HK1169325B - Extract of whole seeds of moringa sp., and use thereof in cosmetic and/or dermatological compositions - Google Patents

Description

Extract of whole seeds of Moringa species and use thereof in cosmetic and/or dermatological compositions

Technical Field

The present invention relates to an extract of whole seeds of Moringa species (Moringa sp.) containing oils (including triglycerides, fatty acids and polar lipids) and polyphenols and its use in cosmetic and/or dermatological compositions.

Background

Moringa is a small tree native to india, but cultivated around the world and transplanted in many of its very popular environments. There are 13 species of Moringa belonging to the Moringa family, Moringa (m.oleifera), the synonym Moringa oleifera (Moringa pterygosperma), being the best known.

Moringa oleifera (Moringa oleifera) is a small tree, 4 to 8 meters high, with open tree crowns spreading in the form of umbrellas. The leaves are deciduous, 30 to 70n cm long, the flowers are white and very fragrant, the fruits are linear elongated sacs, triangular, cortical and pendulous, up to 30 to 40cm in length. The seeds were rounded triangular, 1.2cm long and 1cm wide, and had three membranous petals extending from the seed that were 2cm long.

The trees are wild or cultivated in tropical, humid or very dry climates, which can survive extreme conditions and grow very rapidly. Its extremely deep root system allows it to endure without water for months.

It has very many common names, including the tree (Ben tree), Winged (Winged Ben), Neverdi (e), Anamambobo, horse radish, drumstick, and Neverde or "miracle". In fact, indian herbal medicine (ayurvedic medicine) is known to treat 300 diseases in addition to having very significant nutritional value. The fruits are cooked and the leaves are consumed as vegetables and have a nutritional value to address malnutrition in some countries.

The oleic acid-rich edible oil is derived from seeds that also serve as a flocculant for disinfecting water. The oil is obtained by pressure extraction or hexane extraction of the dehulled seeds. The oil seed cake is recovered after pressing by virtue of the flocculant properties.

When the seeds are still green, they can be eaten as legumes. Mature seeds contain about 40% oil. Oils from moringa species are high quality edible oils (similar to olive oil) which are also used in perfumery, either for the manufacture of soaps or as lamp oils (which are extremely stable to oxidation).

In traditional medicine, oils are used in applications for pain relief during gout or rheumatism attacks (The Indian materials medical, pp 811-816). Seeds for oral administration are used as antipyretics (Hukkeri et al, Indian j.pharm.sci. (2006)68, pp.124-126).

Oil from seeds, obtained by compression or by very apolar extraction (using hexane in particular), is widely used in cosmetology because of its nutritional nature, due to the triglycerides it contains.

The use of aqueous seed extracts in cosmetology is described: it contains peptides and proteins that will have anti-wrinkle and cleansing properties: seeds were first dehulled and defatted (U.S. Pat. Nos. 6500470 and 2006/0275247). The aqueous defatted protein fraction extracted from whole seeds or dehulled seeds of Moringa species has moisturizing, repairing and anti-wrinkling effects on skin (European patent 1064008). The protein fraction of the seed may be characterized as "very polar".

According to the literature, the seeds consist of sterols (campesterol, stigmasterol, β -sitosterol, Δ 5-avenasterol, erythrosterol.) fatty acids (C18: 1-oleic acid-68 to 76%, C16:06 to 7.8%, C18:04 to 7.6%, C20:02.8 to 4% and C22:05 to 6.7%), proteins (26 to 32%), fibres, tocopherols (α, γ: 134, 93 and 48mg/kg of oil, respectively).

The seeds also contain glucosinolates, including 4- (alpha-L-rhamnopyranosyloxy) -benzyl glucosinolate. Leaves and seeds of Moringa species have also been described to contain certain cell stimulating peptides such as zeatin, dihydrozeatin and isopentenyladenine (U.S. Pat. No. 2006/0222682).

Disclosure of Invention

The applicant has demonstrated the use of a specific extract of the whole seed of the moringa species comprising oils (including triglycerides, fatty acids and polar lipids) and polyphenols as active ingredient in cosmetic and/or dermatological compositions.

