WO2022112862A1

WO2022112862A1 - Phytocomplex and extract of a meristematic cell line selected from perilla frutescens

Info

Publication number: WO2022112862A1
Application number: PCT/IB2021/057560
Authority: WO
Inventors: Giovanna Pressi; Oriana BERTAIOLA; Flavia GUZZO; Marco Biagi
Original assignee: Demethra Biotech S.R.L.
Priority date: 2020-11-24
Filing date: 2021-08-17
Publication date: 2022-06-02

Abstract

The present invention relates to a meristematic cell line selected from a plant belonging to the species Perilla frutescens, characterized by a high polyphenol and anthocyanin content, and the cosmetic, nutraceutical and medical use of said cell line or a derivative thereof. The meristematic cell line comprises an amount of polyphenols (in particular rosmarinic acidand salvianolic acid B) greater than 1% w/w, preferably comprised between 2.5 % and 10% w/w, more preferably between 3.2 and 5% w/w; and an amount of anthocyanins (in particular shisonin and malonyl-shisonin) greater than 0.05% w/w, preferably comprised between 0.05% and 1% w/w, more preferably between 0.1 and 0.5% w/w. The invention also relates to a phytocomplex or an extract of the cell line and the use thereof in the treatment and/or prevention of inflammations or as an adjuvant in skin repair.

Description

“Phytocomplex and extract of a meristematic cell line selected from Perilla frutescens”

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a meristematic cell line selected from a plant belonging to the species Perilla frutescens, characterized by a high polyphenol and anthocyanin content, and the cosmetic, nutraceutical and medical use of said cell line or a derivative thereof.

PRIOR ART

Perilla frutescens is an annual herbaceous plant belonging to the family Lamiaceae. It is a medicinal, ornamental and horticultural plant. Perilla has historical importance in Chinese medicine, as it was recorded as a drug around 500 A.D. It is used as an ornamental plant and in the food industry it is commonly used in salads, soups and sushi, as a spice and also as a food colouring. Some Perilla-based products are widely used to treat seasonal allergy symptoms and aphthae. The presence of phenolic compounds, flavonoids and anthocyanins in Perilla frutescens is amply documented in the scientific literature (Hiwa M. Ahmed, 2018, Molecules).

A high variability in the content of anthocyanins and polyphenols in said plants is associated with many factors which are difficult to control: seasons, plant age, geographical growing areas and different parts of the plant used for the preparation of products.

The preparation of standardized plant derivatives (i.e. extracts with a reproducible metabolite of metabolites) poses numerous problems tied to the variability of the metabolite content in different plant tissues, seasonal variability in both the content and type of metabolites, contaminations by plant parasites, differences tied to the geographical growing areas and loss of the molecule’s biological activity during harvest, storage and extraction. The extreme variability in the content of phytoconstituents of plant preparations obtained by extraction directly from a plant, or parts thereof, negatively impacts the effectiveness of the same.

One 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 and having anti-inflammatory activity can be derived.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a meristematic cell line selected from a plant belonging to the species Perilla frutescens, 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 meristematic cell line and a derivative thereof are characterized by a high content of polyphenols, in particular rosmarinic acid, and a high content of anthocyanins.

A third aspect of the invention relates to 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 invention relates to a process for the preparation and selection of plant meristematic cells of Perilla frutescens with a high content of polyphenols, in particular rosmarinic acid, and a high content of anthocyanins.

The Applicant has demonstrated that the meristematic cell line or a derivative thereof is active in inhibiting the activation of the JNK protein (belonging to the mitogen-activated protein kinase - MAPK - family), which translates into a strong ability to inhibit the release of pro-inflammatory cytokines and thus to exert a marked anti-inflammatory activity.

Therefore, the invention also relates to a cosmetic or pharmaceutical use of the meristematic cell line or of a derivative thereof in skin repair, preferably for the treatment of dermatitis due to skin hypersensitivity, allergic dermatitis or reddening.

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 the selected meristematic cell line, PFr09, on a specific solid medium, PFr. Figure 2 shows a magnification (200X) of a portion of figure 1.

Figure 3 shows the UPLC-DAD-MS chromatographic profile, in the negative ionization mode, of the phytocomplex deriving from the PFr09 line.

Figure 4 shows the anti-inflammatory activity of the phytocomplex deriving from the PFr09 line on keratinocytes stimulated with a medium of activated human monocytes. Figure 5 shows the activity of the phytocomplex deriving from the PFr09 line on the activation of the JNK protein in keratinocytes stimulated with a medium of activated human monocytes. Figure 6 shows the cell viability of J774A1 macrophages after treatment with the phytocomplex deriving from the PFr09 line and stimulation with LPS.

Figure 7 shows the anti-inflammatory activity of the phytocomplex deriving from the PFr09 line in J774 A1 macrophages on I L-1 b and TNF-a.

