HK1179173A - Preparation created from an in vitro culture of dedifferentiated, non-elicited cells of the argania tree, use thereof for treating skin ageing, inflammation and scarring, and production thereof - Google Patents

Description

Formulations derived from in vitro cultures of dedifferentiated, non-induced cells of the amantada spinosa, their use for treating skin ageing, inflammation and scars, and their production

Technical Field

The present invention is directed to a formulation derived from an in vitro culture of dedifferentiated, non-induced cells of the argan tree, a cosmetic or dermatological composition containing said formulation, and the use thereof for treating skin aging, inflammation and healing.

Background

The argan tree is a tree belonging to the plant Sapotaceae, and its scientific name is argan tree (Arganiaspinosa (L.) sceles).

Its habit is similar to olive trees; it has a short and twisted trunk. Wood is very hard and dense. Branches are very thorny, and they have small, needle-like, intergrown, short (about 2 cm long) and narrow leaves that tend to bunch. Leaves are evergreen, but in severe drought, the leaves die and fall off. Flowers are hermaphrodite and pentameric. They are concentrated in the clique and flower in july and june. They are yellow-green.

The Argan tree can bear fruits from 5 years old. The fruit is yellow, oval, stemless berry, about 4-5 cm long. It consists of a slurry around nuts that contain 2 to 3 flat seeds stuck together, each wrapped with an oil rich kernel.

Avanga is peculiar to morocco and is located mainly in the southwest portion of morocco between soverra and argader. The African forest covers about 830000 ha.

Due to the particularly hard wood of the amalgar, morocco was the first to be used as a fuel supply. Other major traditional uses are oil that is initially extracted by hand, but now using pressing for extraction. The primary use of such oils is as food; another important application is currently in cosmetics. The pulp and residual meal resulting from the production of oil are commonly used in animal feed.

Many cosmetic products are developed from the tree Argan. There have been several patents of inventions directed to oils derived from seeds, such as oils obtained by solvents (patent Fr 2553788), argan oil rich in non-saponifiable matter (patent Fr 2724663).

Substances other than oil have also been patented, such as peptides derived from seed meal obtained after oil extraction; compositions of peptides from oil and meal are used to treat skin aging-related problems (patent Fr 2756183). Also, there are inventions of proteins and saponins from leaves, meal of argan tree = extract from leaves (patent EP 1213025), meal proteins (patent EP 1213024), meal saponins (patent EP 1430900). Recently, patent application EP 1968536 has disclosed the use of extracts from the pulp of the fruit of the agana tree in anti-ageing cosmetics by preservation.

Thus, compositions of whole argania are of interest for dermatological and/or cosmetic use.

The agana tree is an important resource plant because, first of all, it is an "ecosystem" plant ecologically. It is fully adapted to arid areas and it protects the soil from water and wind erosion, thus preventing the desert from approaching into morocco. But also because economically it is a tree with multiple uses.

It is the leading crop of morocco. Therefore, the African forest is protected by the Zhao (dahir) (statute) issued in 1925, and the written country has the highest right for the African forest, but the local masses enjoy the benefits (fruits, deadwoods, crops under the African tree). UNESCO recently listed ajanine as a biosphere reserve.

This is why the use of wood or leaves for cosmetic use can have serious consequences for the protected plants.

Another means of obtaining molecules of interest from plants is to prepare totipotent dedifferentiated cell cultures. The use of dedifferentiated plant cells also avoids the industrial problems encountered during the development of some cosmetic products. With cell cultures, there is no difference in the concentration of the substance of interest between different plant batches or harvests. It is also a non-destructive technique that is relatively easy and inexpensive to build. Finally, this obviates the need for extraction, since the cells are in culture medium and the active compound is in culture medium or in the intracellular liquid. These compounds can thus be obtained by simple grinding.

Patent application WO03077881 discloses compositions for topical application containing at least one plant cell homogenate dedifferentiated and induced in vitro cultures for the synthesis of at least one phytoalexin. The plant material is preferably from rattan.

Disclosure of Invention

Thus, this document discloses cosmetic applications of plant cells that can be dedifferentiated and induced to produce secondary metabolites in sufficient quantities to enable biological activity in topical applications.

Surprisingly and unexpectedly, the applicant has demonstrated that in vitro cultures derived from dedifferentiated but not induced cells of the argan tree have good cosmetic and/or dermatological activity in the fields of anti-aging, inflammation and healing.

The results obtained indicate that in the specific case of the Argan tree, another application of the Argan tree cell culture (in this case non-induced cells) is possible within the context mentioned above.

Furthermore, obtaining a formulation according to the present disclosure may eliminate the need for an induction step, which is an industrially difficult and expensive step; induction leads to lower biomass production due to slow cell growth.

Application WO03077881 mentions various ways of carrying out this induction, such as UV irradiation for 3 days; carbon dioxide for 24 hours to 2 days; UV irradiation and carbon dioxide for 5 days.

