WO2023021317A1

WO2023021317A1 - Plant cells stabilized with lecithin

Info

Publication number: WO2023021317A1
Application number: PCT/IB2021/057553
Authority: WO
Inventors: Rachid Ennamany; Bruno Guillaume; Rachid ANANE
Original assignee: Rachid Ennamany
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2023-02-23

Abstract

Homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium, said stable plant cells having each a cell wall membrane defining an inner cell volume containing a central vacuole surrounded by a tonoplast membrane, some bi-layered lecithin membrane fragments being fused the wall membrane or the tonoplast membrane for forming a lecithin protecting membrane structure.

Description

PLANT CELLS STABILIZED WITH LECITHIN

The present invention relates to a homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium, said stable enriched living plant cells having each a cell wall membrane defining an inner cell volume containing a central vacuole surrounded by a tonoplast membrane, said central vacuole comprising a plant sap from said first plant species. The wall membrane of plant cells or agglomerates thereof is associated with bi-layered lipidic lecithin based membrane fragments, whereby some bilayered lipidic lecithin based membrane fragments are fused with the plant cell wall or the tonoplast membrane of plant cells forming a lecithin containing protecting membrane structure for the plant cell or the vacuole thereof.

State of the art

The use of plant cells, such as elicited and dedifferentiated plant cells in cosmetic preparations is disclo sed for example in W02016/ 120713.

It has been tested to prepare cosmetic preparations comprising elicited and dedifferentiated plant cells of a first plant species, with one or more cosmetic additives. Said tests made by Applicant have shown the following problems:

- plant cells stability problem when mixing the plant cells with some cosmetic additives, meaning lo ss of plant cell activity and/or variable and/or undesired cosmetic effects of the plant cells, after a storage period, for example from 1 to 6 months ;

- the mixing operation could be problematic, in order to ensure that the small sized marketed cosmetic preparations comprise well the right amount of plant cells of the first plant species, as well as the right amount of possible cosmetic active agent(s) . - it is quite difficult to prepare a homogeneous plant cell mixture with one or more cosmetic active agents, especially for small sized containers. This could lead to variation of cosmetic activity, with a risk of local skin application of an overdose of plant cells and/or active cosmetic agent(s) .

- some stability problems for the plant cells occur when mixing dedifferentiated plant cells with one or more cosmetic excipients.

- it is difficult to obtain a storage stable homogeneous cosmetic preparation.

- when mixing plant cells with some cosmetic excipient(s), the plant cell membrane is damaged.

- when mixing plant cells with some cosmetic excipients, some phase separation can occur during the storage, whereby the cosmetic preparation is improper for use and marketing.

- plant cells agglomerates can be sometimes still visible in the cosmetic preparation.

- for ensuring some dispersion of the plant cells in a cosmetic formulation, high shearing forces are required, whereby breaking the plant cell membrane.

- impossibility to use the plant cells in dry or semi-dry cosmetic formulations.

The invention aims to solve one or more of said problems, and especially to prepare a homogeneous stable plant cells of a first plant species, which can be easily mixed with one or more cosmetic excipient formulations, for making storage stable homogeneous liquid cosmetics, storage stable viscous cosmetics, storage stable semi-dry cosmetics, as well as storage stable dry cosmetics. The invention relates also to stable plant cells with a modified cell wall membrane or tonoplast membrane, forming a protecting barrier for the plant cells, as well as advantageously for the central vacuole of the plant cells, whereby protecting and controlling the release or slow release of phytoalexins present in the vacuoles of the plant cells .

Brief description of the invention

The invention relates to a homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium, said stable plant cells having each a cell wall membrane defining an inner cell volume containing a central vacuole surrounded by a tonoplast membrane, whereby wall membrane of plant cells or agglomerates thereof are associated with bi-layered lipidic lecithin based membrane fragments, whereby some bilayered lipidic lecithin based membrane fragments are fused with the plant cell wall or the tonoplast membrane of plant cells forming a lecithin containing protecting membrane structure for the plant cell or the vacuole thereof.

Such a homogeneous mixture comprises plant cell vacuoles which are protected, whereby enabling a mixing with one or more cosmetic acceptable excipient easier for forming a cosmetic preparation which is stable and in which the plant cells are homogeneously distributed. Even after the mixing step, the plant cell vacuoles are still protected, enabling an improved stability for the compounds present in the vacuoles, a controlled release of the compounds present in the vacuole, and an improved stability of the homogeneity of the fixture. The mixing operation is even facilitated, even at low or ambient temperature. Tests have even shown that by applying the protected plant cells on the skin, a slow and progressive release of cosmetic active agent(s), an improved skin penetration or stratum corneum penetration of one or more active agents, improving the healing of skin micro-cracks, as well as improving the skin microbiote. Advantageously, plant cells or agglomerates thereof of the homogeneous stable mixture are separated the one from another by a multilayered lecithin structure, whereby a first plant cell or a first plant cells agglomerate has a first lecithin containing protecting membrane structure, while a second plant cell or a second plant cells agglomerate has a second lecithin containing protecting membrane structure, whereby the first lecithin containing protecting membrane structure of the first plant cell or the first plant cells agglomerate and the second lecithin containing protecting membrane structure of the second plant cell or the second plant cells agglomerate are spaced the one from the other by a lecithin containing intermediate structure comprising at least one sub-structure consisting of two lecithin layers separated the one with respect to the other by an intermediate aqueous layer or an oil intermediate layer or a glycerol containing layer.

Such a mixture provides a multilayered protection of plant cells or agglomerates thereof, still further improving the stable dispersion of the plant cells in a cosmetic formulation, the controlled release of the plant cell vacuole content.

Details and characteristics of preferred embodiments of the mixture of the invention are one or more of the followings:

- the intermediate aqueous layer comprises at least one cosmetic active agent which is water soluble at 20°C or water dispersible, and/or the oil intermediate layer comprises a vegetal oil which is liquid at 20°C, especially an essential vegetal oil. The said vegetal oil can also comprise one or more cosmetic active agents soluble or dispersible in said vegetal oil or said essential plant or vegetal oil. The cosmetic active agent(s) is/are so homogeneously distributed in the cosmetic excipient formulation. Moreover, the cosmetic active agent can be applied on a skin portion before the plant cell vacuole content is released on said skin portion.

- the first lecithin containing protecting membrane structure of the first plant cell or the first plant cells agglomerate and the second lecithin containing protecting membrane structure of the second plant cell or the second plant cells agglomerate are spaced the one from the other by a lecithin containing intermediate structure comprising at least three superposed sub-structures consisting each of two lecithin layers separated the one with respect to the other by an intermediate aqueous layer or an oil intermediate layer or a glycerol containing layer. Such a mixture is quite easy to mix with cosmetic excipient formulation without damaging the plant cell vacuole and the protecting tonoplast structure of the plant cells

- the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof contains one or more hydrolyzed lecithin containing lysophospholipids. Hydrolyzed lecithin containing lysophospholipids is the most appropriate for ensuring stable homogeneity of the mixture or cosmetic formulation, but also for improving the cosmetic topic treatment.

- the hydrolyzed lecithin containing lysophospholipid is a mixture of lysophospholids, 90% by weight or more, advantageously 95% by weight or more of the lysophospholipids of the said mixture have each a critical packing parameter from 0.5 to 1 , the weight average critical packing parameter of the lysophospholipids from the mixture being preferably comprised between 0.7 and 1 , most preferably between 0.8 and 0.95. Such hydrolyzed lecithin was the mo st adequate for ensuring that some bi-layered lipidic lecithin based membrane fragments are fused with the plant cell membrane, making an excellent membrane protecting structure, which is skin acceptable, with no skin side effect, while when in contact with the skin enabling an excellent controlled release of the vacuole content.