Therefore, the object of the present invention is an extract of the whole seed of moringa species containing oil (including triglycerides, fatty acids and polar lipids) and polyphenols. In the context of the present invention, "whole seed" is understood to be seed in which the outer skin has not been removed.

The extract of whole seeds of moringa species is characterized (in weight% relative to the weight of the dry extract):

-an oil at a content of 5% to 50% comprising (i) 2% to 10% triglycerides and fatty acids and (ii) 5% to 15% polar lipids;

-total polyphenols (expressed in g of pyrogallol of 100g of dry extract) in a content ranging from 0.01% to 5%.

According to a characteristic of the invention, the extract has a content of oil (in% by weight relative to the weight of dry extract) of 25% to 40%.

According to another characteristic of the invention, the species of the genus Moringa is preferably Moringaoleifera (Moringaoleeiera) or Moringa druhardii (Moringa druhardii).

The extract of the moringa species according to the invention, whose cosmetic and dermatological value (valorization) has been explained, is obtained from the whole seeds of the moringa species, which are advantageously dried, ground and then subjected to at least one extraction with a medium-polar solvent.

In the context of the present invention, the term "moderately polar solvent" is understood to mean a solvent selected from the group consisting of C1 to C4 alcohols, acetone, water/alcohol mixtures and water/acetone mixtures, used alone or in combination.

Preferably, it is an ethanol/water mixture. Advantageously, the ethanol/water mixture may be characterized by various ratios of ethanol/water from 9/1 to 7/3 (v/v).

Even more advantageously, the moderately polar solvent is an ethanol/water mixture of 9/1 or 3/1 (v/v).

The extraction is carried out under stirring or under static conditions, at reflux or at ambient temperature, for a duration of 30 minutes to 48 hours, the plant/solvent volume ratio can be from 1/5 to 1/20. The extraction can be repeated 2 or 3 times.

The residue is then separated from the extract by centrifugation or filtration, and the solution can be more or less concentrated until a dry extract is obtained with a yield of 5% to 10%. The extract obtained is not very homogeneous and a carrier (support) can be added during the drying step in a mass proportion relative to the dry matter of extraction, which can be between 1% and 75%. The carrier may be maltodextrin, lactose, silica or any other cosmetically acceptable carrier that solubilizes the extract, such as propylene glycol/ethoxylated oleyl alcohol in various ratios.

The obtained Moringa species extract is characterized by its oil (including triglycerides, fatty acids and polar lipids) and polyphenol content.

Another object of the present invention relates to the use of such moringa species extract as an anti-aging active ingredient.

Preferably, the extract is suitable for combating all signs of skin ageing in a person with mature skin. In the context of the present invention, the term "mature skin" is to be understood as the skin of a human being generally over the age of 55 years, preferably over the age of 60 years.

In particular, the signs of aging of the skin are characterized by a loss of firmness and/or elasticity and/or tonicity and/or suppleness of the skin, as well as the appearance of wrinkles and fine lines.

It is therefore an object of the present invention to provide a novel active ingredient capable of providing protection, moisturization and nourishment to the epidermis/mature skin simultaneously, and thus providing a skin smoothing, anti-sagging, restructuring effect.

Through different types of tests, the applicant evaluated the overall anti-ageing effect of the moringa species extract according to the invention.

The novel extract has been shown to bring about various desired effects simultaneously, namely:

antioxidants, anti-radical action to limit the oxidation processes associated with internal and external ageing;

the effect on the repair of the barrier function of the skin (protein and lipid structure of the epidermis) as a function of age: the use of said extract makes it possible to limit skin dehydration and therefore protect the skin;

the action on the extracellular matrix to improve the mechanical properties (firmness, elasticity, tonicity) of mature skin.

The extract according to the invention promotes moisturization, smoothness, non-sagging and restructuring of the skin. The extract according to the invention thus makes it possible to beautify and to unify the complexion.

Another object of the invention relates to the use of the extract, as defined to enhance and restore the barrier function of the skin.

In the context of the present invention, the term "enhancing the barrier function of the skin" means increasing the barrier function of the skin.