The process, carried out within the framework of the present invention, consisting of the dedifferentiation of cells from plant material derived from the tree argania followed by cell culture in suspension, allows the rapid production of a fine, abundant, homogeneous and sterile biomass of this plant. Cell culture makes it possible to apply the biosynthetic pathways of such plants directly to the cell scale.

The present invention therefore aims at formulations derived from in vitro cultures of dedifferentiated non-induced cells of the argan tree, cosmetic or dermatological compositions comprising said formulations and their use in cosmetics and/or dermatology, preferably for the treatment of skin ageing, inflammation and healing.

More generally, in vitro culture of plant tissue in suspension provides a means to produce active organic compounds directly derived from primary or secondary cell metabolism.

Plant cells are in a dedifferentiated state in suspension, similar to that of stem cells used in animal cell cultures. Thus, these plant cells are theoretically capable of producing all the metabolites observed in the whole plant. Dedifferentiation leads to genetic or epigenetic interference of the biosynthetic pathway, so that the chemical spectrum between the whole plant and the obtained cell line is qualitatively and quantitatively different. Therefore, theoretically, reaction intermediates that are not observed in the whole plant may appear in the cell suspension. This provides a new opportunity to obtain "dormant" chemical biodiversity.

In the current state of the art, typically induction (chemical, physical, biological) of cell cultures can stimulate and produce more secondary metabolites. In our process according to the invention, we will show that, unlike most cases, surprisingly, induction is not necessary but is detrimental to the growth of the biomass and the desired biological activity.

One of the objects of the present invention relates to a preparation derived from an in vitro culture of dedifferentiated, non-induced cells of the Argania tree.

"dedifferentiated plant cell" means any plant cell without any specific specialised properties, in other words in a physiological state similar to that of a plant meristem in its natural state. These cells are able to survive themselves independent of other cells.

Dedifferentiated Argania spinosa cells are obtained from living plant material taken from trees or young shoots, consisting of leaves, petioles, stems, bark, roots, fruits, seeds, flowers and floral organs or buds, in particular from leaves.

Methods for obtaining a dedifferentiated cell culture are obtained by any method known to the person skilled in the art, see for example Murashige, t, Skoog, f.1962.a reviseddmedium for rapid growth and bio assays with a tobaco tissue cultures, physics. plant15: 473-496. Plant Culture Media, Vol-1Formulations and uses E.F. George, D.J.M.Puttock, and H.J.George (1987) experiences Ltd. Edington, Westbury, Wilts, BA134QG UK.

The formulation according to the invention can be obtained by carrying out the following steps in sequence:

a) the sterilization of the plant material is carried out,

b) the de-differentiation of the cells is carried out,

c) placing in cell suspension containing culture medium and no inducer,

d) propagation of biomass and production of cultures with inducer-free medium, and

the preparation is obtained.

If the objective is to produce a small amount of biomass, the formulation can be prepared in an erlenmeyer flask, or for larger amounts, the formulation can be prepared in a bioreactor. For example, the average amount collected in a conical flask with 500ml of cell suspension is 100g of dry biomass (i.e. 200g biomass per L of cell suspension), while the average dry material collected in a 10L bioreactor is 3000 g (300 g/L biomass).

Three main modes encountered in plant cell culture in bioreactors:

1. the culture is carried out discontinuously or in batches,

2. feeding/collecting or fed-batch culturing, and

3. and (4) continuously culturing.

a. Plant and method for producing the sameAnd (3) material sterilization:

taking Acacia spinosa (Argania spinosa) explants, and more particularly explants of leaves, the soil is removed with sodium or calcium hypochlorite solution or mercuric chloride solution for several minutes at ambient temperature. The tissue was rinsed with sterile distilled water and then washed at least once with sterile distilled water at the end of the decontamination.

b. Step of cell dedifferentiation

The decontaminated explants were placed under a laminar flow hood in contact with Murashige & Skoog agar nutrient medium to which sucrose and growth factors (or hormones) had been added. These growth hormones will control the cellular mechanisms of the explant leading to cell division and to cell clusters or dedifferentiated calli (callus regeneration). The resulting calli were transferred to new dedifferentiating nutrient medium every 3-4 weeks. Some agar-rich components of this medium can be metabolized by the callus or degraded by the action of air.