- preferably, the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof comprises at least one phosphatidylcholine compound selected from the group consisting of phosphatidylcholines of formula I

with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, phosphatidylcholines of formula I with Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, and mixtures thereof.

- most preferably, the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof comprises a mixture of (a) phosphatidylcholine compounds consisting of the mix of at least one mono-fatty acid phosphatidylcholine of formula I

with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms), and of (b) at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms), whereby the weight ratio between (a) the said at least one mono-fatty acid phosphatidylcholine of formula I with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms), and (b) the said at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms) in the said mix is from 0.00012 and 0.12, for example 0.001 , 0.005, 0.01 , 0.05 and 0.1. Advantageous ratio's are 0.01 to 0.1 , such as 0.02, 0.03 , 0.05 and 0.07.

- the at least one di-fatty acid phosphatidylcholine of formula I has one of the radicals Ri and R2 being a saturated fatty acid radical with 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms), while the other of the radicals Ri and R2 is an unsaturated fatty acid radical with 12 to 22 carbon atoms (preferably 16, 18, 20 or 22 carbon atoms) .

- The fatty acid radical R I and/or R2 in the mono fatty acid phosphatidylcholine and in the di-fatty acid phosphatidylcholine is for example a saturated fatty acid radical (general formula: - (C = O) - C_n H2n - CH3, with n = 10, 12, 14, 16, 18, 20, i.e. radical from one or more of the following fatty acids : lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid) .

The fatty acid radical can also be an unsaturated fatty acid radical with one or more double bonds (radical of the general formula : - (C = O) - ( (CH₂)_m (C₂H₂)_P ) - CH₃ with m + 2 x p = 10, 12, 14, 16, 18, 20, and with p = 1 , 2 or 3. Examples of unsaturated fatty acid radicals are :

- Mono-unsaturated fatty acid radical, such as radicals of Myristoleic acid, Palmitoleic acid, Sapienic acid, Oleic acid, Elaidic acid, Vaccenic acid, Gadoleic acid, Eicosenoic acid, Erucic acid;

- Di-unsaturated fatty acid radical, such as radicals of Linoleic acid, Eicosadienoic acid, Docosadienoic acid, - Tri-unsaturated fatty acid radical, such as radicals of Linolenic acid, Pinolenic acid, Eleostearic acid, Mead acid, Dihomo-y-linolenic acid, Eicosatrienoic acid,

- Tetra-unsaturated fatty acid radical, such as radicals of Stearidonic acid, Arachidonic acid, Eicosatetraenoic acid, Adrenic acid,

- Pentaunsaturated fatty acid radical, such as radicals of Bosseopentaenoic acid, Eicosapentaenoic acid, Ozubondo acid, Sardine acid, and

- Hexa-unsaturated fatty acid radical, such as radical of Cervonic acid,

- mixtures of radicals of unsaturated fatty acids, advantageously mixtures of radicals of staurated and unsaturaded fatty acids.

The fatty acids have preferably 16, 18 or 20 carbon atoms, and are preferably saturated or mono-unsaturated.

- the lecithin containing protecting membrane structure or/and the lecithin containing intermediate structure is enriched with one or more cosmetic active media, which is liquid at a temperature of 20°C at a pressure of 1 x 10⁵ Pa, or which is able to become liquid when contacting the skin, said one or more cosmetic active media comprising one or more cosmetic active agents being advantageously selected among the group consisting cosmetic acceptable vegetal essential oils, glycerol, salicylic acid and salts and esters thereof, glycolic acid, hyaluronic acid and salts and esters thereof , cosmetically acceptable UV filter agents, vitamin A, vitamin B (like B3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, cosmetically acceptable phytoalexins, cosmetic acceptable perfume, and mixtures thereof. The cosmetic active media or agent(s) is advantageously acid or at least partly acid. Possibly, but advantageously the presence of a cosmetically acceptable weak acid is present in the stable formulation, whereby helping the formation of the bilayered lecithin structure fragments and the homogeneous character of the mixture of the invention.

- said plant cells belongs to a first plant family, in which one or more cosmetic active agents is one or more essential oils or parts thereof of a second plant species not belonging to the first family.

- at least 50% by weight of the stable plant cells or agglomerates thereof provided with lecithin containing protecting membrane structure have a plant cell structure with a central vacuole volume occupying from 30 to 50% of the volume of the total inner plant cell volume, and in which the said central vacuole volume occupying 30 to 50% of the volume of the inner plant cell volume is enriched with one or more cosmetic active agents, said cosmetic active agents being advantageously selected among the group consisting cosmetic acceptable vegetal essential oils, glycerol, salicylic acid and salts and esters thereof, glycolic acid, hyaluronic acid and salts and esters thereof , cosmetically acceptable UV filter agents, vitamin A, vitamin B (like B 3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, cosmetically acceptable phytoalexins, cosmetic acceptable perfume, and mixtures thereof.

- the central vacuole volume of plant cells, as well as the lecithin containing protecting membrane structure and advantageously the lecithin containing intermediate structure are enriched with at least a same cosmetic active agent.

- the lecithin protective structure comprises at least one sub-structure consisting of two lecithin layers separated the one with respect the other by an intermediate layer selected from the group consisting of an aqueous layer, a glycerol containing layer and an oil layer from said vegetal essential oil.

- The homogeneous mixture of stable plant cells comprises a glycerol comprising medium comprising 0.5 to 5% by weight lecithin and 0.5 to 5% of a vegetal essential oil, forming a matrix in which the stable plant cells are homogeneously dispersed.

- the glycerol comprising medium comprises from 5 to 25% by weight water. - the lecithin is present in the mixture at a dose of 0.1 % to 3 % by weight.

- the lecithin is from vegetal origin, said lecithin having a HLB value comprised between 8 and 14.

- the lecithin is from vegetal origin, said lecithin having a HLB value comprised between 9 and 12.

- the lecithin is a food grade soy de-oiled lecithin enriched in lysophospholipid, the lecithin having a lysophospholipid weight content with respect to the total phospholipid weight of the lecithin comprised between 5% and 50% .

- the lecithin is a hydrolyzed lecithin having a lysophospholipid weight content with respect to the total phospholipid weight of the lecithin comprised between 20 and 50% .

- The homogeneous mixture of stable plant cells comprises:

- from 25 to 85% by weight of plant cells with a lecithin containing protecting membrane structure;

- from 5 to 20% by weight free water present outside the plant cells with a lecithin containing protecting membrane structure;

- from 0.5 to 10% by weight free essential oil outside the plant cells with a lecithin containing protecting membrane structure;

- from 10 to 70% by weight of glycerol; and whereby the lecithin content in said homogeneous mixture of stable plant cells is from 0.1 % to 3 % by weight.

- any combinations of said details and characteristics.

Advantageously the said cosmetic active agent is selected among the group consisting of glycolic acid, salicylic acid, hyaluronic acid, UV filter agents, vitamin A, vitamin B (like B3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, phytoalexins, etc. , advantageously from biological origin, especially from plant origin if possible. The said cosmetic active agent is advantageously part or mixed to an essential oil, possibly of the first plant species, but advantageously to an essential oil of a second plant species not belonging to the plant family of the first plant species.

