EP3941188A1 - Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir d'echinacea purpurea - Google Patents

Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir d'echinacea purpurea

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Publication number
EP3941188A1
EP3941188A1 EP20711026.3A EP20711026A EP3941188A1 EP 3941188 A1 EP3941188 A1 EP 3941188A1 EP 20711026 A EP20711026 A EP 20711026A EP 3941188 A1 EP3941188 A1 EP 3941188A1
Authority
EP
European Patent Office
Prior art keywords
acid
cell line
concentration
phytocomplex
comprised
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20711026.3A
Other languages
German (de)
English (en)
Inventor
Elena Sgaravatti
Giovanna Pressi
Flavia GUZZO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aethera Biotech SRL
Original Assignee
Aethera Biotech SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aethera Biotech SRL filed Critical Aethera Biotech SRL
Publication of EP3941188A1 publication Critical patent/EP3941188A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • Plants of the genus Echinacea are widely used, above all because of their known immunostimulating activities.
  • Polyphenols in particular cichoric acid, chlorogenic acid, cynarine and caftaric acid are typical constituents of Echinacea purpurea. Said compounds are capable of inhibiting the production of free radicals and lipid peroxidation (Georgieva S. et al., 2014, J.Exp. Integr. Med.).
  • the content of cichoric acid and the other polyphenolic components of Echinacea purpurea is associated with a high variability tied to multiple factors, which are difficult to control: seasons, plant age, geographical growing areas and tissues used for the preparation of products.
  • the preparation of standardized plant derivatives i.e. with a reproducible content of metabolites, poses numerous problems: variability of the content of the metabolites in different plant tissues, seasonal variability, contaminations by plant parasites, differences tied to the geographical growing areas and loss of the biological activity of the molecule during harvest, storage and extraction.
  • variability of the content of phytoconstituents of plant preparations obtained directly from a plant, or parts thereof, by extraction negatively impacts the effectiveness of the same.
  • An alternative method for obtaining contaminant-free standardized plant phytocomplexes in industrial quantities is to use in vitro cell cultures.
  • This technology makes it possible to solve the problems tied to the variability of plant extracts, since it provides preparations with a content of active substances that can be reproduced in a standardized manner.
  • the present invention falls in the context of this technological platform and provides a selected meristematic cell line from which a phytocomplex with a standardized, reproducible content of active substances can be derived.
  • EP2319914 describes the preparation of meristematic cells from plants of the genus Echinacea, preferably Echinacea angustifolia, with a high content of caffeic acid.
  • the present invention provides a selected meristematic cell line from a plant belonging to the species Echinacea purpurea and derivatives thereof with a high content of polyphenols other than caffeic acid.
  • a first aspect of the present invention relates to a selected meristematic cell line derived from a plant belonging to the species Echinacea purpurea, the cell line being preferably derived from a callus tissue obtained from the plant itself.
  • the selected meristematic cell line and a derivative thereof are characterized by a high content of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof.
  • a third aspect of the invention relates to a composition
  • a composition comprising the selected meristematic cell line or a derivative thereof, in a mixture with excipients that are accepted from a cosmetic and/or pharmaceutical viewpoint.
  • the selected cell line or a derivative thereof is active in inducing the expression of the genes of collagen III and IV and possesses marked anti-inflammatory activity, mediated by inhibition of the expression of iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase2), IL-1 b (Interleukin 1 b) and TNF-a (tumour necrosis factor a), and antioxidant activity, mediated by inhibition of ROS (reactive oxygen species), release of MDA (malondialdehyde) and NO2 .
  • Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Echinacea purpurea with a high content of polyphenols other than caffeic acid.
  • Figure 1 shows a photo, taken with a bright-field optical microscope, of the cell line called Ep-2P, maintained in a solid medium.
  • Figure 2 shows a magnification (200X) of a portion of figure 1.
  • Figure 3 shows a magnification (200X) of a portion of figure 1 after staining with fluorescein diacetate.
  • Figure 4 shows a UV/VIS chromatogram, obtained by means of a diode array detector at 330 nm (A) on the selected clone Ep-2P and (B) meristematic cells before the selection process.
  • Figure 5 shows, for each component of the EchiPure-PC® phytocomplex, the identification of the main peaks obtained in the chromatographic profile in the positive and negative ionization modes.
  • Figure 6 shows a chromatogram, in three dimensions, of the EchiPure- PC® phytocomplex obtained with the negative ionization mode.
  • Figure 7 shows the expression of the genes of collagen III and IV in microtissues, 72 hours after treatment with the EchiPure-PC® phytocomplex. The data are expressed as the number of times more compared to the untreated control, the value of the untreated control being set as 1.
  • Figure 11 shows the results of ELISA tests conducted on supernatants of the cell lysates.
  • Figure 12 shows the effect of the EchiPure-PC® phytocomplex on the production of ROS in human keratinocytes.