In general, hormone compositions based on auxin (2-4 dichloro-phenoxyacetic acid) and cytokinin (kinetin) were successfully tested to obtain a rapid and sufficient tissue dedifferentiation in the form of loose callus (callus regeneration) and to facilitate transfer into liquid medium. Sterile leaf explants can be stored paraxially, contacted with an agar medium consisting of Murashige and Skoog medium (Murashige, T., Skoog, F.1962.Arevised medium for rapid growth and bio assays with a superbase tissue culture, Physiol.plant15: 473-496) with 30g/L sucrose, 8g/L agar, 0.5mg/L kinetin and 0.75 mg/L2-4 dichloro-phenoxyacetic acid (24D) additive, and adjusted to pH6 prior to autoclaving at 121 deg.C (1 bar) for 20 minutes. The plates containing the explants were incubated at 28 ℃ in the dark. The first callus appeared after 2 weeks. Every 3-4 weeks, the resulting callus was transferred to new medium by dividing the callus with a scalpel to keep it at a size of 2 to 3 cm. These transfers were kept for 2 to 6 months until loose callus was obtained.

c. Step of producing cell suspension in inducer-free medium

Cell dedifferentiation for continuous transfer of callus onto agar medium resulted in the formation of loose callus. This decrease in intercellular adhesion is a consequence of dedifferentiation, which can occur between two and six months depending on the plant. This state facilitates the transfer into liquid medium, since it ensures the dispersion of the callus in the cell suspension, while minimizing the induced mechanical stress. Therefore, the collection of loose callus (10-20% by volume) was introduced into a liquid nutrient medium prepared using the same formulation as the agar dedifferentiation medium without gelling agent.

Thus, the loose type callus was dispersed in the liquid medium by the action of the vibration table for 2 to 3 days, and the resulting cell suspension was completely free of undispersed callus portions, thereby forming a uniform cell suspension. This suspension is kept in culture to obtain a sufficiently dense population of cells. At this stage, the suspension is passed (sub-cultured) or diluted in a new nutrient medium and the culture is started in the same way.

The initial cell suspension was started by storing 20 to 40g of unconsolidated callus in a 500ml Erlenmeyer flask containing 200ml of medium. Thus, the loose callus was dispersed in liquid medium by the action of a shaking table at 115rpm in the dark at 29 ℃ for 2 to 3 days. The cell float was then collected using a pipette, leaving residual callus clusters undispersed. The cell suspension thus forms a homogeneous cell suspension. This suspension is kept in culture to obtain a "sufficiently" dense population of cells. The resulting cell suspension was cultured for 15 days and then proliferated by dilution to 1:5 in a new medium for the same period of time. Adjustments to the media composition (nutrients, growth factors, etc.) have been made to maximize biomass productivity. The result was an ARGMS biomass multiplication medium optimized for liquid cell suspensions (see Table 1). This medium is a modified version of the Murashige & Skoog medium for callus regeneration. This medium was adjusted to pH6 by addition of KOH, followed by autoclaving at 121 ℃ (p =1 bar) for 20 minutes or filter sterilization at 0.2 μm.

Table 1: the ARGMS medium is a modified version of Murashige & Skoog (ARGMS) medium used to culture Argan tree cells in suspension in Erlenmeyer flasks or bioreactors under optimal conditions

d. Biomass propagation and production culture with inducer-free medium

After several such subcultures, the cell suspension is stable when the cell density obtained is constant over a period of time. Then, in order to maximize biomass yield, adjustments to the media composition (nutrients, growth factors, etc.) are possible. In a particular embodiment of the invention, the optimized medium used as a biomass production device is the medium described in table 1.

The cell suspension is separated from the extracellular medium or culture suspension by filtration, and the biomass collected is returned to the suspension in distilled water and ground at 0 ℃. The homogenate obtained is freeze-dried or centrifuged for clarification before freeze-drying.

The cell culture thus produced is stable under "optimal" conditions and kept in Erlenmeyer flasks (proliferation culture) with 1:5 dilution of the cell suspension every 15 days. This corresponds to inoculation of a cell culture of about 60g/L fresh biomass, which after 15 days of culture yields about 300g/L cell suspension, or if desired in a bioreactor.

The preparation obtained in the erlenmeyer flask or in the bioreactor may consist of:

-a cell suspension (for the purposes of the present invention, "cell suspension" refers to cells (i.e. biomass) in its culture medium);

biomass (for the purposes of the present invention, "biomass" refers to the clusters of cells isolated from the culture medium, i.e. the suspension of cells after filtration);

-returning (or not) the ground biomass in suspension in distilled water;

-a clarified liquid or a float of ground biomass by centrifugation or filtration;

culture float (for the purposes of the present invention "culture float" means the medium in which the cells maintained reside during the culture, or the extracellular medium).

Whether a cell suspension, biomass or suspension of ground biomass is of interest, they may be kept unchanged in frozen form or by the addition of preserving substances such as phenoxy-2-ethanol, benzyl alcohol or any other preserving product appearing in EU instructions appendix VI on cosmetic products entitled "list of preserving agents that may be present in cosmetics". They can also be diluted in a cosmetically acceptable medium in proportions varying from 10% to 60%, such as glycols (propylene glycol, butylene glycol, polyethylene glycol, etc.). The cell suspension or biomass may also be ground and then stored as such or by addition of a preservation substance or medium as described above.

