JP7708669B2 - Phytocomplex and extracts of selected meristematic cell lines from Echinacea purpurea - Google Patents

Description

The present invention relates to a selected meristematic cell line derived from the Echinacea purpurea plant, characterized by a high polyphenol content, and to cosmetic, functional food and medical uses of said meristematic cell line or its derivatives.

Plants of the Echinacea genus are widely used, among other things, for their well-known immune-stimulating activity.

Polyphenols, especially chicoric acid, chlorogenic acid, cynarin and caftaric acid, are typical components of Echinacea purpurea. Said compounds are able to 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 polyphenolic components of Echinacea purpurea is subject to high variability related to several factors that are difficult to control: season, plant age, growing region and tissue used to prepare the product.

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

For the reasons stated above, E. purpurea-based products available on the market today are often contaminated or often substituted with other, often unspecified, plant species.

The preparation of standardized plant derivatives (i.e. with reproducible contents of metabolites) poses many problems: variations in the content of metabolites in different plant tissues, seasonal variations, contamination with plant parasites, differences related to the cultivation region, and loss of biological activity of molecules during harvesting, storage and extraction. Extreme variations in the content of plant constituents of plant preparations obtained directly from the plant or its parts by extraction have a negative impact on their efficacy.

Another method for obtaining industrial quantities of standardized plant phytocomplexes free of contaminants is the use of in vitro cell cultures. This technique makes it possible to solve the problems related to the variability of plant extracts, since it provides preparations with a reproducible content of active substances in a standardized manner. The present invention relates to this technological platform and provides selected meristem cell lines from which phytocomplexes with a standardized and reproducible content of active substances can be obtained.

EP 2 319 914 describes the preparation of meristematic cells from plants of the genus Echinacea, preferably Echinacea angustifolia, having a high content of caffeic acid.

The present invention provides a selected meristematic cell line and its derivatives from a plant belonging to the species Echinacea purpurea, which contains a high content of polyphenols other than caffeic acid.

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

A second aspect of the invention relates to derivatives of selected meristematic cell lines, i.e. phytocomplexes or extracts of the cell lines.

The selected meristematic cell lines and their derivatives are characterized by a high content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof.

A third aspect of the present invention relates to a composition comprising a selected meristematic cell line or a derivative thereof in admixture with a cosmetically and/or medicamentally acceptable excipient.

The applicant has demonstrated that selected cell lines or their derivatives are active in inducing the expression of collagen III and IV genes and have significant anti-inflammatory activity mediated by 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 inhibition of ROS (reactive oxygen species), MDA (malondialdehyde) and _NO2- release.

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

The invention will now be described in detail, by way of example only, with reference to the accompanying drawings.

Shown is a bright field light micrograph of a cell line, designated Ep-2P, maintained in solid medium. 2 shows an enlargement (200x) of a portion of FIG. A magnification (200x) of a portion of Figure 1 after staining with fluorescein diacetate is shown. Shown are UV/VIS chromatograms obtained at 330 nm with a diode array detector for (A) the selected clone Ep-2 and (B) the meristematic cells before the selection process. The identification of the main peaks obtained in the chromatographic profiles in positive and negative ionization modes for each component of the EchiPure-PC® phytocomplex is given. The chromatogram of EchiPure-PC® phytocomplex obtained in negative ionization mode is shown in three dimensions. Figure 1 shows the expression of collagen III and IV genes in microtissues 72 hours after treatment with EchiPure-PC® phytocomplex. Data are expressed as fold increase compared to untreated control, with the untreated control value set at 1. Cryosections of microtissues treated with anti-collagen III antibody and stained with DAPI: A: negative control; A': magnified image of the area shown in A; B: microtissues treated with aristin (positive control); B': magnified image of the area shown in B; C: microtissues treated with a composition containing 0.03% EchiPure-PC (registered trademark); C': magnified image of the area shown in C. The percentage of cell viability after the indicated treatments is shown. Western blot analysis and associated quantification for the evaluation of IkB-α (A), i-NOS (B) and COX-2 (C) following treatment with LPS and EchiPure-PC® at a concentration of 0.5 mg/mL are shown. The results of an ELISA test performed on the supernatant of the cell lysates are shown. FIG. 1 shows the effect of EchiPure-PC® phytocomplex on the production of ROS in human keratinocytes. Nitrite and MDA levels in the supernatant (A) and cell lysate (B) of a macrophage cell model are shown.

Definitions In the context of the present invention, "meristem lineage" or "meristem cell" refers to a plant lineage or cell that is able to maintain the ability to divide by mitosis to give rise to new cells. All meristematic cells are derived from other meristematic cells. The function of a meristematic cell is equivalent to that of an animal stem cell.

