PC® phytocomplex The anti-inflammatory activity of the EchiPure-PC® phytocomplex was evaluated using the J774 A1 macrophage cell line after stimulation with LPS. The tests used were those described in Paterniti et al., Nutrients, 2017.

[0142] To evaluate the protective effect of EchiPure-PC (registered trademark) in macrophage cells after stimulation with LPS, cell viability was evaluated using the MTT colorimetric assay. Figure 9 shows the cell viability data after treatment with EchiPure-PC (registered trademark) at concentrations of 0.5 to 0.1 and 0.01 mg / mL together with LPS. From these results, it can be seen that the EchiPure-PC (registered trademark) phytocomplex has a significant protective effect.

[0143] Aiming to evaluate the anti-inflammatory activity of the EchiPure-PC (registered trademark) phytocomplex, the following parameters: IkB-α, iNOS and COX-2 were evaluated by Western blot analysis. From the results shown in Figure 10, it can be seen that at a concentration of 0.5 mg / mL, the phytocomplex significantly reduces the expression of the enzymes iNOX and COX-2 and prevents the degradation of IkB-α.

[0144] The levels of the inflammatory cytokines TNFα and IL-1β were evaluated by ELISA analysis. After stimulation with LPS, an increase in the inflammatory cytokines was observed. Treatment with EchiPure-PC (registered trademark) at a concentration of 0.5 mg / mL significantly reduces the levels of these cytokines, as demonstrated by the graph in Figure 11.

[0145] Overall, the results obtained demonstrate that the EchiPure-PC (registered trademark) phytocomplex has significant anti-inflammatory activity through the regulation of the inflammatory enzymes iNOS and COX-2 and the cytokines TNFα and IL-1β. Antioxidant activity of the EchiPure-PC (registered trademark) phytocomplex a) Evaluation of ROS generation in human keratinocytes The intracellular generation of reactive oxygen species (ROS) was evaluated using the dichlorodihydrofluorescein diacetate (H2DCF-DA) probe; this probe is internalized into cells where, in the presence of esterase, after deacetylation, it is oxidized by ROS present in the fluorescent compound. When the fluorescence intensity of DCF is read with a Multilabel Plate Reader VICTOR TM X3 2030, PerkinElmer, USA (λec = 485 nm; λem = 535 nm), it is proportional to the amount of ROS present in the cells themselves. The experimental model used to test the antioxidant activity of EchiPure-PC® phycocomplex was the cell line HaCaT, i.e., a human keratinocyte cell line.

[0146] Figure 12 shows data regarding a 3-hour treatment with EchiPure-PC® at a concentration of 0.1% w / v and N-acetylcysteine (NAC) as a positive control, taking into account its known antioxidant action. The experiments were carried out under both basal conditions and conditions of oxidative stress induced by 200 μM H2O2. These data reveal that under both conditions, the EchiPure-PC® phycocomplex significantly reduced reactive oxygen species. b) Evaluation of antioxidant activity in J774-A1 macrophages The antioxidant activity of EchiPure-PC® was evaluated using a J774-A1 macrophage cell model after stress with LPS. A part of the experiment was carried out on macrophages treated with 1 μg / mL LPS, and another part was carried out using macrophages treated with LPS and 0.5 mg / mL EchiPure-PC®.

[0147] To evaluate the antioxidant activity of the phytocomplex, the levels of nitrite and malondialdehyde (MDA) released into the medium were analyzed. The quantification of nitrite was carried out using the Griess reagent. Cells not treated with LPS release very low levels of nitrite (NO2-), whereas stimulation with LPS significantly increases the level of NO2- (Figure 13). MDA is one of the products of lipid peroxidation of cell membranes and represents the degree of oxidative stress. The evaluation of MDA was carried out using the TBARS (thiobarbituric acid reactive substances) assay after cell lysis. MDA has the ability to chemically bind to thiobarbituric acid (TBA) added to the reaction environment. Under suitable conditions of acidity and temperature, the oxidized substrate forms a colored adduct TBA-MDA-TBA, which can be determined by spectrophotometry. Treatment with EchiPure-PC® significantly prevents lipid peroxidation caused by oxidative stress. From the results obtained, it can be seen that EchiPure-PC® exerts antioxidant activity by reducing the release of nitrite and inhibiting lipid peroxidation.

[0148] Formulation of the EchiPure-PC® phytocomplex into two-phase and multiple emulsions, gels and skin and hair cleansing systems The homogenate of the Ep-2P strain, designated EchiPure-PC®, was dispersed in glycerin at a concentration of 3% w / w (INCI name: glycerin, Echinacea purpurea callus lysate, citric acid). The dispersion was added at 3% to the formulations described below.

