Chamomilla recutita (german chamomile) extract for improving the appearance of blemished skin

Abstract

Disclosed herein is a method for improving the appearance of blemished skin, which comprises applying to the skin a composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier. In one embodiment the method relates to decreasing pimples, sebum expression, oversized pores, post-acne scars and expression lines, improving skin barrier function and improving skin tone by reducing redness, pigmented spots, and post-acne hyperpigmentation.

Description

CHAMOMILLA RECUTITA (GERMAN CHAMOMILE) EXTRACT FOR IMPROVING THE APPEARANCE OF BLEMISHED SKINFIELD OF THE DISCLOSURE

[0001] The present disclosure concerns the field of cosmetics, personal care, cosmeceutics and dermocosmetics. More specifically the present disclosure concerns the use of ingredients obtained from living plants to improve the appearance of blemished skin associated with oily skin and acne-prone skin.BACKGROUND

[0002] Blemished skin is a common skin condition typically associated with excessive sebum production leading to oily skin, oversized pores, pimples, post acne scars, post-acne hyperpigmentation, pigmented spots and redness. Most blemishes of the skin are harmless but responsible for a non-aesthetic appearance which can have psychological impact on daily life.

[0003] Seborrhea or oily skin mainly affects facial skin and scalp and results from an excessive sebum expression by sebaceous glands. Sebum is secreted by sebocytes through a process of cell differentiation and lipid synthesis, or accumulation called lipogenesis.

[0004] Sebum secretion may be affected negatively by individual, biological and environmental factors such as diet, age, gender, ethnicity, life stress and hot humid climates (Yang J, Yang H, Xu A, He L. A Review of Advancement on Influencing Factors of Acne: An Emphasis on Environment Characteristics. Front Public Health. 2020 Sep 17;8:450).

[0005] Acne vulgaris is another common condition affecting the skin. It causes chronic inflammatory of the sebaceous glands and results in the formation of inflamed elevations (papules, pustules, nodules, and cysts), comedones and scars. The pathogenesis of acne involves several factors with interrelated mechanisms including increased sebum production, hyperkeratinization, skin inflammation, and Cutibacterium acnes (C. acnes) proliferation. The inflammation due to C. acnes infection results in hyperpigmentation and scars (Dagnelie, M-A., et al. "Cutibacterium acnes phylotypes diversity loss: a trigger for skin inflammatory process." Journal of the European Academy of Dermatology and Venereology 33.12 (2019): 2340-2348.). Pigmentary complications and scars are important cosmetic concerns for all patients regardless of skin color.

[0006] Gamma-aminobutyric acid (GABA) is an amino acid that serves as an inhibitory neurotransmitter in brain but has also functions in skin. GABA(A) and GABA(B) receptors have been identified in human cutaneous cells and recent studies have shown the activation ofGAB A(A) receptor improves skin barrier function and protects the skin from infection by pathogens such as Cutibacterium acne. The inhibition of melanogenesis into human melanocytes through GABA(A) and GABA(B) receptors has also been described (Molagoda, Ilandarage Menu Neelaka, et al. "Gamma-Aminobutyric Acid (GABA) Inhibits a-melanocyte-stimulating hormone-induced melanogenesis through GABA(A) and GABA(B) receptors." International Journal of Molecular Sciences 22.15 (2021): 8257). GABA has also been shown to provide immediate relaxation to the look of expression lines along with long-time improvement of the skin barrier function (Nguyen, Thu Q., et al. "A randomized, double-blind, placebo-controlled clinical study investigating the efficacy and tolerability of a peptide serum targeting expression lines." The Journal of Clinical and Aesthetic Dermatology 14.5 (2021): 14). Based on these results, GABA may be considered as a promising compound to provide interesting cosmetic effects.

[0007] Numerous active agents intended for improving imperfect skin, oily skin, acne-prone skin, have been disclosed. Among them, the following may be cited: polyphenols, retinoids, isotretinoin, salicylic acid, apigenin, niacinamides, botulinic toxin (Endly DC, Miller RA. Oily Skin: A review of Treatment Options. J Clin Aesthet Dermatol. 2017 Aug;10(8):49-55).

[0008] However, these products have moderate or limited efficacy, and it remains a need to provide new active agents of natural origin to offer a global approach for improving the appearance of blemished skin.

[0009] Chamomilla recutita extracts were described for the treatment of various inflammatory skin conditions and for their skin anti-aging, depigmenting, soothing, antiseptic, spasmolytic, astringent, toning, revitalizing and deodorant properties (Kotnala, Astha, et al. "Indian Medicinal Plants for skin care and cosmeceuticals: A review." Journal of Biomedical and Therapeutic Sciences 6.2 (2019): 24-60.).

[0010] The patent application published under number MX2009001657 disclosed a chamomile extract obtained from dried heads of Chamomilla recutita by alcoholic maceration and rich in flavones and polysaccharides, alpha-bisalolol, levomenol, chamazulene, dicycloethers and spirethers, among others. This extract is used for its calming and anti-inflammatory properties.

[0011] The patent No. US 11,154,493 disclosed a Chamomilla recutita extract obtained from living flowers of Chamomilla recutita (German Chamomile) for mitigating adverse effects of exposure of skin to sunlight and improving skin appearance associated with skin aging. This Chamomilla recutita serum fraction is water-soluble / miscible, not irritant and very well tolerated when tested at 100 % (demonstrated in in vitro skin studies). It showed broad UVA and UVB spectral absorption photostability, inhibitory properties of sun-induced Interleukin 8 (IL8) andInterleukin 6 (IL6), both involved in inflammatory mechanism, antioxidant properties (DPPH quenching capacities and free radical scavenging properties) and inhibitory properties of human elastase, involved in skin aging.

[0012] Thus, to date, no prior art mentions a Chamomilla recutita extract obtained from living plants may provide benefits for blemished skin or acne-prone skin.SUMMARY OF THE INVENTION

[0013] The inventors have now found that a Chamomilla recutita serum fraction rich in GABA, has biological properties of interest in the field of cosmetic treatment for improving the aspect of blemished skin.

[0014] In a first aspect, the disclosure relates to a method of cosmetic treatment for improving the appearance of blemished skin, which comprises applying to the skin a composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) flower serum fraction and a physiologically acceptable carrier.

[0015] The Chamomilla recutita serum fraction used according to the present disclosure is isolated using a fractionation process comprising subjecting plant cell juice (6) derived from fresh plant biomass of the Chamomilla recutita comprising flowers and a small part of leaves to an electromagnetic field at a frequency equal to or greater than 2.45 GHz for a time effective to destabilize the plant cell juice (14) yielding a coagulated cell juice mixture, removing coagulated membrane fraction (20) from said coagulated cell juice mixture in order to yield a cytoplasm / cytosole fraction (30), followed by additional treatments (32 or 33) enabling to separate cytoplasm fraction (36) from cytosole fraction (38) that is stabilized (37) to yield a serum fraction (39).

