WO2022112864A1 - Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir punica granatum - Google Patents

Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir punica granatum Download PDF

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Publication number
WO2022112864A1
WO2022112864A1 PCT/IB2021/057648 IB2021057648W WO2022112864A1 WO 2022112864 A1 WO2022112864 A1 WO 2022112864A1 IB 2021057648 W IB2021057648 W IB 2021057648W WO 2022112864 A1 WO2022112864 A1 WO 2022112864A1
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cell line
phytocomplex
concentration comprised
comprised
concentration
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PCT/IB2021/057648
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English (en)
Inventor
Giovanna Pressi
Oriana BERTAIOLA
Flavia GUZZO
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Demethra Biotech S.R.L.
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Publication of WO2022112864A1 publication Critical patent/WO2022112864A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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

Definitions

  • the present invention relates to a meristematic cell line selected from a plant belonging to the species Punica granatum, characterized by a high ellagitannin content and the cosmetic, nutraceutical and medical use of said cell line or of a derivative thereof.
  • Punica granatum is a plant belonging to the family Lythraceae.
  • the pomegranate shrub is used as an ornamental plant in gardens; it is grown industrially for the production of the edible fruits, pomegranates.
  • Pomegranate seed oil contains phytoestrogen compounds and the fruit is rich in phenolic compounds with high antioxidant activity.
  • the fruit and bark of the pomegranate shrub are used against intestinal parasites, dysentery and diarrhoea. The juice and seeds are considered a tonic for the heart and throat.
  • One method for producing 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 it is possible to derive a phytocomplex (and also an extract) with a standardized, reproducible content of active substances.
  • the standardized phytocomplex obtained from a selected meristematic cell line of Punica granatum was demonstrated to have a high antioxidant activity and an activity of increasing the wellbeing of hair follicles on the scalp.
  • a first aspect of the present invention relates to a meristematic cell line selected from a plant belonging to the species Punica granatum, the cell line being preferably derived from a callus tissue obtained from the plant itself.
  • a second aspect of the present invention relates to a derivative of the selected meristematic cell line, i.e. a phytocomplex or an extract of the cell line.
  • the selected meristematic cell line and a derivative thereof are characterized by a high content of ellagitannins, in particular punicalagin, pedunculagin, catechin and gallic acid hexose.
  • a third aspect of the invention relates to a composition
  • a composition comprising the meristematic cell line and/or a derivative thereof, in a mixture with excipients that are accepted from a cosmetic and/or pharmaceutical viewpoint.
  • Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Punica granatum with a high content of ellagitannins, in particular punicalagin, pedunculagin, catechin and gallic acid hexose.
  • the Applicant has demonstrated that the selected cell line or a derivative thereof exhibits a marked antioxidant activity and an activity of increasing the wellbeing of hair follicles on the scalp.
  • Figure 1 shows the selected cell line PGr64 on a specific solid medium, PGr.
  • Figure 2 shows the UPLC-DAD-MS chromatographic profile of the PGr64 phytocomplex in the negative ionization mode.
  • Figure 3 shows the results of viability, measured with an MTT assay, of primary cultures of dermal papilla fibroblasts following treatment with the Punica granatum phytocomplex; **highly significant versus untreated;
  • Figure 4 shows the results of cell proliferation (KI-67) of primary cultures of dermal papilla fibroblasts following treatment with the Punica granatum phytocomplex; * significant versus untreated.
  • 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 plant meristematic cells is comparable to that of 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 selected species Punica granatum comprising an amount of ellagitannins (in particular punicalagin, pedunculagin, catechin and gallic acid hexose) that is greater than 2% w/w and preferably comprised between 4% and 50% w/w.
  • Punica granatum comprising an amount of ellagitannins (in particular punicalagin, pedunculagin, catechin and gallic acid hexose) that is greater than 2% w/w and preferably comprised between 4% and 50% w/w.
  • said meristematic cell line is obtained by means of a process comprising the steps of:
  • step 1) the tissue obtained from plants of the species Punica granatum is placed in a solid medium in order to obtain an undifferentiated callus tissue.