The cell suspension, biomass or biomass suspension, whether ground or not, can also be dried by freeze-drying or atomization and as such maintained or dried on a maltodextrin, lactose or silica type mediator or any other cosmetically acceptable medium.

Finally, the cell suspension can be enriched with useful compounds by affinity chromatography: absorbed on a resin (Type polystyrene copolymer, etc.) and eluted with a suitable solvent such as ethanol.

The fresh biomass obtained according to the process of the invention represents from about 100 to 500g per litre of suspension and more preferably between 200 and 350g per litre of suspension for an optimal collection time (i.e. on average about 15 days).

The following table shows the yields obtained (yields are expressed as grams of product obtained per cell suspension L):

the invention also relates to a cosmetic or dermatological composition comprising, as active ingredient, a preparation derived from a culture of non-induced dedifferentiated cells of the Argan tree, as described above.

Preferably, the amount of said formulation is between 0.1 and 10% of the total amount of the composition. Even more preferably, the amount of extract is between 0.2% and 5%.

The cosmetic composition according to the invention may advantageously be in any galenic form conventionally used in cosmetics for topical or oral application, preferably topical application. For administration by the topical route, the galenic form may be a cream, gel, ointment or spray. The oral formulation is selected from tablets, capsules and powders for drinkable suspensions.

The cosmetic composition according to the invention also comprises usual cosmetically compatible excipients.

The excipients generally compatible with the cosmetic composition may be any excipients known to the person skilled in the art so as to obtain a cosmetic composition for topical application in the form as those described above.

The cosmetic and/or dermatological compositions according to the invention may contain, inter alia, additives and formulation auxiliaries (aid), such as emulsifiers, detergents, foaming surfactants, etc., complexing agents, thickeners, gelling agents, stabilizers, preservatives including antimicrobials and antioxidants, regulators, acidifying agents, basifying agents, softeners, solvents, colorants and fragrances.

The present inventors also showed that preparations derived from dedifferentiated non-induced cells of the argania tree may have the following activities:

antioxidant, anti-radical activity to limit the oxidative processes associated with intrinsic and extrinsic aging, as well as inflammatory processes.

The activity on the extracellular matrix improves the mechanical properties (firmness, elasticity, tonicity) of mature skin by inhibiting the degradation of collagen by metalloproteinases.

Finally, the present invention relates to the compositions disclosed herein for the treatment of skin aging, inflammation and healing.

Detailed Description

The following examples are given as non-limiting examples.

Examples for the manufacture of formulations according to the invention.

Example 1: method for preparing fresh biomass/conical flask

Preferably 3 to 4 months old leaves of the agave plant are sterilized by several baths in sequence: 70% ethanol for 1 minute, 2% sodium hypochlorite for 3 minutes, then rinsed with two successive demineralised water baths for 8 and 10 minutes.

Sterile leaf explants, kept paraxial, were contacted with an agar medium consisting of Murashige and Skoog medium (Murashige, T., Skoog, F.1962.A reviewed media for rapid growth and bio assays with a tobaco tissue culture medium. physiol. plant15: 473-496) with 30g/L sucrose, 8g/L agar, supplemented with 0.5mg/L kinetin and 0.75 mg/L2-4 dichloro-phenoxyacetic acid (2.4-D), and adjusted to pH6 before autoclaving (1 bar) at 121 ℃ for 20 minutes. The plates containing the explants were left to incubate at 28 ℃ in the dark and proliferate until loose and stable callus was obtained.

The initial cell suspension was generated by storing approximately 40g of unconsolidated callus in a 500ml Erlenmeyer flask containing 200ml of autoclaved medium, the composition of which is described in Table 1 above.

The cultures were left on a shaking table at 115RPM in the dark at 29 ℃ for 1 week. The cell float was then collected using a pipette, leaving a cluster of residual callus. The resulting cell suspension was cultured for 15 days and then proliferated by dilution in a new medium at 1:5 for the same time.

The suspension was then filtered under vacuum to recover the biomass. The yield of fresh biomass obtained was 168 g/L.

The biomass was maintained at-20 ℃.

Example 2: dry biomass

Example 2 a: process for dry biomass/Process for batch cultivation in a bioreactor

Four 500ml conical flasks of cell suspension obtained as described in example 1 were placed together in an inoculation apparatus and formed a 2L inoculum which was aseptically poured into a 10L bioreactor. The bioreactor was filled with 8L of optimal medium (see Table 1), supplemented with 30mg/L of a pre-sterilized antifoam, then cooled and maintained at 29.5 ℃ by a thermostatically controlled water circulation in a closed loop in the bioreactor housing.