[0016] In one aspect, the method comprises decreasing pimples, sebum expression, oversized pores, and expression lines, smoothing the skin, improving skin barrier function, and improving skin tone by reducing redness and pigmented spots.

[0017] The present disclosure also relates to a method of cosmetic treatment for improving the appearance of blemished skin in association with acne-prone skin and for protecting the skin from overproduction of lipids by sebocytes resulting from stresses chosen among airborne pollution, UVA irradiation and lifestyle stresses.

[0018] According to yet another embodiment of the present disclosure, there is provided a composition comprising as an active ingredient a Chamomilla recutita serum fraction and a physiologically acceptable carrier, for its use in the treatment of blemished skin associated with acne.

[0019] These and other objects, features, and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For the purpose of illustrating aspects of the present invention, there are depicted in the drawings of certain embodiments of the invention. However, the invention is not limited to the embodiments depicted in the drawings. Further, if provided, reference numerals contained in the drawings are meant to identify similar or identical elements.

[0021] FIG. l is a schematic drawing demonstrating one embodiment of a process for preparing the serum fraction of the present disclosure.

[0022] FIG. 2 is a chart showing the effect of Chamomilla recutita serum fraction on the secretion of lipids by human primary sebocytes after cortisol stress.

[0023] FIG. 3 is a chart showing the effect of Chamomilla recutita serum fraction on the secretion of lipids by human primary sebocytes after glucose stress.

[0024] FIG. 4 is a chart showing the effect of Chamomilla recutita serum fraction on the secretion of lipids by human primary sebocytes after pollution (PM10) stress.

[0025] FIG. 5 is a chart showing the effect of Chamomilla recutita serum fraction on the secretion of lipids by human primary sebocytes after UVA irradiation.

[0026] FIG. 6 is a chart showing the effect of Chamomilla recutita serum fraction on the expression of melanin after C. acnes stress.

[0027] FIG. 7 is a chart showing the effect of Chamomilla recutita serum fraction on the expression of GAB A(A) receptor in ex vivo skin.

[0028] FIG. 8 is a chart showing the effect of Chamomilla recutita serum fraction on C. acnes lipase activity at concentrations from 0.5 % to 5%

[0029] FIG. 9 is a chart showing the effect of Chamomilla recutita serum fraction on the lipid expression in ex vivo skin.

[0030] FIG 10 is a chart showing the effect of Chamomilla recutita serum fraction on the volume of pores in vivo.

[0031] FIG 11 is a chart showing the effect of Chamomilla recutita serum fraction on the area of pores in vivo.DETAILED DESCRIPTIONDefinitions

[0032] The term “fresh”, “fresh living” or “fresh Chamomilla recutita" as used herein means that live flowers and small part of leaves of German Chamomile are harvested and stored at 4°C until sufficient biomass is collected with storage less than 8 hours before initiating the Serum Fraction process.

[0033] The terms “plant biomass”, “Chamomilla recutita", “Chamomilla recutita flowers” and “Chamomilla recutita (German Chamomile) flowers” are used herein interchangeably with the meaning of Chamomilla recutita (German Chamomile) flowers including a small part of leaves.

[0034] The term “maceration” as used herein has the meaning of mechanically breaking apart the flowers to increase efficiency during pressing. It does not include the addition of liquids or solvents.

[0035] The composition described and used in the present disclosure can comprise, consist essentially of, or consist of, the essential active ingredient as well as physiologically acceptable carrier and optional ingredients described herein.

[0036] As used herein, “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0037] The term “physiologically acceptable” as used herein means that the compositions or components described are suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0038] The term “effective amount” as used herein means an amount of a compound or composition sufficient to significantly induce a positive appearance and / or feel benefit, but low enough to avoid serious side effects (i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan).

[0039] The term “phenolic compounds” as used herein means any molecules having one or more aromatic rings themselves carrying one or more hydroxyl group, such as, phenolic acids, flavonoids, or any other polyphenols.

[0040] The term “blemished skin” as used herein means skin which shows at least one sort of blemish of the skin selected among: pimples, oily skin due to excessive sebum expression by sebocytes, oversized pores, pigmented spots, post-acne scars, post-acne hyperpigmentation, redness, and expression lines.

[0041] The term “acne-prone skin” as used herein means a skin having a propensity to develop pimples in association with excessive sebum production and can degenerate into acne if not properly treated, although the term acne-prone skin refers to a healthy skin without pathology.

[0042] The term “post-acne hyperpigmentation” as used herein means dark spots or patches appearing after acne lesions. It is a consequence of inflammation induced by C. acnes infection resulting in the release of factors increasing melanin production in the skin.

[0043] The term “smoothing the skin” as used herein means attenuating at least one sign selected among wrinkles, expression lines, attenuating post-acne scars and decreasing the size and volume of pores.

[0044] The terms “expressions lines” as used herein has the meaning of the creases that form on the face due to repeated or excessive contraction of the facial muscles. When skin ages expression lines tend to become permanent.

[0045] All numeric ranges described herein are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated.

[0046] The inventors have now found that a Chamomilla recutita (German Chamomile) serum fraction according to the present disclosure has biological properties of interest for improving the aspect of blemished skin and more particularly, for decreasing pimples, sebum expression, oversized pores, post-acne scars and expression lines due to excessive facial muscle tension and for improving skin barrier function and skin tone by reducing redness, pigmented spots and postacne hyperpigmentation.

[0047] The present disclosure draws advantages from these new properties.

[0048] In a first aspect, the disclosure relates to a method of cosmetic treatment for improving the appearance of blemished skin, which comprises applying to the skin a composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier, wherein the Chamomilla recutita (German Chamomile) serum fraction is isolated using a fractionation process comprising subjecting plant cell juice (6) derived from fresh plant biomass of the Chamomilla recutita (German Chamomile)comprising flowers and a small part of leaves to an electromagnetic field at a frequency equal to or greater than 2.45 GHz for a time effective to destabilize the plant cell juice (14) yielding a coagulated cell juice mixture , removing coagulated membrane fraction (20) from said coagulated cell juice mixture in order to yield a cytoplasm / cytosole fraction (30), followed by additional treatments (32 or 33) enabling to separate cytoplasm fraction (36) from cytosole fraction (38) that is stabilized (37) to yield a serum fraction (39).

[0049] The above fractionation process is depicted in FIG. 1.

[0050] In one aspect, the method of the present disclosure provides the step of applying the above composition to blemished skin presenting at least one sign selected among: pimples, oily skin due to excessive sebum expression, oversized pores, expression lines, redness, pigmented spots.

[0051] In accordance with the present disclosure the method applies for decreasing pimples, sebum expression, oversized pores, expression lines, smoothing the skin, improving skin barrier function, and improving skin tone by reducing redness and pigmented spots.

[0052] In another embodiment the present disclosure relates to a method for smoothing the skin, and more particularly for attenuating at least one of sign selected among: wrinkles, expression lines and oversized pores.

