WO2020188533A1

WO2020188533A1 - Phytocomplex and extract of meristematic cell line selected from daucus carota sativa

Info

Publication number: WO2020188533A1
Application number: PCT/IB2020/052588
Authority: WO
Inventors: Elena Sgaravatti; Giovanna Pressi; Flavia GUZZO
Original assignee: Demethra Biotech S.R.L.
Priority date: 2019-03-21
Filing date: 2020-03-20
Publication date: 2020-09-24
Also published as: IT201900004107A1

Abstract

The present invention relates to a meristematic cell line selected from tissue, preferably from callus tissue, of a plant of Daucus carota sativa. The invention also relates to a derivative of the cell line, i.e. a phytocomplex or an extract of the cell line. The selected meristematic cell line is characterized by a high anthocyanin content. Furthermore, the present invention relates to the cosmetic, nutraceutical and medical use of the selected meristematic cell line or a derivative thereof.

Description

“Phytocomplex and extract of meristematic cell line selected from

Daucus carota sativa”

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a meristematic cell line selected from a plant of Daucus carota sativa, characterized by a high anthocyanin content, and the cosmetic, nutraceutical and medical use of said meristematic cell line or a derivative thereof.

PRIOR ART

NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a nuclear transcription factor involved in the response to oxidative stress. The inhibition of the factor NFkB is accompanied by a decrease in the expression of VEGF (vascular endothelial growth factor). Conversely, an increase in the expression of NFkB is accompanied by an increase in the expression of VEGF (Jiali Zeng and Bin Peng BMC Cancer, 2007; Hyun- Mi Ko et al. FEBS Letters, 2006).

Increased levels of VEGF-A are associated with an increased effectiveness of wound healing.

In the presence of oxidative stress, the factor IKK (IkB kinase) phosphorylates IkBa, making NFkB available and enabling the nuclear translocation thereof. In the nuclei, the transcription factor interacts with DNA. The nuclear translocation increases following irradiation/stress (Van den Berg R. et al, Br. J. Nutr. 2001 ).

Plants of the genus Daucus carota sativa are known to contain anthocyanins. A large variability in the anthocyanin content of said plants is associated with 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 tied to the variability of the content of metabolites in different plant tissues, seasonal variability in the content and type of metabolites, 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. The extreme variability in 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 (and also an extract) with a standardized, reproducible content of active substances can be derived.

Ceoldo et al., in Plant Science (176) 2009 p. 553-565, describe meristematic cells obtained from plants of D. carota L.cv. Flakkese. The lines described by Ceoldo et al. show, as maximum levels of anthocyanins, amounts of 200 pg/g of fresh cells, i.e. 0.004% w/w of the dry mass of the cell line.

The present invention provides a selected meristematic cell line of Daucus carota sativa and derivatives thereof with an anthocyanin content greater than the one found by Ceoldo et al.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a selected meristematic cell line derived from a plant belonging to the species Daucus carota sativa, 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 anthocyanin content.

A third aspect of the invention relates to 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.

Another aspect of the present invention relates to a process for the preparation and selection of plant meristematic cells of Daucus carota sativa with a high anthocyanin content.

The Applicant has demonstrated that the selected cell line or a derivative thereof is active in inhibiting the nuclear translocation of NFkB and increasing the gene expression of VEGFA.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described in detail below and illustrated by way of example with reference to the appended figures, in which:

Figure 1 shows a photo, taken with a bright-field optical microscope, of the cell line called Dc-AnA4 maintained in a solid medium.

Figure 2 shows a magnification (400X) of a portion of the figure 1.

Figure 3 shows a magnification (400X) of a portion of the 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 Dc-AnA4 and (B) on meristematic cells before the selection process.

Figure 5 shows the chromatogram of the Dc-AnA4 phytocomplex in 3 dimensions. Figure 6 shows the identification of the main peaks obtained in the chromatographic profile, in the positive and negative ionization modes, for the Dc-AnA4 phytocomplex.

Figure 7 shows the expression of NFkB after irradiation using a secondary antibody (A). The signal intensity was quantified and expressed as the mean number of nuclear translocations observed (B).

Figure 8 shows the increase in the gene expression of VEGF-A after 72h of treatment with the Dc-AnA4 phytocomplex. The results are expressed as the number of times more compared to untreated microtissues.

DEFINITIONS

In the context of the present invention“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 the stem cells in animals.

In the context of the present invention, “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.

In the context of the present invention, unless specified otherwise,“w/w” means a weight/weight amount relative to the dry mass of the cell line.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a meristematic cell line derived from a plant belonging to the species Daucus carota sativa selected so as to comprise an amount of anthocyanins greater than 0.1 % w/w, preferably comprised from 0.5 % to 10% w/w.

In one embodiment, said meristematic cell line is obtained by means of a process comprising the steps of: 1 ) plating a tissue obtained from a plant belonging to the species Daucus carota sativa onto a solid culture medium;

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium; 4) determining the anthocyanin content and preferably the polyphenol and polysaccharide content for each clone;

5) selecting the cellular clone with the highest anthocyanin content and, preferably, polyphenol and polysaccharide content.