Figure 8 shows the skin repair activity of the phytocomplex deriving from the PFr09 line on human keratinocytes. 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 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 Perilla frutescens comprising an amount of polyphenols (in particular rosmarinic acid and salvianolic acid B) greater than 1% w/w, preferably comprised between 2.5 % and 10% w/w, more preferably between 3.2 and 5% w/w; and an amount of anthocyanins (in particular shisonin and malonyl-shisonin) greater than 0.05% w/w, preferably comprised between 0.05% and 1% w/w, more preferably between 0.1 and 0.5% 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 Perilla frutescens onto a solid culture medium (called “PFr solid medium”);

2) isolating a plurality of cellular clones;

3) inoculating each of the isolated clones into a liquid culture medium (called “PFr 1 liquid medium”);

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

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

In step 1), the tissue obtained from plants of the species Perilla frutescens is placed in a solid medium (PFr solid medium) in order to obtain an undifferentiated callus tissue. Preferably, the tissue of Perilla frutescens is at least one young leaf of Perilla frutescens or a plurality of young leaves of Perilla frutescens. The collected tissue is kept in the dark at room temperature (T = 20-25°) until obtaining the undifferentiated callus tissue.

In a preferred embodiment of the invention, the solid (PFr solid medium) and liquid (PFr 1 liquid medium) culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA) and indoleacetic acid (IAA). 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 between 15 and 60 g/L, preferably between 20 and 55 g/L; naphthylacetic acid (NAA) in a concentration of 0.05 to 2 mg/L, preferably 0.1 to 1 mg/L; indoleacetic acid (IAA) in a concentration of 0.3 to 3 mg/L, preferably 0.5 to 2 mg/L.

In a preferred embodiment, the solid culture medium (PFr solid 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 of 0.1 a 2 mg/L, preferably 0.3 a 1 mg/L; indoleacetic acid (IAA) in a concentration of 0.5 to 3 mg/L, preferably 0.8 to 2 mg/L.

Preferably, the solid culture medium comprises 0.7-0.9% of plant agar.

In a preferred embodiment, the liquid culture medium (PFr 1 liquid medium) comprises: sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L, naphthylacetic acid (NAA) in a concentration of 0.05 to 1 mg/L, preferably 0.1 to 0.8 mg/L; and indoleacetic acid (IAA) in a concentration of 0.3 to 2 mg/L, preferably 0.5 to 1 mg/L.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgS0_4, NaFhPCL, (NFU^SCL and combinations thereof. Preferably, in both the solid and liquid culture media the salts suitable for the growth of plant cells are selected from: CoCL-ehhO, CuS0₄-5H₂0, NaEDTA-2H₂0, FeS0₄-7H₂0, H3BO3, Kl, MnSCL-FhO, Na₂Mo0₄-2H₂0, ZnS0₄-7H₂0 and combinations thereof.

Both the solid and liquid culture media further comprise vitamins suitable for the growth of plant cells, preferably selected from: myo-inositol, nicotinic acid, pyridoxine-HCI, thiamine- HCI and combinations thereof.

In one embodiment, in both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgS0_4, NaFhPCL, (NFU^SCL, COCI₂-6H₂0, CUS0₄-5H₂0, NaEDTA-2H₂0, FeS0₄-7H₂0 H3BO3, Kl, MnS0₄-H₂0,

Na₂Mo0₄-2H₂0 and ZnS0₄-7H₂0 and combinations thereof. This combination of compounds is the Gamborg B5 medium.

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 CaCL in a concentration comprised between 120 and 170 mg/L, preferably between 130 and 160 mg/L; KNO₃ in a concentration comprised between 800 and 3000 mg/L, preferably between 1000 and 2600 mg/L; MgS0₄ in a concentration comprised between 220 and 270 mg/L, preferably between 230 and 260 mg/L, NaH₂P0₄ in a concentration comprised between 100 and 180 mg/L, preferably between 110 and 150 mg/L; and (NFU^SCL in a concentration comprised between 100 and 180 g/L, preferably between 110 and 150 mg/L.

The solid and liquid culture media preferably each comprise CoCL-ehhO in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; CUSC>₄-5H₂0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; NaEDTA-2H₂0 in a concentration comprised between 20 and 60 mg/L, preferably between 30 and 45 mg/L; FeSCL-TFhO in a concentration comprised between 15 and 45 mg/L, preferably between 20 and 35 mg/L; H₃BO₃ 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-FhO in a concentration comprised between 5 and 20 mg/L, preferably between 7 and 15 mg/L; Na₂Mo0₄-2H₂0 in a concentration comprised between 0.1 and 0.5 mg/L, preferably between 0.15 and 0.3 mg/L, and ZnSC>₄-7H₂0 in a concentration comprised between 0.5 and 5 mg/L, preferably between 1 and 3 mg/L.

Both 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.

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 (PFr solid medium) (step 1a)) so as to obtain stabilized cells. This step takes the name of stabilization step.

After the stabilization step 1a), the stabilized cells preferably undergo a first “clonal selection". The clonal selection consists in culturing the stabilized cells in the PFr solid medium for an adequate duration, preferably from 5 to 25 days of culture, more preferably 15 to 22 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.