  • the tissue of Punica granatum is at least one young leaf of Punica granatum or a plurality of young leaves of Punica granatum.
  • the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), indoleacetic acid (IAA) and kinetin (K).
  • the solid culture media further comprises agar, whereas the liquid culture medium does not contain agar.
  • the solid and liquid culture media preferably each comprise sucrose in a concentration comprised between 15 and 50 g/L, more preferably between 20 and 45 g/L; naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 0.8 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.08 and 0.5 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 3 mg/L, preferably between 0.3 and 2 mg/L.
  • sucrose in a concentration comprised between 15 and 50 g/L, more preferably between 20 and 45 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the solid culture medium comprises: sucrose in a concentration of 15 to 35 g/L, preferably between 20 and 30 g/L, naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 1 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.1 and 1 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.3 and 1.5 mg/L.
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the liquid culture medium comprises: sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L, naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 0.8 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.08 and 0.5 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.3 and 1 g/L.
  • sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgSCL, NahhPCL, (NhU ⁇ SCL and combinations thereof.
  • the salts suitable for the growth of plant cells are selected from: CoCh-ehhO, CuSCL-ShhO, NaEDTA-2H 2 0, FeSC> 4 -7H 2 0, H3BO3, Kl, MnSCL- ⁇ O, Na2MoC>4-2H20, ZnSC> 4 -7H 2 0 and combinations thereof.
  • the solid and liquid culture media both 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: CaCL, KNO3, MgSCL, NahhPCL, (NhU ⁇ SCL, COCI 2 -6H 2 0, CUS0 4 -5H 2 0, NaEDTA-2H 2 0, FeS0 4 -7H 2 0 H3BO3, Kl, MnS0 4 -H 2 0,
  • 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: myoinositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.
  • the solid and liquid culture media preferably each comprise CaCL in a concentration comprised between 120 and 170 mg/L, preferably between 130 and 160 mg/L; KNO 3 in a concentration comprised between 800 and 3000 mg/L, preferably between 1000 and 2600 mg/L; MgSCL in a concentration comprised between 220 and 270 mg/L, preferably between 230 and 260 mg/L; NaH2PC>4 in a concentration comprised between 100 and 180 mg/L, preferably between 110 and 150 mg/L; and (NH4)2SC>4 in a concentration comprised between 100 and 180 mg/L, preferably between 110 and 150 mg/L.
  • the solid and liquid culture media preferably each comprise CoCl 2 -6H 2 0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; CUSC> 4 -5H 2 0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; NaEDTA-2H 2 0 in a concentration comprised between 20 and 60 mg/L, preferably between 30 and 45 mg/L; FeSC> 4 -7H 2 0 in a concentration comprised between 15 and 45 mg/L, preferably between 20 and 35 mg/L; H 3 BO 3 in a concentration comprised between 1 and 7 mg/L, preferably between 2 and 5 mg/I; Kl in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.4 and 1 mg/L; MnSCL- ⁇ O in a concentration comprised between 5 and 20 mg/L, preferably between 7 and 15 mg/L; Na 2 Mo0 4 -2H 2 0 in
  • the solid and liquid culture media preferably each comprise myo-inositol in a concentration comprised between 70 and 130 mg, preferably between 90 and 110 mg; pyridoxine-HCI between 70 and 130 mg, preferably between 90 and 110 mg; and thiamine-HCI between 5 and 20 mg/L, preferably between 7 and 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 (PGr solid 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 in the solid culture medium for an adequate duration, preferably from 5 to 20 days of culture, more preferably 10 to 18 days (step 1b).
  • the cells are incubated in the dark at a temperature comprised between 15°C and 35°C, preferably between 24°C and 26°C.
  • step 2) a plurality of cellular clones is isolated by taking aggregates of stabilized cells from the solid culture medium (PGr solid 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 18 days, in step 4) the ellagitannin content is determined for each clone.
  • a second clonal selection according to step 1b) is preferably carried out until obtaining a plant cell line of Punica granatum wherein the production of ellagitannins is optimal.
  • the clonal selection of step 5) is repeated until obtaining a cell line of Punica granatum which comprises an amount of ellagitannin derivatives greater than 2% w/w, preferably comprised between 4% and 50% w/w, more preferably between 4% and 20% w/w, relative to the dry mass of the cell line.