In the context of the present invention, "callus tissue" refers to a disorganized mass of undifferentiated or poorly specialized cells with thin cell walls and large vacuoles in which secondary metabolites accumulate.

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

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

In one embodiment, the meristematic cell line comprises:
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) quantifying the content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid and fertaric acid for each clone;
5) Selecting cell clones containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof in an amount greater than 0.05% w/w, preferably between 0.5% and 25% w/w.

In step 1), tissue obtained from a plant of the genus Echinacea purpurea is placed in a solid medium to obtain undifferentiated callus tissue. The Echinacea purpurea tissue 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 media contain salts suitable for plant cell growth, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.

Solid media further contain agar, whereas liquid media do not.

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.

In a preferred embodiment, the solid medium contains the following: sucrose at a concentration of 10-30 g/L, preferably 15-25 g/L, naphthalene acetic acid (NAA) at a concentration of 0.1-2 mg/L, preferably 0.5-1.5 mg/L, indole acetic 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.

In a preferred embodiment, the liquid medium contains the following: sucrose at a concentration of 25-50 g/L, preferably 30-45 g/L, naphthalene acetic acid (NAA) at a concentration of 0.1-2 mg/L, preferably 0.5-1.5 mg/L, indole acetic 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.

Salts suitable for growing _plant cells in both solid and liquid media are selected from the following: _{CaCl2 ,} KNO3, _MgSO4 , _NaH2PO4 , ( _NH4 ) _{2SO4 and} combinations thereof.

Salts suitable for growing plant cells in both solid and liquid _media 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 _.

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

In one embodiment, salts suitable for growing plant cells in both solid and liquid media are _{selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4 , CoCl2.6H2O , CuSO4.5H2O , NaEDTA.2H2O , FeSO4.7H2OH3BO3 , Kl , MnSO4.H2O , Na2MoO4.2H2O , ZnSO4.7H2O , and combinations thereof} . This combination _of compounds is Gamborg _B5 medium.

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

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.

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 _. O; Na ₂ MoO ₄ .2H ₂ O having a concentration of 0.1 to 0.5 mg/L, preferably 0.15 to 0.3 mg/L, and ZnSO ₄ .7H ₂ O having a concentration of 0.5 to 5 mg/L, preferably 1 to 3 mg/L.

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.

After step 1), the callus tissue is preferably divided into several parts, which are stabilized by successively transferring them to a solid medium (step 1a) to obtain stabilized cells. This step is called the stabilization step.

After the stabilization step 1a), a portion of the stabilized cells preferably undergoes a first "clonal selection". Clonal selection consists in culturing the stabilized cells for a suitable period of time, preferably 5-20 days of culture, more preferably 10-15 days (step 1b). These cells are incubated in the dark at a temperature between 15°C and 35°C, preferably between 24°C and 26°C.

In step 2), multiple cell clones are isolated by removing the stabilized cell aggregates 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), after a period of time to achieve adequate proliferation of the cell clones, preferably a proliferation phase of 10 to 15 days, the polyphenol content of each clone is quantified.

In step 5) of the selection of cell clones, it is preferable to carry out a second clonal selection according to step 1b) until a plant cell line of Echinacea purpurea is obtained, which contains polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof in an amount of more than 0.5% w/w, preferably between 0.5% and 25% w/w.

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

The mixture of polyphenols preferably comprises chicoric acid, preferably chicoric acid and cynarin, more preferably chicoric acid, cynarin and chlorogenic acid, more preferably chicoric acid, cynarin, chlorogenic acid, caftaric acid and fertaric acid.

In a preferred embodiment, the polyphenols contain at least 40%, at least 35% or at least 30% w/w chicoric acid, preferably 38% to 50% w/w.

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

The selected meristematic cell lines of the present invention also further comprise polysaccharides in an amount of 30-70% w/w, preferably 40%-65% w/w.

The selected meristematic cell lines of the present invention further comprise protein in an amount of 5-40% w/w, preferably 10-30% w/w.

The selected meristematic cell lines of the present invention further comprise lipids in an amount of 3-30% w/w, preferably 5-10% w/w.

In a preferred embodiment, the selected meristematic cell line is the Ep-2P line, which contains 0.5-20% polyphenols, of which 35-40% is chicoric acid.

A second aspect of the invention relates to cell line derivatives that are phytocomplexes or extracts of selected meristematic cell lines.

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

The phytocomplex comprises polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof in an amount of more than 0.5% w/w, preferably between 0.5% and 25% w/w, based on the dry weight of the phytocomplex. More preferably, the phytocomplex comprises the aforementioned polyphenols in an amount between 3 and 15% w/w, even more preferably between 3.5 and 10% w/w, based on the dry weight of the phytocomplex.