In step 1), the tissue obtained from plants of the species Daucus carota sativa is placed in a solid medium in order to obtain an undifferentiated callus tissue. The tissue of Daucus carota sativa is preferably at least one shoot of Daucus carota sativa or a plurality of shoots of Daucus carota sativa.

In a preferred embodiment of the invention, the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA).

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 55 g/L, more preferably from 20 to 50 g/L; naphthylacetic acid (NAA) in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 4 mg/L.

In a preferred embodiment, the solid culture medium comprises: sucrose in a concentration of from 15 to 35 g/L, preferably from 20 to 30 g/L, and naphthylacetic acid (NAA) in a concentration of from 2 to 5 mg/L, preferably from 2.5 to 4 mg/L.

In a preferred embodiment, the liquid culture medium comprises: sucrose in a concentration of from 20 to 50 g/L, preferably from 30 to 45 g/L, naphthylacetic acid (NAA) in a concentration of from 0.5 to 4 mg/L, preferably from 1 to 3 mg/L. In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCl₂, KNO₃, MgSO₄, NaH₂PO₄, (NH₄)₂SO₄ and combinations thereof.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are preferably selected from: CoCl₂-6H₂O, CuSO₄-5H₂O, NaEDTA-2H₂O, FeSO₄-7H₂O, H₃BO₃, Kl, MnSO₄- H₂O, Na₂MoO₄-2H₂O, ZnSO₄-7H₂O 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.

In one embodiment, in both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCl₂, KNO₃, MgSO₄, NaH₂PO₄, (NH₄)₂SO₄, CoCl₂-6H₂O, CuSO₄-5H₂O, NaEDTA-2H₂O, FeSO₄-7H₂O H₃BO₃, Kl, MnSO₄- H₂O, Na₂MoO₄-2H₂O, ZnSO₄-7H₂O and combinations thereof. This combination of compounds is the medium Gamborg B5.

In one embodiment, 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 CaCl₂ 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; MgSO₄ in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NaH₂PO₄ in a concentration comprised from 100 to 180 mg/L, preferably from 110 to 150 mg/L; and (NH₄)₂SO₄ 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 CoCl₂-6H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuSO₄-5H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H₂O in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSO₄-7H₂O 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; MnSO₄- H₂O in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na₂MoO₄-2H₂O in a concentration comprised from 0.1 to 0.5 mg/L, preferably from 0.15 to 0.3 mg/L and ZnSO₄-7H₂O in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 3 mg/L.

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.

After step 1), 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.

After the stabilization step 1 a), the stabilized cells preferably undergo a first "clonal selection". The clonal selection consists in culturing the stabilized cells for an adequate duration, preferably 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.

In step 2), a plurality of cellular clones is isolated by taking aggregates of stabilized cells from the solid culture medium.

In step 3) the cellular clones are each inoculated into the liquid culture medium described above. According to one embodiment, 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 anthocyanin content of each clone is determined.

In one embodiment, in step 5) a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Daucus carota sativa wherein the production of anthocyanins is optimal.

In a preferred embodiment, the clonal selection of step 5) is repeated until obtaining a cell line of Daucus carota sativa which comprises an amount of anthocyanins greater than 0.1 % w/w, preferably comprised from 0.5 % to 10% w/w relative to the dry mass of the cell line.

Said anthocyanins are preferably selected in the group consisting of cyanidin-O-xylose-glucose-galactose, cyanidin-O-(synaptic acid)-xylose- glucose-galactose, cyanidin-O-(ferulic acid)-xylose-glucose-galactose, cyanidin-O-(coumaric acid)-xylose-glucose-galactose and mixtures thereof.

In one embodiment, said selected meristematic cell line comprises polyphenols in an amount comprised from 0.01 % to 0.5%, preferably from 0.01 to 0.2%, preferably from 0.05 to 0.1 %, said polyphenols being preferably selected in the group comprising caffeoylquinic acid, ferulic acid, chlorogenic acid and mixtures thereof.

The selected meristematic cell line according to the invention also further comprises an amount of polysaccharides of from 30 to 80% w/w, preferably from 40% to 70% 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 1 to 20% w/w, preferably from 2 to In a preferred embodiment, said selected meristematic cell line is the line DcAnA4, which comprises from 0.5% to 10% w/w of anthocyanins, preferably 3.9-4.2% w/w of anthocyanins.

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 anthocyanins greater than 0.1 % w/w, preferably comprised from 0.5 % to 10% w/w relative to the dry mass of the phytocomplex.

Said anthocyanins are preferably selected in the group comprising cyanidin-O-xylose-glucose-galactose, cyanidin-O-(synaptic acid)-xylose- glucose-galactose, cyanidin-O-(ferulic acid)-xylose-glucose-galactose, cyanidin-O-(coumaric acid)-xylose-glucose-galactose and mixtures thereof.