[0053] In accordance with the present disclosure, the method also relates to protecting the skin from overproduction of lipids by sebocytes resulting from diverse stresses such as airborne pollution, UVA irradiation and lifestyle stress such as high glycemic-load diets or mental stress.

[0054] In accordance with the present disclosure, the method also relates to decreasing the quantity of sebum at the skin surface, the number of dilated pores, the volume, and the area of pores.

[0055] In accordance with the present disclosure, the method also relates to improving the appearance of blemished skin in association with acne-prone skin.

[0056] In accordance with the present disclosure, the method also relates to increasing the expression of GABA(A) receptor in cutaneous cells.

[0057] It has been shown, the composition comprising as an active ingredient a Chamomilla recutita serum fraction and a physiologically acceptable carrier when applied for three to four weeks significantly decreased the quantity of sebum at the skin surface.

[0058] For example, the clinical study showed a decrease of 178.5% of the quantity of sebum at the skin surface of volunteers.

[0059] The composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier when applied for three to four weeks significantly decreases the number of dilated pores as well as the volume and the area of pores.

[0060] For example, the clinical study showed a decrease of 113% of the number of dilated pores as well of 90% in average of the volume and 101.5% of the area of pores.

[0061] In another embodiment, the composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier when applied for three to four weeks significantly decreases the number of fine lines.

[0062] For example, the clinical study showed a decrease of 140% of the number of fine lines.

[0063] According to the present disclosure, the above composition showed and improvement of blemishes of the skin in all ethnic skin types (Caucasian, Asian, Latin and dark black skin).

[0064] In another embodiment the present disclosure relates to a method for improving the appearance of blemished skin in association with acne, wherein the skin of the subject in needs presents at least one sign selected among: post-acne scars or post-acne hyperpigmentation.

[0065] In another embodiment the present disclosure relates to a composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier, for its use in the treatment of blemished skin associated with acne, wherein the Chamomilla recutita (German Chamomile) serum fraction is isolated using a fractionation process comprising subjecting plant cell juice (6) derived from fresh plant biomass of the Chamomilla recutita (German Chamomile) comprising flowers and a small part of leaves to an electromagnetic field at a frequency equal to or greater than 2.45 GHz for a time effective to destabilize the plant cell juice (14) yielding a coagulated cell juice mixture, removing coagulated membrane fraction (20) from said coagulated cell juice mixture in order to yield a cytoplasm / cytosole fraction (30), followed by additional treatments (32 or 33) enabling to separate cytoplasm fraction (36) from cytosole fraction (38) that is stabilized (37) to yield the serum fraction (39).

[0066] Advantageously, in this particular embodiment the blemished skin presents at least one sign selected among: post-acne scars or post-acne hyperpigmentation.

[0067] Advantageously, in this particular embodiment, the composition may be used in a treatment for preventing the overexpression of melanin induced by C. acnes and for decreasing the virulence of C. acnes by decreasing its lipase activity.

[0068] For example, it was demonstrated that the composition comprising a Chamomilla recutita serum fraction and a physiologically acceptable carrier could decrease the virulence of C. acnes by decreasing its lipase activity and could prevent the overexpression of melanin induced by C. acnes responsible for post-acne hyperpigmentation.

[0069] In accordance with this particular embodiment, the composition also relates to increasing the expression of GABA(A) receptor in cutaneous cells.

[0070] In accordance with the methods of described above, the Chamomilla recutita serum fraction is isolated from fresh living flowers and a small part of leaves using a fractionation process described in U.S.A, patent Nos. 7,442,391; 11,154,493 and European patent No 2919757, which are all incorporated herein by reference.

[0071] The serum fraction used in the method of the present disclosure are not naturally occurring, but instead are produced using a manmade manufacturing process.

[0072] The overall process for preparing the bioactive serum fraction of the present invention is depicted in FIG. 1.

[0073] Fresh living plants of Chamomilla recutita (German Chamomile) comprising flowers and a small part of leaves are harvested, collected, and washed to yield fresh plant biomass (2). This fresh plant biomass is subjected to grinding, maceration, and pressing (4) to yield intracellular plant material (cell juice) (6) and fiber-enriched material (press-cake) (8). Cell juice (6) is then filtered through nylon mesh (10) to remove solid particles and yield filtered plant cell juice (12). Filtered cell juice is exposed to electromagnetic waves treatment (14) at a frequency equal to or greater than 2.45 GHz for a time effective to trigger its destabilization. The destabilized cell juice is then subjected to centrifugation (18), the precipitate containing membrane fraction (20) is removed and the supernatant which is cytoplasm / cytosole fraction (30) is collected.Cytoplasm / cytosole fraction is subjected to additional treatments enabling to separate cytoplasm fraction (36) from cytosole fraction (38).

[0074] As a non-limiting example, treatment can include (i) isoelectric precipitation (32) or alternatively (ii) additional electromagnetic treatment (33) at frequency > 2.45 GHz followed by centrifugation (34) enabling to separate precipitated cytoplasm fraction from supernatant containing cytosole fraction.

[0075] The stabilizing step (37) can involve incubating the cytosole fraction (38) in a mixture of at least one preservative, at least one chelating agent, and at least one antioxidant to yield a stabilized cell serum fraction to yield a stabilized cell serum fraction (39).

[0076] Advantageously, after washing step (2) the excess of water is removed from plant biomass in order to keep the dry matter content close to natural level.

[0077] Advantageously, in step (4) the fresh plant biomass is not subjected to grinding, but only to maceration for a time ranging from 1 to 5 minutes, then pressed.

[0078] The destabilizing treatment (14) is advantageously performed such that the temperature of said cell juice during said treatment does not exceed 30° C.

[0079] After centrifugation (16) the precipitate containing membrane fraction is removed and the supernatant cytoplasm / cytosole fraction (30) is substantially free from membrane fraction.

[0080] The isolated cytosole fraction contains low molecular weight water soluble components dissolved in the intracellular water.

[0081] The cytosole fraction is a clear liquid which has a yellow color and slight characteristic odor.

[0082] Suitable agents for use in the stabilizing step of the present disclosure include, but are not limited to potassium sorbate, sodium benzoate, ascorbyl glucoside, and sodium phytate.

[0083] An example of a suitable antioxidant for use in the present invention is ascorbyl glucoside.

[0084] An example of a suitable chelating agent is sodium phytate.

[0085] The obtained cell serum fraction can be frozen at -18 °C for storage.

[0086] In accordance with the methods of the present disclosure, the obtained Chamomilla recutita serum fraction is rich in protein, in amino acids, among which asparagine, GABA and proline, in vitamin B5 and in organic acids, among which malic acid and quinic acid.

[0087] The obtained Chamomilla recutita serum fraction has a dry matter ranging from 30 to 80 g / Kg (3 to 8 %) and comprises at least 0.5 g / Kg of amino acids among which at least 50 mg / Kg of GABA, at least 0.5 g / Kg of phenolic compounds and at least 1 g / Kg of organic acids.

[0088] The content of the Chamomilla recutita serum fraction is illustrated in the Table 1.

