JP2022527846A - Phytocomplexes and extracts of meristem cell lines selected from Echinacea purpurea - Google Patents

Abstract

The present invention relates to a meristem cell line selected from a plant tissue, preferably a callus tissue, of Echinacea purpurea. The invention also relates to a derivative of the cell line, i.e., a phytocomplex or extract of the cell line. Meristem cell lines are characterized by high polyphenol content, especially high chicory acid content. Furthermore, the present invention relates to cosmetics, functional foods and medical uses of selected meristem cell lines or derivatives thereof. [Selection diagram] FIG. 5

Description

The present invention relates to selected split tissue cell lines derived from Echinacea purpurea plants characterized by high polyphenol content, as well as cosmetics, functional foods and medical uses of said split tissue cell lines or derivatives thereof. ..

Plants of the genus Echinacea are widely used, among other things, because of their well-known immunostimulatory activity.

Polyphenols, in particular chicory acid, chlorogenic acid, cynarine and kaphthalic acid, are typical components of Echinacea purpurea. The compound can inhibit the production of free radicals and lipid peroxidation (Georgieva S. et al., 2014, J. Exp. Integra Med.). The chicory acid content and other polyphenolic components of Echinacea purpurea have multiple factors that are difficult to control: season, plant age, growing area and high variability associated with the tissues used to prepare the product. Accompany.

In addition, there are many problems associated with the cultivation of Echinacea: fungal infections, seed dormancy, low germination, long maturation times and difficulties in extract standardization.

For the above reasons, the E. et al. Available on the market today. Products based on E. purpurea are often contaminated or often replaced by other plant species not specified.

Preparation of standardized plant derivatives (ie, including metabolites of reproducible content) has many problems: variation in metabolite content in different plant tissues, seasonal variation, contamination with plant parasites, cultivation. Causes regional differences as well as loss of biological activity of molecules during harvesting, storage and extraction. Extreme variations in the content of plant components of plant preparations obtained directly from the plant or parts thereof by extraction have a negative effect on its effectiveness.

Another method for obtaining standardized plant phytocomplexes free of contaminants in industrial quantities is to use in vitro cell cultures. This technique makes it possible to solve problems related to variability of plant extracts by providing preparations containing active substances with contents that can be reproduced in a standardized fashion. INDUSTRIAL APPLICABILITY The present invention relates to this technical foundation and provides a selected meristem cell line from which a phytocomplex containing a standardized and reproducible content of active material can be obtained.

European Patent No. 2319914 describes the preparation of plant-derived dividing tissue cells of the genus Echinacea, preferably Echinacea angustifolia, having a high content of caffeic acid.

The present invention provides selected meristem cell lines and derivatives thereof from plants belonging to the Echinacea purpurea species, which contain a high content of polyphenols other than caffeic acid.

A first aspect of the invention relates to selected meristem cell lines derived from plants belonging to the Echinacea purpurea species, preferably cell lines derived from curls tissue obtained from the plant itself.

A second aspect of the invention relates to a derivative of the selected meristem cell line, i.e., a phytocomplex or extract of the cell line.

The selected dividing tissue cell lines and derivatives thereof are characterized by a high content of polyphenols selected from the group consisting of chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof.

A third aspect of the invention relates to a composition comprising a selected meristem cell line or a derivative thereof, mixed with an excipient acceptable from the point of view of cosmetics and / or pharmaceuticals.

Applicants have selected cell lines or derivatives thereof that are active in inducing the expression of the genes for collagen III and IV, iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase 2), IL-. Significant anti-inflammatory activity mediated by inhibition of expression of 1β (interleukin 1β) and TNFα (tumor necrosis factor α), as well as inhibition of ROS (reactive oxygen species), MDA (malondialdehyde) and NO _2- It has been demonstrated to have antioxidant activity mediated by inhibition of release.

Another aspect of the invention relates to a process for the preparation and selection of Echinacea purpurea plant meristem cells containing high content of polyphenols other than caffeic acid.

The present invention will be described in detail below with reference to the attached figure as an example.

A photograph of a cell line (referred to as Ep-2P) maintained in a solid medium taken with a bright-field optical microscope is shown. A partial enlargement (200 ×) of FIG. 1 is shown. A partial enlargement (200 ×) of FIG. 1 after staining with fluorescein diacetate is shown. The UV / VIS chromatograms obtained at 330 nm by a diode array detector for (A) selected clones Ep-2 and (B) meristem cells prior to the selection process are shown. For each component of the EchiPure-PC® phytocomplex, the identification of the main peaks obtained by chromatographic profiles of positive and negative ionization modes is shown. The chromatogram of the EchiPure-PC® phytocomplex obtained in the negative ionization mode is shown in three dimensions. Shown is the expression of collagen III and IV genes in microtissue 72 hours after treatment with EchiPure-PC® Phytocomplex. The data is expressed as an increment multiple compared to the untreated control, with the value of the untreated control set to 1. Cold section of microtissue treated with anti-collagen III antibody and stained with DAPI: A: negative control; A': magnified image of portion shown in A; B: microtissue treated with Aristin (positive control) B': Enlarged image of the portion shown in B; C: Microstructure treated with a composition containing 0.03% EchiPure-PC®; C': Enlarged image of the portion shown in C Is shown. The displayed percentage of cell viability after treatment is shown. Western blot analysis and Western blot analysis for evaluation of IkB-α (A), i-NOS (B) and COX-2 (C) after treatment with LPS and EchiPure-PC® at concentrations of 0.5 mg / mL. The relevant quantification is shown. The results of an ELISA test performed on the supernatant of cytolysis are shown. The effect of EchiPure-PC® phytocomplex on the generation of ROS in human keratinocytes is shown. The levels of nitrite and MDA in the supernatant (A) and cytolysis (B) of the macrophage cell model are shown.

Definitions In the context of the present invention, "meristem lineage" or "meristem cell" means a plant lineage or cell capable of maintaining the ability to divide by mitosis to produce new cells. All meristem cells are derived from another meristem cell. The function of meristem cells is comparable to that of animal stem cells.

In the context of the present invention, "callus tissue" refers to an undifferentiated or less specialized cell disordered mass with a thin cell wall and large vacuoles with accumulation of secondary metabolites.

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

A first aspect of the present invention relates to a plant-derived meristem cell line belonging to the Echinacea purpurea species.

In one embodiment, the meristem cell line is:
1) A step of planting a tissue obtained from a plant of the genus Echinacea purpurea in a solid medium;
2) Steps to isolate multiple cell clones;
3) 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 chicory acid, chlorogenic acid, cynarine, caphthalic acid and fertal acid;
5) Selected from the group consisting of chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof in an amount of more than 0.05% w / w, preferably 0.5% to 25% w / w. Steps to select cell clones containing polyphenols.