One of the essential functions of the skin is to ensure a barrier between the body and the external environment, on the one hand to make itself resistant to the penetration of the epidermis by the fungi, bacteria and allergens of the environment ("outside-in") and on the other hand to loss of water ("inside-out").

The epidermis is a multilayered epidermis of continuously renewed ectodermal origin. Several layers of different morphological properties and cellular composition can be distinguished from the inside to the outside: the basal layer, the cell layer where keratinocytes have a very high proliferative capacity (which allows for automatic renewal of the epidermis), the basal layer (granular, spinous) and finally the stratum corneum (cornified layer, SC). These stages correspond to increasingly higher levels of keratinocyte differentiation. Once the keratinocytes of the basal layer begin the process of migration towards the surface of the epidermis (a process of programmed cell death), they lose their proliferative capacity, during which they express a differentiation program, leading to keratinization. The skin barrier function is first performed by the stratum corneum, a solid sealed assembly consisting of two compartments (components):

-lipid-rich intercellular keratinocyte adhesive: penetration of lipid-restricted molecules organized in sheets and covalently linked to cells through the stratum corneum;

a layer of keratinocytes dead of the cell unit (keratinocytes), which corresponds to the final stage of differentiation of the keratinocytes.

The integrity of the extracellular lipid binder and all the cellular elements of the stratum corneum, and the balance between proliferation and keratinocyte differentiation are important to maintain a functional epidermal barrier function.

Perturbation of the barrier function, either chronic or acute, makes the body susceptible to external stress and dehydration.

An improvement in barrier function is particularly decisive when the barrier function of the skin is altered and has to be reestablished. This is the case in response time (skin aging) or in certain physiological states associated with hormonal environments or stress. This repair rate, which allows return to homeostasis, is delayed. Furthermore, skin barrier function is altered in the most common pathology of the skin of most people, which is usually accompanied by an inflammatory component (dryness of the skin).

It is also advantageous to improve the barrier function when the initial function of the skin to be consolidated, in particular to provide the body with better resistance to the external stresses to which it is exposed, in particular to the type of environment (uv, humidity level, external temperature, pollution, burning). The barrier function of the skin includes all mechanisms of natural defense against the stresses to which it is subjected. The important components that perform this function are located in the most superficial part of the epidermis (in the stratum corneum region called stratum corneum).

It has been demonstrated by different tests that the extract as defined has an advantageous effect of repairing the lipid and protein structure of the epidermis.

The use of the extract according to the invention is particularly effective for the enhancement and repair of the skin barrier function.

Another object of the present invention relates to a cosmetic and/or dermatological composition comprising, as active ingredient, an extract of the whole seeds of the moringa species according to the invention, together with at least one cosmetically and/or dermatologically acceptable excipient.

The cosmetic and/or dermatological composition according to the invention comprises an amount of dry extract of the whole seeds of the moringa species comprised between 0.1g and 5g for 100g of said composition.

Advantageously, the amount of extract of the moringa species is between 0.25g and 1g for 100g of cosmetic and/or dermatological composition.

More particularly, the present invention relates to anti-aging cosmetic compositions. Preferably, the cosmetic composition is suitable for combating all signs of skin ageing of a person with mature skin.

The anti-aging cosmetic composition according to the invention may also contain one or more active ingredients, such as an active ingredient suitable for sun protection and/or an active ingredient suitable for skin hypopigmentation.

The active ingredient suitable for protection against the sun is further selected from chemically synthesized molecules known for their anti-UVA action, anti-UVB action, such as octocrylene and/or dioctylbutylaminotriazinone and/or bisethylhexyloxyphenylmethoxyphenyl triazine.

In addition, the active ingredients suitable for skin hypopigmentation, lightening and unifying skin tone may be niacinamide, vitamin C and its derivatives.

In view of obtaining an anti-aging cosmetic composition, a cosmetically acceptable excipient is selected to enable topical administration or oral administration.

Advantageously, the topical form is selected from the group consisting of milks, creams, ointments, oils, lotions, gels, foaming gels, hair creams, sprays, and the like.

Advantageously, the oral form is selected from tablets, capsules, lozenges, powders, granules, solutions or oral suspensions.