In step 2), a plurality of cellular clones is isolated by taking aggregates of stabilized cells from the solid culture medium (PFr solid 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 25 days, in step 4) the polyphenol and anthocyanin content of each clone is determined.

In one embodiment, in step 5), a second clonal selection according to step 1b) is preferably carried out until obtaining a plant cell line of Perilla frutescens wherein the production of polyphenols and anthocyanins is optimal. In a preferred embodiment, the clonal selection of step 5) is repeated until obtaining a cell line of Perilla frutescens which comprises an amount of polyphenols greater than 1% w/w, preferably comprised between 2.5 % and 10% w/w, more preferably between 3.2 and 5% w/w, relative to the dry mass of the cell line. Preferably, said polyphenols are selected in the group consisting of rosmarinic acid, salvianolic acid B and mixtures thereof.

In one embodiment, said selected meristematic cell line comprises anthocyanins in an amount greater than 0.05% w/w, preferably comprised between 0.05% and 1% w/w, more preferably between 0.1 and 0.5% w/w, said anthocyanins preferably being selected in the group comprising shisonin, malonyl-shisonin and mixtures thereof.

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 5 and 30% w/w, preferably between 10 and 20% w/w.

The selected meristematic cell line according to the invention further comprises an amount of lipids between 0.5 and 20% w/w, preferably between 1 and 10% w/w.

In a preferred embodiment, said selected meristematic cell line is the PFr09 line, which comprises polyphenols in amounts greater than 1% w/w relative to the dry mass of the cell line and anthocyanins in an amount greater than 0.05% w/w. The polyphenols are preferably comprised between 2.5% and 10% w/w, more preferably between 3.2 and 5% w/w; the anthocyanins are comprised between 0.05% and 1% w/w, more preferably between 0.1% and 0.5% w/w.

A second aspect of the present invention relates to a derivative of the cell line which is a phytocomplex or an extract of the selected meristematic cell line.

Phytocomplex means: dried or lyophilized cells, a cellular homogenate, or the cell walls and the components thereof. The phytocomplex is preferably a cellular homogenate.

Said phytocomplex comprises polyphenols in amounts greater than 1% w/w and anthocyanins in amounts greater than 0.05% w/w.

The polyphenols are preferably comprised between 2.5% and 10% w/w, more preferably between 3.2 and 5% w/w; the anthocyanins are comprised between 0.05% and 1% w/w, more preferably between 0.1% and 0.5% w/w.

Preferably, said polyphenols are selected in the group comprising rosmarinic acid, salvianolic acid B and mixtures thereof.

The anthocyanins are preferably selected in the group comprising shisonin, malonyl-shisonin and mixtures thereof. 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 the phytocomplex.

The phytocomplex further also comprises an amount of proteins between 5 and 30% w/w, preferably between 10 and 20% w/w relative to the dry mass of the phytocomplex.

The phytocomplex further also comprises an amount of lipids between 0.5 and 20% w/w, preferably 1 and 10% w/w, relative to the dry mass of the phytocomplex.

In a preferred embodiment, said phytocomplex is a derivative of the selected meristematic cell line PFr09 and comprises polyphenols in amounts greater than 1% w/w and anthocyanins in an amount greater than 0.05% w/w.

Preferably, the polyphenols are comprised between 2.5% and 10% w/w, more preferably between 3.2 and 5% w/w; the anthocyanins are comprised between 0.05% and 1% w/w, more preferably between 0.1% and 0.5% w/w.

The phytocomplex is preferably a cellular homogenate of the selected meristematic cell line PFr09.

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 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.

In one embodiment, the cell line and/or a derivative thereof is 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 a stick (similar to cocoa butter).

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 derivatives thereof possess anti-inflammatory and skin repair activity.

The subject matter of the present invention relates to the use of the meristematic line or derivatives thereof for the activity of inhibiting the JNK protein, which translates into a strong capacity to inhibit the release of pro-inflammatory cytokines and thus to exert a marked anti inflammatory activity. The invention relates to the use of the meristematic cell line and/or derivatives thereof for skin repair, preferably for the treatment of dermatitis due to skin hypersensitivity, allergic dermatitis and reddening. In this case, the administration is preferably topical.

The invention also relates to a method for treating conditions tied to skin alterations, preferably skin hypersensitivity, allergic dermatitis or reddening, the method comprising the administration of an effective amount of the meristematic cell line and/or a derivative thereof to a patient in need thereof. Preferably, the administration is topical, on the skin.

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 inflammatory states or as an adjuvant in skin repair. The invention relates to a method for treating alterations ascribable to inflammatory states or for accelerating the process of skin repair, which comprises oral administration of a dietary supplement comprising the meristematic cell line and/or a derivative thereof to a patient in need.

Another aspect of the present invention relates to a process for preparing and selecting plant meristematic cells with a high polyphenol content, preferably with a polyphenol content greater than 1% w/w relative to the dry mass of the cell line and with a high anthocyanin content, preferably greater than 0.05% w/w.