The phytocomplex comprises polyphenols in an amount comprised from 0.01 % to 0.5%, preferably from 0.01 to 0.2%, preferably from 0.05% to 0.1 %, said polyphenols being preferably selected in the group comprising caffeoylquinic acid, ferulic acid, chlorogenic acid and mixtures thereof.

The phytocomplex also further comprises an amount of polysaccharides of from 30 to 80% w/w, preferably from 40% to 70% 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%, preferably 1 to 20% w/w, preferably from 2 to 10% w/w, relative to the dry mass of phytocomplex. In a preferred embodiment, said phytocomplex is derived from said selected meristematic cell line DcAnA4 and comprises from 0.5% to 10% w/w of anthocyanins, preferably 3.9-4.2% w/w of anthocyanins. The phytocomplex is preferably a cellular homogenate of the selected meristematic cell line DcAnA4.

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 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.

In one embodiment, 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 relative to the weight of the composition. Said composition preferably comprises a phytocomplex which is a cellular homogenate.

In one embodiment, the cell line and/or a derivative thereof are dispersed before being mixed with the excipients to prepare the composition of the invention. By way of example, suitable dispersing agents are glycerine, propylene glycol or butylene glycol.

The 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.

The 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).

The 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.

In one embodiment, 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 selected cell line or the derivatives thereof possesses an activity of improving microcirculation and an anti-inflammatory activity.

The subject matter of the present invention also relates to the use of the meristematic line or the derivatives thereof to improve endothelial microcirculation and for the treatment of inflammatory pathologies. The present invention further relates to the use of the meristematic line or the derivatives thereof for protection against UV radiation.

The dosage varies, in relation to the age, weight and sex of the subject and the type of treatment, from 0.1 mg to 5 g a day and preferably from 10 to 500 mg a day of the composition, taken in a single administration or in 2-4 doses or in slow-release forms according to the therapeutic needs of the subject and for periods ranging from 1 to 90 days.

The meristematic line or the derivatives thereof can be formulated, in suitable concentrations, as a dietary supplement to be taken orally for the prevention of or as an adjuvant treatment for alterations attributable to inflammatory states or alterations in endothelial microcirculation in humans or animals. 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 species Dacus carota sativa with a high anthocyanin content, preferably with an anthocyanin content greater than 0.1 % w/w relative to the dry mass of the cell line and, preferably, with an amount of polyphenols comprised from 0.01 % to 0.5% w/w and an amount of polysaccharides of from 30 to 80% w/w relative to the dry mass of the cell line.

Said process comprises the steps of:

1 ) plating a tissue obtained from a plant of Daucus carota sativa onto a solid culture medium;

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

4) determining the anthocyanin content and, preferably, the polyphenol and polysaccharide content for each clone;

In one embodiment, the preparation of meristematic cells entails collecting tissue, preferably of shoots from plants selected from the species Daucus carota sativa, 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.

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

In a preferred embodiment of the invention, the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose and naphthylacetic acid (NAA).

The solid and liquid culture media preferably each comprise sucrose in a concentration comprised from 15 to 55 g/L more preferably from 20 to 50 g/L; and naphthylacetic acid (NAA) in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 4 mg/L.

In a preferred embodiment, the liquid culture medium comprises: sucrose in a concentration of from 20 to 55 g/L, preferably from 30 to 45 g/L, and naphthylacetic acid (NAA) in a concentration of from 0.5 to 4 mg/L, preferably from 1 to 3 mg/L.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCL, KNO₃, MgSO₄, NaH₂PO₄, (NH₄)₂SO₄ and combinations thereof.

In one embodiment, 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 CaCl₂ 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; MgSO₄ in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NaH₂PO₄ in a concentration comprised from 100 to 180 mg/L, preferably from 110 to 150 mg/L; and (NH₄)₂SO₄ 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 CoCl₂-6H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuSO₄-5H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H₂O in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSO₄-7H₂O 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; MnSO₄-H₂O in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na₂MoO₄-2H₂O in a concentration comprised from 0.1 to 0.5 mg/L, preferably from 0.15 to 0.3 mg/L and ZnSO₄-7H₂O in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 3 mg/L.

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.

After step 1), 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. After the stabilization step 1 a), the stabilized cells preferably undergo a first "clonal selection". The clonal selection consists in culturing the stabilized cells for an adequate duration, preferably 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.

In step 3) the cellular clones are each inoculated into the liquid culture medium described above.

According to one embodiment, 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 anthocyanin content of each clone is determined.

In one embodiment, in step 5) a second clonal selection according to step 1 b) is preferably carried out until obtaining a plant cell line of Daucus carota sativa wherein the production anthocyanins 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 DC. The liquid growth medium DC is a medium containing the Gamborg salts specified above, the vitamins listed above, sucrose and NAA.

The liquid growth medium DC contains, among 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 20 g/L to 30 g/L. NAA is preferably comprised from 1 mg/L to 4 mg/L.

