EP2355782A2 - A complex of active vegetal stem cells and a cosmetic composition - Google Patents

Abstract

The present invention refers to a complex of vegetal source active stem cells, including stem cells of Malus Domestica, stem cells of Buddleja Davidii amd preferably Teprenone, and to a cosmetic composition for topical use, which contains such complex. The complex of stem cells with Teprenone and the cosmetic composition of the invention perform a regenerating action and a repairing action on the skin cell layers, increasing vitality and longevity of the skin and hair follicle.

Description

A COMPLEX OF ACTIVE VEGETAL STEM CELLS AND A COSMETIC COMPOSITION

TECHNICAL FIELD The present invention relates to a complex based on stem cells from a vegetal source, and to a cosmetic composition containing the complex as active principle.

The present invention falls within the field of cosmetic or dermatological preparations for topical use for skin and scalp follicle treatment.

PRIOR ART

As is known, stem cells are non specialized cells that renew through cell division for an indefinite period of time and which can transform into specialized cells of various tissues of the organism. In the animal organism, stem cells are typically found in the embryo, in some adult's tissues, for example in the bone marrow and in the umbilical cord. Embryonic stem cells (ES) are totipotent, that is, they are capable of differentiating into cells that represent the three primary germinal layers endoderm, ectoderm and mesoderm and they can originate any type of tissue. Embryonic stem cells, moreover, have autorenovation capabilities and are capable of passing through several cycles of cell division while maintaining an undifferentiated status.

While adult animal stem cells have some of the features of ES cells, such as autorenovation, long life and a high proliferative potential, they are multipotent non specialized cells, that is, they can only originate some types of cells or tissues.

The multiplication of animal stem cells can take place both symmetrically, when a cell division originates two identical stem cells, or asymmetrically, when one stem cell produces an identical and more differentiated daughter cell. ES cells divide symmetrically while an asymmetric cell division is used in adult stem cells. These rare and specialized adult cells are necessary for tissue replacement for the entire life of an animal organism. In many tissues, stem cells are largely kept in a quiescence status, but they can be returned to the cell cycle in response to extracellular stimuli. Once the stimulus to division has started, stem cells release undifferentiated progenitors (Transient amplifying cells) that are relatively undifferentiated cell types, derived from the asymmetric division of stem cells and do not have autorenovation capabilities. Progenitors in turn produce effector cells through subsequent proliferation cycles. It is believed that resident autorenovating cells have an important role in the homeostatic maintenance of many organs and tissues. An example thereof is the epidermis, which has a "high cell metabolism cycle", besides a high regeneration potential.

At the level of the deep basal layer of the face and body epidermis there are stem cells that have the capability of differentiating as they proliferate and become superficial, originating the specialized differentiated cells of the epidermis that form the three separate tissue layers: spinous, granular and horny.

As is known, human skin epidermis is a stratified squamous epithelium which provides the first line of defence against harmful environmental agents and which renews constantly during life. The interfollicular epidermis maintains homeostasis by the proliferation of keratinocytes in the basal layer attached to the underlying basal membrane. As basal cells detach from the basal membrane, they withdraw from the cellular cycle and start a terminal differentiation program to gradually move upwards to the skin surface. At the beginning of the differentiation program, the keratinocytes go through various steps to form three different layers of the epidermis: the spinous layer, followed by the granular one and finally, the keratinocytes lose the cores becoming flattened keratinized cells that make up the horny layer. Keratinized cells continuously fall from the skin surface and thus, new differentiated cells must be generated during life.

In the skin, the filling of the differentiation compartment depends on the proliferation of a subpopulation of cells in the basal layer, better known as stem cells that represent the 2 - 7% of all the cells found on the basal membrane of the skin. The most widely acknowledged model consists in the fact that at the time of division, a stem cell can produce a daughter stem cell and an initiator cell (progenitor) called Transient Amplifying cell, or TA cell. The daughter of TA stem cell intended to be subject to terminal differentiation can initially divide a limited number of times (about 5 times). Therefore, of the two daughter cells that derive from the division of a cell of the basal layer, one maintains the features of stem cell and remains in the germinal layer while the other moves towards the upper layer, called spinous.

The epidermis basal layer therefore contains two different types of cellular populations:

- Epidermal Stem Cells;

- The daughters of stem cells (Transiently Amplifying Cells) that are in an active and quick division in order to provide new cells, replacing those lost subsequent to desquamation.

After a predetermined number of divisions, the cells detach from the basal membrane and start the cellular differentiation that in the end will bring them to become cells of the horny layer. The entire process, that is, the time required for a cell to move from the basal to the horny layer, on the average lasts 30 days. If the epithelium is subject to harms of various nature, the keratinization time decreases significantly. Moreover, it has been noted that the mitotic activity of basal cells is much more active overnight. Also the tissue regeneration subsequent to injuries involves epithelial stem cells to replace damaged cells.

The skin adult stem cells grow, live and autoreproduce in a microenvironment called niche, located in the basal layer of the epidermis. This protective niche provides a microenvironment that allows stem cells to show their intrinsic properties while maintaining their stock of differentiated programs.

The niche protects and preserves them from degradation and at the same time regulates their physiological proliferation that is what allows the skin to regenerate and remain toned and compact. The niche consists of an extracellular matrix with a glicoproteic base, other non-cellular constituents and three- dimensional spaces. Contact between these elements allows molecular interactions, that are essential for regulating the stem cell functions. Non-proteic components of the local microenvironment also affect the stem cell functions (D.Scadden 2006).

In stem cell niches there are microenvironmental cells that feed stem ones, allowing them to maintain homeostasis. Niche cells provide a protective environment that defends the cells from the apoptosis stimuli and other stimuli that could impair the reserves of stem cells.

The epidermis therefore is a dynamic epithelium, subject to constant renovation for the entire life. The capability of renovation and repair of the skin epithelium and of the underlying derma is based on the presence of multipotent adult stem cells in each of these compartments.

The decay of the regeneration potential of tissues is a sign of aging and can be caused by changes due to age in the specific stem cells of tissues.

While aging, our stem cells lose their capability of multiplying causing some aspects of aging. At the skin level, photoaging, chronoaging and oxidative stress can damage epidermis and derma, the niche and affect the functions of the epidermal stem cells, causing a reduction of the number and weakening the functionality thereof, that is, the capability of producing progenitors and effector cells.

If a loss of potency occurs, to the skin this means early aging. Skin aging is caused by an intrinsic factor, chronological aging, and by an extrinsic one, photoaging, a consequence of environmental external factors, especially UV radiations but also pollution that specifically involve the skin due to its external position.

The two processes coexist in a different proportion in different people, interlacing and overlapping their effects.

The intrinsic skin aging (chronoaging) is a consequence of genetic processes that cause a reduced functionality of the skin, caused by natural factors such as limited life of skin fibroblasts mutations of the immune system - DNA mutations. From a clinical point of view, the effects of skin aging are seen with the appearance of wrinkles, thin at first, then increasingly deeper, spots and loss of tone and elasticity.

From a histological point of view, the modifications in chronological skin aging are as follows:

- a structural reduction of skin, which becomes thinner in all layers;

- epidermal thinning;

- reduction of derma thickness with decrease in the number of cells (fibroblasts) and their biosynthetic capability; - the connective tissue loses its fibrous structure and its hygroscopic property;

- collagen and elastic fibres degenerate;

- the vascularization decreases;

- the subcutaneous fatty tissue decreases little by little. The exact order of the single layers in the epidermis is lost. Less epidermal cells form and their size decreases. Horn cells remains almost twice the time in the horny layer, thus being exposed to harmful environmental influences for longer. The skin is more subject to injuries.

The activity of sebaceous and sudoriferous glands decreases, with the consequence of a decrease in the skin elasticity and disorders in its function as a barrier.