In step 1), a tissue obtained from a plant belonging to the genus Echinacea purpurea is placed in a solid medium to obtain an undifferentiated callus tissue. The tissue of Echinacea purpurea is preferably at least one shoot of Echinacea purpurea or multiple shoots of Echinacea purpurea.

In a preferred embodiment of the invention, the solid and liquid medium comprises salts suitable for plant cell growth, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.

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

The solid and liquid media are sucrose at a concentration of 15-50 g / L, preferably 18-45 g / L, and naphthylacetic acid at a concentration of 0.1-2.5 mg / L, preferably 0.5-2 mg / L, respectively. (NAA), 0.1-2.5 mg / L, preferably 0.3-1 mg / L concentration of indole acetate (IAA), and 0.1-2 mg / L, preferably 0.2-1 mg / L. Concentrates of kinetin.

In a preferred embodiment, the solid medium is below: sucrose at a concentration of 10-30 g / L, preferably 15-25 g / L, 0.1-2 mg / L, preferably 0.5-1.5 mg / L. Naphthalene acetic acid (NAA), 0.1-2 mg / L, preferably indol acetic acid (IAA) at a concentration of 0.5-1.5 mg / L, and 0.2-1 mg / L, preferably 0.3-. Contains kinetin at a concentration of 0.8 mg / L.

In a preferred embodiment, the liquid medium is below: sucrose at a concentration of 25-50 g / L, preferably 30-45 g / L, 0.1-2 mg / L, preferably 0.5-1.5 mg / L. Naphthalene acetic acid (NAA), 0.1-2 mg / L, preferably indol acetic acid (IAA) at a concentration of 0.5-1.5 mg / L, and 0.2-1 mg / L, preferably 0.3-. It contains kinetin at a concentration of 0.8 mg / L.

Suitable salts for plant cell growth in both solid and liquid media are selected from: CaCl ₂ , KNO ₃ , 0054 ₄ , NaH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ and combinations thereof.

Suitable salts for plant cell growth in both solid and liquid media are preferably: CoCl _{2.6H 2} O, CuSO 4.5H ₂ O, _NaEDTA · 2H ₂ O, FeSO _{4.7H 2} O, H It is selected from ₃ BO ₃ , Kl, MnSO ₄ · H ₂ O, Na ₂ MoO _{4.2 H 2 O, ZnSO 4.7 H 2 O and combinations} thereof.

Both solid and liquid media preferably further include vitamins suitable for plant cell growth selected from: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof.

In one embodiment, the salts suitable for plant cell growth in both solid and liquid media are: CaCl ₂ , KNO ₃ , sulfate 4, NaH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , CoCl _2.6H . ₂ O, CuSO 4.5H ₂ O, _NaEDTA · 2H ₂ O, FeSO _{4.7H 2} O H ₃ BO ₃ , Kl, MnSO ₄ · H ₂ O, Na ₂ MoO _4.2 H ₂ O, ZnSO _4.7 H ₂ It is selected from O and combinations thereof. The combination of this compound is Gamborg B5 medium.

In one embodiment, both solid and liquid media, in addition to the salts listed above, are selected from the following: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof, plant cell growth. Further contains suitable vitamins.

Solid and liquid media are preferably CaCl ₂ at a concentration of 120-170 mg / L, preferably 130-160 mg / L, respectively; KNO ₃ at a concentration of 800-3000 mg / L, preferably 1000-2600 mg / L; 220- NaH ₂ PO ₄ at a concentration of 270 mg / L, preferably 230-260 mg / L, _100-180 mg / L, preferably 110-150 mg / L; and 100-180 mg / L, preferably 110-150 mg. Contains (NH ₄ ) ₂ SO ₄ at a concentration of / L.

The solid and liquid media are preferably CoCl _{2.6H 2 O} at concentrations of 0.01 to 0.05 mg / L, preferably 0.015 to 0.03 mg / L, respectively; 0.01 to 0.05 mg / L. CuSO 4.5H _{2 O at a concentration of 0.015 to 0.03 mg / L; NaEDTA · 2H 2 O at} a concentration of 20 to 60 mg / L, preferably 30 to 45 mg / L; 15 to 45 mg / L, FeSO _{4.7H 2} O at a concentration of 20 to 35 mg / L; H ₃ BO ₃ at a concentration of 1 to 7 mg / L, preferably 2 to 5 mg / L; 0.1 to 2 mg / L, preferably 0. Kl at a concentration of 4 to 1 mg / L; MnSO ₄ · H ₂ O at a concentration of 5 to 20 mg / L, preferably 7 to 15 mg / L; 0.1 to 0.5 mg / L, preferably 0.15 to 0. It contains Na ₂ MoO _{4.2H 2} O at a concentration of 3 mg / L and ZnSO _{4.7H 2} O at a concentration of 0.5-5 mg / L, preferably 1-3 mg / L.

Both solid and liquid media are preferably 70-130 mg, preferably 90-110 mg concentrations of myo-inositol; 70-130 mg, preferably 90-110 mg concentrations of pyridoxine-HCl; and 5-20 mg / L, respectively. It preferably contains thiamine-HCl at a concentration of 7-15 mg / L.

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.

After stabilization step 1a), the portion of stabilized cells preferably goes through the first "clonal selection". Clonal selection involves culturing stabilized cells for an appropriate period, preferably 5-20 days, more preferably 10-15 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.

In step 2), multiple cell clones are isolated by removing aggregates of stabilized cells from the solid medium.

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

According to one embodiment, in step 4), the polyphenol content of each clone is quantified after a period of time, preferably 10-15 days, to achieve proper growth of the cell clones.

In step 5) of cell clone selection, chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and theirs in an amount of more than 0.5% w / w, preferably 0.5% to 25% w / w. It is preferable to carry out a second clone selection according to step 1b) until a plant cell line of Echinacea purpurea containing a polyphenol selected from the group consisting of the mixture is obtained.

The meristem cell line selected in the process described above comprises the polyphenols listed above, more preferably in an amount of 3-15% w / w, even more preferably 3.5-10% w / w.

The mixture of polyphenols preferably contains ticolic acid, preferably chicory acid and cynarine, more preferably chicory acid, cynarine and chlorogenic acid, more preferably ticolic acid, cynarine, chlorogenic acid, caphthalic acid and fertal acid.

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 chicory acid.

The selected cell line preferably comprises an amount of 3.5-10% w / w of polyphenols, the polyphenols being at least 40%, or at least 35% or at least 30% w / w, preferably 38%. Contains ~ 50% w / w chicory acid.

The selected meristem cell line of the present invention also further comprises an amount of 30-70% w / w, preferably 40% -65% w / w of polysaccharide.

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

The selected meristem cell line of the present invention further comprises an amount of 3-30% w / w, preferably 5-10% w / w.