[0089] Digital applications using various specific databases allowed to predict the plausible biological effects of phytochemical compounds of interest contained in the Chamomilla recutita serum fraction such as ferulic acid, chi orogenic acid, malic acid, proline, GABA and vitamin B5. The plausible biological effects highlighted through the identification of Gene Ontology (GO) associated terms were the response to oxidative stress, the response to UVA irradiation, theregulation of lipid metabolism, the modulation of porphyrin activity related to a possible antimicrobial effect and on GABA signaling pathway. More particularly, digital analysis predicted a potential effect of GABA and proline contained in the Chamomilla recutita serum fraction on GABA receptors signaling pathway.

[0090] In more particular embodiments, the composition comprises, by weight of the total composition, as an effective amount of active ingredient, from 0.001 to 10 %, preferably from 0.005 to 5 % and more preferably from 0.01 to 2 % of Chamomilla recutita serum fraction.

[0091] In accordance with the methods of the present disclosure, the compositions may in particular be in the form of an aqueous, hydro-alcoholic or oily solution; and oil-in-water emulsion, a water-in-oil emulsion or multiple emulsions; they may also be in the form of suspensions, or powders, suitable for application on the skin, mucous membranes, lips and / or hair.

[0092] These compositions may be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, a pomade, a gel, a paste or a foam. They may also be in solid form such as a stick or be applied on the skin in the form of aerosol.

[0093] The composition may be applied by any suitable route, in particular by external topical route, and the formulation of the compositions will be adapted by a person skilled in the art.

[0094] In a preferred embodiment, the compositions used according to the present disclosure are in a form suitable for topical application.

[0095] It is obvious that the invention concerns mammals in general, and more specifically human beings.

[0096] These compositions may also include any additive commonly used in the field of application envisaged, as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, coloring agents, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, cosmetic or pharmaceutical active agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, and so on.

[0097] In every case, a person skilled in the art will ensure that said adjuvants (excipients) as well as the proportions thereof are chosen so as not to interfere with the desired advantageous properties of the composition of the invention. These adjuvants may, for example, correspond to 0.01 to 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent 5 to 80% by weight and preferably 5 to 50% by weight with respect to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those conventionally used in the field considered. For example,they may be used in a proportion ranging from 0.3 to 30% by weight, with respect to the total weight of the composition.

[0098] Advantageously, the composition suitable for use in the present disclosure may include, in addition to the active ingredient serum fraction of Chamomilla recutita, at least one other active agent having effects that are similar and / or complementary to those of the invention. According to the invention, this active agent will be defined as an "additional active agent".

[0099] For example, the additional active agent(s) may be chosen from: sunscreen actives, anti- UV, anti-VIS, anti-IR, photo-stabilizers, anti-aging, toning, lightening, hydrating, draining, and microcirculation-promoting agents, pharmaceutical, exfoliating, scrubbing, extracellular matrixstimulating, energy metabolism-activating, antibacterial, antifungal, calming, anti-free radical, and anti-acne agents, anti-inflammatory agents, anesthetics, warming agents, cooling agents and weight-loss agents.

[0100] Such additional agents may be chosen from the groups including: vitamin A and in particular retinoic acid, retinol, retinol propionate, retinol palmitate, vitamin B3 and more specifically niacinamide, tocopherol nicotinate, vitamin B5, vitamin B6, vitamin B 12, vitamin C, in particular ascorbic acid, ascorbyl glucoside, ascorbyl tetrapalmitate, magnesium and sodium ascorbyl phosphate, vitamins E, F, H, K, PP, coenzyme Q10, metalloproteinase inhibitors, a TIMP activator, DHEA, precursors and derivatives thereof; amino acids such as arginine, ornithine, hydroxypropline, hydroxyproline dipalmate, palmitoylglycine, hydroxylysine, methionine and derivatives thereof, N-acyl amino acid compounds, natural or synthetic peptides, including di-, tri-, tetra-, penta- and hexapeptides and the lipophilic derivatives thereof, isomers and complexed with other species such as a metal ion (e.g. copper, zinc, manganese, magnesium, and others), plant-based peptide extracts such as extracts of soy, spelt, grapevine, rapeseed, linseed, rice, com, pea, yeast extracts, Artemia Salina extracts, dehydroacetic acid (DHA), phytosterols of synthetic or natural origin, salicylic acid and derivatives thereof, alpha- and betahydroxyacids, amino sugars, glucosamine, D-glucosamine, N-acetyl-glucosamine, N-acetyl-D- glycosamine, mannosamine, N-acetyl mannosamine, galactosamine, N-acetyl galactosamine extracts of polyphenols, isoflavones, flavonoids, such as grape extracts, pine extracts and olive extracts, lipids such as ceramides or phospholipids, oils of animal origin, such as squalene or squalane; plant oils, such as sweet almond, copra, ricin, jojoba, olive, rapeseed, peanut, sunflower, wheat germ, com germ, soy, cottonseed, alfalfa, poppy, winter squash, evening primrose, millet, barley, rye, safflower, passion fruit, hazelnut, palm, apricot seed, avocado, and calendula oil; ethoxylated plant oils, and shea butter, all UV screens and broad spectrum sunscreens.

[0101] Specific embodiments of this cosmetic treatment method also result from the above description. Other advantages and features of the invention will be more apparent upon reading the examples provided for illustrative and non-limiting purposes.EXAMPLES

[0102] The following examples are intended to illustrate particular embodiments of the present invention but are by no means intended to limit the scope of the present invention.Example 1 Fractionation process as described in U.S.A, patents No. 11,154,493

[0103] The overall process for preparing the bioactive serum fraction of the present invention is depicted in FIG. 1.

[0104] Fresh living plants of Chamomilla recutita (German Chamomile) comprising flowers and a small part of leaves are harvested, collected, and washed to yield fresh plant biomass (2). The excess of water is carefully removed from flowers in order to keep the dry matter content close to natural level.

[0105] This fresh plant biomass is subjected to maceration and pressing (4) using a screw press. The maceration of the plant biomass (4) takes place for 1 to 5 minutes as it rotates through the machine prior to pressing. This step yields intracellular plant material (cell juice) (6) and fiber- enriched material (press-cake) (8). Cell juice (6) is then filtered through nylon mesh (10) to remove solid particles and yield filtered plant cell juice (12). Filtered cell juice is exposed to electromagnetic waves treatment (14) at a frequency equal to or greater than 2.45 GHz for a time effective to trigger its destabilization. The destabilized cell juice has the appearance of a coagulated cell juice mixture. This coagulated cell juice mixture is then subjected to centrifugation (16). The precipitate containing membrane fraction (20) is removed and a supernatant which is cytoplasm / cytosole fraction (30) is collected.

[0106] The destabilizing treatment (14) is advantageously performed such that the temperature of said cell juice during said treatment does not exceed 30° C.