The lack of natural hydration factors in the upper layers of the epidermis and of the horny layer causes marked skin dryness.

Old skin looks pale, thin, flabby, hypoelastic, hyperextensible, finely wrinkled, often suffering from severe xerosis. As one grows old, the basal keratinocytes undergo a reduction of mitosis and a tendency to an early keratinization. The consequence is a thinning of the epidermis ascribable to the decrease of the malpighian layer axes; by the crest and papilla flattening, the contact surface with the derma decreases. The latter also shows a reduced thickness, with decrease of the collagen and proteoglycan contents. With the passing of the years, an increasing population of "aged" keratinocytes and fibroblasts build up in the skin; since at this point they have realized the maximum number of cellular duplications allowed, they have reached the senescence phase, characterized by mitosis block and by a complex series of functional alterations that makes the permanence of senescent elements unproductive, or even disadvantageous for the tissue physiology. Among the other things, senescent fibroblasts express high levels of Matrix Metalloproteinase (MMP), zinc dependent endopeptidases, capable of degrading virtually all the components of the intercellular substance of the connective and of the dermo-epidermal junction, and low activities of the MMP tissue inhibitors, called TIMP, changing from matrix production cells into break-up factors for the same.

The face and neck skin is also chronically photoexposed.

On the other hand, photoexposed skins look tough, rough, spotted by large hyper- and/or hypo-pigmentary dyschromia and furrowed with deep cuts and rough macro-wrinkles.

The texture is stiff, non elastic, and a thickness increase is found at the horny layer, the overall epidermis and the derma. The dermo-epidermal junction is irregular due to crests and papilla development with different shape and dimension. The keratocyne-produced fibrils of collagen VII which group into irregular and fragmented bundles, are strongly reduced, and the relative mRNA is reduced, too. The elastic fibres become disorganized and abnormal in morphology and dyeing properties. This renders the coalescence between derma and epidermis insufficient and promotes the development of wrinkles. Ultraviolet rays that affect the skin also determine the formation of reactive oxygen species (ROS). The free radicals and the ROS are capable of triggering a rapid sequence of radical reactions, which involve not only the phospholipid bilayers of the cytoplasmatic, mitochondrial and nuclear (lipoperoxidation) membranes but also the cellular and extracellular components of a protein nature and all the elements of the matrix.

Aging is therefore accompanied by a decline in cell proliferation, both in the epidermis and the derma, partly linked to a reduced response of the cells to growth factors. This determines an overall reduction in the thickness of the skin and greater fragility and exposure to trauma.

The reduced speed of cicatrization in older skin suggests both the reduced mobilization of stem cells, and that a reduced number of stem cells are able to respond to signals of proliferation. Following chronological aging, the level of stem cells tends to diminish and change functionally. Even if the basic number of stem cells could be maintained for one's entire life, their endogenous replicative potential has been shown to diminish abruptly with age.

Alterations in the number and functionality of epidermal and dermal stem cells are therefore associated with the aging of the skin.

Both the processes are connected to:

- alterations in the Telomeres, nucleoproteins that accumulate in the terminal region of the chromosomes and which do not codify for any protein. - the changes in the activity of the Telomerase, enzyme capable of extending, that is stretching the Telomeres.

The Telomeres are repetitive structures of aminoacidic sequences (TTAGGG)n accumulated at the end of the chromosomes. The average length of the telomeres drops by 20-200 pairs of bases at each cell division and this is due to the inability by the DNA polymerase to replicate from the 3'-terminal extremity of a linear molecule of DNA since synthesis of the DNA can only occur in the direction 5'—>3'. In order to avoid serious damage to the functionality of the chromosome, at the 3' and 5¹ extremities are positioned telomeric nucleotide sequences (Telomeres) repeated several times and that, as previously stated, do not codify for any protein.

The Telomerase is an enzyme used for the elongation of telomeres. It has a decisive role in avoiding the loss of information during the duplication of the chromosomes, promoting their stability. As previously stated, the DNA polymerase, the enzyme used in the replication of the DNA, is not capable of replicating the chromosome until its termination; if there were no Telomeres, which are thus shortened with each replication, the replication of the DNA would involve each time a significant loss of genetic information and consequent cell senescence.

The senescence of the cells can be associated with the continuous shortening of the telomeres. This is because the telomeres act as a sort of biological clock, linked that is to a maximum number of mitoses (and replications of DNA), at the end of which the cell would be too old to be maintained alive and would go towards apoptosis. The Telomerase is expressed in the stem cells of many adult tissues, even if it is not sufficient to prevent the progressive shortening of the telomeres with age. The length of the telomeres and the activity of the Telomerase enzyme are independently decisive on the mobilization and epidermal stem cell proliferative capacity.

The critically short telomeres inhibit the mobilization and the regeneration of epidermal stem cells; in addition, oxidative stress can also shorten the telomeres. It has been observed that a minimum shortening under 20 pairs of bases per cell division occurs in cells with high antioxidant capacity, while the shortening of the telomeres is higher in cells with low antioxidant defenses.

By cultivating cells under increasing oxidative stress such as a light hyperoxia (40% of normobaric oxygen) it can be seen that the telomeres become prematurely shorter and consequently also their replicative life. Many of the parameters of these prematurely aged fibroblasts are identical to those of fibroblasts that have aged normally (for example the morphology, specific changes in the genetic expression). Anti-radical agents are able to lower the phenomenon of the shortening of the telomeres. By measuring both the shortening of the telomeres and the antioxidant capacity (by measuring the fluorescence of the DCF as indicator of the intracellular level of peroxide) one finds a significant inverse correlation between shortening of the telomeres and anti-oxidative capacity.

Fibroblasts with a low anti-oxidative defense shorten their own telomeres faster and vice versa. Therefore the length of the telomeres is principally determined by the relation between oxidative stresses and capacity for defense again the oxidants. It has been observed that the use of active ingredients with anti-oxidant activity can slow the shortening of the telomeres in the epidermal and dermal cells.

The hair follicle also has a reserve of stem cells capable of guaranteeing continuous cell reproduction. The greatest reserve of stem cells capable of stimulating the formation of a new bulb, after the disappearance of the old one in telogen, which is located in the area of the bulge, an area characterized histologically by a type of swelling of a portion of the external epithelial sheath, in the area of insertion of the arrector pili. Another stem cell zone is found in the area underneath the dermal papilla. Alopecia and graying of the hair have been associated with reduction in the stem cells of the bulge and the progenitor cells. The stimulus to proliferation of the stem cells for the formation of the new bulb appears to be determined by the passing of the bulb into Telogen which, having lost adhesion with the derma, runs towards the epidermis, out of which it emerges when it falls out. This process is very probably modulated by a complex series of biochemical mechanisms induced by numerous mediators and growth factors.

The stem cells of the hair follicle, like other adult stem cells, have a slow reproductive cycle, but very high proliferative capacity. The slow cycle of cellular division allows the stem cells of the follicle to retain the proliferative potential, but minimizes the errors correlative to the replication of the DNA.

The stem cells within the hair follicle are normally in a quiescent state, but they start a transient period of cellular proliferation giving rise to cells of amplification of reproduction during the initial phase of anagen and after a stimulation. The stem cells of the hair follicle have multipotent reproduction capabilities, in fact they can give rise to the structures of the follicle (matrix, dermal papilla and internal and external epithelial sheath) and the shaft, but also of the sebaceous gland and the epidermis. The major flow of the cells of the bulge, under physiological conditions, occurs in the formation of the bulb and the hair shaft at the start of the anagen phase.

It has been observed that the area of the bulge of the human hair follicle contains keratinocyte stem cells while the matrix of the skin represents a compartment of TA cells (cell of amplification of transit) during proliferation and differentiation.