In a preferred embodiment, the selected meristem cell line is an Ep-2P strain containing 0.5-20% polyphenols, of which 35-40% is chicory acid.

A second aspect of the invention relates to a phytocomplex or derivative of a cell line that is an extract of the selected meristem cell line.

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

The phytocomplex is in an amount of more than 0.5% w / w, preferably 0.5% to 25% w / w with respect to the dry mass of the phytocomplex, with ticolic acid, chlorogenic acid, cynarine, caphthalic acid and fertal. Contains polyphenols selected from the group consisting of acids and mixtures thereof. More preferably, the phytocomplex comprises the aforementioned polyphenols in an amount of 3-15% w / w, more preferably 3.5-10% w / w relative to the dry mass of the phytocomplex.

The polyphenol mixture preferably contains chicory acid, preferably chicory acid and cynarine, more preferably chicory acid, cynarine and chlorogenic acid, more preferably ticolic acid, cynarine, chlorogenic acid, caphthalic acid and fertal acid.

In a preferred embodiment, the polyphenol contains at least 40%, or at least 35% or at least 30% w / w, preferably 32-40% w / w of chicory acid with respect to the dry mass of the phytocomplex.

The phytocomplex preferably contains 3.5-10% w / w of polyphenols relative to the dry mass of the phytocomplex, which polyphenols are composed of 38-50% w / w of chicory acid. To.

The phytocomplex also further comprises an amount of 30-70% w / w, preferably 40% -65% w / w of polysaccharide with respect to the dry mass of the phytocomplex.

The phytocomplex also further comprises an amount of protein of 5-40% w / w, preferably 10-30% w / w, relative to the dry mass of the phytocomplex.

The phytocomplex also further comprises an amount of 3-30% w / w, preferably 5-10% w / w of lipid relative to the dry mass of the phytocomplex.

In a preferred embodiment, the phytocomplex is derived from the selected meristem cell line Ep-2P and contains 0.5-20% polyphenols, 35-40% of which is chicory acid.

The phytocomplex is preferably a cell homogenate of the selected meristem cell line Ep-2P.

The extract is an extract of the cell line itself or the phytocomplex of the cell line in an alcohol solvent, eg methanol or ethanol, or in a water / ethanol mixture of various proportions: 50:50 or 60:40 or 70:30. Means. The extract is preferably an extract of the cell homogenate of the cell line. The content of the extract is consistent with the content of the phytocomplex or the cell line from which it was obtained and can vary depending on the extraction technique.

A third aspect of the invention is a meristem cell line and / or a derivative thereof (phytocomplex and / or extract) bound to cosmetics, functional foods and / or at least one excipient permitted from a pharmaceutical point of view. ) Containing the composition.

In one embodiment, the composition is cells at a concentration of 0.01% to 30% w / w, preferably 0.03% to 15% w / w, more preferably 0.05% to 10% w / w. Includes strains and / or derivatives thereof. The composition preferably comprises a phytocomplex that is a cellular homogenate.

In one embodiment, the cell line and / or its derivative is dispersed prior to mixing with the excipient to prepare the composition of the invention. As an example, suitable dispersants are glycerin, propylene or butylene glycol.

The compositions of the invention contain at least one excipient acceptable for pharmaceutical and / or cosmetic use, which is useful in the preparation of the composition and is generally biologically safe and non-toxic. ..

The excipient is at least one conditioning agent, moisturizer, or occlusiveing agent, surfactant, stabilizer, preservative or emollient for the skin.

The compositions of the invention are creams, gel creams, gels, serums, oils, emulsions, emulsions-gels (emalgels), ointments, eye drops, mouthwashes, sprays, preferably nasal sprays, or sticks (such as lip balms). Formulated for topical use as.

The composition may also be formulated for oral administration, preferably as pills, capsules, tablets, granules, hardshell capsules, orally disintegrating granules, sachets or lozenges.

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

The experimental data contained herein show that the aforementioned cell lines or derivatives thereof can stimulate the synthesis of collagen III and IV, resulting in improved skin elasticity and density. In addition, the cell line or derivatives thereof have anti-inflammatory activity associated with reduced synthesis of the inflammatory enzymes iNOS and COX-2 and the cytokines TNFα and IL-1β stimulated by LPS, as well as the production of ROS after oxidative stress. It has been demonstrated that it has anti-inflammatory activity associated with the reduction of.

Another aspect of the invention relates to cosmetic uses of cell lines or derivatives thereof.

Cosmetic use means signs of skin aging, loss of skin elasticity and density and prevention, reduction and / or coping with redness, skin hydration, anti-wrinkle activity and antioxidant activity.

Another aspect of the invention relates to the use of cell lines or derivatives thereof for physical care and hygiene; in this case, the cell lines or derivatives thereof, along with suitable excipients, bath foams, shower gels, etc. It is formulated as a soap, shampoo or hair conditioner.

Another aspect of the invention relates to a cell line or derivative thereof for use as a drug or dietary supplement, in particular for the treatment or prevention of inflammation and infections affecting the upper respiratory tract.

Another aspect of the invention relates to a process for the preparation and selection of plant meristem cells of plants belonging to the genus Echinacea purpurea, which is described below:
1) A step of planting a tissue obtained from a plant of the genus Echinacea purpurea in a solid medium;
2) Steps to isolate multiple cell clones;
3) 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 containing chicory acid, chlorogenic acid, cynarine, caphthalic acid and fertal acid;
5) Selected from the group consisting of chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof in an amount of more than 0.05% w / w, preferably 0.5% to 25% w / w. Includes the steps of selecting cell clones containing polyphenols.

In one embodiment, the preparation of a meristem cell line is a step of collecting tissue from a plant of Echinacea purpurea, preferably shoots, washing it with water, for example, shredding it into small pieces. And include, for example, sterilizing it on a plate by continuous treatment with ethanol, sodium hypochlorite and mercury salt.

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

In a preferred embodiment of the invention, the solid and liquid medium comprises salts suitable for plant cell growth, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.

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

The solid and liquid media are preferably 15-50 g / L, more preferably 18-45 g / L concentration of sucrose, 0.1-2.5 mg / L, more preferably 0.5-2 mg / L, respectively. Concentrations of naphthyl acetate (NAA), 0.1-2.5 mg / L, more preferably 0.3-1 mg / L of indol acetate (IAA), and 0.1 mg / L-2 mg / L, more preferred. Contains kinetin at a concentration of 0.2 mg / L to 1 mg / L.

In a preferred embodiment, the solid medium is below: 10-30 g / L, preferably 15-25 g / L concentration of sucrose, 0.1-2 mg / L, preferably 0.5-1.5 mg / L concentration. Naphthalene acetic acid (NAA), 0.1 to 2 mg / L, preferably indol acetic acid (IAA) at a concentration of 0.5 to 1.5 mg / L, and 0.2 mg / L to 1 mg / L, preferably 0. It contains kinetin at a concentration of 3 mg / L to 0.8 mg / L.