The oxygen probe was calibrated by saturation and data was entered into the computerized pO2 conditioning device in real time. This device maintained pO2 at 80% by injecting sterile, pure oxygen into the ventilation system. This bioreactor was also equipped with an on-line measurement of CO2 at the effluent gas (head space) which simultaneously input data to a computerized pCO2 conditioning unit, maintaining the pCO2 at 6%. This is done by injecting sterile ambient air mixed with oxygen into the aeration device. The bioreactor was also equipped with a paddle type stirring system at 75RPM to stir the cell suspension and prevent it from settling. An automated device is installed at the output of the bioreactor to enable sterile sampling and monitoring of the biomass.

The batch culture is maintained under these conditions of constant temperature and dissolved gas for 15 to 17 days until a cell density of fresh biomass of 280 to 320g/L is reached. After completion of the batch culture, the bioreactor was emptied and the biomass was collected by filtration on a filter with a buchner funnel.

The collected fresh biomass dissolved in the same volume of distilled water was cold milled using an "ultrasonic cleaner" and then freeze dried.

Example 2 b: dry Biomass/Process in bioreactor with fed-batch culture 10.0.0.1

Four 500ml conical flasks of cell suspension were used as inoculum as described in example 2 a. The bioreactor was prepared as shown in example 2 a. A dissolved gas, temperature and agitation conditioning system was prepared as shown in example 2 a.

The initial culture is maintained under these conditions of constant temperature and dissolved gas for 15 to 17 days until a cell density of fresh biomass of 280 to 320g/L is reached. After the initial culture was completed, 80% of the contents of the 10L bioreactor were removed. Then 8L of cell suspension was collected. The biomass in this suspension was collected by filtration on a filter with a buchner funnel. 2240g to 2560g of fresh biomass are collected. When cooled, the collected fresh biomass dissolved in the same volume of distilled water was ground using an ultrasonic cleaner and then freeze-dried. 100 to 130g of freeze-dried biomass are obtained.

While 80% of the partial collection was performed, 8L of previously autoclaved and cooled ARGMS medium was poured into the bioreactor, and then 2L of cell suspension was contained, thereby restoring a culture volume of 10L. The fed-batch culture is maintained under these conditions of constant temperature and dissolved gas for 5 to 7 days until a cell density of fresh biomass of 280 to 320g/L is reached. This culture is faster (higher yield) than the initial culture, since the biomass is in a state of continuous physiological cell division, so that the pouring of new nutrient medium is characterized by a latency of less than 24 hours and the biomass expands immediately. At the end of this fed-batch culture, 80% of the 10L bioreactor was removed. Then 8L of cell suspension was collected. The biomass of this suspension was collected by filtration on a filter with a buchner funnel. Then, the culture was restarted as before.

Example 2 c: dry biomass/Process carried out in a bioreactor with continuous culture

Four 500ml conical flasks of cell suspension were used as inoculum as described in example 2 a. The bioreactor was prepared as shown in example 2 a. A dissolved gas, temperature and agitation conditioning system was prepared as shown in example 2 a.

The initial culture was maintained for 10 days under these conditions of constant temperature and dissolved gas until a cell density of 150g/L and a cell density of 0.2d were reached^-1The instantaneous fresh biomass growth rate. At this stage, 1.2% of the contents of the 10L bioreactor were removed every 1 hour for 20 minutes. These samples were automatically compensated by pouring the same volume of fresh ARGMS medium into the bioreactor. This method maintains cells in a constant physiological state and cell density.

Then 100 to 120ml of cell suspension was collected. The biomass in this suspension was collected by filtration on a filter with a buchner funnel. 15g to 18g of fresh biomass was collected per sample. The collected fresh biomass dissolved in the same volume of distilled water was cold milled using an ultrasonic cleaner and then freeze dried. The results obtained were 0.71 to 0.85g of freeze-dried biomass for each extraction. Therefore, the culture was maintained for at least 60 days. It is theoretically possible to maintain this without time limitation.

The advantages of continuous culture over previous mode are that the bioreactor does not need to be prepared again, it requires cleaning and sterilization, and there is no cell latency. A1 to 1.5% cell suspension is automatically withdrawn and subsequently caused to change minimally in the ongoing composition of the medium in the bioreactor by replenishing the bioreactor with fresh medium. Thus, the cell population does not make any metabolic adjustments to latency, which results in the loss in biomass volumetric productivity observed in other culture modes.

Example 3: fresh biomass flotage obtained as described in example 2a

20g of freshly ground biomass obtained as described in example 2a were centrifuged at 10000g for 15 minutes and the flotage collected. And then freeze-dried.

The average yield was 30mg freeze-dried floe per gram of fresh biomass.