While not being bound to any theory, it seems that when using some vegetal essential oil within the intermediate layer structure between two adjacent lecithin containing protecting membrane structures, some essential oil can flow through the membrane structure, with a lecithin layer of the intermediate structure forming then with the cell or tonoplast membrane a new protecting cell or tonoplast membrane structure for the vacuole or vacuoles of plant cells. The vacuoles can in this way be enriched with at least an essential oil (of the first plant species, or from another plant species, such as not belonging to the first plant species), and possibly with one or more cosmetic active agents soluble or dispersible in said essential oil. The vacuole is then advantageously an enriched central vacuole comprising a plant sap from said first plant species (said plant sap comprising phytoalexins and essential oil of the first plant species), said sap being preferably enriched with at least one plant essential oil or part thereof (said essential oil comprising phytoalexins of the second plant species, as well as preferably one or more further cosmetic active agents) which is liquid at least at a temperature of 20°C and at a pressure of 1 x 10⁵ Pa, said plant essential oil or part thereof being advantageously a vegetal essential oil issued from a second plant species not belonging to the first plant family. In this way it is possible to combine in the vacuole or plant cell structure, the cosmetic activity due to the first plant species and the cosmetic activity due to another plant species. Due the homogeneous character of the mixture, and its ability to be mixed in a homogeneous way into a cosmetic excipient preparation or formulation, it is possible to prepare small sized stable homogeneous cosmetic formulation or emulsion. The Critical packing parameter (CPP) used in the present specification is defining a theoretical framework for determining the type of aggregation formed by one surfactant or a mixture of surfactants .

When using a mixture of surfactants, the CPP of the mixture of surfactant can be considered as being equal to an average CPP of the CPP of the different surfactants present in the mixture weighted in function of their weight percentage in the mixture.

CPP = S (CPPi x wi) in which CPPi is the CPP of the surfactant compound i, and wi is the weight percentage of compound i in the surfactant mixture.

Critical packing parameter is defined for example in W02012/08019 - corresponding to US 8,980,818 as being the ratio ( Volume of the hydrophobic tail) / (Length of the hydrophobic tail x area of the head group)

When using a mixture of lecithin compounds, the mixture comprises lecithin compound(s) with a CPP of about 0.5 (corresponding to a substantially truncated cone, suitable for forming bilayer protective coating or vesicle) and lecithin compound(s) with a CPP of about 1 (corresponding to a substantially cylindrical shape, suitable for forming lamellar protective ) . The lecithin mixture is advantageously substantially free of lecithin compounds with a CPP of less than 0.47, as well as lecithin compounds with a CPP of more than 1.05. The lecithin compounds are advantageously selected so as to form a lamellar protective coating with formation of some bilayered structures structure, such as for example two distinct bilayered structure zones being connected therebetween by a lamellar structure zone. The lamellar structure can be a multilayered lamellar structure.

For a fatty acid phosphatidylcholin (lecithin), the hydrophobic tail is formed by the fatty acid radicals attached to the glycerol backbone, while the head group is formed by the quaternary ammonium group - phosphate group - glycerol backbone.

In US 8,980,818, the CPP parameter of surfactants is disclo sed, with a method for determing / calculating the said CPP (see columns 19 and 20 :

" To determine the shape of a single surfactant, it was modelled using the molecular modelling module Discover® in Materials Studio (Materials Studio v4.3.0.0. Copyright 2008, Accelrys Software Inc. ). The surfactant is modelled by defining the atoms and assuming a harmonic potential for the bonds using the pcff forcefield. This forcefield was developed based on CFF91. More details on this type of modelling can be found in the work by Allen and Tildesley (1 M. P. Allen; D. J. Tildesley. Computer Simulation of Liquids, Oxford University Press (1987)).

Discover® was used to find a (local) energy minimum of the surfactant molecular structure, the starting point for the minimization was an extended conformation of the tail. After minimisation, the three necessary parameters to calculate a CPP can be determined: vtail (volume of the hydrophobic tail), Itail (the length of the hydrophobic tail) and ao (the area of the head group). For those materials that are not a pure chemical substance a CPP was calculated for each major homologue of the series and the CPP estimated based on the mole fraction of the component."

The invention relates also to a method for the preparation of a homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium according to the invention, as disclosed here above. Said method comprises at least the following steps:

* Step 1 : in vitro culturing of plant cells from the first plant species belonging to said first genus of said first plant family in a culture medium at a temperature from 20°C up to 45°C ;

* step 2: collecting the plant cells in the form of plant cells agglomerates from the culture medium, washing the plant cells agglomerates at least with water, and filtering for removing free water, so as to prepare collected and washed plant cells agglomerates with a free water content of less than 5% by weight, said steps being operated at a temperature from 20 to 45°C;

* step 3 : mixing at a temperature from 20°C up to 45°C an amount of lecithin in substantially dry form with the collected and washed plant cells agglomerates, whereby the amount of lecithin corresponds to 0.1 % to 3 % of the weight of the collected and washed plant cells agglomerates without the remaining free water, so as to prepare a lecithin containing plant cells medium ; and

* step 4: mixing at a temperature from 20 to 45°C, the lecithin containing plant cells medium with a glycerin containing medium, whereby forming bi-layered lipidic lecithin based membrane fragments, a portion thereof being fused with the cell and/or tonoplast membrane of plant cells forming a lecithin containing protecting cell or tonoplast membrane structure for plant cells or agglomerates thereof in a glycerol comprising medium.

Preferably, an vegetal oil, advantageously an essential vegetal oil, which is liquid at 20°C, lecithin and advantageously one or more cosmetic active agents soluble or dispersible in said vegetal oil or essential vegetal oil are mixed together for preparing a lecithin oily mixture advantageously enriched with one or more cosmetic active agents. The lecithin oily mixture advantageously enriched with one or more cosmetic active agents is then mixed with the lecithin containing plant cells medium, so as to form a lecithin containing intermediate structure spacing lecithin containing protecting cell or tonoplast membrane structure for plant cells or agglomerates thereof, said lecithin containing intermediate structure comprising at least one substructure consisting of two lecithin layers separated the one with respect to the other by an oily intermediate layer advantageously enriched with one or more cosmetic active agents.

Most preferably, the glycerin containing medium is mixed with one or more cosmetic active agents soluble or dispersible in the glycerin containing medium prior its mixing with the lecithin containing plant cells medium.

According to a possible embodiment, an essential vegetal oil or part thereof of a second plant species not belonging to the first plant species of said plant cells or agglomerates thereof, and advantageously one or more cosmetic active agents soluble or dispersible in said essential vegetal oil are mixed together for preparing a lecithin oily mixture advantageously enriched with one or more cosmetic active agents, and in which the lecithin oily mixture advantageously enriched with one or more cosmetic active agents is mixed with the lecithin containing plant cells medium, so as to form a lecithin containing intermediate structure spacing lecithin containing protecting cell or tonoplast membrane structures for plant cells or agglomerates thereof, said lecithin containing intermediate structure comprising at least one sub-structure consisting of two lecithin layers separated the one with respect to the other by an essential oil intermediate layer comprising the said essential oil of the second plant species, said intermediate layer being advantageously enriched with one or more cosmetic active agents.

Advantageously, the lecithin containing oily mixture is prepared by mixing together a vegetal essential oil or part thereof, and lecithin, the dry weight ratio vegetal essential oil / lecithin being comprised between 1 : 1 and 10: 1.