In a preferred embodiment, the liquid medium is below: sucrose at a concentration of 25-50 g / L, preferably 30-45 g / L, 0.1-2 mg / L, preferably 0.5-1.5 mg / L. Naphthalene acetic acid (NAA), 0.1 to 2 mg / L, preferably indol acetic acid (IAA) at a concentration of 0.5 to 1.5 mg / L, and 0.2 mg / L to 1 mg / L, preferably 0. It contains kinetin at a concentration of 3 mg / L to 0.8 mg / L.

Suitable salts for plant cell growth, both in solid and liquid media, are selected from: CaCl ₂ , KNO ₃ , 00544, NaH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ and combinations thereof.

Suitable salts for plant cell growth in both solid and liquid media are preferably: CoCl _{2.6H 2} O, CuSO 4.5H ₂ O, _NaEDTA · 2H ₂ O, FeSO _{4.7H 2} O, H It is selected from ₃ BO ₃ , Kl, MnSO ₄ · H ₂ O, Na ₂ MoO _4.2 H ₂ O, and Zn SO _4.7 H ₂ O and combinations thereof.

Both solid and liquid media further contain vitamins suitable for plant cell growth and are preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof.

In one embodiment, the salts suitable for plant cell growth in both solid and liquid media are: CaCl ₂ , KNO ₃ , sulfate 4, NaH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , CoCl _2.6H . Selected from _{2 O, CuSO 4.5H 2 O} , _NaEDTA · 2H ₂ O, FeSO _{4.7H 2} O H ₃ BO ₃ , Kl, MnSO ₄ · H ₂ O, Na ₂ MoO _4.2 H ₂ O, ZnSO ₄ . To. This salt combination is Gambog B5 medium.

In one embodiment, both solid and liquid media, in addition to the salts listed above, are selected from the following: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof, plant cell growth. Further contains suitable vitamins.

The solid and liquid media are preferably CaCl ₂ at a concentration of 120-170 mg / L, more preferably 130-160 mg / L, respectively; KNO ₃ at a concentration of 800-3000 mg / L, more preferably 1000-2600 mg / L; 220-270 mg / L, more preferably 230-260 mg / L concentrations of marriage ₄ , 100-180 mg / L, more preferably 110-150 mg / L concentrations of NaH ₂ PO ₄ ; and 100-180 mg / L, more. It preferably contains (NH ₄ ) ₂ SO ₄ at a concentration of 110-150 mg / L.

The solid and liquid media are preferably CoCl _{2.6H 2 O} at concentrations of 0.01 to 0.05 mg / L, preferably 0.015 to 0.03 mg / L, respectively; 0.01 to 0.05 mg / L. CuSO 4.5H ₂ O at a concentration of 0.015 to 0.03 mg / L; 20 to 60 mg / L, more preferably _{NaEDTA.2H 2 O} at a concentration of 30 to 45 mg / L; 15 to 45 mg / L. L, more preferably FeSO _{4.7H 2} O at a concentration of 20-35 mg / L; H ₃ BO ₃ at a concentration of 1-7 mg / L, more preferably 2-5 mg / L; 0.1-2 mg / L, More preferably 0.4 to 1 mg / L concentration Kl; 5 to 20 mg / L, more preferably 7 to 15 mg / L concentration MnSO ₄ · H ₂ O; 0.1 to 0.5 mg / L, and more. Na ₂ MoO _{4.2H 2} O at a concentration of 0.15 to 0.3 mg / L and ZnSO _{4.7H 2} O at a concentration of 0.5 to 5 mg / L, more preferably 1 to 3 mg / L. contains.

Both solid and liquid media are preferably 70-130 mg, more preferably 90-110 mg concentrations of myo-inositol; 70-130 mg, more preferably 90-110 mg concentrations of pyridoxine-HCl; and 5-20 mg /, respectively. It contains L, preferably at a concentration of 7-15 mg / L of thiamine-HCl.

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.

After stabilization step 1a), the stabilized cells preferably go through the first "clonal selection". Clonal selection involves culturing stabilized cells for a suitable period, preferably 5 to 20 days, 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.

In step 2), multiple cell clones are isolated by removing aggregates of stabilized cells from the solid medium.

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

According to one embodiment, in step 4), the polyphenol content of each clone is quantified after a period of time, preferably 10-15 days, to achieve proper growth of the cell clones.

Echinacea purpurea plants for which the production of polyphenols selected from the group comprising chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof in step 5) of cell clone selection is optimal. It is preferable to carry out a second clone selection according to step 1b) until a cell line is obtained.

The selected cell lines are then grown in flasks or bioreactors or fermenters to achieve an increase in biomass. Biomass increase occurs at the first stage in a liquid growth medium called EP. The liquid growth medium EP is a medium containing the above-mentioned Gamborg salt, the vitamins listed above, sucrose, NAA, and IAA.

The liquid growth medium EP contains KNO ₃ in an amount of 1.5 g / L to 3.5 g / L, preferably 2 g / L to 3 g / L, among the Gambog salts. Sucrose is preferably contained in an amount of 15 g / L to 25 g / L. NAA is preferably contained in an amount of 0.5 mg / L to 1.5 mg / L. IAA is preferably contained in an amount of 0.2 mg / L to 1 mg / L. Kinetin is preferably contained in an amount of 0.2 mg / L to 1 mg / L.

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

The final medium EP-F contains KNO ₃ in an amount of 0.5 g / L to 2 g / L among the Gambog salts. Sucrose is preferably contained at 30 g / L to 50 g / L. NAA is preferably contained in an amount of 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.

According to a preferred embodiment, in both EP growth medium and final EP-F medium, cell line growth in flasks, bioreactors or fermenters is typically 15 ° C.-35 ° C. under dark conditions. Is carried out at a temperature of 25 ° C. for 7 to 30 days, preferably 14 to 21 days.

At the end of growth in the final EP-F medium, the cell line is filtered and then the cells are harvested and used in the next step in the form of a phytocomplex, or also in a later extraction phase in an alcohol solvent. They can also be attached to produce cell extracts characterized by high polyphenol content.

The phytocomplex can be obtained by lyophilization or drying of living cells; in this case, the phytocomplex is a lyophilized product of dead cells.

In one embodiment, at the end of growth in a flask, bioreactor or fermenter, the cells are preferably in an acidified solution, eg, containing ascorbic acid and / or citric acid and / or acetic acid, eg. After homogenization by mechanical grinding, freeze-dry or dry. In the latter case, the phytocomplex is a cell homogenate in which the cell and its internal structure are disrupted. All of these different types of phytocomplexes are characterized by having high polyphenol content as previously described.