The cells grown in the liquid growth medium DC are transferred, for the final phase of growth, into a final liquid medium, DC-F containing the Gamborg salts listed above, the vitamins listed above, sucrose and NAA, which induces an increase in the anthocyanin content and the biomass. The final liquid medium contains, among the Gamborg salts, KNO₃ in an amount comprised from 0.5 g/L and 2 g/L. Sucrose is preferably comprised from 30 g/L to 50 g/L. NAA is preferably comprised from 1 mg/L to 3 mg/L.

According to a preferred embodiment, the growth of the cell line in the flask, bioreactor or fermenter, both in the liquid growth medium DC and in the final liquid medium DC-F, 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.

At the end of growth in the final liquid medium DC-F, 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 anthocyanin content.

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

In one embodiment, at the end of growth in the flask, bioreactor or fermenter the cells are homogenized, for example by mechanical disintegration, preferably in an acidified solution (for example with ascorbic acid or citric acid or acetic acid) and subsequently lyophilized or dried. In the latter case, the phytocomplex is a cellular homogenate wherein the cells and the internal structures thereof are disintegrated. These different types of phytocomplexes are all characterized in that they have a high anthocyanin content as previously described.

Alternatively, the phytocomplex, preferably in the form of a cellular homogenate, undergoes extraction in an alcohol solvent (for example methanol or ethanol) using conventional techniques. The extract thus obtained is characterized by a high anthocyanin content as detailed above and can be used for the preparation of cosmetic or pharmaceutical compositions as described above.

Alternatively, the live cells as such, following purification, can be directly employed for the preparation of the compositions of the invention. The biological data obtained in the experiments that follow confirm the effectiveness of the cell line of the invention and/or derivatives thereof, which have a surprisingly high anthocyanin content, for the uses described herein. A surprising effectiveness of a derivative of said selected cell line was also demonstrated, where said derivative is a homogenate, that is, a phytocomplex that comprises all of the cellular components, without the cells being exposed to solvents or other extraction procedures that could compromise the integrity of the active ingredients.

The authors of the present invention have in fact surprisingly verified that the cell line of the invention and/or derivatives thereof inhibit the nuclear translocation of NFkB, with a consequent increase in the gene expression of VEGF-A.

ESEMPI

Generation and selection of the meristematic cell lines of Daucus carota sativa

The induction of callus tissue was achieved using standard procedures described in the literature.

The induction of callus tissue was achieved using standard procedures described in the literature. The procedure provides for the collection of young tissues (shoots) from plants of Daucus carota sativa, the cleaning thereof, for example with running water, minute fragmentation into 2-5 cm pieces and sanitization, for example by treatment, in sequence, with 70% ethanol in water for about 15’, 2% sodium hypochlorite and 0.1 % Tween 20 for about 5 minutes and, finally, at least 4 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.

As regards the solid medium, the medium is a Gamborg B5 (Gamborg O.L. et al, 1968, Exp. Cell. Res., 50, 151 ) with the addition of 25 g/L of sucrose, 3 mg/L of NAA and 0.7-0.9% of plant agar, final pH 5.5 (DC medium).

As regards the liquid medium, the medium is a Gamborg B5 (Gamborg O.L. et al, 1968, Exp. Cell. Res., 50, 151 ) with the addition of 45 g/L of sucrose, 2 mg/L of NAA, final pH 5.5 (DC-F medium).

The cell line obtained in this specific culture medium, after clonal selection performed in solid DC medium (with agar) and liquid DC-F medium (without agar), was called Dc-AnA4. The belonging of the meristematic cells obtained to the botanical species Daucus was confirmed by DNA fingerprint analysis.

The selected plant cell lines were multiplied to obtain sufficient amounts of biomass to be transferred into the liquid culture medium.

After growth in liquid DC-F medium, the cell suspensions were transferred into bioreactors containing the liquid DC-F medium for further phases of growth.

At the end of growth in DC-F medium the cell suspensions are filtered and the biomasses are recovered and used for the subsequent steps of preparing the phytocomplexes.

The characteristics of the cell lines of Daucus carota sativa called Dc- AnA4 will be described by way of non-limiting example.

Morphological characteristics of the cell line

The selected cell line of Daucus carota sativa, called Dc-AnA4, is maintained in the solid DC culture medium, is purple in colour and has a friable texture (Figures 1 , 2, 3).

Homogenization procedure 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 Dc-AnA4 cell culture in the DC-F culture 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 1.5% 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 Ultra-Turrax 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.

Using the procedure described, one obtains the phytocomplex called Dc- AnA4.

Description of the content of the homogenate Dc-AnA4:

40-70% carbohydrates

0.5-10% total anthocyanins

0.01 -0.2% total polyphenols (derivatives of caffeic acid)

12-20% proteins

1 -4% lipids

2-5% moisture

4-1 1 % ash

5-30% citric acid.