Said process comprises the steps of: 1 ) plating tissues obtained from plants of Perilla frutescens onto a solid culture medium (called “PFr solid medium”);

2 ) isolating a plurality of cellular clones;

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

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

In one embodiment, the preparation of meristematic cells entails collecting tissue, preferably of leaves from selected plants of the species Perilla frutescens, 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 (“PFr solid medium”) in order to obtain an undifferentiated callus tissue. The collected tissue is kept in the dark at room temperature (T = 20-25°) until obtaining the undifferentiated callus tissue.

In a preferred embodiment of the invention, the solid (“PFr solid medium”) and liquid (“PFr 1 liquid medium”) culture media comprise salts suitable for the growth of plant cells, sucrose, naphthylacetic acid (NAA) and indoleacetic acid (IAA).

The solid culture media further comprises agar, whereas the liquid culture media does not contain agar.

In a preferred embodiment, the solid culture medium (PFr solid 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 of 0.1 to 2 mg/L, preferably 0.3 to 1 mg/L; indoleacetic acid (IAA) in a concentration of 0.5 to 3 mg/L, preferably 0.8 to 2 mg/L. Preferably, the solid culture medium comprises 0.7-0.9% of plant agar.

In a preferred embodiment, the liquid culture medium (PFr 1 liquid medium) comprises: sucrose in a concentration of 15 to 55 g/L, preferably between 20 and 50 g/L; naphthylacetic acid (NAA) in a concentration of 0.05 to 1 mg/L, preferably 0.1 to 0.8 mg/L; indoleacetic acid (IAA) in a concentration of 0.3 to 2 mg/L, preferably 0.5 to 1 mg/L.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are selected from: CaCL, KNO3, MgSCL, NahkPC , (NFU^SCL and combinations thereof.

In both the solid and liquid culture media, the salts suitable for the growth of plant cells are preferably selected from: CoCL-ehhO, CuSCL-SFhO, NaEDTA-2H₂0, FeSCL-TFhO, H₃BO₃, Kl, MnSCL-FhO, Na₂MoC>₄-2H₂0, ZnSCL-TFhO 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: CaCI₂, KNO3, MgS0_4, NaH₂P0₄, (NH₄)₂S0₄, COCI₂-6H₂0, CUS0₄-5H₂0, NaEDTA-2H₂0, FeS0₄-7H₂0 H3BO3, Kl, MnS0₄-H₂0,

The solid and liquid culture media preferably each comprise CaCI₂ in a concentration comprised between 120 and 170 mg/L, preferably between 130 and 160 mg/L; KNO₃ in a concentration comprised between 800 and 3000 mg/L, preferably between 1000 and 2600 mg/L; MgS0₄ in a concentration comprised between 220 and 270 mg/L, preferably between 230 and 260 mg/L, NaH₂P0₄ in a concentration comprised between 100 and 180 mg/L, preferably between 110 and 150 mg/L; and (NH₄)₂S0₄ 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 CoCI₂-6H₂0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; CUS0₄-5H₂0 in a concentration comprised between 0.01 and 0.05 mg/L, preferably between 0.015 and 0.03 mg/L; NaEDTA-2H₂0 in a concentration comprised between 20 and 60 mg/L, preferably between 30 and 45 mg/L; FeS0₄-7H₂0 in a concentration comprised between 15 and 45 mg/L, preferably between 20 and 35 mg/L; H₃BO₃ 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; MnS0₄-H₂0 in a concentration comprised between 5 and 20 mg/L, preferably between 7 and 15 mg/L; Na₂Mo0₄-2H₂0 in a concentration comprised between 0.1 and 0.5 mg/L, preferably between 0.15 and 0.3 mg/L, and ZnS0₄-7H₂0 in a concentration comprised between 0.5 and 5 mg/L, preferably between 1 and 3 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 (PFr solid medium) (step 1a)) so as to obtain stabilized cells. This step takes the name of stabilization step. After the stabilization step 1a), the stabilized cells undergo a first "clonal selection". The clonal selection consists in culturing the stabilized cells in the PFr solid medium for an adequate duration, preferably from 5 to 25 days of culture, more preferably 15 to 22 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.

In one embodiment, in step 5) a second clonal selection according to step 1b) is preferably carried out until obtaining a plant cell line of Perilla frutescens wherein the production of polyphenols and 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 (i.e. the growth of the biomass) takes place in a first step in the same liquid medium as used for the clonal selection (PFr 1 liquid medium). The PFr 1 liquid medium is a medium containing the Gamborg salts specified above, the vitamins listed above, sucrose, naphthylacetic acid and indoleacetic acid at a final pH of 6.5.

The PFr 1 liquid medium contains sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L; naphthylacetic acid (NAA) in a concentration of 0.05 to 1 mg/L, preferably 0.1 to 0.8 mg/L; and indoleacetic acid (IAA) in a concentration of 0.3 to 2 mg/L, preferably 0.5 to 1 mg/L.