The mixture of polyphenols preferably comprises chicoric acid, preferably chicoric acid and cynarin, more preferably chicoric acid, cynarin and chlorogenic acid, more preferably chicoric acid, cynarin, chlorogenic acid, caftaric acid and fertaric acid.

In a preferred embodiment, the polyphenols comprise 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.

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

The phytocomplex also further comprises polysaccharides in an amount of 30-70% w/w, preferably 40%-65% w/w, based on the dry weight of the phytocomplex.

The phytocomplex also further comprises protein in an amount of 5-40% w/w, preferably 10-30% w/w, based on the dry weight of the phytocomplex.

The phytocomplex also further comprises lipids in an amount of 3-30% w/w, preferably 5-10% w/w, based on the dry weight of the phytocomplex.

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

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

Extract means an extract of the cell line itself or of the phytocomplex of the cell line in an alcoholic solvent, for example methanol or ethanol, or a water/ethanol mixture 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 said extract corresponds to that of the phytocomplex or of the cell line from which it was obtained and may vary depending on the extraction technique.

A third aspect of the present invention relates to a cosmetic, functional food and/or composition comprising a meristematic cell line and/or a derivative thereof (phytocomplex and/or extract) in association with at least one excipient acceptable from a pharmacopoeial point of view.

In one embodiment, the composition comprises the cell line and/or its derivatives 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. The composition preferably comprises a phytocomplex that is a cell homogenate.

In one embodiment, the cell line and/or its derivatives are dispersed prior to mixing with the excipient to prepare the composition of the invention. By way of example, suitable dispersing agents are glycerin, propylene or butylene glycol.

The compositions of the present 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, moisturizing agent, or occlusive agent, surfactant, stabilizer, preservative, or emollient for the skin.

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

The compositions may also be formulated for oral administration, preferably as a pill, capsule, tablet, granule powder, hard shell capsule, orally disintegrating granules, sachet or lozenge.

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, and is preferably formulated as a liposome.

The experimental data contained herein show that the aforementioned cell line or its derivatives are able to stimulate the synthesis of collagen III and IV, resulting in improved skin elasticity and firmness. Furthermore, the cell line or its derivatives have been demonstrated to have anti-inflammatory activity associated with the reduction of the synthesis of the inflammatory enzymes iNOS and COX-2 and the LPS-stimulated cytokines TNFα and IL-1β, as well as antioxidant activity associated with the reduction of the generation of ROS following oxidative stress.

Another aspect of the present invention relates to cosmetic uses of the cell line or its derivatives.

By cosmetic use is meant prevention, reduction and/or treatment of signs of skin aging, loss of skin elasticity and firmness and redness, skin hydration, anti-wrinkle activity and antioxidant activity.

Another aspect of the invention relates to the use of the cell line or its derivatives for personal care and hygiene; in this case, the cell line or its derivatives are formulated together with suitable excipients as a bath foam, shower gel, soap, shampoo or hair conditioner.

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

Another aspect of the present invention relates to a process for the preparation and selection of plant meristem cells of a plant belonging to the genus Echinacea purpurea, which comprises:
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) quantifying for each clone the content of polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid and fertaric acid;
5) selecting cell clones containing polyphenols selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof in an amount greater than 0.05% w/w, preferably between 0.5% and 25% w/w.

In one embodiment, the preparation of the meristematic cell line comprises the steps of harvesting tissue, preferably shoots, from an Echinacea purpurea plant, washing it, for example with water, chopping it into small pieces, and sterilizing it on plates, for example by successive treatments with ethanol, sodium hypochlorite and mercuric salts.

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

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

Solid media further contain agar, whereas liquid media do not.

The solid and liquid media each preferably 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 to 2 mg/L, more preferably 0.2 mg/L to 1 mg/L.

In a preferred embodiment, the solid medium contains the following: sucrose at a concentration of 10-30 g/L, preferably 15-25 g/L, naphthalene acetic acid (NAA) at a concentration of 0.1-2 mg/L, preferably 0.5-1.5 mg/L, indole acetic 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 to 1 mg/L, preferably 0.3 mg/L to 0.8 mg/L.

In a preferred embodiment, the liquid medium contains the following: sucrose at a concentration of 25-50 g/L, preferably 30-45 g/L; naphthalene acetic acid (NAA) at a concentration of 0.1-2 mg/L, preferably 0.5-1.5 mg/L; indole acetic 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 to 1 mg/L, preferably 0.3 mg/L to 0.8 mg/L.

Salts suitable for growing plant cells in both solid and liquid media are selected from the following: _{CaCl2 ,} KNO3, _MgSO4 , _NaH2PO4 , ( _NH4 ) _{2SO4 and} combinations thereof.