More particularly, the invention also relates to a cosmetic and/or dermatological composition suitable for strengthening and restoring the barrier function of the skin.

The cosmetically and/or dermatologically acceptable excipients are chosen so as to be able to be administered topically, in view of obtaining a cosmetic and/or dermatological composition that enhances and restores the barrier function of the skin.

Advantageously, the topical form is selected from the group consisting of milks, creams, ointments, oils, lotions, gels, foaming gels, hair creams, sprays, and the like.

Another object of the present invention relates to a cosmetic method of combating all the signs of skin ageing of a person with mature skin, characterized in that it involves the use, by topical or oral administration, of an extract of the whole seeds of the moringa species according to the invention.

The following preparations and compositions are provided by way of non-limiting examples.

Detailed Description

Examples of the preparation of plant extracts

Example 1

2.5kg of whole seeds of dried and ground moringa oleifera were extracted in 17.5L of ethanol 90 (ethanol/water ratio: 9/1) by two counter current extractions at 80 ℃. After cooling at ambient 50 ℃, the extracted solution was recovered by solid/liquid separation. The sample was dried, thereby obtaining 243g of extract. The extract is characterized by an oil content of 36% comprising (i) 10% triglycerides and fatty acids and (ii) 10% polar lipids and a total polyphenol content of 0.02% (expressed in g pyrogallol of 100g dry extract).

Example 2

20g of whole seeds of dried and ground Moringa oleifera were extracted under reflux in 100ml of an ethanol/water mixture 75: 25 for 1 hour. The extracted solution was recovered by solid/liquid separation and dried using a rotary evaporator at 50 ℃. 1.66g of extract are thus obtained in the form of a brown paste titrated to 5% of oil and 0.68% of total polyphenols expressed as pyrogallol.

Example 3

20g of dried and ground whole seeds of drumstick tree were extracted under reflux in 200ml of an ethanol/water 90: 10 mixture for 1 hour. The extracted solution was recovered by solid/liquid separation and dried using a rotary evaporator at 50 ℃. 1.29g of extract was thus obtained in the form of a yellow-brown paste titrated to 35% of oil (triglycerides, fatty acids and polar lipids) and 1.3% of total polyphenols expressed as pyrogallol.

Examples of cosmetic compositions

Example 4: eye care

Example 5: whitening new cream

Evaluation of antioxidant Activity

DPPH test

The antioxidant activity of the seed extract of moringa oleifera according to the present invention was evaluated using the DPPH test. The test is based on the measurement of the antioxidant capturing capacity of the stable free radical 2, 2-diphenyl-1-picrylhydrazino (DPPH). When the stable free radical reacts with a hydrogen donor, the stable free radical with an absorption at 517nm is reduced to the corresponding hydrazine.

As a result:

the results are expressed as IC50, which IC50 corresponds to a concentration providing a 50% reduction in absorbance of a 0.06mM DPPH in methanol.

Test product	IC50(μg/ml)
		Non-polar extraction of intact seedsExtract (Hexane)	> 1000 (inactivated)
Seed extract without husk using EtOH 90%	1000 (deactivated)
		Extract according to example 1	280
Extract of cortex with EtOH 90%	30
		Vitamin E (ref.)	6-10

The extract according to the invention has an antioxidant activity which is provided for the most part by the molecules present in the outer skin.

Chemiluminescence

It is the generation of free radicals (superoxide radical O) by photochemical signals₂°^-) The method of (1). The oxidation strength is 1000 times greater than that obtained under normal conditions. The detection is performed by chemiluminescence and allows the antioxidant oil-soluble or water-soluble extract or molecule to be evaluated. The results are expressed as equivalents of vitamin C or Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), respectively. The sensitivity is in the order of nanomolar.

As a result:

65 μ g of extract according to example 1 was necessary to obtain an activity comparable to that detected for 1 μ g trolox: corresponding to the activity of lycopene, a molecule known for its antioxidant activity.

The test also established that the antioxidant activity observed is mainly brought about by molecules of the outer skin: for 1. mu.g trolox (activity equivalent to genistein), only 4, 7. mu.g of EtOH 90% extract of the outer skin was required. This determines the importance of using the whole seed of the moringa species according to the invention.