  • “meristematic line” or“meristematic cell” means a plant line or cell capable of maintaining the ability to divide by mitosis so as to originate new cells. Every meristematic cell derives from another meristematic cell. The function of meristematic cells is comparable to that of the stem cells in animals.
  • callus tissue means a disorganized mass of undifferentiated or very scarcely specialized cells with thin cell walls and a large vacuole where secondary metabolites are accumulated.
  • “w/w” means a weight/weight amount relative to the dry mass of the cell line.
  • a first aspect of the present invention relates to a meristematic cell line derived from a plant belonging to the species Echinacea purpurea.
  • said meristematic cell line is obtained by means of a process comprising the steps of:
  • step 1) the tissue obtained from plants of the genus Echinacea purpurea is placed in a solid medium in order to obtain an undifferentiated callus tissue.
  • the tissue of Echinacea purpurea is preferably at least one shoot of Echinacea purpurea or a plurality of shoots of Echinacea purpurea.
  • the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.
  • the solid culture media further comprises agar, whereas the liquid culture media does not contain agar.
  • the solid and liquid culture media each comprise sucrose in a concentration comprised from 15 to 50 g/L preferably from 18 to 45 g/L, naphthylacetic acid (NAA) in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.5 to 2 mg/L, indoleacetic acid (IAA) in a concentration comprised from 0.1 to 2.5 mg/L, preferably from 0.3 to 1 mg/L, and kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.
  • the solid culture medium comprises: sucrose in a concentration of from 10 to 30 g/L, preferably from 15 to 25 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • NAA naphthaleneacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • the liquid culture medium comprises: sucrose in a concentration of from 25 to 50 g/L, preferably from 30 to 45 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1.5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • NAA naphthaleneacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgSC , NahtePC , (NH4)2SC>4 and combinations thereof.
  • the salts suitable for the growth of plant cells are preferably selected from: CoCl2-6H20, CuSC>4-5H20, NaEDTA-2H 0, FeS04-7H 0, H3BO3, Kl, MnS04- H 0, Na2Mo04-2H 0, ZnS04-7H20 and combinations thereof.
  • Both the solid and liquid culture media further comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.
  • the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgSC , NaH P04, (NH ) 2 SC>4, CoCI 2 -6H 2 0, CuS0 4 -5H 2 0, NaEDTA-2H 2 0, FeS04-7H 2 0 H3BO3, Kl, MnS0 4 - H 2 0, Na 2 MoC>4-2H 2 0, ZnS0 4 -7H 2 0 and combinations thereof.
  • This combination of compounds is the medium Gamborg B5.
  • both the solid and liquid culture media in addition to the salts specified above, further comprise vitamins suitable for the growth of plant cells selected from: myo-inositol, nicotinic acid, pyridoxine-HCI thiamine-HCI and combinations thereof.
  • the solid and liquid culture media preferably each comprise CaCI 2 in a concentration comprised from 120 to 170 mg/L, preferably from 130 to 160 mg/L; KNO3 in a concentration comprised from 800 to 3000 mg/L, preferably from 1000 to 2600 mg/L; MgSC>4 in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NaH 2 P04 in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L; and (NH4) 2 SC>4 in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L.
  • the solid and liquid culture media preferably each comprise CoCI 2 -6H 2 0 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuS04-5H 2 0 in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H 2 0 in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSC>4-7H 2 0 in a concentration comprised from 15 to 45 mg/L, preferably from 20 to 35 mg/L; H3BO3 in a concentration comprised from 1 to 7 mg/L, preferably from 2 to 5 mg/I; Kl in a concentration comprised from 0.1 to 2 mg/L, preferably from 0.4 to 1 mg/L; MnSC>4- H 2 0 in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na 2 Mo04-2H 2 0 in a concentration comprise
  • Both the solid and liquid culture media preferably each comprise myo inositol in a concentration comprised from 70 to 130 mg, preferably from 90 to 110 mg; pyridoxine-HCI from 70 to 130 mg, preferably from 90 to 110 mg; and thiamine-HCI from 5 to 20 mg/L, preferably from 7 to 15 mg/L.
  • the callus tissue is preferably divided into a plurality of portions that are stabilized through successive transfers into the solid culture medium (step 1a), so as to obtain stabilized cells.
  • This step takes the name of stabilization step.
  • the portions of stabilized cells preferably undergo a first "clonal selection".
  • the clonal selection consists in culturing the stabilized cells for an adequate duration, preferably comprised from 5 to 20 days of culture, more preferably 10 to 15 days (step 1 b).
  • the cells are incubated in the dark at a temperature comprised from 15°C to 35°C, preferably from 24°C to 26°C.
  • step 3 the cellular clones are each inoculated into the liquid culture medium described above.
  • step 4 after a phase of growth for a time such as to obtain an appropriate multiplication of the cellular clone, preferably 10 to 15 days, in step 4) the polyphenol content of each clone is determined.
  • a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Echinacea purpurea comprising an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof, greater than 0.5% w/w, preferably comprised from 0.5% to 25% w/w.