The hair bulb encloses the dermal papilla; this is a fundamental area for the activity of the bulb, with specific connection between activity of the fibroblasts of the papilla and the keratinocytes of the matrix of the hair. The tissue rich in mucopolysaccharides located under the dermal papilla is the area where the stem cells are located, fundamental for it maintenance and stimulation. The hair bulb undergoes a cyclical transformation throughout life. This complex cyclical activity involves continuous rapid remodelling of the various dermal and epithelial structures, determined by numerous cellular mediators that control the growth and morphogenesis of the bulb.

With the onset of the thinning of the hair, inflammatory processes can occur that trigger fibrotic reactions in the derma. The process of cellular ageing of the hair bulb and follicle determines the progressive reduction of the synthesis of the biosynthetic follicle capacity with reduction of the amount of hair and its diameter. The growth of the shaft tends to diminish, probably due to reduction of the synthesis of keratin and modification of the protein composition, and the anagen phase is reduced. In order to prevent and fight the ageing mechanism of the bulb, the application to the scalp of substances with antioxidant properties is attractive in order to reduce to formation of the free radicals and the apoptosis which causes the bulb to go into catagen and telogen.

The stimulation of the anagen phase or its prolongation is fundamental to the health of the hair and the growth of the shaft. This means promoting, with appropriate topical cosmetic type substances, the proliferation of the keratinocytes of the hair bulb and the mechanisms of protein synthesis, or keratin, associated with them.

Plants also have stem cells (or meristematic cells) which are at the origin of the growth of the organism. Plants have two distinct populations of stem cells: one includes the apical meristem of the sprout and the other the apical meristem of the root. All plant cells can differ and become plant stem cells.

Unlike man, adult plants contain totipotent stem cells capable of regenerating new organs (leaves, flowers, etc.) or even the entire plant. More specifically plant stem cells or meristematic cells are undifferentiated plant cells whose sole purpose is reproduction; they take care of the repair and growth of the plant in length (primary or apical meristem) and in width (secondary meristem). Plant stem cells (meristematic cells) have, with few exceptions, a homogeneously slender wall, large nucleus, very few and small vacuoles, plastids at the proplastid stage, abundant ribosomes, mitochondria with few crests.

They are small (10-15μ in diameter), are virtually isodiametric and do not present intercellular spaces. They have a considerable density of cytoplasm and have the highest nucleo-plasmatic ratio since the nucleus can even occupy more than 50% of the cellular volume. This pre-eminence of the nucleus is certainly strictly correlated to the fact that in these cells the proliferative activity is intense, which implies nuclear growth and plasmatic growth to keep producing daughter cells.

Part of this logic is also the very large abundance of ribosomes connected with an intense activity of protein synthesis.

These small organs are also multiple and the mitochondria are particularly numerous (up to more than 100-150 per cell) which moreover are not very large (1-2μm) and have poorly developed crests. The plastids are in the form of proplastids, also very small. Proplastids and mitochondria are in active autoduplication to hold back the plasmatic growth and to distribute themselves in the daughter cells. The meristematic cells do not contain well developed true vacuoles but if anything very small and very few vacuoles. The cellular walls are extremely thin, of a pectin-cellulose nature.

The maintenance of the stem cells in plants depends on signals coming from the microclimate and from an epigenic type control, similar to that of stem cells in mammals.

Currently there are numerous topical cosmetic preparations on the market aimed at improving the aesthetic aspect of the skin and the body and to mitigate the wrinkles of the face.

These preparations contribute mainly to increasing the hydration and the lipidic layer of the skin without however activating the deeper cellular processes which preside over the renewal of the epidermis and the dermis.

Some of the cosmetic products incorporate components of plant origin, typically extracts or mature plant cells. However the use of mature plant cells has not produced as of today appreciable results from a cosmetic point of view since the functionality of these active plant ingredients has not proved satisfactory.

OBJECTS OF THE INVENTION

At present, the need of having topical compositions with a cosmetic action is felt, which should be based on active principles of a natural and typically vegetal source, particularly effective and physiologically compatible with the skin.

One of the primary objects of the present invention therefore consists in providing a complex of vegetal stem cells and a cosmetic composition containing the same, capable of favoring a repair, regenerating, vitality and longevity action on the epidermis and derma cells.

Another object of the present invention consists in providing a complex of vegetal stem cells and a cosmetic composition incorporating said complex, and which should provide a protective action on the skin and on the hair follicles from the action of external aggressive agents without acting aggressively on the skin cells.

A further object of the present invention consists in providing a combination of vegetal stem cells and a cosmetic composition containing the same, which should be capable of preventing or considerably reducing photoaging, chronoaging and oxidative stress that can damage epidermis, derma and follicle.

SUMMARY OF THE INVENTION

In view of these objects, according to a first aspect of the invention there is provided a complex based on vegetal stem cells for regenerating the skin cells and/or stimulating the hair follicle cells comprising stem cells of Malus Domestica and stem cells of Buddleja Davidii. Typically, the vegetal complex of the invention originates from agglomerates of vegetal stem cells also called calli, obtained from in vitro cultivation.

According to an aspect of the invention, this vegetal complex based on stem cells finds application as active component for regenerating the skin and/or hair follicle cells to use as they are or as component rich in active vegetal principles to incorporate in a cosmetic composition or base suitable for skin application.

Typically, the two vegetal active principles contained in the complex of the invention originate from suspensions of dedifferentiated vegetal cells respectively coming from Malus Domestica and Buddleja Davidii. Buddleja Davidii is a plant belonging to the Scrophulariaceae family whereas Malus Domestica belongs to the Rosaceae family.

It has been found that by combining Malus Domestica stem cells with Buddleja Davidii stem cells in a complex suitable for external skin application, a synergic effect of regeneration and renovation is obtained on the skin and hair follicle cells, which produces particularly appreciable effects at a cosmetic level.

In particular, Malus Domestica vegetal stem cells increase the proliferation of epidermal, dermal and follicular cells and their capacity of autorenovation whereas Buddleja Davidii vegetal stem cells protect the skin and follicle cells from degradation and damages due to oxidation, delaying the cellular senescence process.

The combination of these two selected types of vegetal stem cells in a cosmetic formulation produces a regenerating, revitalizing and/or protective synergy action on epithelial, derma and hair bulb cells.

According to a preferred embodiment, the vegetal complex based on vegetal stem cells further comprises Teprenone as active principle. This combination based on three active principles has been found particularly effective in exerting a regenerating or eutrophicating action on the epidermis and/or hair bulb cells, for example keratinocytes.

In particular, it has been seen that by combining vegetal stem cells from Malus Domestica and Buddleja with Teprenone, a synergic cellular regeneration action is activated on epidermis, derma and hair follicle, through action mechanisms different from one another yet complimentary, which delays skin aging, the appearance of age-related skin signs, photoaging damages and early hair loss as well as insufficient growth thereof. Teprenone (Geranylgeranone) exerts this effect mainly by slowing down the reduction of the cellular telomere length, thus contributing to increasing the cellular longevity. Specifically, the combination of said three active principles in a cosmetic composition allows synergizing these activities, allowing epidermis and derma to be kept active and functional, seeing to an adequate cell metabolism and hindering the reactions that damage cells, causing the senescence and early death thereof.

According to another embodiment, the vegetal complex may comprise at least one more type of vegetal stem cells, that is, vegetal stem cells that originate from other types of plants. In particular, the complex based on vegetal stem cells of the invention may further comprise vegetal stem cells from Centella Asiatica. The combination of Malus Domestica and Buddleja Davidii stem cells with Centella Asiatica stem cells and preferably with Teprenone has been found particularly suitable in the cosmetic treatment of the face epidermis, in particular for smoothing out expression wrinkles and filling age-related wrinkles.