  • Said ellagitannins are preferably selected in the group consisting of: punicalagin, pedunculagin, catechin and gallic acid hexose.
  • the selected meristematic cell line according to the invention further also comprises an amount of polysaccharides between 30 and 70% w/w, preferably between 40% and 60% w/w.
  • the selected meristematic cell line according to the invention further comprises an amount of proteins between 8 and 30% w/w, preferably between 12 and 25% w/w.
  • the selected meristematic cell line according to the invention further comprises an amount of lipids between 0.3 and 5% w/w, preferably between 0.5 and 2% w/w.
  • said selected meristematic cell line is the Pgr64 line, which comprises between 2% and 50% w/w of ellagitannins, preferably between 4 and 50% w/w, more preferably between 4 and 20% w/w of ellagitannins.
  • 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 ellagitannins greater than 2% w/w, preferably comprised between 4% and 50% w/w, more preferably between 4% and 20% w/w, relative to the dry mass of the phytocomplex.
  • said ellagitannins are selected in the group comprising punicalagin, pedunculagin, catechin and gallic acid hexose.
  • the phytocomplex further also comprises an amount of polysaccharides between 30 and 70% w/w, preferably between 40% and 60% w/w relative to the dry mass of phytocomplex.
  • the phytocomplex further also comprises an amount of proteins between 8 and 30% w/w, preferably between 12 and 25% w/w relative to the dry mass of phytocomplex.
  • the phytocomplex further also comprises an amount of lipids between 0.3 and 5% w/w, preferably between 0.5 and 2% w/w, relative to the dry mass of phytocomplex.
  • said phytocomplex is a derivative of said selected meristematic cell line PGr64 comprising an amount of ellagitannins greater than 2% w/w, preferably comprised between 4% and 50% w/w, more preferably between 4% and 20% w/w.
  • the phytocomplex is preferably a cellular homogenate of the selected meristematic cell line PGr64.
  • Extract means an extract in an alcoholic solvent, for example in methanol or ethanol, or a water/ethanol mixture, for example 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 the 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 between 0.01% and 30% w/w, preferably between 0.03% and 15% w/w, more preferably between 0.05% and 10% w/w, relative to the weight of the composition.
  • 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, nutraceutical 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 a stick (similar to cocoa butter).
  • 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 Applicant has demonstrated that the cell line selected or the derivatives thereof possess antioxidant activity and an activity of increasing the wellbeing of hair follicles on the scalp.
  • the subject matter of the present invention relates to the use of the meristematic line or derivatives thereof to combat oxidative stress. Prolonged oxidative stress can lead to chronic and inflammatory pathologies.
  • the subject matter of the present invention further relates to the use of the meristematic line and/or derivatives thereof for topical treatments for combatting oxidative stress in skin, which can be a cause of premature skin aging.
  • the subject matter of the present invention further relates to the use of the meristematic line or derivatives thereof to increase the wellbeing of hair follicles on the scalp.
  • the dosage varies in relation to the individual’s age, weight and sex and the type of treatment, ranging between 0.1 mg and 5 g per day and preferably between 10 and 500 mg per day of the composition, applied in a single administration or in 2-4 doses or in slow- release forms depending on the individual’s therapeutic needs and for periods ranging between 1 and 90 days.
  • the meristematic line or derivatives thereof can be formulated, in appropriate concentrations, in the form of a supplement to be taken orally for prevention or as an adjuvant treatment for alterations ascribable to states of oxidative stress and to increase the wellbeing of hair follicles on the scalp.
  • Another aspect of the present invention relates to a process for preparing and selecting plant meristematic cells with a high ellagitannin content, preferably with an ellagitannin content greater than 2% w/w relative to the dry mass of the cell line:
  • Said process comprises the steps of:
  • the preparation of meristematic cells entails collecting tissue, preferably young leaves from selected plants of the species Punica granatum, 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 (K).
  • the solid culture medium further comprises agar, whereas the liquid culture medium does not contain agar.