Examples of preparation of the standardized Dc-AnA4 phytocomplex in DC-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 CROP®-G. Preparation and analysis of the Dc-AnA4 phvtocomplex

Meristematic cells, stabilized and selected as previously described, cultured in solid DC medium (Gamborg B5 with 25 g/L of sucrose, 3 mg/L of NAA and 0.7% plant agar, final pH 5.5) were inoculated into 5 flasks with a 1 -litre capacity, containing 200 ml of DC-F liquid medium (Gamborg B5 with the addition of 45 g/L of sucrose and 2 mg/L of NAA, final pH 5.5). 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 120 RPM. After 14 days of incubation the plant biomass (1 litre of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 mm and washed with 650 mL of sterile saline solution (0.9% W/V). The washed cells (fresh weight 320 g) were supplemented with 3.2 g of citric acid and homogenized with an Ultra-Turrax.

The homogenized cells were lyophilized. 29.5 g of lyophilizate (Dc-AnA4 phytocomplex) with a total anthocyanin content equal to 1.15 g, total polysaccharides of 16,7 g, 4.8 g of proteins, 0.83 g of lipids, 2.06 g of ash and 3.1 g of citric acid was obtained from 1 litre of cell suspension. Table 1 provides the characterization of the phytocomplex:

Table 1

The characterization of the phytocomplex was carried out using the methods described below:

a) Quantification of total anthocyanins in the Dc-AnA4 phytocomplex by UPLC-DAD

100mg of powder of the Dc-AnA4 phytocomplex were weighed into a 15 ml_ test tube and 30 volumes of ethanol-HCI (99:1 ) and water 60:40 (V/V) were added. The suspension was mixed for 30 seconds with a vortex mixer and sonicated for 15 minutes in an ice bath; finally, it was centrifuged at 4000 rpm for 15 minutes at 6°C. At the end of centrifugation, the supernatant was recovered. 15 mL of supernatant were transferred into a new test tube and preserved in ice until loading into the UPLC system. The sample was diluted 1 :10 (first 1 :5 in a solvent and then 1 :2 in water). The diluted sample was filtered over 0.22 mm filters before being loaded into the UPLC system. The chromatography system used for quantification of the anthocyanins consists in an Acquity UPLC BEH C18 1.7 mm column, size 2.1 x 100 mm, coupled to an Acquity UPLC BEH C18 1.7 mm 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 eA 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: Table 2

For quantification of the anthocyanins, the chromatogram associated with the wavelength of 520 nm was used. The anthocyanins were quantified thanks to the calibration curve of the authentic commercial standard, cyanidin-3-O-glucoside (purity ³96%; Extrasynthese). The data analysis was carried out with Empower 3 software. Figure 4 shows the chromatographic profile of the DcAnA4 phytocomplex at 520 nm. The total anthocyanins were calculated as cyanidin-3-O-glucoside equivalents and amounted to 3.9 ± 0.08%. The total polyphenols (derivatives of caffeic acid) were quantified using the chromatogram obtained at the wavelength of 330 nm and thanks to the calibration curve of the commercial standard, chlorogenic acid (purity ³99%; Extrasynthese). The total polyphenols (derivatives of caffeic acid) were calculated as chlorogenic acid equivalents and amounted to 0.07±0.0002%.

b) HPLC-ESI-MS analysis of the DcAnA4 phytocomplex

The powder of the phytocomplex was extracted with 6 volumes of methanol/HCI (1 %V/V):water 90:10 for 15 minutes in a sonicator at 40Flz 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. Prior to the analysis, the sample was suitably diluted with water and filtered.

For the mass spectrometry associated with the chromatographic separation, use was made of an FIPLC system (Beckman Coulter System Gold 1 , Solvent Module provided with auto-sampler) coupled“on-line” with an Esquire 6000 mass spectrometer (Bruker Daltonik GmbH, Germany), provided with an ESI source. The chromatographic and mass data were collected using the Bruker Daltonics Esquire 5.2-EsquireControl 5.2 program and processed using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 program (Bruker Daltonik GmbH, Germany).

Flow: 200 mI/min, 25°C; injection volume: 10 mI.

Column used: Alltima HP C18 3 mm 150x2,1 mm, coupled with a 7.5 x 2.1 mm guard column (Alltech Associates, Inc, Derfield, IL).

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.

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

The untargeted metabolomic analyses were conducted as described by Commisso et al. (2013).

The chromatographic separation method used was the following:

Eluents:

-Solution A: 5% ACN and 0.5% formic acid in water, Solution B: 100% ACN

Total length of the elution: 42 minutes

Gradient:

-Start of analysis: eluent A 100%;

-gradient 1 : 10% of eluent B in 2 minutes;

-gradient 2: at 2 minutes, 20% of B in 10 minutes;

-gradient 3: at 12 minutes, 25% of B in 2 minutes;

-gradient 4: at 14 minutes, 70% of B in 7 minutes;

-isocratic 1 : with 70% of B for 6 minutes;

-gradient 5: at 27 minutes, 90% of B in 5 minutes;

-isocratic 2: 90% of B for 10 minutes;

-final re-equilibration of column: at 42 minutes, 0% of B in 1 minute, end of analysis at 60 minutes. In Figure 5 one may observe the chromatogram in 3 dimensions (retention time, values of m/z in the negative mode, intensity in arbitrary units) and Figure 6 shows the chromatogram of the DcAnA4 phytocomplex in two dimensions.