The lecithin containing oily mixture is mixed with the lecithin containing plant cells medium so as to prepare a plant cells enriching enfleurage medium with a water free weight ratio of the lecithin containing oily mixture / the lecithin containing plant cells medium comprised between 2: 1 and 10: 1 , whereby said plant cells enriching enfleurage medium is hold, advantageously with some mixing, at a temperature from 5 °C and 45°C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for at least 10 days, whereby the lecithin containing protecting cell or tonoplast membrane structure of plant cells is enriched with vegetal essential oil or part thereof from the second plant species forming plant cells or agglomerates thereof with oily enriched cell or tonoplast membrane structure; whereby the plant cells or agglomerates thereof with oily enriched cell or tonoplast membrane structure are collected from the plant cells enriching enfleurage medium; and thereafter mixed with a glycerol comprising medium at a temperature from 20°C to 45°C.

Preferably, at least one cosmetic active agent selected from the group consisting of salicylic acid, glycolic acid, hyaluronic acid, UV filter agent, vitamin A, vitamin B (like B3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, phytoalexin, cosmetic acceptable perfume, and mixtures thereof, is added to the vegetal essential oil of part thereof from the second plant species prior the mixing of said vegetal essential oil to an amount lecithin for the preparation of the lecithin containing oily mixture.

Most preferably, the method comprises the step of holding said plant cells enriching enfleurage medium at a temperature from 20°C and 40°C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for a period from 15 days up to 30 days.

According to advantageous embodiment of the invention, the lecithin mixed to the collected and washed plant cell agglomerates is a hydrolyzed lecithin containing lysophospholipid, said hydrolyzed lecithin being in powder form when mixed to the collected and washed plant cells .

The lecithin is advantageously a lecithin from vegetal origin, said lecithin having a HLB value comprised between 8 and 14. Preferably, the hydrolyzed lecithin containing lysophospholipid is a mixture of (a) phosphatidylcholine compounds consisting of the mix of at least one mono-fatty acid phosphatidylcholine of formula I

Oh G - ^:*£

with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, and of (b) at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms, whereby the weight ratio between (a) the said at least one mono-fatty acid phosphatidylcholine of formula I with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, and (b) the said at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms in the said mix is from 0.00012 and 0.12.

The method of the invention has advantageously one or more of the following details or characteristics for one or several process steps:

* in vitro culturing of plant cells from the first plant species belonging to said first genus of said first plant family in a culture medium; said in vitro culturing being at least partly operated with one or more elicitation step. * collecting the plant cells in the form of plant cells agglomerates from the in vitro culture medium, washing the plant cells agglomerates with water, and filtering the said plant cells agglomerates, the so collected and washed plant cells agglomerates having a free water content of less than 5% by weight, before mixing them with lecithin;

* mixing an amount of lecithin in substantially dry form with the collected and washed and filtered plant cells, whereby the amount of lecithin corresponds to 0.1 % to 3 % of the weight of the collected and washed plant cells agglomerates, so as to prepare a lecithin containing plant cell medium ;

* the method comprises the step of holding said plant cells enriching enfleurage medium at a temperature from 20°C and 40°C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for a period from 15 days up to 30 days.

* the method comprises the step of holding the collected enriched plant cells from said plant cells enriching enfleurage medium mixed with a glycerol comprising medium at a temperature from 30°C to 45°C for at least 10 days.

Details and characteristics of preferred embodiments of the invention (given as example only) will appear from the following description in which reference is made to the attached drawings.

Brief description of the drawings

In said drawings,

- Figure 1 is a schematic view of living elicited and dedifferentiated plant cells, not stabilized with a protective coating structure,

- Figure 2 is an electron micrograph of the cell membrane or a vacuole membrane of an elicited dedifferentiated plant cell,

- Figure 3 is a schematic view of the plant cells stabilized with a multilayered lecithin protective coating structure with a lecithin containing protecting membrane structure and intermediate bi-layered structures,

- Figure 4 is an electron micrograph of the plant cells embedded in a multi layered lecithin protective coating structure,

- Figure 5 is an enlarged electron micrograph of the plant cells of figure 4, showing the presence of a protective lecithin containing membrane structure around the cell or vacuole thereof;

- Figure 6 is an enlarged view of a multilayer lecithin structure embedded into the cell and protecting the inner tonoplast membrane, whereby protecting the enriched vacuole content, as well as the enriched protecting structure.

- Figure 7 is a further enlarged view of the lecithin multi layer protective layer of the cell membrane. The said multi layer structure is in some way fused with the cell wall while forming a new protecting membrane.

- Figure 8 is a schematic view of bi-layered protective structure achieved with the lecithin for the elicited dedifferentiated plant cells,

- Figure 9 is a flow sheet of a method for preparing lecithin protected enriched plant cells of the invention,

Figure 1 is a schematic view of an elicited dedifferentiated plant cell, which is for example prepared in W02016/1207 13 or in WO 2003/077880, prior its crushing.

The plant cell of figure 1 comprises :

- a cell wall 1 ,

- a plasma membrane layer 2 along the inner side of the cell wall 1 ,

- one or more plasmodesmata 3 at the level of the cell wall 1 ,

- a nucleolus 4 with its nucleus and the nuclear membrane 4,

- a vacuole 5 with its tonoplast layer 6, said vacuole containing at least some phytoalexins of the plant,

- a cytoplasm 7,

- a chloroplast 8,

- chromoplast 9, and while not being drawn, also peroxisome, membranous vesicles, mitochodrion, microtubule, ribosome, polyribosome, endoplasmic reticulum (smooth and rough), golgi apparatus and lisosomeus, for simplyfing the representation of the plant cell in figure 1.

More information on the structure of living plant cell can be found on the web page : https://flexbooks.ck l2.org/cbook/ck- 12- bio logy- flexbook- 2.0/sect io n/2.9/primar y/les so n/plant- cell- structure s- bio (which was available on January 21 , 2021 ) . Similar information can also be retrieved on the web, as well as in books and articles.

Figure 2 is an electron micrograph of a portion of an elicited dedifferentiated plant cell, namely showing the thin tonoplast membrane of a plant cell not provided with the lecithin protective structure.

The cell wall and the tonoplast membrane are thus not protected, meaning that when mixing the plant cells with cosmetic excipient(s), the plant cell membrane, as well as the tonoplast can be damaged, by contact with some cosmetic ingredients, especially active cosmetic ingredients, such as plant essential oils, as well as during the mechanical mixing. For trying to get a homogeneous cosmetic formulation, a vigorous mixing is required, meaning a damage of plant cell wall and tonoplast membrane. Without such vigorous mixing, no homogeneous cosmetic preparation can be achieved. In case of damage of the plant cell wall and/or of the tonoplast membrane, the phytoalexins present in the plant cell are no more protected within the plant cell membrane and are released within the cosmetic preparation. Said preparation is then not homogeneously stable, with some phase separation. Due to the lost of homogeneity of the cosmetic preparation, it is difficult to guarantee a correct cosmetic activity when applying on the skin a small quantity of cosmetic formulation from a small sized container. Moreover, some released phytoalexins or essential oils present in the plant cell can further damage the cell wall. The presence of cell wall in a cosmetic formulation is not a guarantee of the presence of phytoalexin. The plant cell distribution in the cosmetic preparation is thus no guaranty of a same distribution of the plant cell. The problem of stability and homogeneity of cosmetic preparation comprising plant cells of a first species and an essential oil of another plant species is not solved at all, even with vigorous mixing, which further causes plant cell wall damages.