The cells cultured in the PFr 1 liquid medium are transferred, for the final phase of growth, into a final PFr 2 liquid medium containing the Gamborg salts, vitamins, sucrose, NAA and IAA, which induce an increase in the polyphenol and anthocyanin content and biomass.

The final PFr 2 liquid medium contains the Gamborg salts, sucrose, which is preferably comprised between 20 g/L and 50 g/L, naphthylacetic acid (NAA), preferably in a concentration of 0.1 to 0.8 mg/L; and indoleacetic acid (IAA), preferably in a concentration of 0.5 to 1 mg/L, with a final pH of 6.5.

According to a preferred embodiment, the growth of the cell line in the flask, bioreactor or fermenter, both in the PFr 1 liquid medium and in the final PFr 2 liquid medium, takes place at a temperature comprised between 15 °C and 35 °C, typically about 25 °C, and for a period comprised from 7 to 30 days, preferably 15 to 22 days, and under conditions of darkness. At the end of growth in the final PFr 2 liquid medium, 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 may 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 and/or citric acid and/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 and/or ethanol) using conventional techniques. The extract thus obtained is characterized by a high polyphenol and 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 of derivatives thereof, which have a surprisingly high polyphenol and anthocyanin content, for the uses described herein. A surprising effectiveness of a derivative of said selected cell line was likewise demonstrated, wherein said derivative is a homogenate, or a phytocomplex that comprises all the cellular components, without the cells being exposed to solvents or other extraction procedures that could compromise the integrity of the active ingredients.

The inventors of the present invention have in fact surprisingly verified that the cell line of the invention and/or the derivatives thereof exhibit a marked anti-inflammatory activity through the inhibition of the JNK protein, which translates into a strong capacity to inhibit pro- inflammatory cytokines, an inhibition of leukocyte infiltration and skin repair activity.

EXAMPLES

Generation and selection of lines of meristematic cells of Perilla frutescens 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 Perilla frutescens, 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 30’, 2% sodium hypochlorite and 0.1% Tween 20 for about 3- 5 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 (PFr solid medium).

As regards 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, 0.5 mg/L of NAA, 1 mg/I of IAA and 0.7-0.9% of plant agar, final pH 6.5 (PFr solid medium).

As regards the 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, 0.5 mg/L of NAA and 1mg/l of IAA, final pH 6.5 (PFr 1 liquid medium).

As regards 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 50 g/L of sucrose, 0.3 mg/ NAA and 0.8mg/L of IAA, final pH 6.5 (final PFr 2 liquid medium).

The cell line obtained in this specific culture medium, after clonal selection in the PFr solid medium (with agar) and PFr 1 liquid medium (without agar), was called PFr09. The belonging of the meristematic cells obtained to the botanical species Perilla frutescens 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 the PFr 1 liquid medium, the cell suspensions were transferred into bioreactors containing the PFr 2 liquid culture medium for further phases of growth.

At the end of growth in PFr 2 medium, the cell suspensions were filtered and the biomasses were recovered and used for the subsequent steps of preparing the phytocomplexes.

The characteristics of the cell line of Perilla frutescens called PFr09 will be described by way of non-limiting example.

Morphological characteristics of the cell line

The selected cell line of Perilla frutescens, called PFr09, is maintained in PFr solid medium, is purple-brown in colour and has a friable texture (Figures 1 and 2).

Homogenization procedure

The procedure for homogenizing the biomasses of cells selected and grown in bioreactors for 21 days at 25°C (±2) comprises the following steps: a) filtration of the biomass obtained from the growth of the PFr09 cell culture in the final PFr 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 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 PFr09.

Description of the content of the PFr09 homogenate:

20-60% polysaccharides

0.1-0.5% total anthocyanins (in particular shisonin and malonyl-shisonin)

2.5-10% total polyphenols (in particular rosmarinic acid and salvianolic acid B)

8-20% proteins 0.6-3% lipids 2-8% moisture 2-10% ash 5-30% citric acid.

Examples of preparation of the standardized PFr09 phytocomplex in PFr 2 liquid 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 PFr09 phvtocomplex

Meristematic cells, stabilized and selected as previously described, cultured in PFr solid medium (Gamborg B5 with 20 g/L of sucrose, 0.5mg/L of NAA, 1mg/L of IAA and 0.8% plant agar, final pH 6.5) were inoculated into 5 flasks with a 1-litre capacity, containing 200 ml of PFr1 liquid medium (Gamborg B5 with the addition of 20 g/L of sucrose, 0.5mg/L of NAA, 1mg/L of IAA, final pH 6.5). The amount of meristematic cells inoculated into the liquid medium was equal to 10% 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 14 days of incubation, every cell suspension (200 ml) contained in a 1 -litre flask was poured into a flask with a 3- litre capacity, containing 800 ml of final PFr2 liquid medium (Gamborg B5 with the addition of 50 g/L of sucrose, 0.3mg/L of NAA and 0.8mg/L of IAA, 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 21 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 2.2 L of sterile saline solution (0.9% W/V). The washed cells (fresh weight 1100 g) were supplemented with 11 g of citric acid and homogenized with an UltraTurrax. The homogenized cells were lyophilized. 140 g of lyophilizate (PFr09 phytocomplex) with a content of polyphenols equal to 5.33 g, anthocyanins equal to 0.29 g, total polysaccharides equal to 65.8 g, proteins equal to 30.8 g, lipids equal to 3.5 g, ash equal to 15.8 g and citric acid equal to 10.6 g were obtained from 5 litres of cell suspension.