In the DcAnA4 phytocomplex one observes chlorogenic acid and several anthocyanins (AC1 : cyanidin-O-xylose-glucose-galactose, AC2: cyanidin- O-xylose-galactose, AC3: cyanidin-O-(ferulic acid)-xylose-glucose- galactose, AC4: cyanidin-O-(synaptic acid)-xylose-glucose-galactose) as the prevalent signals.

The data regarding the FIPLC-ESI-MS analysis in the negative ionization mode, the most suitable for plant extracts rich in acidic substances, were transformed, using the Bruker Daltonics Esquire 5.2-Data Analysis 3.2 software program (Bruker Daltonik GmbFI, Germany), from the proprietary Bruker format to the format .netcdf.

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

1 19 signals were revealed, 55 of which identified based on the retention time, m/z ratio and fragmentation pattern. The signals identified include metabolites plus the isotopes, fragments and adducts thereof. Table 3 below shows the retention times and m/z values of the signals identified.

Table 3

c) Quantitative analysis of polysaccharide content in the Dc-AnA4 phytocomplex

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 567 mg/g of Dc-AnA4 phytocomplex, equivalent to 56.7%.

d) Analysis of the protein content of the Dc-AnA4 phytocomplex

A determination of the total content of protein nitrogen was conducted on the Dc-AnA4 phytocomplex using the Kjeldahl method, as described in Lynch, J M. et al,“Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products” Journal of AOAC International (1999), 82(6), 1389-1398.

The protein content in the Dc-AnA4 phytocomplex was equal to 16.2% w/w.

e) Analysis of the lipid content of the Dc-AnA4 phytocomplex

The extraction of the total lipid fraction was carried out on the Dc-AnA4 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 Dc-AnA4 phytocomplex was equal to 2.8% w/w. f) Analysis of the moisture and ash in the Dc-AnA4 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 phytocomplex was equal to 3.2%, whilst the ash was equal to 9.7%.

Comparison of anthocvanin profile before and after selection of the cell line

Calluses of meristematic cells of Daucus carota sativa were analysed to determine their anthocyanin content prior to selection. The method of analysis used was the UPLC analysis described above. As is evident from figure 4, the selected line Dc-AnA4 (A) shows an anthocyanin content that is about 50 times greater than that of the initial cells (B).

Preparation of the Dc-AnA4 phvtocomplex on an industrial scale

Meristematic cells, stabilized and selected as previously described, cultured in solid DC medium (Gamborg B5 with 25 g/L of sucrose, 3 mg/L of NAA and 0.7% plant agar, final pH 5.5) were inoculated into 10 flasks with a 1 -litre capacity, containing 200 ml of DC liquid medium. 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 used to inoculate 10 flasks with a 3- litre capacity, containing 800 ml of DC liquid medium. 200 ml of the cell suspension was transferred into 800 ml of DC 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 DC-F medium (Gamborg B5 with the addition of 45 g/L of sucrose and 2 mg/L of NAA, final pH 5.5). After 14 days of growth in the bioreactor the plant biomass (90 litres of cell suspension) was collected and filtered over a nylon mesh with a porosity of 50 mm and washed with 50 L of sterile saline solution (0.9% W/V). The washed cells (fresh weight 25 kg) were supplemented with 250 g of citric acid and homogenized with an Ultra-Turrax.

The homogenized cells were dried. 2640 g of Dc-AnA4 phytocomplex were obtained from 90 litres of cell suspension.

Biological test: NFkB modulation with Dc-AnA4 phytocomplex on a model of irradiated reconstructed skin

An evaluation of the modulation of the nuclear transcription factor NFkB was performed on a model of irradiated reconstructed skin (Vitroscreen- Milan).

After arriving in the laboratory, the epidermal tissues are placed in specific culture media and incubated overnight at 37°C and 5% CO₂. After the overnight incubation, the extract to be tested is applied topically (15 mL of saline solution containing 3% w/w of Dc-AnA4 phytocomplex in glycerol) or systemically (0.03% and 0.1 % w/w of Dc-AnA4 phytocomplex in the culture medium). After a further overnight incubation, the tissues are exposed to UV irradiation (2MED=500J/m2 = 0.050J/cm2). After 4 hours of irradiation the tissues are collected, washed with saline solution and half of each tissue is fixed in formalin and embedded in paraffin, whereas the other half is used for PCR. The evaluation of the modulation of NFkB was carried out by immunostaining, using the anti-NFkB antibody (Abeam). The signal was viewed by means of a fluorescence microscope. The results obtained are shown in figure 7.