  • the solid and liquid culture media preferably each comprise sucrose in a concentration comprised between 15 and 50 g/L, more preferably between 20 and 45 g/L; naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 0.8 and 2 mg/L; indoleacetic acid (IAA) in a concentration 0.05 and 1 mg/L, preferably between 0.08 and 0.5 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 3 mg/L, preferably between 0.3 and 2 mg/L.
  • sucrose in a concentration comprised between 15 and 50 g/L, more preferably between 20 and 45 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the solid culture medium comprises: sucrose in a concentration of 15 to 35 g/L, preferably between 20 and 30 g/L, naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 1 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.1 and 1 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.3 and 1.5 mg/L.
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the liquid culture medium comprises: sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L; naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 0.8 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.08 and 0.5 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.3 and 1 mg/L.
  • sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgSCL, NahhPCU, (NhU ⁇ SCU and combinations thereof.
  • the salts suitable for the growth of plant cells are selected from: CoCh-ehhO, CuSC> 4 -5H 2 0, NaEDTA-2H 2 0, FeSC hhO, H3BO3, Kl, MnSCU-FhO, Na 2 MoC> 4 -2H 2 0, ZnSC hhO and combinations thereof.
  • the solid and liquid culture media both 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: CaCL, KNO3, MgSCL, NahhPCU, (NhU ⁇ SCU, COCI 2 -6H 2 0, CUS0 4 -5H 2 0, NaEDTA-2H 2 0, FeS0 4 -7H 2 0 H3BO3, Kl, MnS0 4 -H 2 0,
  • 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 CoCl 2 -6H 2 0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; CUSC> 4 -5H 2 0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; NaEDTA-2H 2 0 in a concentration comprised between 20 and 60 mg/L, preferably between 30 and 45 mg/L; FeSC> 4 -7H 2 0 in a concentration comprised between 15 and 45 mg/L, preferably between 20 and 35 mg/L; H 3 BO 3 in a concentration comprised between 1 and 7 mg/L, preferably between 2 and 5 mg/I; Kl in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.4 and 1 mg/L; MnSCL- ⁇ O in a concentration comprised between 5 and 20 mg/L, preferably between 7 and 15 mg/L; Na 2 Mo0 4 -2H 2 0 in
  • the solid and liquid culture media preferably each comprise myo-inositol in a concentration comprised between 70 and 130 mg, preferably between 90 and 110 mg; pyridoxine-HCI between 70 and 130 mg, preferably between 90 and 110 mg; and thiamine-HCI between 5 and 20 mg/L, preferably between 7 and 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 in the solid culture medium for an adequate duration, preferably from 5 to 20 days of culture, more preferably 10 to 18 days (step 1b).
  • 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 18 days, in step 4) the ellagitannin content of each clone is determined.
  • step 5 a second clonal selection according to step 1b) is preferably carried out until obtaining a plant cell line of Punica granatum wherein the production of ellagitannins 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 the same liquid medium as used for the clonal selection, the PGr 1 liquid medium.
  • the PGr 1 liquid medium is a medium containing the Gamborg salts specified above, the vitamins listed above, sucrose, naphthylacetic acid, indoleacetic acid and kinetin, with a final pH of 6.5.
  • the PGr 1 liquid culture medium 1 contains sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L; naphthylacetic acid (NAA) in a concentration comprised between 0.5 and 4 mg/L, preferably between 0.8 and 2 mg/L; indoleacetic acid (IAA) in a concentration comprised between 0.05 and 1 mg/L, preferably between 0.08 and 0.5 mg/L; and kinetin (K) in a concentration comprised between 0.1 and 2 mg/L, preferably between 0.3 and 1 mg/L.
  • sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L
  • NAA naphthylacetic acid
  • IAA indoleacetic acid
  • K kinetin
  • the cells cultured in the PGr 1 liquid medium are transferred, for the final phase of growth, into a final liquid medium, PGr 2, containing the Gamborg salts, vitamins, sucrose, naphthylacetic acid, indoleacetic acid and kinetin, which induce an increase in the ellagitannin content and biomass.