Alternatively, the phytocomplex, preferably in the form of a cellular homogenate, is subjected to extraction in an alcohol solvent (eg, 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 for the preparation of cosmetics, functional foods or pharmaceutical compositions as described above.

Alternatively, after purification, the living cells can be used directly for the preparation of the composition of the present invention.

Biological data obtained in subsequent experiments will be selected from the group comprising chicory acid, cynarine, chlorogenic acid, caphthalic acid, fertal acid and mixtures thereof for the purposes described herein. Confirm the effectiveness of the cell line and / or derivative containing a surprisingly high content of polyphenols.

The unexpected efficacy of the derivative of the selected cell line was similarly demonstrated, in which case the derivative is whole without exposing the cells to a solvent or other extraction procedure that may compromise the integrity of the active ingredient. It is a homogenate containing cellular components, or a phytocomplex.

Preparation and Selection of Meristem Cell Lines of Echinacea purpurea The induction of callus tissue was achieved using standard procedures described in the literature. By this procedure, collection of juvenile tissues (shoots) from plants of Echinacea purpurea, eg washing them with tap water, shredding into 2-5 cm sections, and eg, in sequence, 70% ethanol. Treatment with aqueous solution for about 15 seconds with 2% sodium hypochlorite and 0.1% Tween 20 for about 5 minutes, and finally sterilization on the plate with at least 4 washes with sterile distilled water. All fragments of further crushed plant tissue (external pieces) are placed in a Petri dish containing a nutrient medium that has been solidified by the addition of agar and supplemented with growth hormone without antibiotics. After a suitable period of incubation in the dark at 25 ° C., undifferentiated callus tissue is formed; it is then transferred to a wider surface containing fresh medium and then grown.

Meristem cell lines obtained from undifferentiated callus tissue are stabilized by transfer to solid medium several times (secondary culture). This medium was supplemented with 20 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA, 0.5 mg / L kinetin and 0.8-1% plant agar (EP medium). It consists of a solid Ganborg B5 type (containing 2.5 g / L KNO ₃ ) with a final pH of 6.5. The cell line obtained in this particular medium after clonal selection was referred to as Ep-2P.

It was confirmed by DNA fingerprint analysis that the obtained meristem cells belonged to the plant species Echinacea purpurea.

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

The production liquid medium (optimized to increase polyphenol content) was supplemented with 40 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA and 0.5 mg / L kinetin, final. Ganborg B5 (containing 1 g / L of KNO ₃ ) having a pH of 6.5 (EP-F medium).

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

Morphological Characteristics of Cell Lines Selected cell lines of Echinacea purpurea, referred to as Ep-2P, are maintained in solid EP medium, beige with a whitish tint and have a brittle texture ( Figures 1 to 3).

Homozygization procedure The procedure for homogenizing the biomass of cells selected and grown in a bioreactor at 25 ° C (± 2) for 14 days is as follows:
a) Steps to obtain only cells by filtration of biomass obtained from proliferation of Ep-2P cell cultures in EP-F medium and discard the medium;
b) Washing the cells with twice the amount of saline (0.9% W / V NaCl in sterile water);
c) The step of 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) The step of homogenizing the mixture, for example using Ultra-Turrax or other instrument suitable for disrupting cells and their internal structures;
e) Includes steps to dry the biomass by freeze-drying or air circulation drying or rotary cylinder drying or fluidized bed drying or spraying.

The procedure described is used to obtain a phytocomplex referred to as EchiPure-PC®.
Details of EchiPure-PC® Homogenate:
40-65% polysaccharide 0.5-20% total polyphenols (of which about 38% is chicory acid)
10-30% Protein 5-10% Lipid 2-6% Moisture 2-17% Ash 5-30% Citric Acid Preparation of Standard EchiPure-PC® A Phyto Complex in EP-F Medium The example of is provided as a non-limiting example.

The phytocomplex thus obtained can be used as 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 referred to as EchiPure CRO®-G.

Preparation and analysis of EchiPure-PC® phytocomplex Solid EP medium (20 g / L sucrose, 1 mg / L NAA, 0.5 mg / L IAA, 0.5 mg / L kinetin and 0.8% 200 ml of the stabilized and selected dividing tissue cells (referred to as Ep-2P) derived from the strain of Echinacea purpurea cultured in Gambog B5) supplemented with plant agar. Liquid EP-F medium (40 g / L sucrose, 1 g / L KNO ₃ , 1 mg / L NAA, 0.5 mg / L IAA and 0.5 mg / L kinetin instead of 2.5 g / L Inoculated into 5 1 liter volumetric flasks containing Ganborg B5) with a final pH of 6.5. The amount of meristem 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 jasmine was added to the cell suspension (95%, Sigma-Aldrich, CAS Number: 39924-52-2). After 7 days of incubation, plant biomass (1 liter cell suspension) was collected, filtered through a nylon mesh with a pore size of 50 μm and washed with 700 ml 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 Ultra-Turrax.

The homogenized cells were lyophilized. From a 1 liter cell suspension, 25 g containing 1.05 g of polyphenol, 13 g of polysaccharide, 3.75 g of protein, 2.2 g of lipid, 0.75 g of ash and 3.5 g of citric acid. (EchiPure-PC® Phytocomplex) was obtained.

Table 1 shows the characterization of the phytocomplex.

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

Chromatograms associated with wavelengths of 330 nm were used for the quantification of polyphenols. Polyphenols are quantified as chicory acid equivalents, and chicory acid is the most representative molecule in the above group. Polyphenols were quantified by a reliable commercial standard calibration curve for chicory acid (CAS 6537-80-0; purity ≥ 95%; Sigma Aldrich). Data analysis was performed using Empor 3 software. The chromatograph profile of the EchiPure-PC® phytocomplex is shown in FIG. UPLC analysis reveals that the EchiPure-PC® phytocomplex contains a percentage of polyphenols equal to 4.2 (represented as chicory acid equivalent). The percentage of chicory acid in the phytocomplex was equal to 1.6.
b) HPLC-ESI-MS analysis of EchiPure-PC® phytocomplex EchiPure-PC® phytocomplex powder is stirred with a vortex mixer for 30 seconds and then under ice for 15 minutes at 40 Hz. Extraction was performed in the treatment apparatus with 20 volumes of methanol: water 90:10; the extract was recovered after centrifugation at 18000 g for 10 minutes at 4 ° C. Prior to the 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.