[0107] Cytoplasm / cytosole fraction (30) is subjected to additional treatments enabling to separate cytoplasm fraction from cytosole fraction. As a non-limiting example, treatment can include (i) isoelectric precipitation (32) or alternatively (ii) additional electromagnetic treatment (33) at frequency > 2.45 GHz with following centrifugation (34) enabling to separate precipitated cytoplasm fraction (36) from supernatant containing cytosole fraction (38).

[0108] The isolated cytosole fraction (38) contains low molecular weight water soluble components dissolved in the intracellular water.

[0109] The cytosole fraction is a clear liquid which has a yellow color and slight characteristic odor. In several hours, the unstable cytosole fraction is irreversibly transformed into dark brown color suspension containing heavy precipitate and strong non-characteristic odor.

[0110] The stabilizing step involves incubating the cytosole (38) fraction in a mixture of at least one preservative, at least one chelating agent, and at least one antioxidant (37) to yield a stabilized cell serum fraction (39).

[0111] Suitable agents for use in the stabilizing step (37) of the present disclosure include, but are not limited to potassium sorbate, sodium benzoate, ascorbyl glucoside, and sodium phytate.

[0112] An example of a suitable antioxidant for use in the present invention is ascorbyl glucoside.

[0113] An example of a suitable chelating agent is sodium phytate.

[0114] The obtained cell serum fraction can be frozen at -18 °C for storage.Example 2: Analysis of Chamomilla recutita serum fraction versus a conventional flower extraction

[0115] Extracts:

[0116] Three batches of Chamomilla recutita serum fraction prepared as described in Example 1 without preservative and stored at minus 18°C for storage prior to be analyzed have been used.

[0117] For comparative purpose a conventional extract was prepared. A mixture of 1% dried flowers (including a small part of leaves) of Chamomilla recutita in distilled water was heated for 1 hour at 25°C, then filtered on filters with a large porosity of 30 pm in order to remove the solid residual vegetable matter from the liquid part. Sequential filtrations on filters of decreasing porosity were then carried out to clarify the plant extract until a final sterilizing filtration at porosity 0.2 pm, to yield a liquid part which is the conventional extract.

[0118] Protocols of analysis:

[0119] Total soluble sugars analysis was based on colorimetry method. 1 mL of each sample or standard was reacted with 5 mL of ice cold 2000 ppm anthrone in 72% sulfuric acid. The reagents were heated to 100°C for 10 minutes and a four-point calibration curve was created from the absorbance values of the solutions at 680 nm and compared against the calibration curve (fructose and glucose) to determine their total sugar concentrations.

[0120] The total phenolic content was measured by spectrophotometry at 760 nm after reduction of the Folin-Ciocalteu reagent by phenols. Quantification was performed using a standard gallic acid curve, the results are expressed in gallic acid equivalent.

[0121] The total protein content was measured by spectrophotometry at 550 nm after colorimetric reaction of Biuret combined with that of the Folin-Ciocalteu reagent. Quantification was performed using a standard BSA (Bovine Serum Albumin) curve.

[0122] Amino acid total content was quantified using a colorimetric assay at 570 nm. Free amino acids react with ninhydrin reagent to yield a colored complex. The total amino acid content was determined using a standard curve of amino acid pool.

[0123] Individual phenolic compound content analysis was carried out by liquid chromatography coupled to a UV detector fixed at 254 nm. The samples were separated on an UPTISPHERE CS EVOLUTION C18-AQ column by an Agilent 1200 HPLC system (Agilent Technologies), using a solution of 0.1% formic acid in water and methanol as mobile phases.

[0124] Individual organic acid content was assayed by high-performance liquid chromatography, coupled with detection by mass spectrometer (Acquity Qda, Waters) equipped with a negative mode electrospray ion source. The samples were separated on a column EC 150 / 4.6 Nucleoshell RP 18plus-5pm (Macherey Nagel: 763236.46) by an HPLC system Agilent 1260 (Agilent Technologies). The flow rate was 0.3 ml / min. The mobile phases consisted of a solution of 0.01% formic acid in water and acetonitrile.

[0125] Individual amino acid content analysis was carried out by liquid chromatography coupled to a UV detector fixed at 254 nm. The samples were separated on a UPTISPHERE strategy C18-2 column by an Agilent 1200 HPLC system (Agilent Technologies) after phenylisothiocyanate derivation. The mobile phases consisted of a solution of 0.1% phosphoric acid in water and acetonitrile.

[0126] Characterization and quantification of B vitamins were carried out by high-performance liquid chromatography, coupled with detection by mass spectrometer (Acquity Qda, Waters) equipped with a negative mode electrospray ion source. The samples were separated on a column EC 150 / 4.6 Nucleoshell RP 18plus-5pm (Macherey Nagel: 763236.46) by an HPLC system Agilent 1260 (Agilent Technologies). The flow rate was 0.5 ml / min. The mobile phases consisted of a solution of 20 mmol ammonium acetate and 0.1% formic acid in water and in methanol / acetonitrile (65 / 35).

[0127] Results

[0128] Table 2: Quantitative results of Chamomilla recutita serum fraction versus conventional extract.LQ: limit of quantification, can vary according to the method.

[0129] Chamomilla recutita serum fraction contains 33 times more proteins, 7 times more phenolic compounds (including 2 times more ferulic acid and derivatives and at least 17 times more apigenine and derivatives), 30 times more B5 vitamin and 80 times more organic acids (including 74 times more malic acid and at least 100 times more quinic acid) than a conventional extract. Amino acids (in particular, asparagine, proline and GABA) were highly detected in Chamomilla recutita serum fraction and not in conventional extract.Example 3: Prediction of biological activities of Chamomilla recutita serum fraction using digital applications

[0130] Several digital tools were used to predict biological activities of the Chamomilla recutita serum fraction of Example 1, based on its content in the following phytochemical compounds of biological interest: ferulic acid, chi orogenic acid, malic acid, proline, GABA and vitamin B5.

[0131] Target predictions were performed using SMILES files and using a published database SwissTargetPrediction (Daina A., Michielin O. and Zoete V, 2019. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res, 47: W357-W364) based on the target / ligand similarity principle. Then, predicted targets were pooled and target enrichment was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Sherman B.T., Hao M., Qiu J., Jiao X., Baseler M.W., Lane H.C., Imamichi T. and Chang W, 2022. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Research, 50(Wl): W216-W221; Huang DW, Sherman BT, Lempicki RA, 2009.Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc., 4(1): 44-57). This process allowed to classify the predicted target proteinsaccording to their involvement in different biological functions. Indeed, the DAVID database gives enriched biological themes, particularly Gene Ontology (GO) terms for the global predicted targets.

[0132] Table 3__

[0133] Digital applications using various specific databases allowed to predict, through the identification of Gene Ontology (GO) associated terms, the following plausible biological effects:the response to oxidative stress, the response to UVA irradiation, the regulation of lipid metabolism, the modulation of porphyrin activity related to a possible anti-microbial effect and on GABA signaling pathway.