  • the meristematic cell line selected with the process described above more preferably comprises an amount of polyphenols, as indicated above, comprised from 3 to 15% w/w, even more preferably from 3.5 to 10% w/w.
  • Said mixture of polyphenols preferably comprises cichoric acid, preferably cichoric acid and cynarine, even more preferably cichoric acid, cynarine and chlorogenic acid, more preferably, cichoric acid, cynarine, chlorogenic acid, caftaric acid and fertaric acid.
  • said polyphenols comprise at least 40%, at least 35% or at least 30% w/w of cichoric acid, preferably from 38% to 50% w/w.
  • Said selected cell line preferably comprises an amount of polyphenols comprised from 3.5 to 10% w/w and the polyphenols comprise at least 40%, or at least 35% or at least 30% w/w of cichoric acid, preferably from 38% to 50% w/w.
  • the selected meristematic cell line according to the invention also further comprises an amount of polysaccharides of from 30 to 70% w/w, preferably from 40% to 65% w/w.
  • the selected meristematic cell line according to the invention further comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w.
  • the selected meristematic cell line according to the invention further comprises an amount of lipids of from 3 to 30% w/w, preferably from 5 to 10% w/w.
  • a second aspect of the present invention relates to a derivative of the cell line which is a phytocomplex or an extract of the selected meristematic cell line.
  • Phytocomplex means: dried or lyophilized cells, a cellular homogenate, or the cell walls and the components thereof.
  • the phytocomplex is preferably a cellular homogenate.
  • Said phytocomplex comprises an amount of polyphenols selected in the group consisting of cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof, greater than 0.5% w/w, preferably comprised from 0.5% to 25% w/w relative to the dry mass of the phytocomplex. More preferably, the phytocomplex comprises an amount of polyphenols, as indicated above, of from 3 to 15% w/w, even more preferably from 3.5 to 10% w/w relative to the dry mass of the phytocomplex.
  • Said mixture of polyphenols preferably comprises cichoric acid, preferably cichoric acid and cynarine, even more preferably cichoric acid, cynarine and chlorogenic acid, more preferably, cichoric acid, cynarine, chlorogenic acid, caftaric acid and fertaric acid.
  • said polyphenols comprise at least 40%, or at least 35% or at least 30% w/w of cichoric acid, preferably from 32 to 40% w/w relative to the dry mass of the phytocomplex.
  • Said phytocomplex preferably comprises an amount of polyphenols comprised from 3.5 to 10% w/w relative to the dry mass of the phytocomplex and said polyphenols consist of 38-50% w/w cichoric acid.
  • the phytocomplex also further comprises an amount of polysaccharides of from 30 to 70% w/w, preferably from 40% to 65% w/w relative to the dry mass of phytocomplex.
  • the phytocomplex also further comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w relative to the dry mass of phytocomplex.
  • the phytocomplex also further comprises an amount of lipids of from 3 to 30% w/w, preferably from 5 to 10% w/w relative to the dry mass of phytocomplex.
  • said phytocomplex is derived from said selected meristematic cell line Ep-2P and comprises 0.5-20% of polyphenols, of which 35-40% is cichoric acid.
  • the phytocomplex is preferably a cellular homogenate of the selected meristematic cell line Ep-2P.
  • Extract means an extract in an alcoholic solvent, for example in methanol or ethanol, or a water/ethanol mixture in different proportions: 50:50 or 60:40 or 70:30, of the cell line itself or a phytocomplex of the cell line.
  • the extract is preferably an extract of a cellular homogenate of the line.
  • the content of said extract corresponds to the content of the phytocomplex or cell line from which it was derived, with the variability due to the extraction technique.
  • a third aspect of the present invention relates to a composition
  • a composition comprising the meristematic cell line and/or a derivative thereof (phytocomplex and/or extract) in association with at least one excipient that is accepted from a cosmetic, nutraceutical and/or pharmaceutical viewpoint.
  • the composition comprises the cell line and/or a derivative thereof in a concentration comprised from 0.01 % to 30% w/w, preferably from 0.03% to 15 % w/w, more preferably from 0.05% to 10% w/w.
  • Said composition preferably comprises a phytocomplex which is a cellular homogenate.
  • the cell line and/or a derivative thereof is dispersed before being mixed with the excipients to prepare the composition of the invention.
  • suitable dispersing agents are glycerine, propylene glycol or butylene glycol.
  • composition of the present invention comprises 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 nontoxic.
  • Said excipient can be at least one conditioning, humectant, or occlusive agent, a surfactant, a stabilizing agent, a preservative or an emollient for the skin.
  • composition of the invention is formulated for topical use as a cream, gel-cream, gel, serum, oil, emulsion, emulsion-gel (emulgel), ointment, eye drops, mouthwash, spray, preferably nasal spray or stick (such as lip balm).
  • composition can also be formulated for oral administration, preferably as a pill, capsule, tablet, granular powder, hard-shelled capsule, orally dissolving granule, sachet or lozenge.