For mass spectrometry with chromatograph separation, an autosampler with an ESI source, an Esquire 6000 mass spectrometer (Bruker Daltonik GmbH, Germany) and an "online" coupled HPLC system (Beckman Coulter System Gold 1, autosampler). A solvent module (Solvent Module) was used. The Bruker Daltonics Esquire 5.2-Esquire Control 5.2 program was used to collect chromatographs and mass data, and the following parameters were applied to the Bruker Daltonics Esquire 5.2-Data Analysis 3.2Data Analysis 3.2 It was processed using Germany).
Flow rate: 200 μl / min, 25 ° C; Injection volume: 10 μl.
Columns used: Alltima HP C18 3 μm 150 × 2.1 mm (Alltech Associates, Inc, Defield, IL) coupled with a 7.5 × 2.1 mm guard column.
ESI: Nebulizer gas N2, pressure 50 psi, temperature 350 ° C., dry gas 10 l / min Mass acquisition: AC full scan in the range of 50-1500 m / z; Vacuum pressure: 1.4 x 10-5 m bar.

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

The chromatograph 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: Eluent A 100%;
-Gradient 1: 10% eluent B for 2 minutes;
-Gradient 2: At 2 minutes, 20% B for 10 minutes;
-At a gradient of 3:12 minutes, 25% B for 2 minutes;
-At a gradient of 4:14 minutes, 70% B for 7 minutes;
-Isocratic 1: 70% B for 6 minutes;
-At a gradient of 5:27 minutes, 90% B for 5 minutes;
-Isocratic 2: 90% B for 10 minutes;
-Final re-equilibration of the column: At 42 minutes, 0% B at 1 minute, 60 minutes to complete the analysis.

Bruker Daltonics Esquire 5.2-Data Analysis 3.2 Using a software program (Bruker Daltonik GmbH, Germany), data on chromatograms and mass analysis from the proprietary Bruker format. Converted to netcdf format.

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

FIG. 5 shows a chromatogram of the EchiPure-PC® phytocomplex of Echinacea purpurea meristem cells.

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

FIG. 6 shows in three dimensions the chromatogram of the analysis performed on the EchiPure-PC® phytocomplex using the negative ionization mode.
c) Quantitative analysis of the polysaccharide content in the EchiPure-PC® phytocomplex The analysis was performed by modifying the phenol sulphate method (Segarra et al., 1995, Am J Enol Vitic.). This method involves acid hydrolysis of the polysaccharide, which releases the monosaccharide. The monosaccharide reacts with phenol to develop a yellow color, which can be measured at 490 nm using a spectrophotometer. The results obtained show an amount of polysaccharide equivalent to 520 mg / g EchiPure-PC® phytocomplex, which corresponds to 52%.
d) Analysis of protein content of EchiPure-PC® phytocomplex Lynch, JM. et al. , "Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products" Journal of AOAC International (1999), p. ) The total content of proteinaceous nitrogen was measured for the phytocomplex.

The protein content in the EchiPure-PC® phytocomplex was 15% w / w.
e) Analysis of Lipid Content of EchiPure-PC® Phytocomplex Martinez M. et al. et al. , "Soxhlet lipids extraction from cotton from dichloromethane dividing areas. Comparison of dichloromethane or successive dichlomethane-methan" Extraction of the total lipid fraction by Soxhlet extraction with dichloromethane for at least 12 hours according to the method described in Grassy Elements (1997), 48 (4), 226-230, EchiPure-PC® Phyto. Performed on the complex.

The lipid content in the EchiPure-PC® phytocomplex was equal to 8.8% w / w.
f) Analysis of Moisture and Ash in EchiPure-PC® Phytocomplex Moisture measurements were performed on the phytocomplex by leaving the material on a stove at 40 ° C. for 12 hours. Ash measurements were taken by processing the material in a muffle furnace at 300 ° C. until a constant weight was reached.

The water content of the phytocomplex was equal to 3% and the ash was equal to 3%.

Comparison of Polyphenols Before and After Selection of Cell Lines Callus of Echinacea purpurea meristem cells was analyzed to quantify their polyphenol content, especially their ticolic acid content, before selection. The analysis method used was the UPLC analysis described above. As is clear from FIG. 4, the selected strain Ep-2P (A) exhibits a total polyphenol content (expressed as chicory acid equivalent) 39 times higher than that of the early cells (B).

Preparation of EchiPure-PC® Phytocomplex on an Industrial Scale Stabilized as described above, derived from a strain of Echinacea purpurea called Ep-2P cultured in solid EP medium. And selected split tissue cells were inoculated into 10 flasks with a volume of 1 liter containing 200 ml of liquid EP medium. The amount of meristem 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 inoculated into 10 flasks with a volume of 3 liters containing 800 ml of liquid EP medium. The 200 ml cell suspension was transferred into 800 ml EP medium in a 3 liter volume 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 inoculated with a bioreactor containing 80 liters of EP-F medium. After growing in a bioreactor for 7 days in the dark at 25 ° C., 5 mg / L methyl jasmine was added to the cell suspension.

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

The homogenized cells were dried. From 90 liters of cell suspension, 2950 g of EchiPure-PC® phytocomplex was obtained.
Biological studies: Increased gene expression of collagen III and IV Evaluation of stimulation of collagen III and IV biosynthesis was performed using microtissues of human fibroblasts from elderly donors. The assay was set up with EchiPure-PC® phytocomplexes at 0.03% and 0.1% concentrations. The concentration used in the assay is comparable to the final concentration of phytocomplex in the cosmetic formulation that will be used by the consumer.

0.03% and 0.1% EchiPure-PC® phytocomplex was contacted with microtissue and left for 72 hours. Skin microtissues were prepared from primary skin fibroblasts taken from a 60 year old donor. Microtissues were collected to perform PCR and histological analysis at the end of the 72 hour incubation in specific medium filtered through a 0.22 μm filter (Fibroblast Culture Medium). PCR analysis was performed by extracting RNA using the kit. The extracted RNA was used to synthesize cDNA and tested for biomarker gene expression using PCR techniques. Fluctuations in gene expression were assessed by comparing the samples treated with the extract with those untreated. FIG. 7 shows the results regarding gene expression of collagen III and collagen IV.

The results show that the EchiPure-PC® phytocomplex induces increased gene expression of collagen IV, whereas the effect is less pronounced for collagen III. Cold sections of microtissue prepared 72 hours after treatment with the phytocomplex were treated with anti-collagen III antibody (Abcam) and the nuclei were stained with DAPI. Signals were observed using a Leica DM 2500 fluorescence microscope. In FIG. 8, an increased deposition of collagen III in the microtissue treated with EchiPure-PC® Phytocomplex is observed as compared to the untreated microtissue.
Anti-inflammatory activity of EchiPure-PC® phytocomplex The anti-inflammatory activity of EchiPure-PC® phytocomplex was evaluated using the J774 A1 macrophage cell line after stimulation with LPS. The tests used were described in Paterniti et al. , Nutrients, 2017.