Table 1 shows the characterization of the phytocomplex:

Table 1

The characterization of the phytocomplex was carried out using the methods described below: l. UPLC-ESI-MS analysis of the PFr09 phytocomplex

The powder of the phytocomplex was extracted with methanol/HCI 99/1 (V/V) 100% for 15 minutes in a sonicator bath, and the methanolic extract was recovered by centrifugation. The volume of the extraction solvent relative to the sample weight was 1:10.

Just before the analysis, the samples were further diluted with water in a 1:4 ratio and filtered.

The instruments used to carry out reversed-phase chromatography consisted in an Acquity I- class Ultra Performance Liquid Chromatography (UPLC) system (Waters) equipped with a BEH C-18 column (100 x 2.1 mm; 2.7 pm) temperature controlled at 30°C and an FTN autosampler (Waters) refrigerated at 10°C. The chromatographic method used adopts a 20- minute-long “gradient”-type elution with the use of two solvents: water with the addition of 0.1% formic acid (A) and acetonitrile (B). The imposed flow rate was 0.350 ml/min. Details of the method are shown in the table below. Table 2

Once eluted from the chromatographic column, 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 chromatograms, in the positive and negative ionization modes, were viewed and analysed by means of MassLynx v.4.2. The data matrix, in the negative ionization mode, was generated by means of ProgenesisQI software (http://www. nonlinear.com/ ).

Extrapolation of the fragmentation spectra (ms/ms) for the various metabolites was conducted with ProgenesisQI. The metabolites were identified by comparing the exact mass (rounded to 3 decimal places), the isotopic pattern and the fragmentation pattern with those of a proprietary in silico library of mass structures. Figure 3 shows the chromatographic profile of the PFr09 phytocomplex in the negative ionization mode. The main peaks that were identified are indicated in the chromatographic profile: rosmarinic acid, salvianolic acid, a caffeic acid derivative, caffeoyl hexoside and rosmarinic acid methyl ester. The group indicated as AC refers to the anthocyanins, of which shisonin and malonyl- shisonin were identified. a) UPLC-DAD quantification of total polyphenols and total anthocyanins in the PFr09 phytocomplex

100mg of powder of the PFr09 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 in 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 the supernatant were transferred into a new test tube and kept in ice until being loaded into the UPLC column. The sample was diluted 1:10 (first 1:5 in solvent and then 1:2 in water). The diluted sample was filtered over 0.22 pm filters before being loaded into the UPLC column. The chromatographic system used to quantify the polyphenols and anthocyanins 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 3:

Table 3

For the quantification of the total polyphenols, the chromatogram for the wavelength of 330 nm was used. The total polyphenols were quantified thanks to the calibration curve of the authentic commercial standard of rosmarinic acid (CAS 20283-92-5; purity³95%; Sigma Aldrich).

The anthocyanins were quantified thanks to the calibration curve of the authentic commercial standard of cyanidin-3-O-glucoside (purity ³96%; Extrasynthese).

The data analysis was carried out with Empower 3 software. The total polyphenols were calculated as rosmarinic acid equivalents and were found to be equal to 3.8 ± 0.07%.

The total anthocyanins were calculated as cyanidin-3-O-glucoside equivalents and were found to be equal to 0.2 ± 0.03% c) Quantitative analysis of the polysaccharide content in the PFr09 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 470 mg/g of PFr09 phytocomplex, equivalent to 47%. d) Analysis of the protein content of the PFr09 phytocomplex

A determination of the total content of protein nitrogen was conducted on the PFr09 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 PFr09 phytocomplex was equal to 22% w/w. e) Analysis of the lipid content of the PFr09 phytocomplex

The extraction of the total lipid fraction was carried out on the PFr09 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 PFr09 phytocomplex was equal to 2.5% w/w. f) Analysis of the moisture and ash in the PFr09 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 5.5%, while the ash was equal to 11.3%. Biological test: anti-inflammatory activity on skin

Non-tumoral immortalized keratinocytes originating from adult human skin (HaCat) were used in the study. The test of anti-inflammatory activity was carried out by treating human keratinocytes for 24 hours with the samples of the PFr09 phytocomplex; the keratinocytes were simultaneously stimulated with a medium derived from the culture of human monocytes (THP-1) activated with bacterial LPS, as a simulation of a condition of acute dermatitis. The inflammatory parameters, assayed by means of a new-generation ELISA kit, were the cytokines TNF-a, I L-1 b and IL-60. The PFr09 phytocomplex was also studied to assess its ability to inhibit cytokine release by human monocytes stimulated with LPS.