Salts suitable for growing plant cells, both in solid and liquid media, 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 .}

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

In one embodiment, salts suitable for growing plant cells in both solid and liquid media are _{selected from the following: CaCl2, KNO3, MgSO4, NaH2PO4, (NH4)2SO4 , CoCl2.6H2O , CuSO4.5H2O , NaEDTA.2H2O , FeSO4.7H2OH3BO3 , Kl , MnSO4.H2O , Na2MoO4.2H2O , ZnSO4 . This} combination _{of salts} is Gamborg B5 _medium .

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

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.

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 _. O; Na ₂ MoO ₄ .2H ₂ O having a concentration of 0.1 to 0.5 mg/L, more preferably 0.15 to 0.3 mg/L, and ZnSO ₄ .7H ₂ O having a concentration of 0.5 to 5 mg/L, more preferably 1 to 3 mg/L.

Both the solid and liquid media preferably contain myo-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.

After step 1), the callus tissue is preferably divided into several parts, which are stabilized by successively transferring them to a solid medium (step 1a) to obtain stabilized cells. This step is called the stabilization step.

After the stabilization step 1a), the stabilized cells preferably undergo a first "clonal selection". Clonal selection consists in culturing the stabilized cells for a suitable period of time, preferably 5-20 days of culture, more preferably 10-15 days (step 1b). The cells are incubated in the dark at a temperature between 15°C and 35°C, preferably 24°C and 26°C.

In step 2), multiple cell clones are isolated by removing the stabilized cell aggregates 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), after a period of time to achieve adequate proliferation of the cell clones, preferably a proliferation phase of 10 to 15 days, the polyphenol content of each clone is quantified.

In step 5) of the selection of cell clones, it is preferable to carry out a second round of clonal selection according to step 1b) until a plant cell line of Echinacea purpurea is obtained which is optimal in the production of polyphenols selected from the group comprising chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof.

The selected cell line is then grown in flasks or bioreactors or fermenters to achieve biomass growth, which occurs in the first stage in a liquid growth medium called EP. The liquid growth medium EP is a medium containing the Gamborg salts mentioned above, the vitamins listed above, sucrose, NAA, and IAA.

The liquid growth medium EP contains, among the 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.

Cells grown in liquid medium EP were transferred into final medium EP-F containing Gamborg salts, vitamins, sucrose, NAA, and IAA for the final growth phase, which induces an increase in polyphenol content and biomass.

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.

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

At the end of the 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 they can also be subjected to a subsequent extraction phase in an alcoholic solvent to produce a cell extract characterized by a high polyphenol content.

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

In one embodiment, at the end of the growth in flasks, bioreactors or fermenters, the cells are homogenized, for example by mechanical grinding, preferably in an acidifying solution (containing, for example, 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 structure are disintegrated. These different types of phytocomplexes are all characterized by a high polyphenol content, as already mentioned.

Alternatively, the phytocomplex, preferably in the form of a cell homogenate, is subjected to extraction in an alcoholic solvent (e.g. 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 previously mentioned, for the preparation of a cosmetic, functional food or pharmaceutical composition.

Alternatively, after purification, the viable cells can be used directly in the preparation of the compositions of the invention.

The biological data obtained in the subsequent experiments confirms the efficacy of said cell lines and/or derivatives containing surprisingly high contents of polyphenols selected from the group comprising chicoric acid, cynarin, chlorogenic acid, caftaric acid, fertaric acid and mixtures thereof for the uses described herein.

The unexpected efficacy of derivatives of the selected cell lines was also demonstrated, in this case homogenates or phytocomplexes containing all cellular components without exposing the cells to solvents or other extraction procedures that may compromise the integrity of the active ingredients.

Creation and Selection of Meristem Cell Lines of Echinacea purpurea The induction of callus tissue was achieved using standard procedures described in the literature, which involve the collection of young tissues (shoots) from Echinacea purpurea plants, e.g. washing them with tap water, chopping into 2-5 cm pieces, and sterilization on plates, e.g. treating sequentially with 70% aqueous ethanol for about 15 seconds, 2% sodium hypochlorite and 0.1% Tween 20 for about 5 minutes, and finally washing at least four times with sterile distilled water. All pieces of further crushed plant tissue (explants) are placed in Petri dishes containing a nutrient medium made solid by the addition of agar and without antibiotics and supplemented with growth hormones. After a suitable period of incubation in the dark at 25° C., undifferentiated callus tissue is formed; this is then transferred to a larger surface containing fresh medium and then propagated.

Meristem cell lines obtained from undifferentiated callus tissue were stabilized by several transfers (subcultures) to solid medium, consisting of solid Gamborg B5 type (containing 2.5 g/L KNO3) with a final pH of 6.5, 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). The cell line obtained in this particular medium after clonal selection was designated Ep-2P.