Free radicals cause alterations in the DNA of skin cells and cell membranes and mitochondria, and the production of free radicals is increased by external stress (cold, pollution, tobacco, UV). The anti-radical activity of the extract prepared according to example 1 makes it possible to combat both internal and external skin ageing.

Evaluation of Activity for repair of Barrier function

The epidermis serves a primary protective role by providing a chemical and mechanical barrier to the body. Which ensures that a seal is maintained, i.e. a skin barrier function. The keratinocytes (keratinocytes of the stratum corneum) in combination with the lipid matrix ensure most of this function. However, deeper layers assist in setting the functional agent in place. The differentiation capacity of keratinocytes of the epidermis ensures that a functionally selective permeable barrier is set in place (Elias PM. J Invest Dermatol 125183-200 (2005)).

In terms of proteins, epidermal differentiation is mainly focused on the evolution of structural proteins, which are keratins and contribute to the structural integrity of the epidermis. Their expression varies with the degree of maturation of the keratinocytes. Basal keratin 1(base keratin 1) and acidic keratin 10 are early markers of keratinocyte differentiation, which are present in the basal layer of the epidermis. Expression of other markers (late markers) of the biological process can be followed, such as expression of proteins of the cuticle envelope (e.g. involucrin), and expression of certain major enzymes, starting at the cross-linking of structural proteins with each other and with keratinocyte lipids, transglutaminase (e.g. transglutaminase 1(TG1)) (Houben E et al, Skin Pharmacol physiol.; 20 (3): 122-32 (2007)).

At the same time, keratinocyte lipid synthesis and transport begins at the onset of the intercellular keratinocyte lipid cement, essential for the skin barrier, whose formation represents the final stage of terminal epidermal differentiation. This extracellular lipid matrix provides a major barrier to the transdermal movement of water and electrolytes (Mizutani Y. et al, FEBS Lett.563: 93 (2004)). Thus, keratinocyte expression of a certain number of enzymes and lipid transporters increases with differentiation. In particular, certain members of the ABC (adenosine triphosphate binding cassette transporter) family of transporters play a major role in the step of placing the lipid barrier in place. Therefore, ABC G1 and ABC a12, which are essential for the transfer of lipid precursors in lamellar bodies, are sensitive markers, and ABC G1 specifically transports glycerol. Epidermal ceramides play a major specific role and are an essential marker for the functional level of the skin barrier. Thus, the expression and activity of enzymes that assist in skin ceramide synthesis increase specifically with the level of epidermal differentiation during the disruption of epidermal barrier function. That is, particularly, in the case of sphingolipid C4-hydrogenase/delta-4 desaturase or hDES2, the dihydroxyceramide hydrogenase activity of the sphingolipid C4-hydrogenase/delta-4 desaturase or hDES2 contributes to the synthesis of phytoceramide (phytoceramide) in human skin (Feingold, K.R.J. Lipid Res 48: 2531-2546 (2007)).

First, the effect of the extracts according to the invention was first studied by means of a differentiated Normal Human Keratinocyte (NHK) model.

This effect was then studied by a model of keratinocyte senescence.

Differentiation model of normal human keratinocytes

Extracts of moringa species were analyzed for their effect on gene expression of different proteins involved in the synthesis or transport of epidermal lipids, ABC G1 and hDES 2.

As a result:

results are expressed as the percentage of stimulation of gene expression (mRNA) of different markers of epidermal differentiation expressed by NHK (relative to untreated cells). A significant positive effect was considered as 100% stimulation. The results obtained are shown in the table below.

Cell processing	ABC G1	hDES2
			Vit D35μM	170％	780％
10 μ M rosiglitazone	330％	190％
			Extract example 1 (20. mu.g/ml)	150％	100％

Table 1: effect of vitamin D3, rosiglitazone and the extract according to the invention according to the differentiated Normal Human Keratinocyte (NHK) model

20 μ G/ml of the extract prepared according to example 1 resulted in gene expression of hDES2 and ABC G1. The extract prepared according to example 1 allows to restore the lipid epidermal differentiation starting from the positioning in place of the hydrophobic barrier which allows to limit the skin dehydration encountered in particular in mature skin.