  • the final PGr 2 liquid medium contains the Gamborg salts, sucrose, which is preferably comprised between 35 g/L and 50 g/L, naphthylacetic acid, preferably comprised between 0.8 and 1.2 mg/L, indoleacetic acid, preferably comprised from 0.05 to 0.3 mg/L and kinetin, preferably comprised between 0.3 mg/L and 0.8 mg/L, with a final pH of 6.5.
  • Gamborg salts sucrose, which is preferably comprised between 35 g/L and 50 g/L, naphthylacetic acid, preferably comprised between 0.8 and 1.2 mg/L, indoleacetic acid, preferably comprised from 0.05 to 0.3 mg/L and kinetin, preferably comprised between 0.3 mg/L and 0.8 mg/L, with a final pH of 6.5.
  • the growth of the cell line in the flask, bioreactor or fermenter, both in the PGr 1 liquid medium and in the final PGr 2 liquid medium takes place at a temperature comprised between 15 °C and 35 °C, typically about 25 °C, for a period comprised from 5 to 22 days, preferably 10 to 18 days, and 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 ellagitannin content.
  • the phytocomplex may 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) using conventional techniques.
  • an alcohol solvent for example methanol and/or ethanol
  • the extract thus obtained is characterized by a high ellagitannin content as detailed above and can be used for the preparation of cosmetic 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.
  • the inventors of the present invention have verified that the cell line of the invention and/or the derivatives thereof exhibit a marked antioxidant activity and an ability to increase the wellbeing of hair follicles on the scalp.
  • the induction of callus tissue was obtained using standard procedures described in the literature.
  • the procedure provides for the collection of young tissues (leaves) from plants of Punica granatum, the cleaning thereof, for example with running water, minute fragmentation into 2-5 cm pieces and sanitization by means, for example, of a treatment in sequence with 70% ethanol in water for about 1 minute, 2% sodium hypochlorite and 0.1% Tween 20 for about 2-4 minutes and, finally, at least 5 washes with sterile distilled 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. 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 meristematic cells obtained are stabilized by means of a certain number of transfers (sub-cultures) onto solid culture media.
  • the solid medium it is a Gamborg B5 medium (Gamborg O.L. et al., 1968, Exp. Cell. Res., 50, 151) with the addition of 20 g/L of sucrose, 1.5 mg/L of naphthylacetic acid, 0.25 mg/I of indoleacetic acid, 1 mg/L of kinetin and 0.8% of plant agar, final pH 6.5 (PGr solid medium).
  • liquid medium it is a Gamborg B5 medium (Gamborg O.L. et al., 1968, Exp. Cell. Res., 50, 151) with the addition of 20 g/L of sucrose, 1.5 mg/L of naphthylacetic acid, 0.25 mg/I of indoleacetic acid and 1 mg/L of kinetin, final pH 6.5 (PGr 1 liquid medium).
  • the final liquid medium it is a Gamborg B5 medium (Gamborg O.L. et al., 1968, Exp. Cell. Res., 50, 151) with the addition of 40 g/L of sucrose, 1 mg/L of naphthylacetic acid, 0.1 mg/L of indoleacetic acid and 0.5 mg/L of kinetin, final pH 6.5 (PGr 2 liquid medium).
  • the selected plant cell lines were multiplied to obtain sufficient amounts of biomass to be transferred into the liquid culture medium.
  • the cell suspensions were transferred into bioreactors containing the PGr 2 liquid medium for further phases of growth.
  • the cell suspensions were filtered and the biomasses were recovered and used for the subsequent steps of preparing the phytocomplexes.
  • the selected cell line of Punica granatum called PGr64, is maintained in PGr solid culture medium, is light brown in colour and has a friable texture ( Figure 1).
  • the procedure for homogenizing the biomasses of cells selected and grown in bioreactors for 14 days at 25°C ( ⁇ 2) comprises the following steps: a) filtration of the biomass obtained from the growth of the PGr64 cell culture in the PGr 2 liquid medium in order to have only cells and discard the medium; b) washing of the cells with a double volume, relative to the cells, of saline solution (0.9% w/V NaCI in sterile water); c) addition of 1% w/w (from 0.5 to 2% w/w) of citric acid (or ascorbic acid or a mixture of citric and ascorbic acid) to the filtered, washed biomass; d) homogenization of the mixture, for example with an UltraTurrax or any other instrument suitable for breaking down the cells and the internal structures thereof; e) drying of the biomass by lyophilization or air circulation drying or rotating cylinder drying or fluid bed drying or atomization.