To evaluate the protective effect of EchiPure-PC® in macrophage cells after stimulation with LPS, MTT colorimetric analysis was used to assess cell viability. FIG. 9 shows cell viability data after treatment with EchiPure-PC® at concentrations of 0.5-0.1 and 0.01 mg / mL with LPS. From these results, it can be seen that the EchiPure-PC® phytocomplex exerts a significant protective effect.

The following parameters: IkB-α, iNOS and COX-2 were evaluated by Western blot analysis with the goal of assessing the anti-inflammatory activity of the EchiPure-PC® phytocomplex. From the results shown in FIG. 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.

Levels of the inflammatory cytokines TNFα and IL-1β were assessed by ELISA analysis. After stimulation with LPS, an increase in inflammatory cytokines was observed. Treatment with EchiPure-PC® at a concentration of 0.5 mg / mL significantly reduces the levels of these cytokines, as evidenced by the graph in FIG.

Overall, the results obtained demonstrate that the EchiPure-PC® phytocomplex exerts significant anti-inflammatory activity through the regulation of the inflammatory enzymes iNOS and COX-2 and the cytokines TNFα and IL-1β. Is.
Antioxidant activity of EchiPure-PC® phytocomplex a) Evaluation of ROS production in human keratinocytes Intracellular production of reactive oxygen species (ROS) using a dichlorodihydrofluorescein diacetate (H2DCF-DA) probe. Evaluated; this probe is intracellularly internalized, where it is deacetylated in the presence of esterase and then oxidized by ROS present in the fluorescent compound. When the fluorescence intensity of DCF is read by 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 the EchiPure-PC® phytocomplex was the cell line HaCaT, a strain of human keratinocytes.

FIG. 12 shows EchiPure-PC® at a concentration of 0.1% w / v, and 3 hours of treatment with N-acetylcysteine (NAC) as a positive control in view of its known antioxidant activity. Shows data about. Experiments were performed under both basic conditions and conditions of oxidative stress induced by 200 μM H ₂ O ₂ . These data reveal that under both conditions, the EchiPure-PC® phytocomplex significantly reduced reactive oxygen species.
b) Evaluation of antioxidant activity in J774-A1 macrophages Using the J7744-A1 macrophage cell model after stress by LPS, the antioxidant activity of EchiPure-PC® was evaluated. Part of the experiment was performed on macrophages treated with 1 μg / mL LPS and another part was performed with macrophages treated with LPS and EchiPure-PC® 0.5 mg / mL.

To assess 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 a grease reagent. Cells not treated with LPS release very low levels of nitrite (NO _2- ), whereas stimulation with LPS significantly increases levels of NO _2- (FIG. 13). MDA is one of the products of lipid peroxidation of cell membranes and represents the degree of oxidative stress. Evaluation of MDA was performed using the TBARS (thiobarbituric acid reactive substance) assay after cytolysis. MDA has the ability to chemically bind to thiobarbituric acid (TBA) added to the reaction environment. Under favorable conditions of acidity and temperature, the oxidizing substrate forms the dye-forming adduct TBA-MDA-TBA, which can be determined by spectrophotometry. Treatment with EchiPure-PC® significantly prevents lipid peroxidation due to 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.

EchiPure-PC® Phytocomplex Two-Phase and Multiple Emulsions, Gels and Formulations for Skin and Hair Cleaning Systems 3% w / w homogenate of Ep-2P strain called EchiPure-PC® Dispersed in glycerin at the concentration of (INCI name: glycerin, Echinacea purpurea carus lysate, citric acid). The dispersion was added at 3% to the formulations described below.

In the case of emulsion and gel preparations, 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. Under the action of a turbo emulsifier, the two phases are combined. After cooling to about 40 ° C., EchiPure-PC® phytocomplex dispersed in glycerin is added with gentle stirring.

For wash systems, mix the water and supernatant with gentle agitation. After heating to 70 ° C, the oil phase is added at 75 ° C, if present. EchiPure-PC® phytocomplex dispersed in glycerin is added with gentle stirring below 40 ° C.

Claims (27)