DNA fingerprint analysis confirmed that the resulting 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 further growth phases.

The production liquid medium (optimized for increased polyphenol content) was Gamborg B5 (containing 1 g/L KNO3) supplemented with 40 g/L sucrose, 1 mg/L NAA, 0.5 mg/L IAA and 0.5 mg/L kinetin, with a final 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 the cell line. The selected cell line of Echinacea purpurea, designated Ep-2P, was maintained on solid EP medium and is beige in color with a whitish tint and has a brittle texture (Figures 1-3).

Homogenization Procedure The procedure for homogenizing the selected cell biomass grown in a bioreactor at 25° C. (±2) for 14 days was as follows:
a) filtration of the biomass obtained from the growth of an Ep-2P cell culture in EP-F medium to obtain only the cells and discard 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) citric acid (or ascorbic acid or a mixture of citric acid and ascorbic acid) to the filtered and washed biomass;
d) homogenizing the mixture, for example using 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.

Using the procedure described, a phytocomplex called EchiPure-PC® is obtained.
EchiPure-PC® Homogenate Contents:
40-65% polysaccharides 0.5-20% total polyphenols (of which about 38% is chicoric acid)
10-30% Protein 5-10% Lipid 2-6% Moisture 2-17% Ash 5-30% Citric Acid An example of a preparation of standard EchiPure-PC® A phytocomplex in EP-F medium is provided as a non-limiting example.

The phytocomplex thus obtained is either used as is or dispersed in a suitable dispersion medium at a concentration ranging from 0.5 to 5% w/w. The suspension thus obtained is called EchiPure CRO®-G.

Preparation and Analysis of EchiPure-PC® Phytocomplex Meristem cells (designated Ep-2P) from a strain of Echinacea purpurea cultivated in solid EP medium (Gamborg B5 supplemented with 20 g/L sucrose, 1 mg/L NAA, 0.5 mg/L IAA, 0.5 mg/L kinetin and 0.8% plant agar), stabilized and selected as described above, were inoculated into five 1-liter volumetric flasks containing 200 ml of liquid EP-F medium (Gamborg B5 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, final pH 6.5). 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 of methyl jasmate 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 harvested, 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.

The homogenized cells were lyophilized. From 1 liter of cell suspension, 25 g of lyophilizate (EchiPure-PC® Phytocomplex) was obtained containing the following: 1.05 g polyphenols, 13 g polysaccharides, 3.75 g protein, 2.2 g lipids, 0.75 g ash and 3.5 g citric acid.

Table 1 shows the characterization of the phytocomplex.

The phytocomplex was characterized using the methods described below:
a) Quantification of polyphenols in EchiPure-PC® Phytocomplex by UPLC-DAD 100 mg of powder of EchiPure-PC® Phytocomplex was weighed into a 15 mL test tube and 30 volumes of ethanol and water 60:40 (V/V) were added. The suspension was mixed with a vortex mixer for 30 seconds, then 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 the centrifugation, the supernatant was collected. 15 mL of the supernatant was transferred to a new tube and kept in ice until it was 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 before being loaded into the UPLC system. The chromatographic system used for the quantification of polyphenols is an Acquity UPLC BEH C18 1.7 μm column, size 2.1×100 mm, coupled to an Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 3/Pk, size 2.1×5 mm. The platform used for the UPLC-DAD analysis includes a UPLC system (Waters) consisting of an eluent management module, Binary Solvent Manager model I Class, and an autosampler, Sample Manager-FTN model I Class, coupled to a PDA eλ diode array detector. Data were acquired and analyzed using Empower 3 (Waters) software. The chromatographic method used was as follows: Solvent A: water, 0.1% formic acid; Solvent B: 100% acetonitrile. The initial conditions were 99% solvent A; and the flow rate remained constant at 0.350 ml/min throughout the analysis period. The chromatographic column was thermostated at 30° C. Elution of the molecules was performed by alternating gradient and isocratic phases, as shown in Table 2.

For the quantification of polyphenols, the chromatogram associated with a wavelength of 330 nm was used. Polyphenols were quantified as chicoric acid equivalents, chicoric acid being the most representative molecule in the group. Polyphenols were quantified by a calibration curve of an authentic commercial standard of chicoric acid (CAS 6537-80-0; purity ≧95%; Sigma Aldrich). Data analysis was carried out with Empower 3 software. The chromatographic profile of EchiPure-PC® phytocomplex is shown in FIG. 4. The UPLC analysis shows that EchiPure-PC® phytocomplex contains % polyphenols (expressed as chicoric acid equivalents) equal to 4.2. The percentage of chicoric acid in the phytocomplex was equal to 1.6.
b) HPLC-ESI-MS Analysis of EchiPure-PC® Phytocomplex EchiPure-PC® Phytocomplex powder was extracted with 20 volumes of methanol:water 90:10 in a sonicator at 40 Hz under ice for 15 min after vortexing for 30 s; the extract was collected after centrifugation at 18000 g for 10 min at 4° C. Prior to analysis, the samples were 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 samples were further diluted with water and analyzed by HPLC-ESI-MS.