[0134] The predictions obtained with the Swiss Target Prediction tool evidenced the potential effect of GABA and proline, contained in the extract, on GABA receptors signaling pathway.Example 4 Chamomilla recutita serum fraction protected the overproduction of lipids in sebocytes induced by several stresses applied in vitro to sebocytes

[0135] Objective: Evaluate the benefits of a Chamomilla recutita serum fraction on the overproduction of lipids in sebocytes induced by several stresses. There is evidence that various environmental stresses such as exposure to airborne pollutants or UV exposure, and lifestyle stresses including high glycemic-load diets induce an overproduction of lipids by sebaceous gland cells (sebocytes). Moreover, Cortisol is a major steroid hormone secreted by the adrenal cortex (HPA pathway) and is a possible candidate as an index for mental stress. The production of lipids is monitored by a standard procedure using nile red staining (a selective fluorescent dye of intracellular lipid droplets).

[0136] Protocol:

[0137] Sebocytes: Normal human primary sebocytes were isolated from plastic surgery. An enzymatic digestion by dispase was used to dissociate the epidermis from the dermis. Sebaceous glands were isolated by microdissection under microscope from the skin (dermis and epidermis) and their ducts were removed. After a second enzymatic digestion by trypsin, isolated sebocytes were cultivated for several days.

[0138] After several days of culture, sebocytes were treated for 48h with the non-preserved Chamomilla recutita serum fraction of Example 1 (0.1% (vol. / vol.) diluted in PBS). At the same time, sebocytes were stressed with a solution of cortisol (10 pM), a solution of glucose (3g / L), pollution (PM10; 50 pg / mL) or UVA irradiation (2J / cm2). Non-treated and non-treated nonstressed controls were performed. After the stress, sebocytes were stained with nile red solution and lipids droplets were observed under fluorescent microscope to quantify the lipogenesis.

[0139] Image quantification: Nile red is a phenoxazone dye that intensely stains hydrophobic lipids in red. The staining of intracellular lipid droplets by Nile Red fluorescent probe red filter (excitation 515-560 nm; emission > 590 nm) can evidence all the lipids. Pictures were acquired with a Nikon Eclipse Ni-E microscope equipped with a DS-Fi3 Nikon camera, using NiS-AR (Nikon) acquisition software. Six photos per condition were analyzed with Volocity image analysis software (Improvision). The fluorescence intensity was used to select the representative zone. The software was quantifying the level of fluorescence intensity which was adjusted by considering the number of cells.

[0140] Statistical analyses: Statistical testing was used to assess the significance of the treatment versus the absence of treatment (100% expression reference), using Student’s t test for independent samples with one-tailed direction of rejection. p<0.05 = * were considered as significant, p<0.01 = ** as very significant and p<0.005 = *** as highly significant. Six photos per condition were analyzed.

[0141] Results: As depicted at Fig. 2 cortisol (mental stress), at Fig. 3 glucose (high glycemic load diet), at Fig. 4 PM10 (airborne pollution) or at Fig. 5 UVA irradiation increased the lipid production produced by sebocytes by +93%***, +41%***, +24%***, +32%** respectively, when compared to negative control (no stress). Treatment of sebocytes with Chamomilla recutita preserved cells from stress and the lipid production was decreased (-35%*, -19%*, -17%***, - 38%*** respectively compared to stress condition)

[0142] Conclusion: Treatment of sebocytes with a Chamomilla recutita serum fraction at 0.1% protected the cells from stresses induced by cortisol (mental stress), glucose (high glycemic load diet), PM10 (airborne pollution) or UVA irradiation and mitigated the production of lipids by human sebocytes.Example 5: Chamomilla recutita serum fraction prevented the hyperpigmentation induced by C. acnes

[0143] Objective: The bacteria mainly involved in acne is Cutibacterium acnes which is also present at the surface of normal skin. C. acnes may induce an inflammation which can lead to a hyperpigmentation due to the release of propigmentary cytokines which stimulates melanogenesis. To evaluate the protective effect of Chamomilla recutita serum fraction, in vitro experiments were designed to mimic the hyperpigmentation of the skin induced by C. acnes.

[0144] Protocol:

[0145] Ex vivo skin: Normal human skin was obtained from plastic surgery (abdomen, female donor 30 years old).

[0146] Skin biopsies were prepared with a 6 mm diameter punch (PFM medical) and maintained in organotypic culture medium (DMEM / Ham’s-F12 50 / 50) supplemented with 10% FBS, 2 mM L-glutamine and 100 pg / ml Primocin. Skin biopsies were maintained at 37°C and 5% CO2 in a humidified atmosphere. C. acnes were inactivated by heating at 80°C for 30 min then protein content was quantified by BCA assay. Skin biopsies were treated for 48h with the non-preserved Chamomilla recutita serum fraction of Example 1 at 1% (vol. / vol.) then stressed with 100 pg / ml of inactivated bacteria for 24 h. After the stress, the skin biopsies were stained with Fontana- Masson solution and the melanin was assayed by the reduction of solutions of ammoniacal silver nitrate to metallic silver (brown) without the use of an external reducing agent.

[0147] Image quantification: Pictures were acquired with a Nikon Eclipse Ni-E microscope equipped with a DS-Fi3 Nikon camera, using NiS-AR (Nikon) acquisition software. Six images per condition were analyzed. ImageJ software allowed to count the number of brown / dark pixels in the manually selected epidermis zone for each photo. The number of pixels obtained was adjusted to the length of the examined epidermis zone.

[0148] Statistical analyses: Statistical testing was used to assess the significance of the treatment versus the absence of treatment (100% expression reference), using Student’s t test for independent samples with one-tailed direction of rejection. p<0.05 =* were considered as significant, p<0.01= ** as very significant and p<0.005= *** as highly significant. Six photos per condition were analyzed.

[0149] Results: The stress with C. acnes induced the overexpression of melanin (+59%*** compared to untreated condition) quantified by Fontana-Masson staining (Fig. 6). The pretreatment of skin biopsies with kojic acid (positive control) or Chamomilla recutita serum fraction at 1% prevented the overexpression of melanin induced by C. acnes (-49%***, -36%*** respectively compared to stress condition).

[0150] Conclusion: Treatment of skin biopsies with Chamomilla recutita serum fraction at 1% allowed to prevent the consequences of C. acnes infection on the overexpression of melanin by melanocytes.

[0151] Example 6: Chamomilla recutita serum fraction increased the expression of GABA(A) receptor

[0152] Objective: Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in brain but it has also functions in skin. Indeed, GABA receptors are present in cutaneous cells such as keratinocytes. In this experiment, the modulation of the expression of GABA(A) receptor in ex vivo skin was observed by immunodetection.

[0153] Protocol:

[0154] Normal human skin came from the same plastic surgery than in Example 5. Skin biopsies were obtained with a 6 mm diameter punch (PFM medical) and maintained in organotypic culture with medium (DMEM / Ham’s-F12 50 / 50) supplemented with 10% FBS, 2 mM L-glutamine and 100 pg / ml Primocin. Skin biopsies were maintained at 37°C and 5% CO2 in a humidified atmosphere. Skin biopsies were treated for 48h with non-preserved Chamomilla recutita serum fraction of Example 1 at 1 % (vol. / vol.) or GABA at 1 mM, 10 mM or 20 mM. After the treatment, GAB A(A) receptors were detected using specific antibodies and quantified using fluorescent microscopy.