The Dc-AnA4 phytocomplex significantly decreases nuclear translocation after topical application (figure 7B). No significant decrease in nuclear translocation is observed with the systemic treatment. Biological test: modulation of VEGF-A gene expression with Dc-AnA4 phytocomplex on a model of microtissue of fibroblasts and endothelial cells

An evaluation of the modulation of the biosynthesis of VEGF-A gene expression was performed using a microtissue of fibroblasts and endothelial cells. The microtissues were prepared from dermal fibroblasts (from a 60-year-old donor) and endothelial cells of dermal microcirculation were subsequently added (the assay was performed in the VitroScreen laboratories-Milan). The assay was set up using the Dc-AnA4 phytocomplex in concentrations of 0.03% and 0.1 %. The Dc-AnA4 phytocomplex was added to the culture medium where the microtissues were incubated. After 72 hours of incubation at 37°C in 5% CO2, the microtissues were collected for PCR analysis. The microtissues were lysed in specific buffers and the extraction of nucleic acids was carried out using an RNA-kit (Life Technologies). A cDNA transcription kit was used to synthesize cDNA from the extracted RNA. A Fast Real Time PCR instrument (Applied Biosystems) was used to study the gene expression of VEGF-A. The results obtained are summarized in figure 8, expressed as an increase in gene expression. Both of the tested concentrations of the Dc-AnA4 phytocomplex produce an increase in the gene expression of VEGF-A. This result is correlated with a positive effect on endothelial microcirculation.

Formulation of the Dc-AnA4 phvtocomplex in two-phase and multiple emulsions and in gel form

The homogenate of the line Dc-AnA4 was dispersed in glycerine in a concentration equal to 3% w/w (INCI NAME: Glycerin (and) Daucus Carota Sativa Callus Lysate (and) Citric Acid). The dispersion was added at 3% to the formulas described below.

For the emulsion and the gel formulation, 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. Combine the two phases under the action of a turbo emulsifier. After cooling to about 40°C add the Dc-AnA4 phytocomplex dispersed in glycerine under gentle stirring. Table 4: Emulsions (two-phase and multiple O/A, A/O, A/S, A/O/A)

Table 5: Gel formulations

Claims

1. A meristematic cell line derived from a plant of Daucus carota sativa, wherein said cell line is obtained by means of a process comprising the steps of:

1 ) plating a tissue obtained from a plant belonging to the species

Daucus carota sativa onto a solid culture medium;

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium;

4) determining the anthocyanin, polyphenol and polysaccharide content for each clone;

5) selecting the cellular clone with the highest anthocyanin, polyphenol and polysaccharide content.

wherein the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA), and wherein the meristematic cell line comprises an amount of anthocyanins greater than 0.1% w/w, preferably comprised from 0.5 % to 10% w/w, an amount of polyphenols comprised from 0.01 % to 0.5% and an amount of polysaccharides of from 30 to 80% w/w, relative to the dry mass of the cell line.

2. The meristematic cell line according to claim 1 , wherein the solid and liquid culture media each comprise sucrose in a concentration comprised from 15 to 55 g/L preferably from 20 to 50 g/L, and NAA in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 4 mg/L.

3. The meristematic cell line according to claim 1 or 2, wherein the liquid culture medium comprises: sucrose in a concentration of from 20 to 55 g/L, preferably from 30 to 45 g/L, and naphthylacetic acid (NAA) in a concentration of from 0.5 to 4 mg/L, preferably from 1 to 3 mg/L.

4. The meristematic cell line according to any one of claims 1 to 3, wherein the solid culture medium comprises: sucrose in a concentration of from 15 to 35 g/L, preferably from 20 to 30 g/L, and naphthylacetic acid (NAA) in a concentration of from 2 to 5 mg/L, preferably from 2.5 to 4 mg/L.

5. The meristematic cell line according to any one of claims 1 to 4, wherein the salts suitable for the growth of plant cells are selected from: CaCl₂, KNO₃, MgSO₄ NaH₂PO₄, (NH₄)₂SO₄ and combinations thereof.

6. The meristematic cell line according to any one of claims 1 to 5, wherein the salts suitable for the growth of plant cells are selected from:

CoCl₂-6H₂O, CuSO₄-5H₂O, NaEDTA-2H₂O, FeSO₄-7H₂O, H₃BO₃, Kl, MnSO₄- H₂O, Na₂MoO₄-2H₂O, ZnSO₄-7H₂O and combinations thereof.

7. The meristematic cell line according to claim 5, wherein the solid and liquid culture media preferably each comprise CaCl₂ 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; MgSO₄ in a concentration comprised from 220 to 270 mg/L, preferably from 230 to 260 mg/L, NaH₂PO₄ in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L; and (NH₄)₂SO₄ in a concentration comprised from 100 to 180 mg/L, preferably from 1 10 to 150 mg/L.