Stem cells from Centella Asiatica are mainly used for compacting skin, thanks to their capability of increasing the skin tone and elasticity.

Hyaluronic acid is the major constituent of the derma extracellular matrix and is responsible for the skin hydration and elasticity. A decrease in hyaluronic acid due to the hyaluronidase enzyme causes a loss of the skin tone and elasticity.

Thanks to the action of its constituents, the active vegetal stem cells from Centella Asiatica inhibit hyaluronidase up to 90%. A re-densifying action on the derma occurs in vivo which is particularly useful for compacting the face skin when it becomes relaxed.

Centella stem cells further contain several specific substances provided with biological activity such as caffeoylquinic acid in large amounts, with an antioxidant, anti-inflammatory and brightening/lightening action. Phytosterols, amino acids and polysaccharides with a hydrating and nourishing action.

It has been found that Centella Asiatica stem cells have a higher antioxidant activity than that of common natural antioxidants, for example Rutina. The caffeoylquinic acid contained therein exerts a significant inhibition of the production of TNF-alpha, cytokine involved in the promotion of the inflammation.

Centella Asiatica stem cells, moreover, control tone and permeability of the derma blood vessels, the integrity whereof may be affected subsequent to the intensive exposure to the sun and to chemical agents.

According to another embodiment, the complex based on vegetal stem cells of the invention may further comprise vegetal stem cells from Echinacea Angustifolia. This combination is particularly useful in the regeneration or activation of the hair bulb cells in control of the hair growth. According to an embodiment, therefore, there is provided the use of stem cells from Echinacea Angustifolia, in combination with active vegetal stem cells from Malus Domestica and Buddleja Davidii. In fact, it has been found that using stem cells from Echinacea Angustifolia, which are rich in Echinacoside, can help preventing death by apoptosis induced on the dermal papilla by DHT (Dehydrotestosterone).

According to an embodiment of the invention, these cosmetic formulations find specific application in the field of trichology.

Recent researches, in fact, have shown that the thinning out of the scalp is highly worsened by the programmed death (apoptosis) of the dermal papilla cells, with consequent stop of the hair growth cycle.

Preventing and reducing both the inflammation and its effects due to the associated oxidative stress, but also preventing and reducing the apoptosis effects of DHT on the papilla are important actions for safeguarding the normal hair physiology. The dermal papilla (DP) of the hair follicle is a key organ for regulating the hair growth cycle and regulating the various growth steps, from anagen to telogen. DP is the main way to supply nutrients and oxygen required for the cellular multiplication that determines the hair life.

DP alterations unavoidably cause the loss of the growing hair or its transformation into fleece. Androgenetic alopecia is associated to a regression by apoptotic death of the DP as final effect of the DHT action, which forms in the hair follicles by the transformation of testosterone by the 5-alpha-reductase. Recent studies, in fact, have shown that the main chemical mediator of androgenic alopecia, dehydrotestosterone (DHT), induces the apoptosis of the cells constituting the dermal papilla, with consequent entry of the follicle in the catagen step, and subsequent hair loss.

Since it has been proved by in vitro tests that Echinacea Angustifolia stem cells exert an anti-5-alpha-reductase action, it comes out that such cells can reduce the cell death rate of the dermal papilla induced by DHT. Moreover, the Echinacoside contained in the Echinacea stem cells has an antioxidant activity, and this property brings about the reduction of oxygen radical forming, among which lipoperoxides, which trigger a series of damages due to oxidative stress that contribute to an early end of the hair growth step.

According to another embodiment, the complex based on vegetal stem cells of the invention, in the various embodiments described above or in yet more possible ones, can further comprise hydrolyzed algin. It is a polysaccharide extract obtained by the enzymatic depolymerization of membrane of dark algae belonging to the Laminaria Digitata species.

The complex based on vegetal stem cells of the invention can be either in solid form or in liquid form, both miscible with other cosmetic bases or formulations. Typically, the liquid form is a solution or suspension with aqueous or hydroalcoholic base wherein the active principles and any excipients are dissolved in the solvent.

According to an embodiment, the complex based on vegetal stem cells may further comprise one or more cosmetically acceptable carriers, excipients or active substances.

By way of an example, a complex based on vegetal stem cells as an aqueous solution, suitable for cosmetic skin or trichological use, comprises water, an extract from a culture of Malus Domestica and Buddleja davidii stem cells, PPG-1-PEG 9 ether lauryl glycol, glycerin, sodium sulfite, imidazolidinyl urea, lactic acid, caprylic/capric acid triglyceride, alcohol, xanthan rubber, lecithin, teprenone, soy isoflavones, methylchloroisothiazolinone, methylisothiazolinone.

According to another aspect of the invention, the complex of vegetal stem cells described above may be incorporated in a cosmetic formulation or base for making a composition for cosmetic use which allows repairing and regenerating the epidermis, derma and follicle cells, regenerating the skin in its different cell layers and extending the life thereof.

Said cosmetic formulation or base may comprise one or more excipients or carriers suitable for carrying the complex of stem cells of the invention to the application site, typically the face or the scalp, or it may comprise a cosmetic formulation containing one or more further components with a cosmetic action. Typically, the cosmetic formulation or base wherein the complex of stem cells of the invention can be dispersed may comprise one or more excipients or substances commonly used for cosmetic applications and for the formulation of creams, for example glycerin, substances with a fatty base such as fatty acids and derivatives thereof, oils, thickeners, liposomes, glycols, alcohols, and also active principles or vitamins commonly used in the cosmetic field such as vitamin C, vitamin E and derivatives thereof, hyaluronic acid, fructose, peptides, ribonucleic acids and derivatives thereof.

The cosmetic base or formulation the complex of stem cells may be added to may be in solid, semisolid or fluid form and may therefore appear as a cream, lotion, gel, solution or suspension.

By way of an example, the complex of vegetal stem cells of the invention may be dispersed, prior to application, in a cosmetic formulation of the type described in the international patent application WO 2006/069608, by the same Applicant, and then applied to the epidermis. According to an embodiment of the invention, a cosmetic kit is provided which comprises:

A) a vegetal complex comprising Malus Domestica stem cells, Buddleja Davidii stem cells and preferably teprenone, too;

B) a cosmetic formulation or base comprising one or more cosmetically active principles and one or more cosmetically acceptable carriers to disperse complex A in. According to an embodiment, complex A) of the kit is provided in liquid form, typically a solution with an aqueous or hydroalcoholic base.

According to another aspect of the invention, a cosmetic composition is provided for regenerating epidermal and/or hair bulb cells, comprising a cosmetically effective amount of vegetal stem cells from Malus Domestica and vegetal stem cells from Buddleja Davidii in a cosmetically acceptable carrier.

This cosmetic composition is suitable for treating expression lines, wrinkles, smoothness and tone loss, dull complexion, loss of the face oval features due to lower density of the derma, skin relaxation, dark spots, sun spots, skin or trichological depigmentation, skin microcirculation disorders, dry skin, couperose, reduced sebaceous secretion, hair loss, lack or insufficient hair growth, thinning out of the scalp.

Further embodiments of the cosmetic composition of the invention are shown in the appended dependent claims 6-11.

According to a further embodiment, there are provided suntan and UVA and/or UVB (sun) ray protective creams comprising a complex of stem cells of the type described above.

Typically, the sun protective creams of the invention comprise one or more substances having a photoprotective action against UVA and/or UVB rays, Malus Domestica and Buddleja Davidii stem cells preferably in combination with Teprenone and one or more cosmetically acceptable carriers.

The vegetal stem cells used in the invention contain active substances like proteins, lipids, carbohydrates, minerals and portions of other specific active compounds, elaborated by the stem cells for protecting from external chemical- physical stress, in the physiologically required amounts for contributing to protect the skin cells.