  • the composition is formulated to release the active ingredients contained therein rapidly, or in a delayed and/or controlled manner after administration, preferably formulated as a liposome.
  • the experimental data included herein indicate that the cell line or a derivative thereof as described above is capable of stimulating the synthesis of collagen III and IV, with a consequent improvement in skin elasticity and compactness. Furthermore, said cell line or a derivative thereof has been demonstrated to possess an anti-inflammatory activity tied to a decrease in the synthesis of the pro-inflammatory enzymes iNOS and COX-2 and the cytokines TNF-a and IL-1 b stimulated with LPS, as well as an antioxidant activity tied to a decrease in the production of ROS after oxidative stress.
  • a further aspect of the present invention relates to the cosmetic use of the cell line or a derivative thereof.
  • Cosmetic use means the prevention, attenuation and/or combatting of the signs of skin aging, the loss of skin elasticity and compactness and reddening, skin hydration, anti-wrinkle activity and antioxidant activity
  • Another aspect of the present invention relates to the use of the cell line or a derivative thereof for personal care and hygiene; in this case the line or a derivative thereof is formulated as a bath foam, shower gel, soap, shampoo or hair conditioner, together with suitable excipients.
  • a further aspect of the present 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 inflammations and infections affecting the upper respiratory tract.
  • Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of a plant belonging to the genus Echinacea purpurea, which comprises the steps of:
  • the preparation of meristematic entails collecting tissue, preferably of shoots from plants of Echinacea purpurea, washing it, for example with water, fragmenting it into small pieces and sterilizing it on plates, for example with successive treatments with ethanol, sodium hypochlorite and a mercury salt.
  • step 1 the collected tissue is placed in a solid culture medium in order to obtain an undifferentiated callus tissue.
  • the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin.
  • the solid culture media further comprises agar, whereas the liquid culture media does not contain agar.
  • the solid and liquid culture media preferably each comprise sucrose in a concentration comprised from 15 to 50 g/L more preferably from 18 to 45 g/L, naphthylacetic acid (NAA) in a concentration comprised from 0.1 to 2.5 mg/L, more preferably from 0.5 to 2 mg/L, indoleacetic acid (IAA) in a concentration comprised from 0.1 to 2.5 mg/L, more preferably from 0.3 to 1 mg/L, and kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.
  • sucrose in a concentration comprised from 15 to 50 g/L more preferably from 18 to 45 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration comprised from 0.1 mg/L to 2 mg/L preferably from 0.2 mg/L to 1 mg/L.
  • the solid culture medium comprises: sucrose in a concentration of from 10 to 30 g/L, preferably from 15 to 25 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • NAA naphthaleneacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • the liquid culture medium comprises: sucrose in a concentration of from 25 to 50 g/L, preferably from 30 to 45 g/L, naphthaleneacetic acid (NAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, indoleacetic acid (IAA) in a concentration of from 0.1 to 2 mg/L, preferably from 0.5 to 1 .5 mg/L, and kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • NAA naphthaleneacetic acid
  • IAA indoleacetic acid
  • kinetin in a concentration of from 0.2 mg/L to 1 mg/L, preferably from 0.3 mg/L to 0.8 mg/L.
  • the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgSC , NahtePC , (NH4)2SC>4 and combinations thereof.
  • the salts suitable for the growth of plant cells are preferably selected from: CoCl2-6H20, CuSC>4-5H20, NaEDTA-2H 0, FeS04-7H 0, H3BO3, Kl, MnS04- H 0, Na2Mo04-2H 0, and ZnS04-7H20 and combinations thereof.
  • Both the solid and liquid culture media further comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.
  • the salts suitable for the growth of plant cells are selected from: CaCl2, KNO3, MgS04, NaH P04, (NH ) 2 S04, CoCl2-6H 0, CuS04-5H 0, NaEDTA-2H 0, FeS04-7H 0 H3BO3, Kl, MnS0 4 - H 0, Na 2 Mo0 4 -2H 0, ZnS0 4 .
  • This combination of salts is the medium Gamborg B5.
  • both the solid and liquid culture media in addition to the salts specified above, further comprise vitamins suitable for the growth of plant cells selected from: myo-inositol, nicotinic acid, pyridoxine-HCI thiamine-HCI and combinations thereof.
  • the solid and liquid culture media preferably each comprise CaCI 2 in a concentration comprised from 120 to 170 mg/L, more preferably from 130 to 160 mg/L; KNO3 in a concentration comprised from 800 to 3000 mg/L, more preferably from 1000 to 2600 mg/L; MgSC>4 in a concentration comprised from 220 to 270 mg/L, more preferably from 230 to 260 mg/L, NaH 2 PC>4 in a concentration comprised from 100 to 180 mg/L, more preferably from 110 to 150 mg/L; and (NH4) 2 SC>4 in a concentration comprised from 100 to 180 mg/L, more preferably from 110 to 150 mg/L.