  • 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.
  • Meristematic cells stabilized and selected as previously described, cultured in PGr solid medium (Gamborg B5 with the addition of 20 g/L of sucrose, 1.5 mg/L of naphthylacetic acid, 0.25 mg/I of indoleacetic acid, 1 mg/L of kinetin and 0.8% of plant agar, final pH 6.5 ) were inoculated into 5 flasks with a 1 -litre capacity, containing 250 ml of PGr 1 liquid medium (Gamborg B5 with the addition of 20 g/L of sucrose, 1.5 mg/L of naphthylacetic acid, 0.25 mg/I of indoleacetic acid and 1 mg/L of kinetin and with 0.8% of plant agar, final pH 6.5).
  • the amount of meristematic cells inoculated into the liquid medium was equal to 8% w/V.
  • the suspensions thus obtained were incubated in the dark at 25°C and placed on top of an orbital shaker set on 110 RPM. After 7 days of incubation, 200 mL of cell suspension contained in a 1 -litre flask was transferred into a flask with a 3-litre capacity, containing 800 ml of PGr 2 liquid medium (40 g/L of sucrose, 1 mg/L of naphthylacetic acid, 0.1 mg/L of indoleacetic acid and 0.5 mg/L of kinetin, final pH 6.5).
  • 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 14 days of incubation, the plant biomass (total 5 litres of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 pm and washed with 1.9 L of sterile saline solution (0.9% W/V). The washed cells (fresh weight 950 g) were supplemented with 9.5 g of citric acid and homogenized with an UltraTurrax.
  • the homogenized cells were lyophilized. 105 g of lyophilizate (PGr64 phytocomplex) with an ellagitannin content equal to 5.25 g, total polysaccharides equal to 55.3 g, proteins equal to 16.3 g, lipids equal to 0.84 g, ash equal to 12 g and citric acid equal to 8.4 g were obtained from 5 litres of cell suspension.
  • Table 1 shows the characterization of the PGr64 phytocomplex.
  • the characterization of the phytocomplex was carried out using the methods described below: a ) UPLC-ESI-MS analysis of the PGr64 phytocomplex
  • the powder of the phytocomplex was extracted with 6 volumes of 100% methanol for 15 minutes in a sonicator at 40 kHz, under ice (Falc Instruments, Bergamo, Italy), after shaking with a vortex-type mixer for 30 seconds; the extract was recovered after centrifugation at 18000 g per 10 minutes at 4°C.
  • the extract was then diluted 1:5 with 100% methanol and subsequently diluted 1:2 with double-distilled water.
  • the extract was filtered, introduced into “LC-MS-grade” glass autosampler vials and analysed by UPLC-QqTOF.
  • the platform used consisted in an Acquity UPLC l-class system (Waters) provided with a refrigerated autosampler and coupled on line with a UV/VIS detector of the diode array type and a Xevo G2-XS QTof 4k high-resolution mass spectrometer (Waters).
  • the molecules were detected by means of a Xevo G2-XS mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source and a QTOF (quadrupole-time of flight) analyzer.
  • the source parameters were as follows: capillary voltage 0.8 KV, cone voltage 30 V and source temperature 120°C. Nitrogen was used both as the nebulizer gas, at a flow rate of 50 L/h, and as the desolvation gas, at a temperature of 500°C and flow rate of 1000 L/h.
  • the analyses were conducted in the positive and negative ionization modes.
  • the instrument in question is a high resolution mass spectrometer and the calculation of the exact mass and maintenance of calibration were achieved by direct infusion of Leucine Enkephalin 100 pg/mI at a speed of 10 mI/min. Fragmentation was achieved using argon gas in the collision cell and applying a voltage of 35V. The scan was comprised between 50 and 2000 m/z and the scan time set at 0.3 s.