[0155] Image quantification: Pictures were acquired with a Nikon Eclipse Ni-E microscope equipped with a DS-Fi3 Nikon camera, using NiS-AR (Nikon) acquisition software. Six photos per condition were analyzed with Volocity image analysis software (Improvision). The software was quantifying the level of fluorescence intensity which was adjusted by considering the area of the examined epidermis zone.

[0156] Statistical analyses: Statistical testing was used to assess the significance of the treatment versus the absence of treatment (100% expression reference), using Student’s t test for independent samples with one-tailed direction of rejection. p<0.05 = * were considered as significant, p<0.01 = ** as very significant and p<0.005 = *** as highly significant. Six photos per condition were analyzed.

[0157] Results: the treatment of skin biopsies with Chamomilla recutita serum fraction at 1% or GABA solution at 1 mM, 10 mM or 20 mM (Fig. 7) induced an increase of GABA(A) receptor expression (+ 14%***, +8%***, +9%*** and +11%*** respectively).

[0158] Conclusion: Treatment of skin biopsies with Chamomilla recutita serum fraction allowed to increase the GABA(A) receptor expression in skin biopsies.Example 7: Chamomilla recutita serum fraction decreased C. acnes lipase activity

[0159] Objective: C. acnes secretes enzymes such as lipase, involved in the pathogenesis of acne. Lipase from C. acnes metabolizes sebum and releases inflammatory metabolites in the skin (Platsidaki, Eftychia, and Clio Dessinioti. "Recent advances in understanding Propionib acterium acnes (Cutib acterium acnes) in acne." FlOOOResearch 7 (2018). Ketoconazole is a well-known antifungal which induces a decrease of C. acnes activity so as a decrease of lipase activity.

[0160] Lipase activity of C. acnes was measured to demonstrate the protective effect of Chamomilla recutita serum fraction.

[0161] Protocol:

[0162] C. acnes'. Cutibacterium acnes strain NCTC 737 [VPI 0389] is a whole-genome sequenced bacterial type strain that was isolated from facial acne (ATCC-6919).

[0163] In the 96-well plates, 100 pl C. acnes at DO 0.5 was grown in Reinforced Clostridial Medium supplemented with ketoconazole (an anti-fungal drug, used as positive control) at final concentration 16 pg / mL or supplemented with non-preserved Chamomilla recutita serum fraction at final concentrations of 0.5%, 1%, 2% and 5%. The plates were incubated anaerobically at 37°C for 24 h. The plate was centrifuged at 4700 rpm for 10 minutes. 50 pl Supernatants (half diluted) were mixed with 50 pL 0.4 mM 4-methyl umbelliferyl oleate (MUO) dissolved in 13 mM Tris- HC1, 0.15 M NaCl, and 1.3 mM CaC12 (pH 8.0) and dropped in the black 96-well plates. The mixtures were incubated for 30 min at 25°C. Enzymatic reactions were stopped by adding 100 pL 0.1 M sodium citrate (pH 4.2). The levels of 4-methylumbelliferone released by the lipase were measured using a fluorometric microplate reader (Synergy 2, Biotek) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

[0164] Lipase activity quantification: Fluorescence values of blanks, corresponding to wells without C. acnes but treated with Chamomilla recutita serum fraction or ketoconazole (16 pg / mL), were subtracted to fluorescence values of Chamomilla recutita serum fraction or ketoconazole (containing C. acnes). Relative percentage of fluorescence for each sample were calculated relative to the average untreated control wells without ketoconazole.

[0165] Statistical analyses: Statistical analyses were performed using Student’s t test for independent samples with one-tailed direction of rejection. p<0.05 = * were considered as significant, p<0.01 = ** as very significant and p<0.005 = *** as highly significant. Experiments were performed in three replicates.

[0166] Results: the treatment with ketoconazole induced a decrease of lipase activity (- 64%*** compared to untreated condition) (Fig.8). The treatment with Chamomilla recutita serum fraction at final concentrations of 0.5%, 1%, 2% and 5% induced a decrease of C. acnes lipase activity ( - 5%, -18%***, -25%***, -54%*** respectively compared to stress condition).

[0167] Conclusion: the treatment of C. acnes with Chamomilla recutita serum fraction at different concentrations inhibited the lipase activity. Chamomilla recutita serum fraction at 5% inhibited C. acnes lipase activity in a similar extend than ketoconazole.Example 8: Chamomilla recutita serum fraction improved the barrier function

[0168] Objective: The skin barrier function is important to protect the skin from the external aggressions and pathogens. In this experiment, the lipid expression was measured in the epidermis to evaluate the effect of Chamomilla recutita serum fraction on the barrier function.

[0169] Protocol: Normal human skin came from the same plastic surgery than in Example 5. Skin biopsies were obtained with a 6 mm diameter punch (PFM medical) and maintained in organotypic culture with medium (DMEM / Ham’s-F12 50 / 50) supplemented with 10% FBS, 2 mM L-glutamine and 100 pg / ml Primocin. Skin biopsies were maintained at 37°C and 5% CO2 in a humidified atmosphere. Skin biopsies were treated for 48h with non-preserved Chamomilla recutita serum fraction of Example 1, at 1% and 2%. After the treatment, the effect on the barrier function was detected by Nile Red staining and observed under fluorescent microscope to quantify the expression of total lipids.

[0170] Nile red is a phenoxazone dye that intensely stains hydrophobic lipids in red. The staining of intracellular lipid droplets by Nile Red fluorescent probe red filter (excitation 515- 560 nm; emission > 590 nm) can evidence all the lipids. Pictures were acquired with a Nikon Eclipse Ni-E microscope equipped with a DS-Fi3 Nikon camera, using NiS-AR (Nikon) acquisition software. Six photos per condition were analyzed with Volocity image analysis software (Improvision). The fluorescence intensity was used to select the representative zone. The software was quantifying the level of fluorescence intensity which was adjusted by considering the area of the examined epidermis zone.

[0171] Statistical analyses: Statistical analyses were performed using Student’s t test for independent samples with one-tailed direction of rejection. p<0.05 = * were considered as significant, p<0.01 = ** as very significant and p<0.005= *** as highly significant. Six photos per condition were analyzed.

[0172] Results: The treatment with Chamomilla recutita serum fraction at final concentrations of 1% or, 2% induced an increase of epidermal lipid expression (+40%***, +33%*** respectively) (Fig. 9).

[0173] Conclusion: The treatment with Chamomilla recutita serum fraction at different concentrations allowed an increase in the expression of lipids in the epidermis, resulting in an improvement of the barrier function of the skin.Example 9: Chamomilla recutita serum fraction improved blemished skin in clinical study

[0174] Objective: A clinical study was conducted on Caucasian skin to evaluate the effect of Chamomilla recutita serum fraction formulated at 1% on skin with blemishes.