  • the solid and liquid culture media preferably each comprise CoCI 2 -6H 2 0 in a concentration comprised from 0.01 to 0.05 mg/L, more preferably from 0.015 to 0.03 mg/L; CuSC>4-5H 2 0 in a concentration comprised from 0.01 to 0.05 mg/L, more preferably from 0.015 to 0.03 mg/L; NaEDTA-2H 2 0 in a concentration comprised from 20 to 60 mg/L, more preferably from 30 to 45 mg/L; FeSC>4-7H 2 0 in a concentration comprised from 15 to 45 mg/L, more preferably from 20 to 35 mg/L; H3BO3 in a concentration comprised from 1 to 7 mg/L, more preferably from 2 to 5 mg/I; Kl in a concentration comprised from 0.1 to 2 mg/L, more preferably from 0.4 to 1 mg/L; MnS04- H 2 0 in a concentration comprised from 5 to 20 mg/L, more preferably from 7 to 15 mg/L; Na 2 MoC>4-2H
  • Both the solid and liquid culture media preferably each comprise myo inositol in a concentration comprised from 70 to 130 mg, more preferably from 90 to 1 10 mg; pyridoxine-HCI from 70 to 130 mg, more preferably from 90 to 1 10 mg; and thiamine-HCI from 5 to 20 mg/L, preferably from 7 to 15 mg/L.
  • the callus tissue is preferably divided into a plurality of portions that are stabilized through successive transfers into the solid culture medium (step 1a), so as to obtain stabilized cells.
  • This step takes the name of stabilization step.
  • the stabilized cells preferably undergo a first "clonal selection".
  • the clonal selection consists in culturing the stabilized cells for an adequate duration, preferably comprised from 5 to 20 days of culture, more preferably 10 to 15 days (step 1 b).
  • the cells are incubated in the dark at a temperature comprised from 15°C to 35°C, preferably from 24°C to 26°C.
  • step 2) a plurality of cellular clones is isolated by taking aggregates of stabilized cells from the solid culture medium.
  • step 3 the cellular clones are each inoculated into the liquid culture medium described above.
  • step 4 after a phase of growth for a time such as to obtain an appropriate multiplication of the cellular clone, preferably 10 to 15 days, in step 4) the polyphenol content of each clone is determined.
  • step 5 of selection of the cellular clone, a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Echinacea purpurea wherein the production of polyphenols selected in the group comprising cichoric acid, chlorogenic acid, cynarine, caftaric acid, fertaric acid and mixtures thereof is optimal.
  • the selected cell line is then multiplied, in a flask or bioreactor or fermenter, so as to obtain an increase in the biomass.
  • the multiplication of the biomass takes place in a first step in a liquid growth medium called EP.
  • the liquid growth medium EP is a medium containing the Gamborg salts specified above, the vitamins listed above, sucrose, NAA and IAA.
  • the liquid growth medium EP contains, among the Gamborg salts, KNO3 in an amount comprised from 1.5 g/L to 3.5 g/L, preferably from 2 g/L to 3 g/L.
  • Sucrose is preferably comprised from 15 g/L to 25 g/L.
  • NAA is preferably comprised from 0.5 mg/L to 1.5 mg/L.
  • IAA is preferably comprised from 0.2 mg/L and 1 mg/L.
  • Kinetin is preferably comprised from 0.2 mg/L to 1 mg/L.
  • the cells grown in the liquid medium EP are transferred, for the final phase of growth, into a final medium EP-F containing the Gamborg salts, vitamins, sucrose, NAA and IAA, which induces an increase in the polyphenol content and biomass.
  • the final medium EP-F contains, among the Gamborg salts, KNO3 in an amount comprised from 0.5 g/L to 2 g/L.
  • Sucrose is preferably comprised from 30 g/L to 50 g/L.
  • NAA is preferably comprised from 0.2 mg/L to 1 mg/L.
  • IAA is preferably comprised from 0.2 mg/L to 1 mg/L;
  • kinetin is preferably comprised from 0.2 mg/L to 1 mg/L.
  • the growth of the cell line in the flask, bioreactor or fermenter, both in the EP growth medium and in the final EP-F medium is carried out at a temperature comprised from 15 °C to 35 °C, typically about 25 °C, for a period comprised from 7 to 30 days, preferably from 14 to 21 days, under conditions of darkness.
  • the cell line is filtered and the cells are recovered in order to be used in the subsequent steps in the form of a phytocomplex, or else they may undergo a subsequent extraction phase in an alcohol solvent in order to produce a cell extract characterized by a high polyphenol content.
  • the phytocomplex can be obtained by lyophilization or drying of live cells; in this case, the phytocomplex is a lyophilizate of dead cells.
  • the cells are homogenized, for example by mechanical disintegration, preferably in an acidified solution (for example with ascorbic acid and/or citric acid and/or acetic acid) and subsequently lyophilized or dried.
  • the phytocomplex is a cellular homogenate wherein the cells and the internal structures thereof are disintegrated.
  • the phytocomplex preferably in the form of a cellular homogenate, undergoes extraction in an alcohol solvent (for example methanol and/or ethanol and/or water) using conventional techniques.