The plant cell of figure 2 can not be incorporated in dry or semi-dry cosmetic formulation, by simple mixing.

For cosmetic preparations comprising plant cells of different plant species, it is also quite difficult to prepare homogeneous cosmetic preparations comprising plant cells of a first plant species and plant cell of a second plant species. For achieving some homogeneity, vigorous mechanical mixing is required, meaning then problem of cell wall damages and release of phytoalexins and essential oils within the cosmetic preparation, causing then problem of stability and homogeneity of the composition with the time. No controlled or slow release of phytoalexins can be obtained.

Figure 3 is an enlarged schematic view of a stable plant cells from a first plant species belonging to a first genus of first plant family, which is enriched with an essential oil issued form a second plant species not belonging to the first plant family, said essential oil issued from the second plant species being advantageously further enriched with one or more further cosmetic active agents, such as glycolic acid, hyaluronic acid, UV filter agent, vitamins (A,B , B3 , B6, B9, C, D, D2, D3 and mixtures thereof as example) .

The first plant family and the second plant family distinct from the first plant family belong to the list available on the web : http://www.theplantlist.org/browse/-Z said list of plant families is given in the following table.

TABLE 1 : List of Plant families

All said plant families have each a list of a series of genera, while a series of plant species belong for each genus of said genera of a plant family. The web site www.theplantlist.org lists for each plant family a list of genera, and for each genus of said genera list of species.

The first plant family and the second plant family belong advantageously to the Angio sperms group ( group of Flowering plants or seed-bearing vascular plants) . Further information about plant families of said Angio sperms group and genera and species thereof can be found on the web site www.theplantlist.org.

As shown in Figure 3 , the structure of the plant cell provided with the lecithin protective coating structure of the invention, having the shape of superposed bi layered lamellar platelet structures with some rounded bilayered portions. The vacuole 5 and/or the tonoplast protecting membrane structure 6 is/are enriched with some part of essential oil of the second plant species, and with some of the further one or more cosmetic active agent(s) possibly added to the essential oil of the second plant species.

The tonoplast membrane protecting structure 6 of said enriched vacuole 5 comprises one or more lecithin protective layer 1 1 , said protective layer embedding also some compounds of the essential oil of the second plant species, and possibly of the further cosmetic active agent(s) added to said essential oil. The tonoplast membrane 6 and the lecithin protective layer 1 1 can be fused altogether, forming then a lamellar or platelet protective structure around the enriched vacuole 5.

The cell wall 1 is also covered with the lecithin protective layer, said layer having the shape of a lamellar structure embedding the cell, possibly a plurality of cells or cells agglomerates. The protective layer 10, 10' is advantageously enriched with some essential oil of the second plant species, as well as of the possibly added further cosmetic active agent(s) . The said protective layer 10 can fuse with the cell wall or membrane 2, while clo sing the plasmodesmata 3 (represented in Figure 1 , but no more visible in Figure 3 due to the protective layers 10, 10'), whereby the plant cell is fully protected by the lecithin protective structure (multi-layered 10, 10', etc.) .

The enriched vacuole 5 with the protective lecithin layer occupies a larger percentage (35% to 50% , or more) of the inner volume of the plant cell provided with the lecithin protective layer, with respect to the percentage ( 15% to 25%) of the volume of the vacuole in the inner chamber of the plant cell prior being provided with the lecithin containing protecting membrane structure, and its enrichment with said essential oil.

The lecithin protective layer around the vacuole forms also an intermediate protection for the vacuole with respect to the plasma membrane 2 and the cell wall 1 , which is also provided with a lecithin protecting structure.

Figure 4 is an electron micrograph of the enriched plant cells embedded in a lecithin protective coating structure, with at least partial fusing of the wall membrane with one or more bi-layered lecithin based membrane. In said figure, the multilamellar layer structure of the lecithin protective structure can be seen. Said multi layer structure enables to ensure to achieve a full embedment of the plant cell, whereby the plant cells are stable and well protected for the further mixing thereof with cosmetic excipients. The intermediate bilayered sub structures spacing the walls of adjacent cells facilitate the mixing of the plant cells into a cosmetic formulation, as well as improvement the stability of the so prepared homogeneous cosmetic formulation.

Figure 5 is an enlarged electron micrograph of the plant cells of figure 4, showing the prescence of a protective lecithin structure for the membrane around the cell (cell wall) .

One can see clearly a multi-layer structure made of aqueous and oily layers embedded into and around the plant cell membrane. From said figure, it seems that at least a portion of the membrane of the oily containing vacuole or of the plant cell is fused with lecithin, for forming a protected essential oil reservoir within the cell and/or around the plant cell, such a reservoir being suitable for a controlled release of the essential oil(s) when applied on the skin, and for ensuring a homogeneous distribution of the plant cell and oil in a cosmetic formulation.

Figure 6 is an enlarged view of a multilayer lecithin structure embedding the plant cell and protecting the plant cell content and the tonoplast membrane, whereby protecting the enriched vacuole content.

Figure 7 is a further enlarged view of the lecithin multi layer protective layer of the plant cell wall or tonoplast membrane. The said multi layer structure is in some way fused with the plant cell wall or tonoplast membrane.

Figure 8 is a schematic view of bi-layered protective structure achieved with the lecithin for the elicited dedifferentiated plant cells .

The structure has several bi-layered components 15, 15', each bi-layered element being form by two lecithin layers L1 ,L2, L1 ',L2', each lecithin layer being formed by individual units defining substantially cylindrical unit C with a CPP of about 1 or truncated unit with a CPP of about 0.5.

The lecithin compounds have a hydrophilic head H and one or two hydrophobic fatty acid chains F 1 ,F2.

The hydrophilic head is advantageously formed by a phosphatidylcholine group with a glycerol backbone. The fatty acid chain is for example a saturated or unsaturated fatty acid radical with 16 to 22 carbon atoms.

Figure 9 is a flow sheet of a preferred method for preparing lecithin protected living enriched plant cells of the invention.

The represented preferred method (given as example only) for the preparation of a homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium, said stable plant cells having each a cell wall membrane defining an inner cell volume containing a central vacuole surrounded by a tonoplast membrane, said central vacuole comprising a plant sap from said first plant species. The plant cells being advantageously enriched at the level of the plant cell wall and/or at the level of the tonoplast membrane and/or at the level of the vacuole. The enrichment is advantageously made by using a plant essential oil of the first species or preferably of another species (such as another species belonging to another plant family) . Said essential oil can be further enriched with one or more active cosmetic agents. , The said plant essential oil or part thereof is liquid at least at a temperature of 20°C and at a pressure of 1 x 10⁵ Pa, said plant essential oil or part thereof being a vegetal / plant essential oil being advantageously issued from a second plant species not belonging to the first plant family. The cell wall membrane of the stable plant cells is provided with a lecithin protective coating structure, and being enriched with said essential oil.

The method of the invention comprises preferably the following steps:

* in vitro culture of elicited dedifferentiated plant cells : in vitro culturing of plant cells from the first plant species belonging to said first genus of said first plant family in a culture medium. Said in-vitro culture is preferably carried out for preparing elicited dedifferentiated plant cells of a first plant species of one of the family listed in table 1. The process for preparing elicited and dedifferentiated plant cells of the first plant family is for example a method as disclo sed in WO 2016/120713 or in WO 03/077880 (without crushing step) . The dedifferentiated plant cells are elicited while growing in the in-vitro culture medium. The elicitation is for example carried out by a UV lamp (254nm), at a temperature of 25-30°C under a humid air atmosphere enriched with CO2 (relative humidity level of about 100% - saturated/ CO2 content about 5% by volume) . The elicitation time was about 10 to 24 hours. For some plant species, the elicitation was carried out with illumination steps, with a dark step between two successive illumination steps.