For mass spectrometry with chromatographic separation, an HPLC system (Beckman Coulter System Gold 1, Solvent Module with autosampler) coupled "on-line" with 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 and processed using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 program (Bruker Daltonik GmbH, Germany) applying the following parameters:
Flow rate: 200μl/min, 25°C; injection volume: 10μl.
Column used: Alltima HP C18 3 μm 150×2.1 mm (Alltech Associates, Inc, Delfield, IL) coupled with 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: alternating full scan in the range 50-1500 m/z; vacuum pressure: 1.4×10-5 mbar.

For tandem mass spectrometry: fragmentation amplitude: 1 V; collision gas: helium.

The chromatographic separation methods used were as follows:
Eluent:
- Solution A: 5% ACN and 0.5% formic acid in water, Solution B: 100% ACN
Total time of elution: 42 min. Gradient:
- Start of analysis: 100% eluent A;
- Gradient 1: 10% eluent B in 2 min;
- Gradient 2: 20% B in 10 min at 2 min;
- Gradient 3: 25% B in 2 min at 12 min;
- Gradient 4: 70% B in 7 min at 14 min;
- Isocratic 1: 6 min at 70% B;
- Gradient 5: 90% B in 5 min at 27 min;
- Isocratic 2: 90% B for 10 min;
- Final re-equilibration of the column: 42 min, 1 min, 0% B, end of analysis at 60 min.

Chromatograms and mass spectrometry data were converted from proprietary Bruker format to .netcdf format using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 software program (Bruker Daltonik GmbH, Germany).

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

Figure 5 shows a chromatogram of EchiPure-PC® phytocomplex from meristematic cells of Echinacea purpurea.

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

FIG. 6 shows in three dimensions the chromatogram of the analysis carried out on the EchiPure-PC® phytocomplex using negative ionization mode.
c) Quantitative Analysis of Polysaccharide Content in EchiPure-PC® Phytocomplex The analysis was performed by modifying the phenol-sulfuric acid method (Segarra et al., 1995, Am J Enol Vitic.). This method involves acid hydrolysis of polysaccharides, which releases monosaccharides. The monosaccharides react with phenol to produce a yellow color, which can be measured spectrophotometrically at 490 nm. The results obtained show an amount of polysaccharides equivalent to 520 mg/g of EchiPure-PC® phytocomplex, which corresponds to 52%.
d) Analysis of Protein Content of EchiPure-PC® Phytocomplex The total protein nitrogen content of EchiPure-PC® phytocomplex was determined using the Kjeldahl method as described in Lynch, J M. et al., “Kjeldahl nitrogen analysis as a reference method for protein determination in daily products” Journal of AOAC International (1999), 82(6), 1389-1398.

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., "Soxhlet lipids extraction from cotton from different producing areas. Comparison of dichloromethane or successive dichloromethane-methanol extractions". Extraction of the total lipid fraction was performed on the EchiPure-PC® phytocomplex by Soxhlet extraction with dichloromethane for at least 12 hours according to the method described in Grasas y Aceites (1997), 48(4), 226-230.

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

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

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

Preparation of EchiPure-PC® Phytocomplex on an Industrial Scale Meristem cells, stabilized and selected as described above, originating from a strain of Echinacea purpurea, designated Ep-2P, cultivated in solid EP medium, were inoculated into 10 flasks of 1 liter volume 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 used to inoculate 10 flasks of 3 liter volume containing 800 ml of liquid EP medium. 200 ml of the cell suspension was transferred into 800 ml of EP medium in a flask of 3 liter volume. 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 jasmate was added to the cell suspension.

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

The homogenized cells were dried. From 90 liters of cell suspension, 2950 g of EchiPure-PC® phytocomplex was obtained.
Biological Test: Increased Gene Expression of Collagen III and IV Evaluation of stimulation of collagen III and IV biosynthesis was performed using human fibroblast microtissues from elderly donors. The assay was set up using EchiPure-PC® phytocomplex at concentrations of 0.03% and 0.1%. The concentrations used in the assay are equivalent to the final concentration of phytocomplex in cosmetic formulations that will be used by consumers.