[0149] For emulsion and gel formulations, the components of the aqueous phase are mixed and heated to 70 °C. The components of the oil phase are mixed and heated to 75 °C. The two phases are combined under the action of a turbo emulsifier. After cooling to approximately 40 °C, the EchiPure-PC® phytocomplex dispersed in glycerin is added while gently stirring.

[0150] In the case of the washing system, mix water and supernatant while gently stirring. After heating to 70 °C, if present, add the oil phase at 75 °C. Add EchiPure-PC (registered trademark) phytocomplex dispersed in glycerin while gently stirring at less than 40 °C. [Table 3] [Table 4] [Table 5]

Claims

A process for obtaining a meristematic cell line derived from a plant of Echinacea purpurea, said process comprising the following: 1) planting tissue obtained from a plant of the genus Echinacea purpurea on a solid medium; 2) isolating a plurality of cell clones; 3) inoculating each of said isolated clones into a liquid medium; 4) for each clone, quantifying the content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caffeic acid, ferulic acid and mixtures thereof; 5) selecting said cell clones containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caffeic acid, ferulic acid and mixtures thereof in an amount greater than 0.05% w / w; 6) transferring said cell clones to a final liquid medium containing salts, sucrose, naphthylacetic acid (NAA) and indoleacetic acid (IAA) used in Gamborg B5 medium to induce an increase in the content of said polyphenols and biomass comprising, said solid and liquid media consisting of said salts, sucrose, NAA, IAA, and kinetin, said cell line containing polyphenols in an amount greater than 0.05% w / w relative to the dry mass of said cell line, wherein said polyphenols are selected from the group consisting of chicoric acid, chlorogenic acid, cichoric acid, caffeic acid, ferulic acid and mixtures thereof, said solid medium being Gamborg B5 medium with 20 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA, 0.5 mg / L kinetin and 0.8% plant agar added, having a final pH of 6.5 and containing 2.5 g / L KNO3, said liquid medium being Gamborg B5 medium with 20 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA, 0.5 mg / L kinetin added, having a final pH of 6.5 and containing 2.5 g / L KNO3, said final liquid medium being Gamborg B5 medium with 40 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA and 0.5 mg / L kinetin added, having a final pH of 6.5 and containing 1 g / L KNO3, process. Claim 2 The process according to claim 1, wherein the polyphenol contains at least 30% of chicoric acid. **Claim 3** The process according to claim 1 or 2, wherein the meristematic cell line contains a polysaccharide in an amount of 30 to 70% w / w. **Claim 4** The process according to any one of claims 1 to 3, wherein the meristematic cell line contains a protein in an amount of 5 to 40% w / w. **Claim 5** The process according to any one of claims 1 to 4, wherein the meristematic cell line contains a lipid in an amount of 3 to 30% w / w. **Claim 6** A process for obtaining a phytocomplex or extract of a meristematic cell line using the meristematic cell line obtained by the process according to any one of claims 1 to 5. **Claim 7** The process according to claim 6, wherein the phytocomplex is a dried or lyophilized cell, or a cell homogenate, or a cell wall and their components. **Claim 8** The process according to claim 6 or 7, wherein the phytocomplex is a cell homogenate containing a polyphenol in an amount exceeding 0.5% w / w based on the dry mass of the cell line, wherein the polyphenol is selected from the group consisting of chicoric acid, chlorogenic acid, caffeoylquinic acid, caffeic acid, ferulic acid and mixtures thereof. **Claim 9** A process for obtaining a composition comprising a meristematic cell line obtained by the process according to any one of claims 1 to 5 and / or a phytocomplex and / or an extract obtained by the process according to any one of claims 6 to 8. **Claim 10** The process according to claim 9, wherein the composition contains the meristematic cell line and / or the phytocomplex 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 phytocomplex is a homogenate at a concentration of 0.05 to 0.2%. **Claim 12** The process according to any one of claims 9 to 11, wherein the composition is formulated as a cream, a gel cream, a gel, a serum, an oil, an emulsion, an emulsion gel (emagel), an ointment, an eye drop, a mouthwash, a spray, a stick, a pill, a capsule, a tablet, a granular powder, a hard shell capsule, an orally disintegrating granule, a sachet, a lozenge, or a liposome. **Claim 13** The split tissue cell line obtained by the process according to any one of claims 1 to 5, the phytocomplex and / or extract obtained by the process according to any one of claims 6 to 8, and / or the composition obtained by the process according to any one of claims 9 to 12 are used for the treatment and / or prevention of inflammation or upper respiratory tract infection, the process according to any one of claims 1 to 12.

14. The split tissue cell line obtained by the process according to any one of claims 1 to 5, the phytocomplex and / or extract obtained by the process according to any one of claims 6 to 8, and / or the composition obtained by the process according to any one of claims 9 to 12 are used as a functional food, the process according to any one of claims 1 to 12.

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