The topical application of vegetal stem cells dispersed in a cosmetically acceptable carrier provides the skin cells with not just molecules or active compounds typical of vegetal stem cells, but also nourishing substances that bring about the skin restructuring and renewal, obtaining synergic effects that could not be obtained using a simple raw extract of the plant. Moreover, it has been found that using vegetal stem cells allows obtaining a greater bioavailability of the active components compared to vegetal cells. In fact, in vegetal stem cells all the active substances are easily accessible by the skin cells whereas in mature vegetal cells many substances change into unusable products, such as cellulose or lignin. According to an embodiment, the vegetal stem cells used in the invention derive from cultured plant explants. According to this procedure, a sort of cicatrization reaction occurs, so new cells form on the cut explant surface. The cells slowly split to form a colorless mass of cells called callus. The callus cells are stem cells comparable to those of the plant meristematic regions. Typically, the plant stem cells, like the skin stem cells, contain proteins

(10OkDa) that interact with the nucleotides (both DNA and RNA) and control development through gene expression.

Within the scope of the present invention, stem cells are used instead of mature vegetal cells because stem cells are rich in epigenetic factors the function whereof is to maintain the autoreplication capability, and in metabolites that contribute to increasing the longevity of the cells they interact with.

Moreover, the vegetal stem cells used according to the present invention provide the skin with the active substances produced thereby, along with proteins, lipids, amino acids, carbohydrates that carry out a nourishing function. As a consequence, using stem cells from a vegetal source, the cosmetic restructuring and cell renewal of the skin is higher than that obtainable using a simple raw extract of the plant.

It has also been found that using vegetal stem cells instead of mature vegetal cells allows obtaining significant advantages in terms of bioavailability of the main components. In fact, the cosmetic use of vegetal stem cells makes the active substances present therein easily accessible by the skin cells, whereas in the case of mature vegetal cells many substances change into unusable products, such as cellulose or lignin.

Moreover, the lack of chloriphylla and therefore of green color in vegetal stem cells simplifies the formulation of a cosmetic composition based on stem cells compared to the preparation of a composition based on differentiated vegetal tissues.

According to an embodiment of the invention, the composition of the invention may be made using whole or fragmented vegetal stem cells, in the form of extracts, homogenized products, derivatives, complexes, mixtures thereof or portions isolated therefrom. In particular, it has been noted that stem cells from Malus Domestica are capable of keeping the autoreplication potential of skin cells and exerting a protective action on the various skin structures.

Typically, stem cells from Malus Domestica are obtained by growing the vegetal tissue. In particular, the tissue material obtained from the plant called explant, is cultured. As a sort of cicatrization reaction, new cells form on the cut explant surface. The cells slowly split forming a colorless mass called "callus".

These cells are dedifferentiated into cells without the distinctive features of normal vegetal cells. The callus cells are stem cells comparable to those of the meristematic regions. In order to obtain a high production, the callus cells can be grown as individual cells or as cellular groups in a culture medium.

According to an embodiment of the invention, the production process comprises the following steps:

- Pick up of a small plant portion/element - Dissection of a part of the plant material for inducing the forming of the callus (cicatrization tissue consisting of dedifferentiated cells)

- Cultivation on agarized medium

- Collection of the callus cells

- Cultivation in suspension to total dedifferentiation to obtain stem cells - Transfer of the stem cells to a liquid medium, and optionally,

- Homogenization, for example at 1200 bars, with liposomes for encapsulating the stem cells and the liposome contents thereof.

The resulting stem cells may be used as liquid suspension in water and/or glycerin or other suitable suspending agent or in freeze dried form. This procedure allows producing vegetal material in standardized and conveniently sterile conditions, irrespective of the season and of the environmental restrictions.

The end product is rich in epigenetic factors and metabolites that contribute to extending the cellular longevity, improve the cell protection and slow down cellular senescence and chronological aging, revitalize and preserve the skin vitality.

According to an embodiment, the method for obtaining extracts of vegetal stem cells of the invention comprises a preliminary step wherein a steady dedifferentiated cell line is established on laboratory scale, the bulk cultivation of the cells in a reactor, for example of the Wave reactor type, the recovery of the total extract, for example by decomposing the plant cells by high pressure homogenization and extracting and stabilizing the extract substances by empty liposomes.

A detailed description of a method for preparing suitable extracts of vegetal stem cells usable within the scope of the present invention is shown in U.S. Patent Application No. US2008/0299092 A1 which is fully incorporated herein by reference.

The Applicant has found that by associating stem cells from B. Davidii to stem cells from Malus Domestica, an increase of regeneration activities is obtained on epithelial tissues, particularly appreciable for the applications in the cosmetic field.

Stem cells from B. Davidii were selected for their high verbascoside contents, an active principle with antioxidant action, along with β-sitosterol, amino acids and polysaccharides that considerably contribute to imparting hydrating and nourishing properties to the cosmetic composition containing them.

In particular, stem cells from B. Davidii, thanks to the presence of verbascoside, have an antioxidant activity higher than rutina and resveratrol, ascorbic acid, alpha-tocopherol, rosmarinic acid, quercetin.

Verbascoside belongs to the large family of phenylpropanoids, natural water-soluble molecules widely distributed in the vegetal reign, formed by caffeic acid and by a portion 2,4-dihydroxyphenylethanol both linked to a same glucose molecule. Verbascoside contains one unit of rhamnose linked to glucose which acts as a bridge.

According to an embodiment, stem cells from Buddleja Davidii can be obtained through a method that comprises the steps of: - sterilization of the vegetal tissue,

- crumbling of the same into small fragments subsequently deposited into Petri plates containing solid nutrient medium,

- forming of a cicatritial tissue consisting of aggregates of undifferentiated and non organized cells (meristematic cells), called calli, - selection of the cells with a high growth speed and higher contents of metabolites of interest,

- growth in liquid medium,

- increase of the biomass in liquid medium in bioreactor.

The verbascoside found in B. Davidii stem cells has scavenger capability against superoxide anion, hydroxyl radical and nitric oxide and of inhibiting the lipid peroxidation, besides that of repairing the DNA radicals generated from ionizing radiations.

Typically, the verbacoside contained in B. Davidii stem cells further exhibits a photoprotective activity against UVB radiations and an anti- inflammatory activity correlated to both the inhibition of the release of arachidonic acid, PGE2 and histamine and to the inhibition of LPS-induced nitroxyde synthase. The demonstration of the high anti-inflammatory activity of verbascoside was obtained on primary cell cultures of human keratinocytes stimulated by TNF-alpha alone or in association with gamma interferon. Moreover, verbacoside exhibits an inhibition activity on both isoforms of 5- α reductase, with higher efficacy than that of the alcoholic extract of Serenoa repens.

Conveniently, stem cells from B. Davidii also have the property of inhibiting the collagenase enzyme, main enzyme responsible for collagen degradation, thus limiting the structural modifications thereof and the consequent skin aging.

In fact, it is known that the major environmental factors that cause skin aging, such as UV radiations and free radicals, also induce the activation of collagenase with consequent loss of the skin elasticity and forming of wrinkles. Teprenone or Geranylgeranone (GGA) usable within the scope of the invention is an active principle from a synthetic source, similar to natural isoprene and described in WO/2002/003981.

Since Teprenone is mainly known for its protective and healing properties on the gastric mucosa, in the present invention it finds a new application in the cosmetic field, in combination with vegetal stem cells, as agent that delays the signs of skin aging such as the appearance of skin wrinkles and face wrinkles.

In particular, the action of prevention of skin aging signs, of regeneration of skin cells and of protection from stress is only partly ascribable to its stabilization activity (WO/2006/120646) of telomeres, a terminal region of the chromosome, consisting of highly repeated DNA, which does not encode for any protein product and which carries out an essential role in avoiding the loss of information during chromosome duplication.