Keratinocyte senescence model

The regenerative capacity of the skin barrier is reduced in mature subjects (Tagami, arch.derm.res.2007), the applicant has used transdermal patches H₂O₂The lineage of the treated human keratinocytes HaCaT mimics a model of the keratinocyte senescence process. The applicant has analyzed the effect of extracts of moringa species on the repair of the expression (mRNA) of markers of protein differentiation, which is inhibited by senescence of cells such as K1 and involucrin.

As a result:

1 and 10. mu.g/ml of the extract according to example 1 allowed to restore the expression of K1 (11 and 35%, respectively) and involucre (15% for 10. mu.g/ml).

Conclusion

The results obtained from the two models show well the complementarity of the action of the extract of the invention (repair of the lipid structure and the protein structure of the epidermis).

Evaluation of the Activity of the extracellular matrix

Extracellular matrix (ECM) is a dynamic structure of tissue with structural and regulatory roles. It gives the skin swelling (turgescience) and mechanical properties: firmness, elasticity and tonicity. In the epidermal region, extracellular matrix occupies the intercellular spaces and plays a role in maintaining the epidermal structure. It also provides for exchange between epidermal cells and plays a role in cell activity. The ECM of the epidermis is in particular composed of type IV collagen (fibrin). As cells age, the components of the ECM are degraded primarily by enzymes of the zinc endopeptidase type known as matrix metalloproteinases or MMPs. The latter are actively involved in the scarring process, but they also contribute to the appearance of skin laxity and wrinkles, which are the first signs of skin aging. Among them, MMP-9 is gelatinase, which has activity against denatured collagen molecules (collagen), but can also split natural molecules of type IV, V and VII collagen.

Menses H in mimicking cellular senescence process₂O₂Lineage of treated human keratinocytes HaCaT the effect of Moringa species extracts on the expression of MMP-9 mRNA was analyzed.

As a result:

extracts prepared according to example 1 inhibited MMP-9 gene expression at three concentrations, notably at 1, 10 and 30 μ g/ml.

The extract prepared according to example 1 makes it possible to restore the mechanical properties of the ECM: the firmness, elasticity and tonicity of the ECM of the skin also make it possible to restore the protein structure of the epidermis.

Claims (7)

1. An extract of whole seeds of a moringa species, wherein the extract is prepared by a process comprising:

2.5kg of whole seeds of dried and ground moringa oleifera were extracted by two counter current extractions at 80 ℃ in 17.5L of ethanol 90, i.e. ethanol/water ratio 9/1;

recovering the extracted solution by solid/liquid separation after cooling at ambient temperature at 50 ℃;

the sample was dried, thereby obtaining 243g of extract,

wherein the extract is characterized by an oil content of 36% and a total polyphenol content of 0.02%, expressed in g of pyrogallol of 100g of dry extract, said oil comprising (i) 10% triglycerides and fatty acids and (ii) 10% polar lipids,

wherein the extract of whole seeds of the Moringa species is an extract of whole seeds of Moringa, wherein the whole seeds are seeds from which the outer skin has not been removed.

2. Use of an extract of whole seeds of the moringa species according to claim 1 for the preparation of an anti-aging cosmetic and/or dermatological composition.

3. Use according to claim 2, characterized in that said anti-aging cosmetic and/or dermatological composition is suitable for counteracting all the signs of skin aging of a person with mature skin.

4. Use of an extract of whole seeds of the moringa species according to claim 1 for the preparation of an anti-aging cosmetic and/or dermatological composition for enhancing and restoring the barrier function of the skin.

5. Cosmetic and/or dermatological composition comprising an extract of whole seeds of moringa species according to claim 1 with active ingredient capacity and at least one cosmetically and/or dermatologically acceptable excipient.

6. The composition according to claim 5, comprising an amount of dry extract of whole seeds of Moringa species of between 0.1g and 5g for 100g of the composition.

7. The composition according to claim 5, further comprising one or more active ingredients suitable for sun protection and/or for skin hypopigmentation.