  • an alcohol solvent for example methanol and/or ethanol and/or water
  • the extract thus obtained is characterized by a high polyphenol content as detailed above and can be used for the preparation of cosmetic, nutraceutical or pharmaceutical compositions as described above.
  • live cells as such following purification, can be directly employed for the preparation of the compositions of the invention.
  • a surprising effectiveness of a derivative of said selected cell line was likewise demonstrated, wherein said derivative is a homogenate, or a phytocomplex that comprises all the cellular components, without the cells being exposed to solvents or other extraction procedures that could compromise the integrity of the active ingredients.
  • the induction of callus tissue was obtained using standard procedures described in the literature. This procedure provides for the collection of young tissues (shoots) from plants of Echinacea purpurea, the cleaning thereof, for example with running water, minute fragmentation into 2-5 cm pieces and sterilization on plates by means, for example, of treatment in sequence with 70% ethanol in water for about 15 seconds, 2% sodium hypochlorite and 0.1 % Tween 20 for about 5 minutes and, finally, at least 4 washes with sterile water. Every fragment of plant tissue, broken down further (explants), is placed in Petri dishes containing a nutrient medium rendered solid by adding agar and supplemented with growth hormones, without antibiotics. After a suitable period of incubation in the dark at 25°C, the undifferentiated callus tissue forms; it is then multiplied after transfer onto a larger surface with fresh medium.
  • the line of meristematic cells deriving from undifferentiated callus tissue is stabilized by means of a certain number of transfers (sub-cultures) onto solid culture media.
  • the medium is of the solid Gamborg B5 type (with 2.5 g/L of KNO3) with the addition of 20 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA, 0.5 mg/L of kinetin and 0.8-1 % plant agar (EP medium), final pH 6.5.
  • Ep-2P The cell line obtained in this specific culture medium, after clonal selection, was called Ep-2P.
  • the belonging of the meristematic cells obtained to the botanical species Echinacea purpurea was confirmed by DNA fingerprint analysis.
  • the cell line Ep-2P is multiplied to obtain sufficient amounts of biomass to transfer the cells into the liquid culture medium (EP medium without agar). After growth in the liquid medium, the cell suspensions can be transferred into bioreactors which can contain the final productive medium or the EP growth medium for further phases of growth.
  • the productive liquid medium (optimized to increase the polyphenol content) is a Gamborg B5 (with 1 g/L of KNO3) with the addition of 40 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA and 0.5 mg/L of kinetin, final pH 6.5 (EP-F medium).
  • the characteristics of the cell line of Echinacea purpurea Ep-2P are described by way of example.
  • Ep-2P The selected cell line of Echinacea purpurea, called Ep-2P, is maintained in solid EP culture medium, is beige-coloured with whitish tinges and has a friable texture ( Figure 1 -3).
  • the procedure for homogenizing the biomasses of cells selected and grown in bioreactors for 14 days at 25°C ( ⁇ 2) comprises the following steps:
  • Examples of preparation of the standardized EchiPure-PC®A phytocomplex in EP-F medium are provided by way of non-limiting example.
  • the phytocomplex thus obtained is used as such or dispersed in a suitable dispersion medium in concentrations ranging from 0.5 to 5% w/w.
  • the suspensions thus obtained are called EchiPure CROP®-G.
  • Meristematic cells stabilized and selected as previously described, deriving from the line of Echinacea purpurea and called Ep-2P, cultured in solid EP medium (Gamborg B5 with the addition of 20 g/L of sucrose, 1 mg/L of NAA, 0.5 mg/L of IAA, 0.5 mg/L of kinetin and 0.8% plant agar) were inoculated into 5 flasks with a 1 -litre capacity, containing 200 ml of liquid EP-F medium (Gamborg B5 with the addition of 40 g/L of sucrose, 1 g/L of KNO3 rather than 2.5 g/L ,1 mg/L of NAA, 0.5 mg/L of IAA and 0.5 mg/L of kinetin, final pH 6.5).
  • the amount of meristematic cells inoculated into the liquid medium was equal to 6% W/V.
  • the suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM. After 7 days of incubation, 5 mg/L of methyl jasmonate were added to the cell suspensions (95%, Sigma- Aldrich, CAS Number: 39924-52-2). After another 7 days of incubation, the plant biomass (1 litre of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 pm and washed with 700 ml of sterile saline solution (0.9% W/V). The washed cells (fresh weight 350 g) were supplemented with 3.5 g of citric acid and homogenized with an Ultra- Turrax.
  • the homogenized cells were lyophilized. 25 g of lyophilizate (EchiPure- PC® phytocomplex) with a content of polyphenols equal to 1.05g, 13 g of polysaccharides, 3.75 g of proteins, 2.2 g of lipids, 0.75 g of ash and 3.5 g of citric acid were obtained from 1 litre of cell suspension.