* collecting elicited dedifferentiated plant cells : collecting the plant cells in the form of plant cells agglomerates from the culture medium, washing the plant cells with water, and filtering the washed plant cells, the so collected, washed and filtered plant cells agglomerates having a free water content of less than 5% by weight. The plant cells are not submitted to a crushing step or a grinding step, whereby said plant cells are still plant cells with their cell wall and with inner vacuole defined by a tonoplast membrane.

* adding and mixing lecithin to the plant cells : mixing an amount of lecithin in substantially dry form with the collected, washed and dried plant cells, whereby the amount of added lecithin corresponds to 0.1 % to 3 % of the weight of the collected and washed plant cells agglomerates, so as to prepare a lecithin containing plant cell medium. During said mixing, the plant cell walls are covered with a lecithin structure with bilayered lipidic lecithin based membrane fragments, at least partly fused the plant cell walls and/or with the tonoplast, whereby forming a lecithin containing protecting membrane structure, while clo sing the one or more plasmodesmata of the plant cells . The mixing enables also to space the plant cells the one from the other, by some intermediate aqueous lecithin structure, with two lecithin substructures separated by an aqueous layer.

The lecithin compound added to the plant cells was a mixture of lecithin compounds of formula I.

As preferred examples, the mixture was a mixture of phosphatidylcholine compounds consisting of :

* mono-fatty acid phosphatidylcholine(s) of formula I with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a saturated or a monounsaturated fatty acid radical comprising 16 to 20 carbon atoms, and

* di-fatty acid phosphatidylcholine(s) of formula I (see above) with Ri and R2 being each a saturated or unsaturated fatty acid radical comprising 16 to 22 carbon atoms, whereby the weight ratio between the said mono-fatty acid phosphatidylcholine(s) of formula I and the said di-fatty acid phosphatidylcholine(s) of formula I in the said mixture of lecithin compounds is about 0.05. The mono-fatty acid phosphatidylcholine compounds have a molar average carbon atom number in the fatty acid radical Ri and R2 , which is substantially equal to the molar average number of carbon atoms present in the fatty acid radical Ri and R2 of di-fatty acid phosphatidylcholine compounds, Ri and R2 of said di-fatty acid phosphatidylcholine compounds having the same number of carbon atoms or a difference of carbon atoms of 2.

Said lecithin dry mixing is operated in a clean room at room temperature (20°C), with gentle mixing with a thin rubber or teflon coated spatula. The mixing step is carried out for incorporating the lecithin compounds between plant cells, without destroying the plant cell wall. The lecithin is folded to the plant cells. The plant cells are in this way provided with a small protection around at least a portion of the wall cell, in order to prevent some damage when mixing the plant cell with an essential oil of another plant species.

* preparation of a lecithin containing essential oil from another plant species : preparing a lecithin containing oily mixture by mixing together the vegetal essential oil or part thereof from the second plant species not belonging to the first plant family, and lecithin, the dry weight ratio vegetal essential oil / lecithin being comprised between 1 : 1 and 10: 1.

The essential oil is preferably issued from a plant species different from the first plant species to which belong the elicited and dedifferentiated plant cells.

Prior the said mixing step, the essential oil of the second plant species was advantageously enriched with one or more cosmetic active agents selected from the group consisting of salicylic acid, glycolic acid, hyaluronic acid, UV filter agent, vitamin A, vitamin B (like B3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, phytoalexin, cosmetic acceptable perfume, and mixtures thereof. For example, the enrichment is made so that the weight content of said active agent in the essential oil (prior its mixing with lecithin) is from 0.01 % up to 5% , advantageously from 0.1 to 1 % . The cosmetic active agents are advantageously soluble or dispersible in said essential oil.

The lecithin used in said step is advantageously the lecithin used in the previous step of adding it to the elicited dedifferentiated plant cells.

Said step is carried out in a clean room at room temperature with gentle mixing with a thin rubber or teflon coated spatula. During said mixing step some bilayered structures are formed with lecithin layers spaced with an oil intermediate layer.

* mixing the lecithin containing essential oil with plant cells mixed with lecithin : mixing the lecithin containing oily mixture with the lecithin containing plant medium so as to prepare a plant cells enriching enfleurage medium with a water free weight ratio of the lecithin containing oily mixture / the lecithin containing plant medium comprised between 2: 1 and 10: 1 , and holding said plant cells enriching enfleurage medium at a temperature from 5 °C and 45 °C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for at least 10 days, whereby the plant cells or the protecting membrane structure or the vacuoles are enriched with vegetal essential oil and possible cosmetic active agents. Said enfleurage step is carried out with gentle mixing. By said enfleurage step, the plant cells are separated the one from the other by a series of intermediate structures consisting each by two lecithin containing layers with an intermediate oil layer.

The mixing step is also operated with thin rubber or teflon coated spatula, so as to fold the lecithin oily mixture to the protected plant cells . Mixing is operated with successive mixing step for 5 to 15 minutes, separated the one from another by a rest period without mixing for 60 minutes to 240 minutes.

Said step is carried out in a clean room, advantageously at 25°C and at atmospheric pressure, for preferably 20 to 30days. * collecting the enriched plant cells from said plant cells enriching enfleurage medium : the enriched stable and protected plant cells are collected by filtering, so as to remove possible excess of water or oil present in the previous mixing step.

* glycerol mixing : mixing the collected enriched plant cells from said plant cells enriching enfleurage medium with a glycerol comprising medium at a temperature from 20°C to 45°C. Said mixing step with glycerol is preferably operated for 15 to 30 days. Said addition of glycerol enables the preparation of a stable homogeneous suspension of the shelled stable oil enriched plant cells . Such homogeneous suspension can be used as such as cosmetics, but is a ready to use composition suitable for preparing liquid, viscous, dry or semi-dry cosmetic formulation or to be incorporated into a liquid or viscous or dry or semi-dry cosmetic formulations, like creams, lotions, gels, sticks, etc.

* adding and mixing cosmetic excipients and cosmetic active agent(s) or adding to a cosmetic formulation. The homogeneous stable mixture of plant cells can be added as such to cosmetic formulations. It has even been observed that the so prepared cosmetic formulation containing the stable homogeneous mixture of plant cells had a homogeneous distribution of the plant cells and had also an improved stability, especially a long term stability of the cosmetic activity.

For example, the homogeneous stable mixture can be used in replacement of the elicited plant cells in the examples of formulations of cosmetic cream, lotions, gels, lipstick, etc. disclosed in WO 03/077881. In said formulations disclosed in WO 03/077881 , the dedifferentiated elicited plant cells are replaced by the homogeneous stable lecithin protected plant cell mixtures, at a rate adapted so that the plant cell content remains the same as in WO 03/077881.

The so prepared cosmetic compositions had a homogeneous dispersion of stable plant cells, said plant cells are shelled with a lecithin protective structure, while comprising within the vacuole and their lecithin protective shell essential oil and phytoalexins from the first plant species or from a second species, as well as possible further one or more cosmetic active agent(s) .