Less than:
1) A step of planting a tissue obtained from a plant of the genus Echinacea purpurea in a solid medium;
2) Steps to isolate multiple cell clones;
3) 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 containing chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof;
5) Selected from the group containing chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof in an amount of more than 0.05% w / w, preferably 0.5% to 25% w / w. A split tissue cell line derived from a plant of Echinacea purpurea obtained by a process comprising the step of selecting the cell clone containing the polyphenol.
The solid and liquid medium contains salts suitable for plant cell growth, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.
The cell line contains more than 0.05% w / w, preferably 0.5% to 25% w / w of polyphenols with respect to the dry mass of the cell line, wherein the polyphenols are: A dividing tissue cell line selected from the group consisting of ticolic acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof. The solid and liquid media are each of 15-50 g / L, preferably 18-45 g / L sucrose, 0.1-2.5 mg / L, preferably 0.5-2 mg / L NAA. , 0.1-2.5 mg / L, preferably 0.3-1 mg / L concentration of indole acetic acid (IAA), and 0.1-2 mg / L, preferably 0.2-1 mg / L concentration. The dividing tissue cell line according to claim 1, which contains kinetin. The liquid medium contains the following: sucrose at a concentration of 25 to 50 g / L, preferably 30 to 45 g / L, and naphthylene acetic acid at a concentration of 0.1 to 2 mg / L, preferably 0.5 to 1.5 mg / L. NAA), 0.1-2 mg / L, preferably 0.5-1.5 mg / L concentration of indol acetic acid (IAA), and 0.2-1 mg / L, preferably 0.3-0.8 mg / L. The dividing tissue cell line according to claim 1 or 2, which contains L concentration of kinetin. The solid medium contains the following: sucrose at a concentration of 10 to 30 g / L, preferably 15 to 25 g / L, and naphthylene acetic acid at a concentration of 0.1 to 2 mg / L, preferably 0.5 to 1.5 mg / L. NAA), 0.1-2 mg / L, preferably 0.5-1.5 mg / L concentration of indole acetate (IAA), and 0.2-1 mg / L, preferably 0.3-0.8 mg / L. The dividing tissue cell line according to any one of claims 1 to 3, which contains kinetin at a concentration of L. The salt suitable for the growth of the plant cells is selected from the following: CaCl ₂ , KNO ₃ , ו ₄ , NaH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , and combinations thereof, any of claims 1 to 4. The meristem cell line according to item 1. The salts suitable for the growth of the plant cells are as follows: CoCl 2.6H ₂ O, CuSO 4.5H ₂ O, _NaEDTA · 2H ₂ O, FeSO _{4.7H 2} O, H ₃ BO _{3 ,} Kl, MnSO ₄ ·. The dividing tissue cell line according to any one of claims 1 to 5, which is selected from H ₂ O, Na ₂ MoO _{4.2H 2} O, ZnSO _{4.7H 2} O and a combination thereof. The solid and liquid media are preferably CaCl ₂ at a concentration of 120-170 mg / L, preferably 130-160 mg / L, respectively; KNO ₃ at a concentration of 800-3000 mg / L, preferably 1000-2600 mg / L; 220. NaH ₂ PO ₄ at a concentration of ~ 270 mg / L, preferably 230-260 mg / L, 100-180 mg / L, preferably 110-150 mg / L; and 100-180 mg / L, preferably _110- The dividing tissue cell line according to claim 5, which contains (NH ₄ ) ₂ SO ₄ at a concentration of 150 mg / L. The solid and liquid media are preferably CoCl _{2.6H 2 O} at a concentration of 0.01 to 0.05 mg / L, preferably 0.015 to 0.03 mg / L, respectively; 0.01 to 0.05 mg / L. L, preferably CuSO 4.5H _{2 O at a concentration of 0.015 to 0.03 mg / L; NaEDTA · 2H 2 O at} a concentration of 20 to 60 mg / L, preferably 30 to 45 mg / L; 15 to 45 mg / L. FeSO _{4.7H 2} O at a concentration of 20 to 35 mg / L; H ₃ BO ₃ at a concentration of 1 to 7 mg / L, preferably 2 to 5 mg / L; 0.1 to 2 mg / L, preferably 0. .4 to 1 mg / L concentration of Kl; 5 to 20 mg / L, preferably 7 to 15 mg / L concentration of MnSO ₄ · H ₂ O; 0.1 to 0.5 mg / L, preferably 0.15 to Claim 6 contains Na ₂ MoO _{4.2H 2} O at a concentration of 0.3 mg / L and ZnSO _{4.7H 2} O at a concentration of 0.5 to 5 mg / L, preferably 1 to 3 mg / L. The dividing tissue cell line described. Both the solid and liquid media preferably contain vitamins suitable for plant cell growth, selected from: myoinositol, nicotinic acid, pyridoxin-HCl, thiamine-HCl and combinations thereof: 1. Item 8. The meristem cell line according to any one of 8. Myoinositol is present at a concentration of 70-130 mg, preferably 90-110 mg; pyridoxine-HCl is present at a concentration of 70-130 mg, preferably 90-110 mg; thiamine-HCl is present at a concentration of 5-20 mg / L, The meristem cell line according to claim 9, preferably present at a concentration of 7 to 15 mg / L. The fission according to any one of claims 1 to 10, wherein the polyphenol contains at least 30%, preferably at least 35%, more preferably at least 45%, still more preferably 38% to 50% chicory acid. Tissue cell line. The meristem cell line according to any one of claims 1 to 11, comprising a polysaccharide in an amount of 30 to 70% w / w, preferably 40 to 65% w / w. The meristem cell line according to any one of claims 1 to 12, which comprises an amount of protein of 5 to 40% w / w, preferably 10 to 30% w / w. The meristem cell line according to any one of claims 1 to 13, which comprises an amount of 3 to 30% w / w, preferably 5 to 10% w / w of lipid. The phytocomplex or extract of the meristem cell line according to any one of claims 1 to 14. 15. The phytocomplex of claim 15, which is a dried or freeze-dried cell, or a cell homogenate, or a cell wall and components thereof, preferably a homogenate of the meristem cell line. A cell homogenate containing a polyphenol in an amount of more than 0.5% w / w, preferably 0.5% to 25% w / w with respect to the dry mass of the cell line, wherein the polyphenol is chlorogenic acid. The phytocomplex according to claim 15 or 16, selected from the group consisting of chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof. A composition comprising the meristem cell line according to any one of claims 1 to 14 and / or the phytocomplex and / or the extract according to any one of claims 15 to 17. The splits at concentrations of 0.01% to 1% by weight, preferably 0.03% to 0.5% by weight, more preferably 0.05% to 0.2% by weight, based on the total composition. 18. The composition of claim 18, comprising a tissue cell line and / or the phytocomplex and / or extract. The composition according to claim 18 or 19, wherein the phytocomplex is a homogenate at a concentration of 0.05 to 0.2%. Cream, gel cream, gel, serum, oil, emulsion, emulsion gel (emalgel), ointment, eye drops, mouthwash, spray, preferably nasal spray, sticks, pills, capsules, tablets, granular powder, hard shell capsules, The composition according to any one of claims 18 to 20, which is formulated as an orally disintegrating granule, sachet, lozenge, or liposome. Any of claims 1 to 14, for the purpose of cosmetics, preferably for the purpose of preventing or reducing or coping with signs of skin aging and / or increase of free radicals in the skin, or preventing or reducing wrinkles due to its antioxidant activity. The dividing tissue cell line according to claim 1, the phytocomplex and / or the extract according to any one of claims 15 to 17, and / or the composition according to any one of claims 18 to 21. Use of. The fission tissue cell line according to any one of claims 1 to 14, and the phyto according to any one of claims 15 to 17, for use in the treatment and / or prevention of inflammation or upper respiratory tract infection. The composition according to any one of claims 18 to 21 as well as a complex and / or an extract. The meristem cell line according to any one of claims 1 to 14, the phytocomplex and / or the extract according to any one of claims 15 to 17, and / or the claim as a functional food. Use of the composition according to any one of 18 to 21. A process for the preparation and selection of plant dividing tissue cells of Echinacea purpurea, wherein the cells are> 0.05% w / w, preferably 0.5% -25% w /. The content of w comprises a polyphenol selected from the group consisting of chicory acid, chlorogenic acid, cynarine, kaphthalic acid, fertal acid and mixtures thereof, the process comprising:
1) A step of planting a tissue obtained from a plant of the genus Echinacea purpurea in a solid medium;
2) Steps to isolate multiple cell clones;
3) 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 containing chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof;
5) Selected from the group comprising chicory acid, chlorogenic acid, cynarine, caphthalic acid, fertal acid and mixtures thereof in an amount of more than 0.05% w / w, preferably 0.5% to 25% w / w. Including the step of selecting the cell clone containing the polyphenol.
Here, a process in which the solid and liquid medium contains salts suitable for plant cell growth, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin. 25. The solid and liquid medium preferably comprises the following vitamins suitable for plant cell growth, selected from: myo-inositol, nicotinic acid, pyridoxine-HCl, thiamine-HCl and combinations thereof. Described process. After growing the strain in a liquid growth medium containing salts, vitamins, sucrose, NAA, IAA and kinetin suitable for the growth of the plant cells, the salts, vitamins, sucrose, NAA suitable for the growth of the plant cells. 25 or 26, the process of claim 25 or 26, comprising a proliferative phase of the dividing tissue cell line by transferring the cells to a final liquid medium containing IAA and kinetin.

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