  • the chromatographic system used for the quantification of ellagitannins consists in a 2.1 x 100 mm Acquity UPLC BEH C18 1.7 pm column coupled to a 2.1 x 5 mm Acquity UPLC BEH C18 1.7 pm VanGuard Pre-Column 3/Pk.
  • the platform used for the UPLC-DAD analysis comprises a UPLC system (Waters) consisting of an eluent management module - I Class Binary Solvent Manager - and an autosampler - I Class Sample Manager FTN - coupled to a Diode Array PDA eA detector.
  • the data acquisition and analysis software is Empower 3 (Waters).
  • the chromatographic method used was as follows: solvent A: water, 0.1% formic acid; solvent B: 100% acetonitrile. The initial condition is 99% solvent A; in addition, the flow remains constant at 0.350 ml/min throughout the duration of the analysis.
  • the chromatography column was temperature controlled at 30°C. The elution of the molecules was conducted by alternating gradient phases and isocratic phases, as indicated in table 4: Table 4
  • the chromatogram for the wavelength of 377 nm was used.
  • the ellagitannins were quantified thanks to the calibration curve of the authentic commercial standard of punicalagin (Sigma, purity>98%).
  • the protein content in the PGr64 phytocomplex was equal to 15.5% w/w. e) Analysis of the lipid content of the PGr64 phytocomplex
  • the extraction of the total lipid fraction was carried out on the PGr64 phytocomplex by Soxhlet extraction with dichloromethane, extended for at least 12 hours according to the method described in Martinez M. et al., “Soxhlet lipids extraction from cotton from different producing areas. Comparison of dichloromethane or successive dichloromethane-methanol extractions”. Grasas y Aceites (1997), 48 (4), 226-230. The lipid content in the PGr64 phytocomplex was equal to 0.8% w/w. f) Analysis of the moisture and ash of the PGr64 phytocomplex
  • 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 PGr64 phytocomplex was equal to 6.5%, while the ash was equal to 11.5%.
  • Biological test antioxidant activity of the phytocomplex evaluated by DPPH assay
  • the assay with the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical has the purpose of evaluating, in vitro, the antioxidant activity of the tested extract on a stable coloured radical and measuring its disappearance by spectrophotometry.
  • the methanolic solution of DPPH is purple coloured and exhibits maximum absorption at a wavelength of 517 nm.
  • the PGr64 phytocomplex was extracted in methanol and placed in an ultrasound bath for 20 minutes. Different aliquots of the extract solution were drawn, added to a same amount of DPPH solution and brought to volume with methanol so as to evaluate the antioxidant capacity of the extract at different concentrations. The results are expressed as EC50, i.e. as the concentration of the tested extract capable of decreasing the initial absorbance of the radical by 50% at 517 nm. The result is then compared with the one obtained from a methanolic solution of ascorbic acid, used as a reference.
  • human dermal papilla-derived fibroblasts were cultured.
  • the primary cultures of human fibroblasts were obtained from brow and forehead lift samples deriving from aesthetic surgery interventions.
  • the dermal papilla fibroblasts thus obtained were amplified and seeded in a 96-well plate, left to acclimatise for 24 hours in a culture medium (DM EM) and finally incubated with the PGr64 phytocomplex for 24 hours. At the end of the incubation, an MTT assay was performed directly in the culture wells.
  • DM EM culture medium
  • the NHDPF cells were processed with 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
  • the cellular enzymes prevalently mitochondrial dehydrogenases, reduce MTT to a blue/purplish-coloured formazan salt.
  • the blue crystals of formazan are dissolved with an isopropanol-based solution and the resulting colour intensity is measured in absorbance at a wavelength of 570 nm; the values obtained are directly proportional to cell viability.
  • the PGr64 phytocomplex demonstrated a significant action on the viability of human dermal papilla-derived fibroblasts, equal to +22% at a concentration of 0.01%.
  • the human dermal papilla fibroblasts were obtained as described in the previous example. After the fibroblasts had been obtained from the dermal papilla, they were seeded in a 96- well plate, left to acclimatise for 24 hours in a culture medium (DM EM) and finally incubated with the PGr64 phytocomplex at concentrations of 0.01% and 0.005% for 24 hours.