Typically, the Teprenone found in the composition of the invention improves the quality of tissues improving the intercellular communication, delays the cell senescence and extends the cell life by 1/3.

The action of Teprenone appears to be partly ascribable to the stimulation of the thioredoxin synthesis, a small protein with a strong antioxidant power that controls the intracellular redox status, increases the cell and tissue resistance to stress and increases the functionality of prenylated proteins, allowing the increase of telomere proteins.

These properties of Teprenone are enhanced by its association with at least one of the two types of stem cells from Malus Domestica and Buddleja Davidii. These combinations of active principles are particularly suitable for the production of cosmetic preparations for topical use suitable for treating body and face skin flaws, such as spots, wrinkles, ptosis (lowering of eyelids) and erythrosis (permanent redness of cheeks).

According to an embodiment of the invention, there is provided a cosmetic composition comprising vegetal stem cells from Malus Domestica and/or stem cells from Buddleja Davidii with a Vitex Agnus Castus Berry extract and Teprenone. The formulation containing Vitex Agnus Castus Berry extract is particularly useful for the cosmetic treatment of the face or body skin and for giving more action specificity and functionality to the cosmetic composition described.

The Vitex Agnus Castus Berry extract contributes to stimulating the proliferation of fibroblasts and keratinocytes, carrying out an important role in renewing the skin's cell assets, both from a number and from a functional point of view, in particular when the skin is aged, damaged by sun and by pollution.

According to an embodiment, the composition of the invention may contain Vitex Agnus Castus Berry extract in an amount comprised from 0.01 to 30% by weight, more preferably from 0.1 to 10% by weight, even more preferably from 1 to 5% by weight.

The Vitex Agnus Castus Berry activity is partly ascribed to the production of lipophylic substances by the plant, such as Casticin, called phyto-endorphyns, which link to the MU opioid receptors located at the level of the derma and of the epidermis and stimulate the in vivo production of Beta-Endorphyns. These are peptides consisting of 31 amino acids that act as neurotransmitters and are generated by the pro-opio-melanocortin (POMC) precursor, a protein made up of 267 amino acid residues.

The processing of the POMC precursor leads to the forming of different hormonal peptides with different functions, among which Beta-Endorphyns. Beta- Endorphyns synthesized by the skin contribute to attenuating or suppressing painful phenomena that follow the skin aggression by external agents. This action is appreciable within the scope of a cosmetic formulation. Moreover, the Beta-Endorphyns induced by the Vitex Agnus Castus Berry extract stimulate the proliferation of fibroblasts, the migration of keratinocytes, the skin differentiation, epthelization and cicatrization. These effects are also ascribable to the fact that Beta-Endorphyns link to opioid receptors, in particular to receptor μ (mu), which are located at the level of the derma and of the epidermis. The Beta-Endorphyns/mu opioid receptor system modulates the migration of keratinocytes involved in cicatrization processes.

The presence of Vitex Agnus Castus Berry extract in the composition of the invention contributes to favoring both the cell proliferation and the epithelial cicatrization processes, thus increasing the revitalizing, regenerating and repairing effect of the cosmetic preparation.

According to another embodiment of the invention, there is provided a cosmetic composition based on vegetal stem cells further comprising soy isoflavones. Isoflavones, for their resemblance with human estrogens, are also classified as phytoestrogens.

The formulation containing soy isoflavones is particularly useful for the topical treatment of the scalp and for giving, also in this case, more specificity and functionality to the cosmetic composition described.

The presence of soy isoflavones, for example in an amount comprised from 0.01 to 30% by weight, preferably from 0.1 to 5%, in the composition of the invention determines a considerable stimulating action on hair follicles.

According to this embodiment, the composition may be in the form of solution or fluid lotion to apply, preferably after dilution in a suitable solution for thricological use, to the scalp for stimulating hair growth. Soy isoflavones act within the scope of the composition in a manner similar to human oestrogen hormones potentially linking to the same receptors. For this reason they are commonly called phytoestrogens. A particularly suitable isoflavone within the scope of the invention is Genisteine, typically used in the amounts described above. Within the scope of the invention it is interesting to note the action of

Genisteine for assisting the regenerating action at the level of the scalp affected by hair loss and/or thinning. In fact, Genisteine is an inhibitor of the 5-alpha reductase isozyme, in particular type 2. 5-alpha reductase is an enzyme that exists in two isoforms, 1 and 2, which catalyzes at follicular level the NAPDH- dependant conversion of testosterone into dehydrotestosterone, responsible for the follicular miniaturization phenomenon. In fact, the growth of a progressively thinner, shorter and scarcely pigmented hair and a progressive hair reduction derives from here: the thinning of the hair.

Form (isozyme) 1 has a low affinity for testosterone, whereas form 2 has a high affinity for testosterone.

Genisteine is an inhibitor of the 5-alpha reductase isozyme, in particular type 2. The presence of Genisteine in the invention, in combination with active vegetal stem cells with Teprenone, acting on the 5-alpha reductase type 2 enzyme, contributes to the vitality and longevity of the hair follicle, delaying the aging thereof. This translates into an anagen phase, that is, a longer growth phase. Since the follicle miniaturization and the onset of the thinning are connected to a continuous shortening of the anagen phase, it is clear that genisteine is a positive stimulus to hair growth.

The presence of Genisteine in the formulation of the invention therefore contributes to the repair, regeneration, vitality and longevity of the hair follicle, delaying the follicular senescence and thus extending the hair life. According to an embodiment, the composition of the invention can further comprise Genisteine also for treating the face and body skin.

Two different receptors have been determined in man for estrogens, called ERa and ERb. Towards such receptors that is a different and very interesting link affinity by the phytoestrogens, and in particular of genisteine. In fact, genisteine has a high affinity for ERb, estrogenic-like and 20 times higher than the affinity for Era receptors. An original safety and efficacy profile results from this differentiated action of genisteine. The high safety profile of genisteine is correlated to the low activity on estrogenic type a receptors (ERa) which abound in the reproductive tissues (uterus, breast), whereas efficacy is due to the high affinity of genisteine for estrogenic type b receptors (ERb) abounding at the level of the bone, of the cardiovascular system.

Moreover, it is interesting to note the action of Genisteine for fighting skin aging signs.

In fact, it is known that an acceleration of the aging process occurs with the menopause, with a progressive damaging of the derma components, in particular elastin and collagen and reduction of hyaluronic acid, which have the consequence of a decrease in the skin tone and elasticity that may evolve into deeper wrinkles and furrows. Genisteine has proved to be a powerful antioxidant and to have a protective action against sun radiation damages (5-6). Moreover, interesting results also at a histological level, have shown that with the reduction of estrogens, typical of female menopause, the oral intake of genisteine allows maintaining the derma thickness significantly compared to untreated controls, wherein on the other hand such thickness reduces progressively. The use of genisteine in the composition of the invention is suitable for fighting in a natural yet effective manner the skin aging process, in particular in menopausal women.

According to an embodiment, the cosmetic composition or complex of the invention may further comprise vegetal stem cells from other botanical species, for example selected from stem cells from Aiuga reptans, Centella Asiatica,

Echinacea Angustifolia, Gardenia jasminoides, Magnolia grandiflora, Echinacea pallida, Echinacea purpurea, Leontopodium alpinum, Lippia ciriodora, Syringa vulgaris, Glycyrrhiza glabra, Lamium album, Plantago lanceolata, Marrubium vulgare, Stachys officinalis and/or mixtures thereof.