The stable homogeneous cosmetic preparation is in this way able to release homogeneously and locally the essential oil of the first plant species and/or the essential oil of the second plant species, possibly with one or more further cosmetic active agents, and thereafter the content of vacuoles meaning a better cosmetic treatment of the skin. As the lecithin protecting layer ensures a protection for the essential oil and the content of the vacuole content against oxidation, better stability of the cosmetic activity can be ensured, even after a long storage period of for example 12 months. Phase separation could be also prevented, despite the presence of essential oil in the formulation.

Tests have shown that the shelled enriched plant cells were stable for more than 1 year, in a humid atmosphere (relative humidity of 75%) at a temperature of 20°C, meaning that the shelled enriched plant cells of the first species had an excellent stability over the time.

Advantages of the mixture of the invention are one or more of the followings:

* the plant cells are encapsulated with one or more lecithin containing structures or intermediate structures, which can be enriched with hydrophilic compound(s) or hydrophilic cosmetic active agent(s) (for example solubilized in an intermediate aqueous layer located between two lecithin containing layers, and/or hydrophobic compound(s) or hydrophobic cosmetic active agent(s) located between two lecithin containing layers, the said lecithin containing structure(s) or intermediate structure fragment(s) being partly fused with the plant cell wall membrane and/or the tonoplast membrane, or are forming part of a protecting layer of the plant cell wall or tonoplast membrane fused with some lecithin containing structure fragments.

* the release of said cosmetic active agent(s) is operated when applying the cosmetic formulation on the skin. Up to the application on the skin, there is a smell and chemical neutralization for the cosmetic active agent(s), helping in this way to the storage stability of the formulation, even in contact with air. The cosmetic formulation is in this way storage stable.

* slow and controlled release of the cosmetic active agent embedded in the lecithin containing structures.

* improved skin/stratum corneum affinity and skin / stratum corneum penetration.

* improved healing of skin micro-cracks, especially by enzymatic path.

* stable homogeneous composition without shaking requirement.

* easy to mix the stable homogeneous plant cell to cosmetic excipients or easy to incorporate the stable homogeneous plant cell into a cosmetic formulation. The so obtained cosmetic formulation has an improved stability.

* the stable homogeneous plant cell mixture can be incorporate into makeup formulation, especially makeup foundation powder. The so makeup skin formulation has an improved shade coloring, stable for at least 12hours. some lecithin interaction with the foundation pigments, especially in powder form.

* improved skin microbiome activity.

Claims

37 WHAT I CLAIM IS :

1. Homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium, said stable plant cells having each a cell wall membrane defining an inner cell volume containing a central vacuole surrounded by a tonoplast membrane, whereby wall membrane of plant cells or agglomerates thereof are associated with bi-layered lipidic lecithin based membrane fragments, whereby some bilayered lipidic lecithin based membrane fragments are fused with the plant cell wall or the tonoplast membrane of plant cells forming a lecithin containing protecting membrane structure for the plant cell or the vacuole thereof.

2. The homogeneous mixture of stable plant cells of claim

1 , in which plant cells or agglomerates thereof are separated the one from another by a multilayered lecithin structure, whereby a first plant cell or a first plant cells agglomerate has a first lecithin containing protecting membrane structure, while a second plant cell or a second plant cells agglomerate has a second lecithin containing protecting membrane structure, whereby the first lecithin containing protecting membrane structure of the first plant cell or the first plant cells agglomerate and the second lecithin containing protecting membrane structure of the second plant cell or the second plant cells agglomerate are spaced the one from the other by a lecithin containing intermediate structure comprising at least one sub-structure consisting of two lecithin layers separated the one with respect to the other by an intermediate aqueous layer or an oil intermediate layer or a glycerol containing layer.

3. The homogeneous mixture of stable plant cells of claim

2, in which the intermediate aqueous layer comprises at least one cosmetic active agent which is water soluble at 20°C or water dispersible, or in which the oil intermediate layer comprises a vegetal oil which is liquid at 20°C, especially an essential vegetal oil. 38

4. The homogeneous mixture of stable plant cells of claim 2 or 3 , in which the first lecithin containing protecting membrane structure of the first plant cell or the first plant cells agglomerate and the second lecithin containing protecting membrane structure of the second plant cell or the second plant cells agglomerate are spaced the one from the other by a lecithin containing intermediate structure comprising at least three superposed sub-structures consisting each of two lecithin layers separated the one with respect to the other by an intermediate aqueous layer or an oil intermediate layer or a glycerol containing layer.

5. The homogeneous mixture of stable plant cells of any one of the preceding claims, in which the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof contains one or more hydrolyzed lecithin containing lysophospholipids.

6. The homogeneous mixture of stable enriched plant cells of claim 5, in which the hydrolyzed lecithin containing lysophospholipid is a mixture of lysophospholids, 90% by weight or more, advantageously 95% by weight or more of the lysophospholipids of the said mixture have each a critical packing parameter from 0.5 to 1 , the weight average critical packing parameter of the lysophospholipids from the mixture being preferably comprised between 0.7 and 1 , most preferably between 0.8 and 0.95.

7. The homogeneous mixture of stable plant cells of claim 5 or 6, in which the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof comprises at least one phosphatidylcholine compound selected from the group consisting of phosphatidylcholines of formula I

8. The homogeneous mixture of stable plant cells of claim 1 , in which the lecithin containing protecting membrane structure of the plant cells or agglomerates thereof comprises a mixture of (a) phosphatidylcholine compounds consisting of the mix of at least one mono-fatty acid phosphatidylcholine of formula I

9. The homogeneous mixture of stable plant cell of claim 8, in which the at least one di-fatty acid pho sphatidylcholine of formula I has one of the radicals Ri and R2 being a saturated fatty acid radical with 12 to 22 carbon atoms, while the other of the radicals Ri and R2 is an unsaturated fatty acid radical with 12 to 22 carbon atoms.

10. The homogeneous mixture of stable plant cells of any one of the preceding claims, in which the lecithin containing protecting membrane structure or/and the lecithin containing intermediate structure is enriched with one or more cosmetic active media, which is liquid at a temperature of 20°C at a pressure of 1 x 10⁵ Pa, or which is able to become liquid when contacting the skin, said one or more cosmetic active media comprising one or more cosmetic active agents being advantageously selected among the group consisting cosmetic acceptable vegetal essential oils, glycerol, salicylic acid and salts and esters thereof, glycolic acid, hyaluronic acid and salts and esters thereof , cosmetically acceptable UV filter agents, vitamin A, vitamin B (like B3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, cosmetically acceptable phytoalexins, cosmetic acceptable perfume, and mixtures thereof.

1 1. The homogeneous mixture of stable plant cells of claim 10, whereby said plant cells belongs to a first plant family, in which one or more cosmetic active agents is one or more essential oils or parts thereof of a second plant species not belonging to the first family.

12. The homogeneous mixture of the stable plant cells of claim 1 , in which at least 50% by weight of the stable plant cells or agglomerates thereof provided with lecithin containing protecting membrane structure have a plant cell structure with a central vacuole volume occupying from 30 to 50% of the volume of the total inner plant cell volume, and in which the said central vacuole volume occupying 30 to 50% of the volume of the inner plant cell volume is enriched with one or more cosmetic active agents, said cosmetic active agents being advantageously selected among the group consisting cosmetic acceptable vegetal essential oils, glycerol, salicylic acid and salts and esters thereof, glycolic acid, hyaluronic acid and salts and esters thereof , cosmetically acceptable UV filter agents, vitamin A, vitamin B (like B 3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, cosmetically acceptable phytoalexins, cosmetic acceptable perfume, and mixtures thereof.