  • DM EM culture medium
  • the cells were fixed and immunostained with an antibody that recognises the antigen of interest (KI-67, a nuclear protein closely associated with cell proliferation).
  • the cell nuclei were marked using DAPI (4’,6-Diaminodino-2-Phenylindole, Dihydrochloride). Proliferation was evaluated by calculating the number of KI-67-positive cells out of the total number of cells.
  • the PGr64 phytocomplex significantly stimulated proliferation in the primary fibroblasts derived from human hair follicle dermal papilla both at the concentration of 0.005% and at 0.01% (+20% and +24% versus untreated, respectively).
  • the PGr64 phytocomplex was dispersed in glycerine in a concentration of 3% W/W (PGr64- G). 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.
  • Table 6 Emulsions (two-phase and multiple O/A, A/O, A/S, A/O/A)

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Abstract

L'invention concerne une lignée cellulaire méristématique choisie à partir d'une plante appartenant à l'espèce Punica granatum, la lignée cellulaire méristématique étant caractérisée en ce qu'elle présente une teneur élevée en ellagitannin, et l'utilisation cosmétique, nutraceutique et médicale de ladite lignée cellulaire ou d'un dérivé de celle-ci. La lignée cellulaire comprend une certaine quantité d'ellagitannin (en particulier la punicalagine, la pedunculagin, la catéchine et l'hexose d'acide gallique) supérieure à 2 % poids/poids, de préférence comprise entre 4 % et 50 % poids/poids. L'invention concerne également un phytocomplexe ou un extrait obtenu à partir de la lignée cellulaire pour une utilisation dans le traitement du stress oxydatif, une cause de pathologies inflammatoires chroniques ou d'un vieillissement prématuré de la peau, ou en tant que produit cosmétique pour améliorer la santé des follicules pileux sur le cuir chevelu.
PCT/IB2021/057648 2020-11-24 2021-08-19 Phytocomplexe et extrait d'une lignée cellulaire méristématique sélectionnée à partir punica granatum WO2022112864A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
EP2703003A1 (fr) * 2012-08-31 2014-03-05 Probelte Biotecnologia S.L. Extrait du grenade et compositions avec des polyphenols des grenades pour le traitement et la prevention des maladies ou des conditions physiopathologiques associées avec un excès d'expression des genes et/ou de l'activité physiologique de l'interleukine-6
WO2015102003A1 (fr) * 2014-01-05 2015-07-09 Bioharvest Ltd. Culture cellulaire dérivée de la grenade et ses méthodes de préparation et d'utilisation
WO2015140470A2 (fr) * 2014-03-18 2015-09-24 Greenpharma Applications cosmétiques et pharmaceutiques de l'acide gallique et de dérivés de l'acide gallique
CN108018255A (zh) * 2017-12-30 2018-05-11 杭州纽贝生物科技有限公司 一种石榴细胞的悬浮培养方法
WO2020188533A1 (fr) * 2019-03-21 2020-09-24 Demethra Biotech S.R.L. Phytocomplexe et extrait de lignée cellulaire méristématique sélectionné à partir de daucus carota sativa

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EP2703003A1 (fr) * 2012-08-31 2014-03-05 Probelte Biotecnologia S.L. Extrait du grenade et compositions avec des polyphenols des grenades pour le traitement et la prevention des maladies ou des conditions physiopathologiques associées avec un excès d'expression des genes et/ou de l'activité physiologique de l'interleukine-6
WO2015102003A1 (fr) * 2014-01-05 2015-07-09 Bioharvest Ltd. Culture cellulaire dérivée de la grenade et ses méthodes de préparation et d'utilisation
WO2015140470A2 (fr) * 2014-03-18 2015-09-24 Greenpharma Applications cosmétiques et pharmaceutiques de l'acide gallique et de dérivés de l'acide gallique
CN108018255A (zh) * 2017-12-30 2018-05-11 杭州纽贝生物科技有限公司 一种石榴细胞的悬浮培养方法
WO2020188533A1 (fr) * 2019-03-21 2020-09-24 Demethra Biotech S.R.L. Phytocomplexe et extrait de lignée cellulaire méristématique sélectionné à partir de daucus carota sativa

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