According to another embodiment, the composition comprises one or more suitable cosmetically acceptable carriers and adjunct substances, cosmetic agents such as for example oils, thickeners, preservatives, water, alcohols, glycerin, stabilizers, antioxidants and antibacterials. According to an embodiment, the composition of the invention may be produced in solid form, for example in the form of cream like face or body creams, sun creams, sticks, trans-dermal patches, make-up (foundation, powder, blush, eye shadow, mascara, eye pencil, lip pencil, lipstick, etc.), or in liquid form, for example as hydrophilic lotions, lotions, hydroalcoholic lotions, milks, oleolites, shampoos, bath foams, sprays, dispersions, suspensions, solutions, or in semisolid form, for example as emulsions oil in water or water in oil, serum, hydrophilic or lipophile gels, hydrophilic or oily make up removers. Typically, the compositions of the invention may be used both for treating the face and/or body skin, and for the scalp.

In the first case, typical formulations for cosmetic skin application may be used, for example in the form of cream, emulsion or serum. These formulations are suitable for example for being used for regenerating the face skin stressed by skin aging, photo-aging and by environmental stress such as smog, wind, differences in temperature, etc.

In the second case, the formulations are suitable for being used as lotion, gel solution, shampoo, emulsion that may be applied easily to the areas covered with hair.

The active principles may be provided on variable amounts in the compositions of the invention, for example the extract of Malus Domestica stem cells may be provided as 0.01 to 30% by weight, more preferably from 0.5 to 10%, even more preferably from 1 to 5% by weight, the extract of Buddleja Davidii stem cells may be provided as 0.01 to 30% by weight, more preferably from 0.5 to 10%, even more preferably from 1 to 5% by weight, Teprenone may be provided as 0.01 to 30% by weight, more preferably from 0.5 to 10%, even more preferably from 1 to 5% by weight.

In the composition of the invention, excipients may be incorporated typically used in the base formulation of cosmetic products such as oils, glycerine, emollients, emulsifiers, dispersants in typical amounts for the cosmetic compositions.

In the case of formulations in the form of solution, suspension, lotion, water is present as diluent or solvent, optionally mixed with other liquids used within the scope of cosmetic formulations such as for example alcohols (e.g., ethyl alcohol) and glycols.

The following examples are given by way of an illustration of the present invention only, and are not to be intended as limiting of the scope of protection as it appears from the annexed claims.

EXAMPLE 1 Cosmetic formulation comprising stem cells from Malus Domestica, Buddleja

Davidii and Teprenone.

EXAMPLE 2

Cosmetic formulation comprising stem cells from Malus Domestica, Buddleja Davidii and Teprenone for the application to the scalp.

EXAMPLE 3

Cosmetic formulation comprising stem cells from Malus Domestica, Buddleja Davidii and Teprenone for the application for skin care.

EXAMPLE 4

Formulation of a cosmetic composition for skin care consisting of 0.01- 10 ml of Formula 1 or 3 mixed with 1 - 200 ml of Formula A. The mixing of the two phases should preferably be carried out upon use.

Formula A

EXAMPLE 5

Formulation of a cosmetic composition for application to the scalp skin consisting of 0.01-10 ml of Formula 1 or 2 mixed with 1 - 200 ml of Formula B.

Formula B

EXAMPLE 6

Cosmetic formulation comprising the association of stem cells from Malus Domestica and Buddleja Davidii to be applied to the face skin, typically 1 to 3 times a day.

EXAMPLE 7

Assessment of the mitogenic activity of a 1 :1 mixture of stem cells from Malus Domestica and stem cells from Buddleja Davidii.

An assessment of the effectiveness of a mixture as shown above was carried out using fibroblast models and the assessment of their proliferation and increase of total proteins in suitable experimental conditions.

1.1 Assayed samples

1.2 TEST

Cell proliferation at different times through the MTT test: cell vitality test after cycle synchronization with fibroblasts cultivated in adhesion for assessing the cell proliferation potential induced by the assayed sample.

Increase of total proteins through the Bradford test: test for the assessment of the total protein contents in fibroblasts exposed to the sample at 2.2 Execution of the tests:

2.2.1 Models adopted: The cell model used for in vitro test is represented by: normal human fibroblasts from human skin (HFFcen2tel clone 19 p18)

Treatment and exposure

The fibroblasts were placed into 24 well plates, 8000 cells/well for 24h in culture medium (Dulbecco's Minimum Essential Medium, DMEM) + 15% Fetal Bovine Serum, FBS. The cells were deprived of the serum 24 h before the experiment. Fresh culture medium was added without serum, but containing the sample to be assayed so as to reach the various final dilutions indicated. The medium containing the samples was replaced every day for 2 days. Both raw materials had been previously dissolved in water at the concentration of 10mg/ml. The solutions thus obtained were then filtered and diluted in the culture medium to obtain the concentration of 2mg/ml and then mixed to a 1 :1 ratio.

After 24h and 48h exposure, the MTT cell vitality test and the measurement of total proteins were carried out by the Bradford method, on separate plates. Non-treated cells were used as negative control, whereas the cells treated with human insulin to the indicated dose were used as positive control.

2.2.2 MTT cell vitality test

The MTT test, originally developed by Mossman, was used. The key reagent is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide o MTT, a substance that imparts a yellow color in aqueous solution. The mitochondrial dehydrogenase of vital cells cuts the tetrazole ring, bringing about the forming of water-insoluble purple formazane crystals. Crystals are dissolved with an MMT- solubilizing solution.

The resulting purple solution is then measured spectrophotometrically. An increase or decrease of vital cells results in a concurrent change in the amount of formazane formed and that can be regarded as an indicator of the cytotoxicity level caused by the exposure to irritating substances.

After 24h and 48h treatment, the cell vitality is assessed by incubating the cells with MTT for 3 hours (300μl/well). The precipitated formazane crystals are then extracted using isopropanol or DMSO (300 μl/well) and spectrophotometrically quantified at 550 nm.

The plate is stirred on a plate stirrer ensuring that all the crystals have dissolved and have formed a homogeneous solution. The absorbance is read by a colorimeter (Tecan model Sunrise remote) fitted with a plate reader, subtracting the background readout. The result is expressed as cell vitality in percentage according to formula:

% cell vitality = [OD(550 nm - 690 nm) tested product/OD(550 nm - 690 nm) negative control] x 100

2.2.3 Dosage of total proteins The test is conducted according to the Bradford method with the Bio-Rad protein assay kit. At the end of the treatment, cells are washed with PBS and lysated. The samples are then titrated with a protein-specific dye. The readout is made spectrophotometrically at 595 nm. The protein dosage is calculated based on the optical density (O. D.) by comparison with a titration curve made with albumin.

3. Results and conclusions

3.1 Cell growth test (mitogenesis) After 24h and 48h treatment, the cell proliferation of fibroblasts was assessed, expressed as vitality percentage compared to non-treated cells.

Cell proliferation test on fibroblasts

Dosage of total proteins

After 24h and 48h treatment, the total proteins on fibroblasts were dosed and expressed as percentage compared to the non-treated control.

Dosage of total proteins on fibroblasts

The assayed sample was capable of stimulating the cell proliferation on human fibroblasts at both the concentrations assayed. The maximum effect is observed after 48h exposure to the concentration of 0.05mg/ml (+20.42%). The sample if effective in stimulating the total protein synthesis at both concentrations tested. The maximum effect is observed after 48h exposure to the concentration of 0.05mg/ml (+37,04%).

4. Conclusions Based on the above results obtained, the association of Malus Domestica and Buddleja Davidii stem cells in a 1 :1 mixture was capable of significantly stimulating the total protein synthesis and the cell proliferation relative to cells not treated on human fibroblasts.

EXAMPLE 8

In vitro study for assessing the action synergy between vegetal stem cells from Malus Domestica, Buddleja Davidii and Teprenone.

In order to prove the action synergy of the vegetal stem cell + Teprenone complex, an in vitro study was conducted which shows how a composition based on stem cells from Malus Domestica, Buddleja Davidii with Teprenone is even more active than a composition containing only Malus Domestica and Buddleja

Davidii stem cells.