Examples of cosmetic compositions:

example 4: H/E formulation

Composition (I)	%
		Fresh biomass (example 1)	5

Glycine	10.0
		Na2EDTA	0.1
Xanthan gum	0.3
		C12-C15 alkyl benzoates	10.0
Palmitic acid octyl ester	5.0
		Preservative agent	qs
Stearic acid alcohol	2.5
		Glyceryl monostearate	2.5
Potassium cetyl phosphate	1.8
		Softened water	QSP 100

Example 5: E/H formulation

Composition (I)	%
		Floating material (example 3)	0.5
Glycine	4.0
		Na2EDTA	0.1
MgSO4	1.0
		Xanthan gum	0.1
C12-C15 alkyl benzoates	12.5
		Isohexadecane	3.5
Cyclomethicone	3.0
		Preservative agent	qs
Polyglycerol and sorbitol esters	4.0
		Myristic-3 myristate	2.0
Softened water	QSP 100

Example 6: evaluation of antioxidant Activity

Chemiluminescence

This method generates free radicals (superoxide radical O) by photochemical signal₂Degree-). The strength of oxidation is 1000 times higher than that obtained under normal conditions. Detection by chemiluminescence was used to evaluate extracts or molecules of fat-soluble or water-soluble antioxidants. The results are expressed as equal amounts of vitamin C or Trolox (6-hydroxy-2, 5,7, 8-tetramethyl chroman-2-carboxylic acid). The sensitivity is in the order of 1 nanomolar.

The analysis of the results relies on two criteria, namely the shape of the curve (integral) and the values given by the software in nanomolar. (Igor Popov and Gudrun Lewis. methods informalogy [44] Vol 300.437-456; Maibach I Howard and col. journal of cosmetic Dermatology Vol 7(2)96-100 (2008)).

The results will be expressed as μ g of sample necessary to obtain an activity corresponding to that detected by the 1 μ g standard (trolox (no corresponding translation)).

As a result:

the antioxidant activity studied in this test represents the capacity to specifically capture superoxide anions by chemiluminescence.

Table 2: evaluation and quantification of antioxidant capacity in trolox equivalent

The floaters of the freeze-dried biomass prepared according to example 2a and of the ground freeze-dried biomass prepared according to example 3 have an overall equivalent anti-radical capture activity.

278 μ g of freeze-dried biomass was necessary to obtain an activity equivalent to that detected by 1 μ g trolox: corresponding to the activity of coenzyme Q10 (referred to as antioxidant molecule).

171. mu.g of ground freeze-dried biomass flotage was necessary to obtain an activity corresponding to the activity detected by 1. mu.g trolox.

Free radicals, whose production increases as a result of external attack (cold, pollution, tobacco, UV), can lead to damage to skin cell DNA, as well as to cellular and mitochondrial membrane DNA. These free radicals also play a very important role in the inflammatory process. It is these active metabolites that are the second messengers of cellular oxidative stress signals and are therefore early mediators of inflammation (A. VanDer Vliet and coll, Chem Biol Interaction 85: 95-1161992).

The anti-radical activity of the formulations described in examples 2a and 3 helps to combat intrinsic and extrinsic skin aging and inflammation.

Example 7: evaluation of the inhibition of collagen formation of the extracellular matrix by Metalloprotease Activity

Extracellular Matrix (MEC) is a dynamic structure that has structural and regulatory effects on tissues. It imparts skin tone (turgescience) and mechanical properties. In the epidermis, it occupies the intercellular spaces and provides support for the epidermal structures. It also controls communication between epidermal cells and plays a role in cell activity. It is composed of fibers, especially collagen, and basic substances (water, salts, glycoproteins, glycosaminoglycans). Collagen is a fibrous protein formed by three polypeptide chains, which may be the same or different, hydrogen bonded by covalent bonds. Collagen forms the main component of the fibrous network structure and provides resistance and elasticity to the skin to exert mechanical action.

When cells are senescent, most components of MEC are degraded by zinc-rich endopeptidase-type enzymes called Matrix Metalloproteinases (MMP) (Hideoki Nagase § and J.Frederick Wessenner.JBiol Chem, Vol.274, Issue 31,21491-21494, July 30,1999). They are either membrane or secreted. All MMPs share strong sequence and structural homology, but differ in their specificity for the substrate. MMP1 or "interstitial collagenase" degrades primarily type I collagen (80% of the content of normal skin dermis) and also degrades II VII VIII and type X collagen.

We analyzed the effect of extracts on the activity of the ligase by fluorescence quantification using the specific peptide substrate Mca-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH 2 on a model of human recombinase (David Leppertd and coll, Analytical Biochemistry 328(2004) 166-17).

The activated enzyme was pre-incubated with different formulations and then placed in the presence of the substrate. The enzyme cleaves the peptide, separating the Mca fluorophore (7 methoxycoumarin-4-yl) acetyl) from the quencher Dpa (N-3- (2, 4-dinitrophenyl) -L-2,3 diaminopropionyl). Then, the peptide emits fluorescence with a wavelength of 405nm when excited at 320 nm. Thus, MMP-1 enzyme activity is measured, which is proportional to the fluorescence emitted.