13. The homogeneous mixture of stable plant cells of claims 10 and 12, in which the central vacuole volume of plant cells, as well as the lecithin containing protecting membrane structure and advantageously the lecithin containing intermediate structure are enriched with at least a same cosmetic active agent.

14. The homogeneous mixture of stable plant cells of claim 1 , in which the lecithin protective structure comprises at least one sub- structure consisting of two lecithin layers separated the one with respect the other by an intermediate layer selected from the group consisting of an aqueous layer, a glycerol containing layer and an oil layer from said vegetal essential oil.

15. The homogeneous mixture of stable plant cells of any one of the preceding claims, which comprises a glycerol comprising medium comprising 0.5 to 5% by weight lecithin and 0.5 to 5% of a vegetal essential oil, forming a matrix in which the stable plant cells are homogeneously dispersed.

16. The homogeneous mixture of stable plant cells of claim 15, in which the glycerol comprising medium comprises from 5 to 25% by weight water.

17. The homogeneous mixture of stable plant cells of claim 15 or 16, in which the lecithin is present in the mixture at a dose of 0.1 % to 3 % by weight.

18. The homogeneous mixture of stable plant cells of any one of the preceding claims, in which the lecithin is from vegetal origin, said lecithin having a HLB value comprised between 8 and 14.

19. The homogeneous mixture of stable plant cells of claim 18, in which the lecithin is from vegetal origin, said lecithin having a HLB value comprised between 9 and 12.

20. The homogeneous mixture of stable plant cells of claim 1 , in which the lecithin is a food grade soy de-oiled lecithin enriched in lysophospholipid, the lecithin having a lysophospholipid weight content with respect to the total phospholipid weight of the lecithin comprised between 5% and 50% .

21. The homogeneous mixture of stable plant cells of claim 20, in which the lecithin is a hydrolyzed lecithin having a lysophospholipid weight content with respect to the total phospholipid weight of the lecithin comprised between 20 and 50% .

22. The homogeneous mixture of stable plant cells of claim 1 , which comprises:

- from 10 to 70% by weight of glycerol; and whereby the lecithin content in said homogeneous mixture of stable plant cells is from 0.1 % to 3 % by weight. 43

23. A method for the preparation of a homogeneous mixture of stable plant cells from a first plant species belonging to a first genus of first plant family in a glycerol comprising medium according to claim 1 , said method comprising at least the following steps:

* step 4: mixing at a temperature from 20 to 45°C, the lecithin containing plant cells medium with a glycerin containing medium, whereby forming bi-layered lipidic lecithin based membrane fragments, a portion thereof being fused with the membrane of plant cells forming a lecithin containing protecting membrane structure for plant cells or agglomerates thereof in a glycerol comprising medium.

24. The method of claim 23 , in which an vegetal oil, advantageously an essential vegetal oil, which is liquid at 20°C, lecithin and advantageously one or more cosmetic active agents soluble or dispersible in said vegetal oil or essential vegetal oil are mixed together for preparing a lecithin oily mixture advantageously enriched with one or more cosmetic active agents, and in which the lecithin oily mixture advantageously enriched with one or more cosmetic active agents is mixed with the lecithin containing plant cells medium, so as to form a lecithin containing intermediate structure spacing lecithin containing protecting membrane structure for plant cells or agglomerates thereof, said lecithin containing intermediate structure comprising at least one sub-structure consisting of two lecithin layers separated the one with respect to the other by an oily intermediate layer advantageously enriched with one or more cosmetic active agents.

25. The method of claim 23 or 24, in which the glycerin containing medium is mixed with one or more cosmetic active agents soluble or dispersible in the glycerin containing medium prior its mixing with the lecithin containing plant cells medium.

26. The method of claim 23 , in which an essential vegetal oil or part thereof of a second plant species not belonging to the first plant species of said plant cells or agglomerates thereof, and advantageously one or more cosmetic active agents soluble or dispersible in said essential vegetal oil are mixed together for preparing a lecithin oily mixture advantageously enriched with one or more cosmetic active agents, and in which the lecithin oily mixture advantageously enriched with one or more cosmetic active agents is mixed with the lecithin containing plant cells medium, so as to form a lecithin containing intermediate structure spacing lecithin containing protecting membrane structure for plant cells or agglomerates thereof, said lecithin containing intermediate structure comprising at least one sub-structure consisting of two lecithin layers separated the one with respect to the other by an essential oil intermediate layer comprising the said essential oil of the second plant species, said intermediate layer being advantageously enriched with one or more cosmetic active agents.

27. The method of claim 26, in which the lecithin containing oily mixture is prepared by mixing together a vegetal essential oil or part thereof, and lecithin, the dry weight ratio vegetal essential oil / lecithin being comprised between 1 : 1 and 10: 1 , while the lecithin containing oily mixture is mixed with the lecithin containing plant cells medium so as to prepare a plant cells enriching enfleurage medium with a water free weight ratio of the lecithin containing oily mixture / the lecithin containing plant cells medium comprised between 2: 1 and 10: 1 , whereby said plant cells enriching enfleurage medium is hold, advantageously with some mixing, at a temperature from 5 °C and 45 °C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for at least 10 days, whereby the lecithin containing protecting membrane structure of plant cells is enriched with vegetal essential oil or part thereof from the second plant species forming plant cells or agglomerates thereof with oily enriched membrane structure; whereby the plant cells or agglomerates thereof with oily enriched membrane structure are collected from the plant cells enriching enfleurage medium; and thereafter mixed with a glycerol comprising medium at a temperature from 20°C to 45°C.

28. The method of claim 27, in which at least one cosmetic active agent selected from the group consisting of salicylic acid, glycolic acid, hyaluronic acid, UV filter agent, vitamin A, vitamin B (like B 3 , B6, B9 and mixtures thereof), vitamin C, vitamin D, phytoalexin, cosmetic acceptable perfume, and mixtures thereof, is added to the vegetal essential oil of part thereof from the second plant species prior the mixing of said vegetal essential oil to an amount lecithin for the preparation of the lecithin containing oily mixture.

29. The method of claim 27 or 28, comprising the step of holding said plant cells enriching enfleurage medium at a temperature from 20°C and 40°C and at a pressure comprised between 0.5 x 10⁵ Pa and 5 x 10⁵ Pa, for a period from 15 days up to 30 days.

30. The method of any one of the claims 23 to 29, in which the lecithin mixed to the collected and washed plant cell agglomerates is a hydrolyzed lecithin containing lysophospholipid, said hydrolyzed lecithin being in powder form when mixed to the collected and washed plant cells.

31. The method of claim 30, in which the lecithin is a lecithin from vegetal origin, said lecithin having a HLB value comprised between 8 and 14.

32. The method of claim 30 or 31 , in which the hydrolyzed lecithin containing lysophospholipid is a mixture of (a) phosphatidylcholine compounds consisting of the mix of at least one mono-fatty acid phosphatidylcholine of formula I

with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, and of (b) at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms, whereby the weight ratio between (a) the said at least one monofatty acid phosphatidylcholine of formula I with one radical of the radicals Ri and R2 being hydrogen, while the other radical of the radicals Ri and R2 being a fatty acid radical comprising 12 to 22 carbon atoms, and (b) the said at least one di-fatty acid phosphatidylcholine of formula I with Ri and R2 being each a fatty acid radical comprising 12 to 22 carbon atoms in the said mix is from 0.00012 and 0.12.