0.03% and 0.1% EchiPure-PC® phytocomplex were left in contact with the microtissues for 72 hours. Skin microtissues were prepared from primary dermal fibroblasts taken from a 60-year-old donor. At the end of 72 hours of incubation in a specific medium (Fibroblast Culture Medium) filtered through a 0.22 μm filter, the microtissues were collected for PCR and histological analysis. PCR analysis was performed by extracting RNA using a kit. The extracted RNA was used to synthesize cDNA and test the expression of biomarker genes using PCR technology. The variation in gene expression was evaluated by comparing the samples treated with the extracts with the untreated ones. Figure 7 shows the results for gene expression of collagen III and collagen IV.

The results show that EchiPure-PC® phytocomplex induces an increase in the gene expression of collagen IV, whereas the effect is less pronounced in the case of collagen III. Cryosections of microtissues prepared 72 hours after treatment with the phytocomplex were treated with anti-collagen III antibody (Abeam) and nuclei were stained with DAPI. The signals were observed using a Leica DM 2500 fluorescence microscope. In Figure 8, an increase in collagen III deposition can be seen in microtissues treated with EchiPure-PC® phytocomplex compared to untreated microtissues.
Anti-inflammatory activity of EchiPure-PC® phytocomplex Evaluation of the anti-inflammatory activity of EchiPure-PC® phytocomplex was carried out using the J774 A1 macrophage cell line after stimulation with LPS. The assay used is described in Paterniti et al., Nutrients, 2017.

To evaluate the protective effect of EchiPure-PC® in macrophage cells after stimulation with LPS, cell viability was assessed using the MTT colorimetric assay. Figure 9 shows cell viability data after treatment with EchiPure-PC® at concentrations of 0.5-0.1 and 0.01 mg/mL along with LPS. These results indicate that the EchiPure-PC® phytocomplex exerts a significant protective effect.

Aiming to evaluate the anti-inflammatory activity of the EchiPure-PC® phytocomplex, the following parameters were evaluated by Western blot analysis: IkB-α, iNOS and COX-2. The results shown in Figure 10 show that at a concentration of 0.5 mg/mL, the phytocomplex significantly reduces the expression of the enzymes iNOX and COX-2, as well as prevents the degradation of IkB-α.

The levels of the inflammatory cytokines TNFα and IL-1β were assessed by ELISA analysis. After stimulation with LPS, an increase in the 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 Figure 11.

Overall, the results obtained demonstrate that the EchiPure-PC® phytocomplex exerts significant anti-inflammatory activity through the modulation of the inflammatory enzymes iNOS and COX-2 and the cytokines TNFα and IL-1β.
Antioxidant activity of EchiPure-PC® phytocomplex a) Evaluation of the generation of ROS in human keratinocytes The intracellular generation of reactive oxygen species (ROS) was evaluated using the dichlorodihydrofluorescein diacetate (H2DCF-DA) probe; this probe is internalized in the cells, where it is deacetylated in the presence of esterases and then oxidized by the ROS present in a fluorescent compound. The intensity of the fluorescence of DCF is read on a MultilabelPlate Reader VICTOR™ X3 2030, PerkinElmer, USA (λec=485 nm; λem=535 nm) and 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 line of human keratinocytes.

Figure 12 shows data for a 3 hour treatment with EchiPure-PC® at a concentration of 0.1% w/v, as well as N-acetylcysteine (NAC) as a positive control, given its known antioxidant activity. Experiments were performed under both basal conditions and conditions of oxidative stress induced _by 200 μM _H2O2 . These data demonstrate that under both conditions, the EchiPure-PC® phytocomplex significantly reduced reactive oxygen species.
b) Evaluation of antioxidant activity in J774-A1 macrophages Evaluation of the antioxidant activity of EchiPure-PC® was performed using the J774-A1 macrophage cell model after stress with LPS. 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 0.5 mg/mL EchiPure-PC®.

To evaluate the antioxidant activity of the phytocomplex, the levels of nitrite and malondialdehyde (MDA) released in the medium were analyzed. The nitrite quantification was performed using the Griess reagent. Cells not treated with LPS released very low levels of nitrite (NO ₂ -), whereas upon stimulation with LPS the levels of NO ₂ - increased significantly (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 performed using the TBARS (thiobarbituric acid reactive substances) assay after cell lysis. MDA has the ability to chemically bind with thiobarbituric acid (TBA) added to the reaction environment. Under suitable conditions of acidity and temperature, the oxidized substrate forms a chromogenic adduct TBA-MDA-TBA that can be determined spectrophotometrically. Treatment with EchiPure-PC® significantly prevents lipid peroxidation caused by oxidative stress. The results show that EchiPure-PC® exerts its antioxidant activity by reducing nitrite release and inhibiting lipid peroxidation.

Formulation of EchiPure-PC® Phytocomplex into Two-Phase and Multiple Emulsions, Gels, and Skin and Hair Cleansing Systems A 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.