  • lyophilizate EchiPure- PC® phytocomplex
  • Table 1 shows the characterization of the phytocomplex: Table 1 :
  • the diluted sample was filtered over 0.22 pm filters before being loaded into the UPLC system.
  • the chromatography system used for quantification of polyphenols consists in an Acquity UPLC BEH C18 1.7 pm column, size 2.1 x 100 mm, coupled to an Acquity UPLC BEH C18 1.7 pm VanGuard Pre-Column 3/Pk, size 2.1 x 5 mm.
  • the platform used for the UPLC-DAD analysis comprises a UPLC system (Waters) consisting of an eluent management module, Binary Solvent Manager model I Class, and an auto-sampler, Sample Manager - FTN model I Class, coupled to a PDA eh diode array detector.
  • Empower 3 (Waters) software was used to acquire and analyse the data.
  • the chromatography method used was the following: solvent A: water, 0.1 % formic acid; solvent B: 100% acetonitrile.
  • the initial condition is 99% solvent A; moreover, the flow remains constant at 0.350 ml/min throughout the duration of the analysis.
  • the chromatography column was temperature controlled at 30° C. Elution of the molecules was conducted by alternating gradient and isocratic phases, as indicated in table 2:
  • the chromatogram associated with the wavelength of 330 nm was used.
  • the polyphenols were quantified as cichoric acid equivalents, cichoric acid being the most represented molecule in the group.
  • the polyphenols were quantified thanks to the calibration curve of the authentic commercial standard of cichoric acid (CAS 6537-80-0; purity>95%; Sigma Aldrich).
  • the data analysis was carried out with Empower 3 software.
  • the chromatographic profile of the EchiPure-PC® phytocomplex is shown in figure 4. From the UPLC analysis it emerges that the EchiPure-PC® phytocomplex comprises a % of polyphenols, expressed as cichoric acid equivalents, equal to 4.2.
  • the percentage of cichoric acid in the phytocomplex was equal to 1.6.
  • the powder of the EchiPure-PC® phytocomplex was extracted with 20 volumes of methanoLwater 90:10 for 15 minutes in a sonicator at 40Hz under ice, after being stirred in a vortex-type mixer for 30 seconds; the extract was recovered after centrifugation at 18000g for 10 minutes at 4°C.
  • the sample Prior to the analysis, the sample was diluted with suitable amounts of the same solvent (methanoLwater 90:10) so as to obtain a spectrophotometric absorbance of between 0.4 and 0.6 at 320 nm. The sample was further diluted with water and analysed by HPLC-ESI-MS.
  • ESI nebulizer gas N2, pressure 50 psi, temp. 350°C, drying gas 10 l/min Mass acquisition: Full Scan alternated in the range 50-1500 m/z; vacuum pressure: 1.4x10-5 mbar.
  • the chromatographic separation method used was the following:
  • Solution A 5% ACN and 0.5% formic acid in water
  • Solution B 100%
  • Figure 5 shows the chromatogram of the EchiPure-PC® phytocomplex from meristematic cells of Echinacea purpurea.
  • the profile shows a dominant peak at 25 minutes of retention time. This peak has an m/z value and a fragmentation profile (MS/MS) equal to those of cichoric acid (commercial standard, Sigma Aldrich).
  • Figure 6 shows the chromatogram, in three dimensions, of the analysis conducted on the EchiPure-PC® phytocomplex with the negative ionization mode.
  • the analysis was conducted by adapting the phenol-sulphuric method (Segarra et al., 1995, Am J Enol Vitic.). This method entails an acid hydrolysis of the polysaccharides, which release monosaccharides. The monosaccharides react with the phenol, producing a yellow colour that can be measured with a spectrophotometer at 490 nm. The results obtained indicate an amount of polysaccharides equal to 520 mg/g of EchiPure-PC® phytocomplex, equivalent to 52%.
  • the protein content in the EchiPure-PC® phytocomplex was equal to 15% W/W.
  • the lipid content in the EchiPure-PC® phytocomplex was equal to 8.8% W/W.
  • a determination of moisture was carried out on the phytocomplex by leaving the material in a stove at 40°C for 12 hours.
  • the determination of ash was obtained by treating the material in a muffle furnace at 300°C until arriving at a constant weight.
  • the moisture of the phytocomplex was equal to 3%, and the ash was equal to 3%.
  • EchiPure-PC® phvtocomplex Preparation of the EchiPure-PC® phvtocomplex on an industrial scale
  • Meristematic cells, stabilized and selected as previously described, deriving from the line of Echinacea purpurea called Ep-2P, cultured in solid EP medium were inoculated into 10 flasks with a 1 -litre capacity, containing 200 ml of liquid EP medium.
  • the amount of meristematic cells inoculated into the liquid medium was equal to 6% W/V.
  • the suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM.
  • the cell suspensions were used to inoculate 10 flasks with a 3-litre capacity, containing 800 ml of liquid EP medium. 200 ml of the cell suspension was transferred into 800 ml of EP medium contained in a flask with a 3-litre capacity. The suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 120 RPM. After 7 days of incubation the cell suspensions were used to inoculate a bioreactor containing 80 litres of EP-F medium. After 7 days of growth in the bioreactor in the dark at 25°C, 5mg/L of methyl jasmonate was added to the cell suspension.