The study described in this report was intended to assess the proliferation and the protein synthesis in an in vitro system, in cultures of fibroblasts and keratinocytes of skin origin, exposed to 2 different mixtures of active principles based on the active vegetal stem cells described above at different concentrations. Cell proliferation, an index of the re-densifying action on the skin, and protein synthesis index of the production of the extracellular matrix components, and thus of the strengthening action, were measured by a colorimetric assay.

The products subject to test are represented by two different mixtures of active principles in aqueous solution:

Solution 1 : Buddleja Davidii stem cells + Malus Domestica stem cells Solution 2: Buddleja Davidii stem cells + Malus Domestica stem cells + Teprenone

Buddleja Davidii and Malus Domestica stem cells were tested at the concentrations of 0.10%, 0.15% and 0.20%.

Teprenone was tested only at the concentration of 0.0025%.

Each active principle is shown with its concentration in the solution. FIBROBLASTS

* 3 concentrations 0.1% Buddleja Davidii stem cells + 0.1% Malus

0.1%: assayed: Domestica stem cells + 0.0025% Teprenone

0,15% Buddleja Davidii stem cells + 0,15% Malus

0.15%: Domestica stem cells + 0.0025% Teprenone

0.2% Buddleja Davidii stem cells + 0.2% Malus

0.2%: Domestica stem cells + 0.0025% Teprenone

KERATINOCYTES

^* 3 concentrations assayed:

The results obtained in the assay confirm that the treatment of cell cultures of fibroblasts and keratinocytes with Solution 2 (consisting of Malus Domestica and Buddleja Davidii stem cells with Teprenone) is more effective than Solution 1 (vegetal stem cells without Teprenone) at all the concentrations assayed. Both the assayed solutions have further increased vitality, growth speed and ex novo synthesis of cell proteins compared to control cultures. The solution of stem cells containing Teprenone too (solution 2), both at 24h and at 48h, has increased the cell proliferation and the protein synthesis compared to the solution of stem cells only, solution 1.

A synergic effect is thus seen in increasing the cell trophism due to the presence of Teprenone in association with the vegetal stem cells mentioned above.

EXAMPLE 9 A comparison of the mitogenic activity was carried out between a solution containing a 1 :1 mixture of Malus Domestica stem cells and Buddleja Davidii stem cells and a solution containing a 1 :1 mixture of Malus Domestica stem cells, Buddleja Davidii stem cells and Teprenone.

A comparative assessment of the effectiveness of a mixture as shown above was carried out using fibroblast models and the assessment of the increase of total proteins in suitable experimental conditions.

1.1 Assayed samples

1.2 TEST

Increase of total proteins through the Bradford test: test for the assessment of the total protein contents in fibroblasts exposed to the sample at issue using scalar concentrations and different treatment times.

2.1 PURPOSE OF THE TEST The purpose of this test is to quantitatively determine the effects of the assayed sample on the increase of total protein synthesis through protein dosage at different exposure times.

2.2 Execution of the tests:

2.2.1 Models adopted:

The cell model used for in vitro test is represented by: normal human fibroblasts from human skin (HFFcen2tel clone 19 p18)

2.2.2 Treatment and exposure

In this study, the fibroblasts were placed into 24 well plates, 8000 cells/well for 24h in culture medium (Dulbecco's Minimum Essential Medium, DMEM) + 15% Fetal Bovine Serum, FBS. The cells were deprived of the serum 24 h before the experiment. Fresh culture medium was added without serum, but containing the sample to be assayed so as to reach the various final dilutions indicated. The medium containing the samples was replaced every day for 2 days.

For solution 1 , raw materials had been previously dissolved in water at the concentration of 10mg/ml. The solutions thus obtained were then filtered and diluted in the culture medium to obtain the concentration of 2mg/ml and then mixed to a 1 :1 ratio. Solution 2 was prepared by adding a teprenone solution 10 mM (10 mg sample dissolved in 3 ml ethanol) to the 1 :1 mixture of the other samples so as to reach the final concentration of 10μM. After 48h exposure, the measurement of total proteins was carried out by the Bradford method, on separate plates. Non-treated cells were used as negative control, whereas the cells treated with human insulin to the indicated dose were used as positive control.

2.2.3 Dosage of total proteins The test is conducted according to the Bradford method with the Bio-Rad protein assay kit. At the end of the treatment, cells are washed with PBS and lysated. The samples are then titrated with a protein-specific dye. The readout is made spectrophotometrically at 595 nm. The protein dosage is calculated based on the optical density (O. D.) by comparisong with a titration curve made with albumin.

3.1 Dosage of total proteins

After 48h treatment, the total proteins on fibroblasts were dosed and expressed as percentage compared to the non-treated control.

From the above results it is clear that both mixtures are capable of stimulating the protein synthesis on human fibroblasts at all the assayed concentrations, but solution 2 exhibits higher efficacy. The maximum effect is seen at the lowest concentration assayed, where after 48h exposure to solution 2, a 43.82% increase of the protein synthesis is obtained.

3.2 Conclusion Based on the results obtained it was seen that Solution 1 (mixture of

Malus Domestica and Buddleja Davidii stem cells) and Solution 2 (mixture of Malus Domestica and Buddleja Davidii stem cells and Teprenone) were capable of stimulating the protein synthesis on human fibroblasts compared to non treated cells. This effect is considerably stronger in Solution 2 containing the association of the three active principles.

Claims

1. A complex based on vegetal stem cells for regenerating the skin and/or hair follicle cells, characterized in that it comprises stem cells of Malus Domestica and stem cells of Buddleja Davidii in a cosmetically effective amount.

2. A complex based on vegetal stem cells according to claim 1 , further comprising teprenone.

3. A complex based on vegetal stem cells according to claim 1 or 2, characterized by further comprising stem cells, preferably selected from Centella Asiatica stem cells, Echinacea Angustifolia stem cells and mixtures thereof.

4. A complex based on vegetal stem cells according to any one of claims

1 -3, characterized by further comprising hydrolyzed algin.

5. A cosmetic composition for regenerating epidermal and/or hair bulb cells, comprising a cosmetically effective amount of vegetal stem cells from Malus Domestica and vegetal stem cells from Buddleja Davidii in a cosmetically acceptable carrier.

6. A cosmetic composition according to claim 5 characterized by further comprising teprenone as active principle.

7. A composition according to claim 5 or 6, characterized in that said stem cells are in a form selected among suspension, extract, homogenized, complex and mixtures thereof.

8. A cosmetic composition according to any one of claims 5-7, characterized by comprising further vegetal stem cells, preferably selected from Centella Asiatica stem cells, Echinacea Angustifolia stem cells and mixtures thereof.

9. A cosmetic composition according to any one of claims 5-8 further comprising soy isoflavones.

10. A cosmetic composition according to claim 9 characterized in that said soy isoflavones are genisteine.

11. A cosmetic composition according to any one of claims 5-10, characterized by further comprising an extract of Vitex Agnus Castus Berry containing phyto-endorphyns.

12. Use of a cosmetic composition or complex according to any one of claims 1-11 , for the cosmetic skin, face or body treatment, for regenerating, revitalizing, increasing longevity, repairing and protecting the skin cell layers.

13. Use of a cosmetic composition or complex according to any one of claims 1-11 , for preventing or reducing photoaging, chronoaging and/or oxidative stress that damage epidermis, derma or follicle.

14. Trichological use of a cosmetic composition or complex according to any one of claims 1 -11 , for promoting hair growth or prevent loss thereof.

15. Use of a complex based on vegetal stem cells according to any one of claims 1-4 as active component for regenerating the epidermis and/or hair bulb cells to incorporate in a cosmetic formulation for local application.