For emulsion and gel formulations, the water phase ingredients are mixed and heated to 70°C. The oil phase ingredients are mixed and heated to 75°C. Under the action of a turbo emulsifier, the two phases are combined. After cooling to approximately 40°C, EchiPure-PC® Phytocomplex dispersed in glycerin is added with gentle stirring.

For the washing system, mix the water and supernatant with gentle stirring. Heat to 70° C., then add the oil phase, if present, at 75° C. Add EchiPure-PC® phytocomplex dispersed in glycerin with gentle stirring below 40° C.

Claims (14)

1. A process for obtaining a meristematic cell line derived from an Echinacea purpurea plant, the process comprising :
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 a liquid culture medium with each of the isolated clones;
4) quantifying for each clone the content of polyphenols selected from the group comprising chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof;
5) selecting said cell clones containing polyphenols selected from the group comprising chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertaric acid and mixtures thereof in an amount greater than 0.05% w/w;
6) transferring the cell clones to a final liquid medium containing the salts used in Gamborg B5 medium, sucrose, naphthylacetic acid (NAA) and indoleacetic acid (IAA) to induce an increase in the polyphenol content and biomass;
The solid and liquid media consist of the salts , sucrose, NAA, IAA, and kinetin;
the cell line comprises polyphenols in an amount greater than 0.05% w/w relative to the dry weight of the cell line, wherein the polyphenols are selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertalic acid and mixtures thereof ;
The solid medium is Gamborg B5 medium supplemented with 20 g/L sucrose, 1 mg/L NAA, 0.5 mg/L IAA, 0.5 mg/L kinetin and 0.8% plant agar, with a final pH of 6.5 and containing 2.5 g/L _KNO3 ;
The liquid medium is Gamborg B5 medium supplemented with 20 g/L sucrose, 1 mg/L NAA, 0.5 mg/L IAA, 0.5 mg/L kinetin, final pH 6.5, and containing 2.5 g/L KNO3 _;
The final liquid medium is Gamborg B5 medium supplemented with 40 g/L sucrose, 1 mg/L NAA, 0.5 mg/L IAA and 0.5 mg/L kinetin, with a final pH of 6.5 and containing 1 g/L KNO3 _;
process . 2. The process of claim 1 , wherein the polyphenols contain at least 30% chicoric acid. The process according to claim 1 or 2 , wherein the meristematic cell line comprises polysaccharides in an amount of 30-70% w/w. The process according to any one of claims 1 to 3 , wherein the meristematic cell line comprises protein in an amount of 5-40% w/w. The process according to any one of claims 1 to 4 , wherein the meristematic cell line comprises lipids in an amount of 3 to 30% w/w. A process for obtaining a phytocomplex or extract of a meristematic cell line using a meristematic cell line obtainable by a process according to any one of claims 1 to 5 . 7. The process according to claim 6 , wherein the phytocomplex is a dried or freeze-dried cell, or a cell homogenate, or a cell wall and its components. 8. The process according to claim 6 or 7, wherein the phytocomplex is a cell homogenate containing polyphenols in an amount of more than 0.5% w/w relative to the dry mass of the cell line, wherein the polyphenols are selected from the group consisting of chicoric acid, chlorogenic acid, cynarin, caftaric acid, fertalic acid and mixtures thereof. A process for obtaining a composition comprising a meristematic cell line obtainable by a process according to any one of claims 1 to 5 and/or a phytocomplex and/or an extract obtainable by a process according to any one of claims 6 to 8 . 10. The process according to claim 9, wherein the composition comprises the meristematic cell line and/or the phytocomplex and/or extract in a concentration of 0.01% to 1% by weight relative to the total composition. 11. The process according to claim 9 or 10 , wherein the phytocomplex is a homogenate at a concentration of 0.05 to 0.2%. 12. The process according to any one of claims 9 to 11, wherein the composition is formulated as a cream, gel-cream, gel, serum, oil, emulsion, emulsion gel (emulgel), ointment, eye drop, mouthwash, spray, stick, pill, capsule, tablet, granular powder, hard shell capsule , orally disintegrating granules, sachet , lozenge, or liposome. The process according to any one of claims 1 to 12 , wherein the meristematic cell line obtainable by the process according to any one of claims 1 to 5, the phytocomplex and/or the extract obtainable by the process according to any one of claims 6 to 8 and/or the composition obtainable 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 infections . The process according to any one of claims 1 to 12, wherein the meristematic cell line obtainable by the process according to any one of claims 1 to 5 , the phytocomplex and/or extract obtainable by the process according to any one of claims 6 to 8 and/or the composition obtainable by the process according to any one of claims 9 to 12 are used as functional food.