  • the plant biomass (90 litres of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 pm and washed with 56 L of sterile saline solution (0.9% W/V).
  • the washed cells (fresh weight 28 Kg) were supplemented with 280 g of citric acid and homogenized with an Ultra-Turrax.
  • the homogenized cells were dried. 2950g of EchiPure-PC® phytocomplex were obtained from 90 litres of cell suspension.
  • the EchiPure-PC® phytocomplex at 0.03% and 0.1 % was left in contact with the microtissues for 72 hours.
  • the dermal microtissues were prepared from primary dermal fibroblasts taken from donors aged 60 years. At the end of a 72h incubation in specific culture media filtered over 0.22pm filters (Fibroblast Culture Medium), the microtissues were collected in order to perform PCR and histological analyses.
  • the PCR analyses were performed by extracting RNA using a kit. The extracted RNA was used to synthesize cDNA and the expressions of the biomarker genes were studied by means of the PCR technique. The variations in gene expression were evaluated by comparing the sample treated with the extract against the untreated one.
  • Figure 7 shows the results relating to the gene expression of collagen III and collagen IV.
  • the levels of the pro-inflammatory cytokines TNF-a and IL-1 b were evaluated by means of an ELISA analysis. After stimulation with LPS, an increase in pro-inflammatory cytokines is observed. The treatment with EchiPure-PC® at a concentration of 0.5 mg/mL significantly decreases the level of these cytokines, as evidenced by the graphs in figure 11.
  • ROS radical oxygen species
  • H2DCF-DA dichlorodihydrofluorescein diacetate
  • the experimental model used to study the antioxidant activity of the EchiPure-PC® phytocomplex was the cell line HaCaT, a line of human keratinocytes.
  • Figure 12 shows the data relating to the 3-hour treatment with EchiPure- PC® at a concentration of 0.1 % w/v and with N-acetyl cysteine (NAC) as the positive control, given its well-known antioxidant action.
  • the experiment was conducted under both basal conditions and conditions of oxidative stress induced by 200mM of H2O2. The data reveal that, under both conditions, the EchiPure-PC® phytocomplex significantly reduced radical oxygen species.
  • EchiPure- PC® An evaluation of the antioxidant activity of EchiPure- PC® was carried out using a J774-A1 macrophage cell model after stress with LPS. Part of the experiment was carried out on macrophages treated with 1 pg/mL LPS and another part was performed using macrophages treated with LPS and EchiPure- PC® 0.5mg/mL.
  • MDA malondialdehyde
  • MDA has the ability to bind chemically to the thiobarbituric acid (TBA) added to the reaction environment.
  • TBA thiobarbituric acid
  • the oxidized substrates form a chromogenic adduct TBA-MDA-TBA which can be determined by spectrophotometry.
  • the treatment with EchiPure- PC® significantly prevents lipid peroxidation caused by oxidative stress.
  • the results obtained indicate that EchiPure-PC® exerts an antioxidant activity by reducing the release of nitrites and inhibiting lipid peroxidation.
  • EchiPure-PC® The homogenate of the line Ep-2P, called EchiPure-PC®, was dispersed in glycerine in a concentration equal to 3% w/w (INCI NAME: Glycerin, Echinacea purpurea Callus Lysate, Citric Acid). The dispersion was added at 3% to the formulas described below.
  • the components of the aqueous phase are mixed and heated to 70°C.
  • the components of the oily phase are mixed and heated to 75°C.
  • water and surfactants are mixed under gentle stirring. Fleat to 70°C and add the oily phase, if present, at 75°C. Add the EchiPure-PC® phytocomplex dispersed in glycerine under gentle stirring below 40°C.

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Abstract

La présente invention concerne une lignée cellulaire méristématique sélectionnée à partir d'un tissu, de préférence un tissu de calle, d'une plante d'Echinacea purpurea. L'invention concerne également un dérivé de la lignée cellulaire, c'est-à-dire un phytocomplexe ou un extrait de la lignée cellulaire. La lignée cellulaire méristématique est caractérisée par une teneur élevée en polyphénols, en particulier une teneur élevée en acide cichorique. En outre, la présente invention concerne l'utilisation cosmétique, nutraceutique et médicale de la lignée cellulaire méristématique sélectionnée ou d'un dérivé de celle-ci.
EP20711026.3A 2019-03-21 2020-03-20 Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir d'echinacea purpurea Pending EP3941188A1 (fr)

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IT102019000004119A IT201900004119A1 (it) 2019-03-21 2019-03-21 Fitocomplesso ed estratto di linea cellulare meristematica selezionata da Echinacea purpurea
PCT/IB2020/052591 WO2020188535A1 (fr) 2019-03-21 2020-03-20 Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir d'echinacea purpurea

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