Using this in the tubo assay, we were able to detect a potential inhibitor of MMP1 activity, an enzyme that has a crucial role in initiating collagen degradation. We measured the percentage of inhibition of MMP1 activity.

Calculation of percent enzyme inhibition associated with inhibitor or product:

% inhibition =100 × (maximum net enzyme activity)Net enzyme activity in the presence of inhibitor)/maximum net enzyme activity

As a result:

the float prepared according to example 3 significantly inhibited MMP1 activity and was dependent on a dose from 60 to 500 μ g/ml.

Table 3: lyophilization of ground biomass floaters (prepared according to example 3), results of MMP1 inhibition expressed as a percentage (%)

Average inhibition%	Concentration in μ g/ml
		100	500
58	300
		41	150
20	60

From 60 to 1000. mu.g/ml of freeze-dried biomass prepared according to example 2a was tested. We were unable to measure inhibitory activity due to physicochemical interference of the biomass as a whole. However, a very similar extract was tested and showed significant inhibition from 60 μ g/ml to 1000 μ g/ml.

The extract prepared according to example 3 can resist increased activity of MMPs when aging occurs, and can help maintain the mechanical action of collagen, thereby providing resistance and elasticity to the skin.

Example 8: measurement of TGF-. beta.1 Synthesis in HaCaT keratinocytes

TGF-beta 1 (transforming growth factor-beta 1) belongs to a TGF-beta superfamily secreted by different cell types and plays an important role in controlling cell growth and regulating various cell responses and biological processes. The major activity of cytokines in this superfamily is that they regulate the proliferation of most cells, they stimulate fibroblast proliferation and increase extracellular matrix formation (Lawrence, 1996). TGF-. beta.1 is also involved in the process of injury repair, healing, especially by inducing recombination of the cytoskeleton (actin) and promoting epithelial cell migration (Boland et al, 1996) (Cullen et al, 1997). The most representative cell population in skin tissue is the keratinocyte cell population. It constitutes an important source of growth factors that can control and influence the behavior of skin cells (i.e., fibroblasts) (Ghahary et al, 2001).

Apparatus and method

Production of non-induced biomass: according to example 2a

Induced biomass production

Murashige & Skoog medium was prepared without growth factors (kinetin and 24D). This medium was adjusted to pH6 by adding KOH, followed by autoclaving at 121 ℃ (p =1 bar) for 20 minutes. The medium was then inoculated with a cell suspension from the propagation culture at a volume of 1: 5. The induction conditions were generated immediately after sterile addition of concentrated 6-benzylaminopurine (BAP or (N- (benzyl) -7H-purin-6-amine) and inducer (acetylsalicylic acid and methyl jasmonate) solutions to kinetin DMSO, the result of which was EMS induction medium (see Table 4).

Table 4 EMS medium, i.e. Murashige & Skoog modified medium for culturing a suspension of agave tree cells in Erlenmeyer flasks under induction conditions

HaCaT keratinocytes were treated with different extracts for 5h, and then the cells were incubated in DMEM for 24 h at 37 ℃. TGF-. beta.1 was dosed to the culture float using an ELISA kit.

The effect of non-induced A.crambe biomass prepared according to example 2a and the A.crambe biomass obtained after induction on TGF-. beta.1 synthesis in human HaCaT keratinocytes is shown in FIG. 1. They show that non-evoked avalia biomass (50. mu.g/mL) prepared according to example 2a stimulates 48% of TGF-. beta.1 synthesis, while evoked avalia biomass inhibits 26% of TGF-. beta.1 synthesis in HaCaT keratinocytes.

Example 9: measurement of human keratinocyte proliferation and cell migration

Example 9. a: measurement of cell proliferation of human keratinocytes

Wound healing is a complex, dynamic, biological process involving the interaction of many local and systemic factors in normal tissue repair. Healing progression includes four interdependent stages: hemostasis, inflammation, proliferation and remodeling. Proliferation means three clearly visible processes, namely granulation, shrinkage and neoepithelialization.

During granulation, cell proliferation is observed, which will be involved in the remaining repair processes, with migration of these cells to the damaged bed. These cells include macrophages, fibroblasts and endothelial cells. Macrophages release chemokines and growth factors continuously. Fibroblasts construct a new cell matrix necessary for cell growth at the damaged substrate. Such scaffolds promote cell migration.

The contraction of the lesion is a mechanism for reducing the size of the lesion, and fibroblasts play a dominant role in this contraction.

Epidermal regeneration consists of epithelial neogenesis, which covers the lesion to form an effective barrier to the external environment, can be coloured and restore its sensory and immunological functions. This therefore implies not only the cell migration and proliferation process of keratinocytes, but also the differentiation of such neoepithelial cells and the restoration of the basement membrane which rejoins the dermis and epidermis. When basal cells migrate toward the center of the lesion, allowing the two sides of the lesion to join, a wave of mitosis occurs to fill the space left by migration and to provide cells for epithelial tissue in three-dimensional regeneration.