The experimental model set up showed that all of the assayed inflammatory cytokines undergo upregulation when the keratinocytes are stimulated with a conditioned medium. The expression of TNF-a undergoes an approximately 10-fold increase compared to the control, whilst I L-1 b and IL-6 increases about 2 times.

The PFr09 phytocomplex demonstrated to be surprisingly effective in inhibiting the release of pro-inflammatory cytokines in keratinocytes treated with a dose of 100 pg/ml. The PFr09 phytocomplex demonstrated to possess a high anti-inflammatory capacity and the excessive release of TNF-a, I L-1 b and IL-6 was almost completely nullified by treatment with the phytocomplex.

Figure 4 shows the overall data of the cytokine assay on stimulated keratinocytes treated with the PFr09 phytocomplex.

Biological test: modulation of the JNK protein in keratinocytes

The activation of the mitogen-activated protein kinase (MAPK) JNK was studied in this test by measuring, by means of an ELISA kit, the ratio between the phosphorylated JNK protein (p-JNK) and the total JNK protein in human keratinocytes stimulated for 30, 60 and 90 minutes with a medium derived from a culture of human monocytes activated with bacterial LPS. The test was carried out to assess the ability of the PFr09 phytocomplex to reduce the activation of JNK, which is a signal upstream of the NF-kB signalling pathway, connected with the transcription and synthesis of pro-inflammatory cytokines.

In the skin inflammation model used, it was possible to monitor upstream signals such as MAPKs, which trigger cellular events leading to the release of pro-inflammatory cytokines. In particular, JNK activation showed to be marked after 60 minutes of stimulation with the medium of monocytes stimulated with LPS. As already shown in similar experiments, MAPK activation has a typical time-dependent pattern and at 30 and 90 minutes the effect is almost nil.

As shown in figure 5, the PFr09 phytocomplex at 100 pg/ml is capable of completely countering the activation of JNK stimulated by the medium at 60 minutes and is able to reduce the activation of this MAPK also in non-stimulated keratinocytes. The PFr09 phytocomplex inhibits signal transduction pathways such as JNK, and this translates into a strong ability to inhibit the release of pro-inflammatory cytokines.

Biological test: leukocyte infiltration

The infiltration test of was carried out according to the protocol published by Governa et al. , 2018 (doi: 10.3390/ph11040126), modified: the keratinocytes were seeded onto a prepared slide and cultured until obtaining a monolayer. The cells were treated with the PFr09 phytocomplex and then with bacterial LPS for 24 hours. The THP-1 cells (monocytes) were subsequently co-cultured. After washing, the slide was stained with eosin-haematoxylin and the images were microscopically analysed.

The infiltration test provided visual confirmation that the PFr09 phytocomplex, at the effective concentration of 100 pg/ml, was able to maintain the integrity of the monolayer of keratinocytes, altered in inflammatory conditions.

Biological test: anti-inflammatory activity on J774 A1 macrophages

The anti-inflammatory activity of the PFr09 phytocomplex was assessed using a J774 A1 macrophage cell line after stimulation with LPS. The test used is described in Paterniti et al., Nutrients, 2017.

In order to assess the protective effect of the PFr09 phytocomplex (ShisoPure-PC) in macrophage cells after stimulation with LPS, cell viability was determined with the MTT colorimetric method. Figure 6 shows the cell viability data after treatment with the PFr09 phytocomplex at concentrations of 0.005, 0.01 and 0.05 mg/mL and with LPS. The results indicate that the PFr09 phytocomplex exerts a significant protective effect against LPS at the concentrations of 0.01 and 0.05 mg/mL.

For the purpose of assessing the anti-inflammatory activity of the PFr09 phytocomplex (ShisoPure-PC), the following parameters were determined by western blot analysis: IkB-o, iNOS and COX-2. The results obtained indicate that, at a concentration of 0.01 mg/mL and 0.05 mg/mL, the PFr09 phytocomplex (ShisoPure-PC) significantly decreases the expression of the enzymes iNOX and COX-2 and prevents the degradation of IkB-o.

The levels of the pro-inflammatory cytokines TNF-o and I L-1 b were evaluated by ELISA. After stimulation with LPS, an increase in pro-inflammatory cytokines is observed. Treatment with the PFr09 phytocomplex (Shiso-Pure-PC) at a concentration of 0.01 mg/mL and 0.05 mg/mL significantly decreases the level of these cytokines, as shown by the graphs in figure 7.

The overall results obtained demonstrate that the PFr09 phytocomplex (ShisoPure-PC) exerts a marked anti-inflammatory activity through the modulation of the pro-inflammatory enzymes iNOS and COX-2 and the cytokines TNF-o and I L- 1 b . Biological test: skin repair test

For the skin repair test, a wound was simulated by making a cross-shaped cut on the monolayer of keratinocytes. The cut was made with a sterile tip.