8. The meristematic cell line according to claim 6, wherein the solid and liquid culture media preferably each comprise CoCl₂-6H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; CuSO₄-5H₂O in a concentration comprised from 0.01 to 0.05 mg/L, preferably from 0.015 to 0.03 mg/L; NaEDTA-2H₂O in a concentration comprised from 20 to 60 mg/L, preferably from 30 to 45 mg/L; FeSO₄-7H₂O 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; MnSO₄- H₂O in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L; Na₂MoO₄-2H₂O in a concentration comprised from 0.1 to 0.5 mg/L, preferably from 0.15 to 0.3 mg/L and ZnSO₄-7H₂O in a concentration comprised from 0.5 to 5 mg/L, preferably from 1 to 3 mg/L.

9. The meristematic cell line according to any one of claims 1 to 8, wherein both the solid and liquid culture media comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.

10. The meristematic cell line according to claim 9, wherein myo-inositol is present in a concentration comprised from 70 to 130 mg, preferably from 90 to 1 10 mg; pyridoxine-HCI is present in a concentration comprised from

70 to 130 mg, preferably from 90 to 1 10 mg; and thiamine-HCI is present in a concentration comprised from 5 to 20 mg/L, preferably from 7 to 15 mg/L.

1 1. The meristematic cell line according to any one of claims 1 -10, wherein said anthocyanins are selected in the group consisting of cyanidin-O-xylose-glucose-galactose, cyanidin-O-(synaptic acid)-xylose- glucose-galactose, cyanidin-O-(ferulic acid)-xylose-glucose-galactose, cyanidin-O-(coumaric acid)-xylose-glucose-galactose and mixtures thereof.

12. The meristematic cell line according to one of claims 1 to 11 , which comprises an amount of polyphenols comprised from 0.01 % to 0.2%, preferably from 0.05% to 0.1 %, said polyphenols being preferably selected in the group consisting of caffeoylquinic acid, ferulic acid, chlorogenic acid and mixtures thereof.

13. The meristematic cell line according to one of claims 1 to 12, which comprises an amount of polysaccharides of from 40% to 70% w/w.

14. The meristematic cell line according to one of claims 1 to 13, which comprises an amount of proteins of from 5 to 40% w/w, preferably from 10 to 30% w/w.

15. The meristematic cell line according to one of claims 1 to 14, which comprises an amount of lipids of from 1 to 20% w/w, preferably from 2 to

10% w/w.

16. A phytocomplex or an extract of the meristematic cell line according to any one of claims 1 -15.

17. A phytocomplex according to claim 16, which consists in dried or lyophilized cells, or a cellular homogenate, or cell walls and the components thereof, preferably a homogenate of said meristematic cell line.

18. A phytocomplex according to claim 16, which is a cellular homogenate and comprises an amount of anthocyanins greater than 0.1 % w/w, preferably comprised from 0.5% to 10% w/w relative to the dry mass of the phytocomplex.

19. A composition which comprises the meristematic cell line according to any one of claims 1 to 15 and/or the phytocomplex and/or the extract according to any one of claims 16 to 18.

20. The composition according to claim 19, comprising the meristematic cell line and/or the phytocomplex and/or the extract in a concentration comprised from 0.01 % to 1 % by weight, preferably from 0.03% to 0.5% by weight, more preferably from 0.05% to 0.2% by weight relative to the total composition.

21. The composition according to claim 19 or 20 which comprises a phytocomplex that is a homogenate in a concentration comprised from 0.05 to 0.2 %.

22. The composition according to any one of claims 19 to 21 , formulated as a cream, gel cream, gel, serum, oil, emulsion, emulsion gel (emulgel), ointment, eye drops, mouthwash, spray, preferably nasal spray, stick, pill, capsule, tablet, granular powder, hard-shelled capsule, orally dissolving granule, sachet, lozenge or liposome.

23. The meristematic cell line according to any one of claims 1 to 15, and/or the phytocomplex and/or the extract according to any one of claims 16 to 18 or the composition according to any one of claims 20-22, for use in the treatment and/or prevention of inflammations or microcirculation disorders and/or protection against UV radiation.

24. Use of the meristematic cell line according to any one of claims 1 -15, the phytocomplex and/or the extract according to any one of claims 16 to 18 or the composition according to any one of claims 20 to 22 as a nutraceutical.

25. A process for the preparation and selection of plant meristematic cells derived from a plant of Dacus carota sativa, said cells having an anthocyanin content greater than 0.1 % w/w, an amount of polyphenols comprised from 0.01 % to 0.5% w/w and an amount of polysaccharides of from 30 to 80% w/w relative to the dry mass of the cell line, said process comprising the steps of: 1 ) plating a tissue obtained from a plant of the species Daucus carota sativa onto a solid culture medium;

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium; 4) determining the anthocyanin, polyphenol and polysaccharide content for each clone;

wherein the solid and liquid culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA).

26. The process according to claim 25, wherein both the solid and liquid culture media comprise vitamins preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI and combinations thereof.

27. The process according to claim 25 or 26, comprising a phase of growth of the meristematic cell line by multiplication of the line in a liquid growth medium comprising salts suitable for the growth of plant cells, vitamins, sucrose, and NAA and subsequent transfer of the cells into a final liquid medium comprising salts suitable for the growth of plant cells, vitamins, sucrose, and NAA.