The proliferation steps of keratinocytes, fibroblasts and endothelial cells can be considered as one of the functional phenomena confirming the healing activity of the active ingredients. Increased proliferation of fibroblasts or endothelial cells will be involved in dermal healing, while increased proliferation of keratinocytes will be involved in epithelial neogenesis.

The equipment and the method are as follows: cell proliferation

The technique used measures incorporation of the nucleotide, 5-bromo-2' -deoxyuridine (BrdU), a thymidine analogue, in the DNA of S phase cells.

Keratinocytes isolated from post-operative discarded skin were cultured in complete KSFM (BPE 25. mu.g/ml; EGF 1.5 mg/ml). Cells were incubated at 37 ℃ for 48 hours in an atmosphere of 5% CO2 in the presence of the molecule to be assessed.

BrdU spiking, which is proportional to the cell proliferation rate, was evaluated by a system of anti-BrdU antibodies coupled to peroxidase. Addition of peroxidase substrate showed a colored reaction (Biotrak Elisa system). The corresponding absorbance (OD (DO for French abbreviation)) was measured at 450 nm. Thus, this data is proportional to the cell proliferation rate.

The percent proliferation was then determined using the following formula:

% proliferation = (OD (treated) -OD (t control)_{Minimum size}) 100/(OD (t control))_{Most preferably Big (a)}) OD (t control)_{Minimum size})）

Note:

control_{Minimum size}= cells incubated with minimal media

Control_{Maximum of}= cells incubated with complete medium

Thus, control_{Minimum size}Corresponding to 0% proliferation, control_{Maximum of}Corresponding to 100% proliferation.

Results on cell proliferation are shown in fig. 2, demonstrating the effect of the agana biomass prepared according to example 2a on human keratinocyte proliferation.

They show that the biomass of the Argan tree prepared according to example 2a stimulates the proliferation of 25% of human keratinocytes at 0.1. mu.g/ml. No effect was measured when tested at 0.01. mu.g/ml.

Example 9. b: measurement of cell migration in human keratinocytes

The equipment and the method are as follows: cell migration of HaCaT keratinocytes

The protocol used to study cell migration was based on the use of a 96-well kit. The principle of this assay consists of studying the migration of cells to the center of the well (96-well plate). To do this, a plug is placed in the centre of each well, thereby creating a 2mm diameter detection zone. HaCaT cells were then seeded around this plug. Once the cells are well bound at the surface surrounding the plug, the plug is withdrawn and the cells can thus migrate to the detection zone. The plates without plugs and with active ingredients were incubated in DMEM 0% SVF for 24 hours at 37 ℃. The number of cells located in the area of the plug is then analyzed to assess cell migration. Cells were labeled with Hoechst 33342 and cells located only in this region were observed and counted using cache. An average of 8 wells was obtained for each condition.

The results are expressed as

Fluorescence intensity (IF-proportional to the number of cells that have migrated)

Percentage of activity of 0% SVF relative to control:

（IF_{treated of}-IF_{0% transferred}）×100/（IF_{t control 0% SVF}-IF_{0% transferred}）

Note:

IF (0% migrated) corresponds to IF (fluorescence intensity) of the plugged wells and thus to background noise.

FIG. 3 shows the results of cell migration and illustrates the effect of the Ajan tree biomass prepared according to example 2a on HaCaT keratinocyte migration.

They show that the biomass of the agana tree prepared according to example 2a, at 0.01 or 0.03 μ g/ml, stimulates migration of 79% and 73% of HaCaT keratinocytes, respectively.

Claims (7)

1. Formulations derived from de-differentiated, non-induced cultures of cells of the Argania tree in vitro for the treatment of skin aging, inflammation and healing.

2. The formulation of claim 1 in the form of a cell suspension, biomass, ground biomass floe, or culture floe.

3. A cosmetic or dermatological composition comprising, as active ingredient, a preparation derived from a culture of non-dedifferentiated argan cells such as defined in claim 1 or 2, in association with a cosmetically or dermatologically acceptable excipient.

4. A composition according to claim 3, characterized in that the amount of the formulation is between 0.1 and 10% of the total amount of the composition.

5. The composition according to claim 3 or 4 for use in the treatment of skin aging, inflammation and healing.

6. A method for obtaining a formulation, characterized by comprising the steps of:

a) sterilization of plant material

b) Dedifferentiation of cells

c) Placing in cell suspension containing culture medium and no inducer,

d) propagation of biomass and production of cultures using a medium without elicitors, and

the preparation is obtained.

7. Method for the cosmetic treatment of skin ageing, comprising the use of a composition as defined according to any one of claims 3 to 5.