[0175] Volunteers: 18 volunteers aged from 21 to 67, with oily skin and dilated pores; phototype II to IV.

[0176] Protocol: skin blemishes were evaluated on the face, before and after 1 month of application with 1% Chamomilla recutita serum fraction of Example 1 formulated in a cream at different time point (DO, D7, D21 and D28). This study was a randomized, double-blind, and split-face test versus placebo.

[0177] [Table 4]: Care formula containing Chamomilla recutita serum fraction at 1% used in the clinical test.Example 1.

[0179] The following parameters were evaluated during the study:

[0180] -quantity of sebum at the surface of the skin with Sebumeter® SM 815 (Courage & Khazaka)

[0181] - pores size (from 0.35 to 2.5 mm) and number of dilated pores by image analysis

[0182] - Color pictures of the face

[0183] Statistical analysis: Student’s t-test or Wilcoxon test depending on whether the data followed a normal distribution or not. The results have been considered as significant when p < 0.05, as very significant when p < 0.01 and as highly significant when p < 0.005.

[0184] Results:

[0185] After 3 and 4 weeks of application with 1% Chamomilla recutita serum fraction containing cream a significant decrease of sebum at skin surface has been observed compared to placebo cream. This first result demonstrates that Chamomilla recutita serum fraction diminished the secretion of sebum at the surface of the skin leading to a less oily skin appearance.

[0186] [Table 5]: Statistical result of sebum quantity at skin surface for side treated with Chamomilla recutita serum fraction at 1% and placebo side.

[0187] Moreover, after 3 and 4 weeks of application with 1% Chamomilla recutita serum fraction containing-cream, number of dilated pores as well as the volume of pores and area weresignificantly less enlarged compared to placebo sides (Fig. 10 and 11). This result demonstrated that the pores are less dilated showing the astringent effect (or pore tightening effect) of Chamomilla recutita serum fraction.

[0188] Fine lines present on the cheek has been also carried out to evaluate the effect of Chamomilla recutita serum fraction on skin expression lines that could be associated with relaxed skin. A relaxed skin can be defined as a skin with less expression lines and fine lines. After 4 weeks of application with 1% Chamomilla recutita serum fraction containing-cream fine lines decreased significantly compared to placebo sides. The skin is smoother, relaxed with less visible fine lines.

[0189] Conclusion: the application of 1% Chamomilla recutita serum fraction containing cream significantly decreases the quantity of sebum at skin surface and reduces the number and size of pores, showing the astringent effect (or pore tightening effect) of Chamomilla recutita serum fraction. In addition, fine lines were less numerous and smaller after application of the cream with 1% Chamomilla recutita serum fraction. Under the present condition, 1% Chamomilla recutita serum fraction formulated in a cream allows to counteract skin blemishing improving oily skin appearance and the size of pores.

Claims

WHAT IS CLAIMED IS:

1. A method of cosmetic treatment for improving the appearance of blemished skin, comprising applying to the skin a composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) flower serum fraction and a physiologically acceptable carrier, wherein the said Chamomilla recutita serum fraction is isolated using a fractionation process comprising subjecting plant cell juice (6) derived from fresh plant biomass of the Chamomilla recutita comprising flowers and a small part of leaves to an electromagnetic field at a frequency equal to or greater than 2.45 GHz for a time effective to destabilize the plant cell juice (14) yielding a coagulated cell juice mixture, removing coagulated membrane fraction (20) from said coagulated cell juice mixture in order to yield a cytoplasm / cytosole fraction (30), followed by additional treatments (32 or 33) enabling to separate cytoplasm fraction (36) from cytosole fraction (38) that is stabilized (37) to yield a serum fraction (39).

2. The method of claim 1, wherein improving blemished skin comprises decreasing pimples, sebum expression, oversized pores, and expression lines, smoothing the skin, improving skin barrier function, and improving skin tone by reducing redness and pigmented spots.

3. The method of claim 1 for protecting the skin from overproduction of lipids by sebocytes resulting from stresses chosen among airborne pollution, UVA irradiation and lifestyle stresses.

4. The method of claim 1 wherein the expression of GABA(A) receptor is increased in cutaneous cells.

5. The method of claim 1 for decreasing the quantity of sebum at the skin surface, the number of dilated pores, the volume, and the area of pores.

6. The method of claim 1 for improving the appearance of blemished skin in association with acne-prone skin.

7. The method of claim 1 wherein the Chamomilla recutita serum fraction has a dry matter ranging from 30 to 80 g / Kg (3 to 8 %) and comprises at least 0.5 g / Kg of amino acids,among which at least 50 mg / Kg of GABA, at least 0.5 g / Kg of phenolic compounds and at least 1 g / Kg of organic acids.

8. The method of claim 1, wherein the composition comprises, by weight of the total composition, from 0.001 to 10 %, preferably from 0.005 to 5 % and more preferably from 0.01 to 2 % of a Chamomilla recutita serum fraction.

9. The method of claim 1, wherein the composition further comprises an additional ingredient selected from the group consisting of a sunscreen active, an anti-inflammatory agent, and a skin tone agent.

10. A composition comprising as an active ingredient a Chamomilla recutita (German Chamomile) serum fraction and a physiologically acceptable carrier, for its use in the treatment of blemished skin associated with acne, wherein the Chamomilla recutita serum fraction is isolated using a fractionation process comprising subjecting plant cell juice (6) derived from fresh plant biomass of the Chamomilla recutita flowers comprising flowers and a small part of leaves to an electromagnetic field at a frequency equal to or greater than 2.45 GHz for a time effective to destabilize the plant cell juice (14) yielding a coagulated cell juice mixture, removing coagulated membrane fraction (20) from said coagulated cell juice mixture in order to yield a cytoplasm / cytosole fraction (30), followed by additional treatments (32 or 33) enabling to separate cytoplasm fraction (36) from cytosole fraction (38) that is stabilized (37) to yield the serum fraction (39).

11. The composition of claim 10 wherein the blemished skin presents at least one sign selected among: post-acne scars or post-acne hyperpigmentation.

12. The composition of claim 10 for its use in preventing the overexpression of melanin induced by C. acnes and for decreasing the virulence of C. acnes by decreasing its lipase activity.

13. The composition of claim 10 wherein the expression of GABA(A) receptor is increased in cutaneous cells.

14. The composition of claim 10 wherein the Chamomilla recutita serum fraction has a dry matter ranging from 30 to 80 g / Kg (3 to 8 %) and comprises at least 0.5 g / Kg of aminoacids, among which at least 50 mg / Kg of GABA, at least 0.5 g / Kg of phenolic compounds and at least 1 g / Kg of organic acids.

15. The composition of claim 10 wherein the composition comprises, by weight of the total composition, from 0.001 to 10 %, preferably from 0.005 to 5 % and more preferably from0.01 to 2 % of a Chamomilla recutita serum fraction.

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