The cells were treated with the PFr09 phytocomplex. At time 0 (tO), after 6 and 24 hours (t6 and t24), digital photographic analyses were performed on the cells and the diagonals of the cut were measured, with a double measurement per sample. All of the tests were conducted in triplicate. The analyses were conducted by means of ImageJ software.

The skin repair test conducted with the PFr09 phytocomplex showed that the plant sample is effective at 100 pg/ml, in particular after a 24-hour treatment. At 24 hours, the increase in the rate of healing of the monolayer of keratinocytes produced by the PFr09 phytocomplex at 100 pg/mL was over 40% compared to the control (p<0.05 vs Ctrl); after 6 hours the healing rate improved by about 35%.

Figure 8 shows the graph of the test results recorded at 24 hours.

Formulation of the phvtocomplex of Perilla frutescens PFr09 in two-phase and multiple emulsions and in gels.

The PFr09 phytocomplex was dispersed in glycerine in a concentration of 3% w/w (PFr09- G). 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 PFr09 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 selected from a plant belonging to the species Perilla frutescens and obtained by means of a process comprising the steps of:

1 ) plating a tissue obtained from a plant belonging to the species Perilla frutescens 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 polyphenol and anthocyanin content for each clone;

5) selecting the cellular clone with an amount of polyphenols greater than 1% w/w, preferably comprised between 2.5 % and 10% w/w, more preferably between 3.2 and 5% w/w, and with an amount of anthocyanins greater than 0.05% w/w, preferably comprised between 0.05% and 1%w/w, more preferably between 0.1 and 0.5% w/w, relative to the dry mass of the cell line; wherein the solid and liquid media comprise: salts suitable for plant cell growth, sucrose, naphthyl acetic acid (NAA) and indoleacetic acid (IAA).

2. The meristematic cell line according to claim 1, wherein the solid and liquid media each comprise sucrose in a concentration comprised between 15 and 60 g/L, preferably between 20 and 55 g/L; naphthyl acetic acid (NAA) in a concentration of 0.05 to 2 mg/L, preferably 0.1 to 1 mg/L; and indoleacetic acid (IAA) in a concentration of 0.3 to 3 mg/L, preferably 0.5 to 2 mg/L.

3. The meristematic cell line according to claim 1 or 2, wherein the liquid culture medium comprises: sucrose in a concentration of 15 to 55 g/L, preferably 20 to 50 g/L, naphthyl acetic acid (NAA) in a concentration of 0.05 to 1 mg/L, preferably 0.1 to 0.8 mg/L; and indoleacetic acid (IAA) in a concentration of 0.3 to 2 mg/L, preferably 0.5 to 1 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 15 to 35 g/L, preferably between 20 and 30 g/L, naphthyl acetic acid (NAA) in a concentration of 0.1 to 2 mg/L, preferably 0.3 to 1 mg/L; and indoleacetic acid (IAA) in a concentration of 0.5 to 3 mg/L, preferably 0.8 to 2 mg/L.

5. The meristematic cell line according to any one of claims 1 to 4, wherein said polyphenols are selected in the group consisting of rosmarinic acid, salvianolic acid B and mixtures thereof.

6. The meristematic cell line according to any one of claims 1 to 5, wherein said anthocyanins are selected in the group comprising shisonin, malonyl-shisonin and mixtures thereof.

7. A phytocomplex or an extract of the meristematic cell line according to any one of claims 1-6.

8. A phytocomplex according to claim 7, which consists in dried or lyophilised cells, or in a cellular homogenate, or in cell walls and components thereof; it is preferably a homogenate of the meristematic cell line.

9. A phytocomplex according to claim 7 or 8, which is a cellular homogenate comprising an amount of polyphenols greater than 1% w/w, preferably comprised between 2.5 % and 10% w/w, more preferably between 3.2 and 5% w/w, and an amount of anthocyanins greater than 0.05% w/w, preferably comprised between 0.05% and 1% w/w, more preferably between 0.1 and 0.5% w/w, relative to the dry mass of the cell line.

10. A composition comprising the meristematic cell line according to any one of claims 1 to 6, and/or the phytocomplex and/or the extract according to any one of claims 7 to 9.

11. The meristematic cell line according to any one of claims 1-6, the phytocomplex and/or the extract according to any one of claims 7 - 9, the composition according to claim 10, for use in skin repair, preferably for the treatment of dermatitis due to skin hypersensitivity, allergic dermatitis and reddening.

12. The meristematic cell line according to any one of claims 1-6, the phytocomplex and/or the extract according to any one of claims 7 - 9, the composition according to claim 10, for use in the treatment and/or prevention of inflammations or as an adjuvant in skin repair.

13. Use of the meristematic cell line according to any one of claims 1-6, the phytocomplex and/or the extract according to any one of claims 7 - 9, the composition according to claim 10, as a cosmetic by application on the skin or as a dietary supplement to be taken orally for the prevention of or as an adjuvant treatment of alterations ascribable to inflammatory states or as an adjuvant in skin repair.