Plant-based components

Abstract

The present invention relates to a botanical composition having antioxidant, antiproliferative, anti-inflammatory, regenerative, and cicatrogenic activity in the skin and dermis. The present invention also relates to a dermatological preparation comprising the botanical composition and a suitable pharmaceutically acceptable excipient. Furthermore, the present invention relates to the botanical composition and dermatological preparation for medical use in the treatment of dermatological disorders and / or skin lesions in humans or animals. The present invention also relates to the cosmetic use of the botanical composition and dermatological preparation of the present invention. Furthermore, the present invention relates to an ophthalmological product comprising the botanical composition.

Description

[Technical Field]

[0001] The present invention relates to a botanical composition having antioxidant, antiproliferative, anti-inflammatory, regenerative, and scar-forming activity in the skin and dermis. The present invention also relates to a dermatological preparation comprising the botanical composition and a suitable pharmaceutically acceptable excipient. Furthermore, the present invention relates to a botanical composition and a dermatological preparation for the treatment of dermatological disorders and / or skin lesions. The present invention also relates to the cosmetic use of the botanical composition and the dermatological preparation. [Background technology]

[0002] Skin disorders are primarily characterized by abnormal cell proliferation and reproduction and / or inflammation. Many skin disorders cause thickening of the stratum corneum of the epidermis (a phenomenon also known as hyperkeratosis). These diseases are also called ichthyosis. Typical examples of ichthyosis include pityriasis rosea, dermatitis rosacea, seborrheic dermatitis, and psoriasis.

[0003] Psoriasis is a common, long-term (chronic) disease that does not heal. Although no wound is present in psoriatic lesions, keratinocytes behave as if a wound is present, switching from a normal proliferative program to regenerative maturation. In the event of skin injury, a wound-healing program, also known as regenerative maturation, is initiated. The normal wound-healing process involves a highly coordinated, regulated series of events, including blood congestion, inflammation, proliferation, and extracellular matrix (ECM) remodeling. In theory, cells are produced at a much faster rate to replace and repair the wound. Increased blood supply and localized inflammation also occur. However, in pathological conditions, including traumatic wounds resulting from burns or accidents, this normal healing process becomes highly dysregulated.

[0004] Also, in psoriatic lesions, cell production pushes the skin up, but the skin cannot shed the cells quickly enough. Excess skin cells build up, forming raised, scaly lesions. The white scales (called "plaques") that usually cover the lesions are made up of dead skin cells, and the redness of the lesions is due to an increased blood supply to the area of ​​rapidly dividing skin cells and may be accompanied by inflammation.

[0005] These skin disorders are typically treated symptomatically with keratolytic agents to remove plaques and glucocorticoids to reduce inflammation.

[0006] The thickened skin layer may be accompanied by an underlying infection. Therefore, antibacterial and antiseptic agents are typically used to treat such disorders. Antibacterial agents, antiseptics, and keratolytic agents (such as salicylic acid or retinoic acid) are also used to treat skin disorders caused by hormonal imbalances, such as acne, or severe skin inflammation, typically characterized by redness, swelling, oozing, scaling, and itching, such as eczema and dermatitis. In people who are naturally "atopic" or allergic, eczema can take the form of contact dermatitis or atopic dermatitis. When this disorder also affects the scalp, it is called seborrheic dermatitis. Dermatitis can be caused by chemicals, plants, fabrics, metal materials, and even medications. Atopic dermatitis is a chronic or chronically recurrent eczema caused by immune and non-immune factors. The former consists of food allergies, inhaled allergens, or contact with allergens; the latter consists of exogenous irritants, infections, autonomic nervous system disorders, lipid metabolism disorders, sweating, and stress. Atopic dermatitis is characterized by a decrease in epidermal ceramides, which weakens the skin's barrier function, increases irritability, and increases transepidermal water loss; concurrent epidermal inflammatory conditions can increase transepidermal water loss. This facilitates the penetration of allergens and haptens into the skin, which bind to keratinocytes and Langerhans cells, leading to their activation. Skin affected by dermatitis is more susceptible to infection due to increased levels of proinflammatory cytokines.

[0007] Within the epidermis, keratinocytes, along with Langerhans cells, dermal mast cells, dendritic cells, and macrophages, are major sources of cytokines. Quiescent keratinocytes constitutively produce multiple cytokines, but various environmental stimuli, including tumor promoters, ultraviolet light, and chemicals, can induce epidermal keratinocytes to release proinflammatory cytokines (e.g., IL-1, TNF-α), chemotactic cytokines (e.g., IL-8), and growth-promoting cytokines (e.g., IL-6 and IL-7).

[0008] Interleukin-1α (IL-1α) is constitutively produced in high amounts by keratinocytes; under normal conditions, IL-1α is retained inside the cell, but injury or various stimuli can trigger its release.

[0009] The primary role of IL-1α is to maintain the skin barrier function, preventing pathogenic microorganisms from invading the body; damage to keratinocytes leads to the release of IL-1α, which in turn stimulates the release of additional IL-1α and the production and release of other cytokines, such as IL-8 and IL-6. IL-1α also induces the production of several inflammatory mediators, including cyclooxygenase (COX)-2, type 2 phospholipase A, and inducible nitric oxide synthase (iNOS). IL-1α is used as a marker to compare the mildness of topical preparations and to assess skin inflammation; mild cleansers do not upregulate or worsen the release of IL-1α from keratinocytes, thereby maintaining the integrity of the skin barrier.

[0010] Interleukin-6 (IL-6) is a cytokine that contributes to innate immune defense; it is also essential for antibody production by B cells. In the skin, IL-6 is produced constitutively by fibroblasts and keratinocytes and after injury, but can also be synthesized by monocytes and macrophages after antigen activation. After mechanical injury, IL-6 messenger RNA (mRNA) expression is upregulated in the skin and exerts its effects on dendritic cells. Interleukin-8 (IL-8) is produced by fibroblasts and keratinocytes after stimulation with inflammatory cytokines, including IL-1α and TNF-α. IL-8 is a potent activator of chemotaxis and can be used as a useful marker for assessing early inflammation or tissue damage in the skin.

[0011] Inflammation, when present, is a sign of an abnormal skin condition, but low-level inflammation can occur in the absence of clinical symptoms such as erythema (skin redness).

[0012] Multiple drugs are currently used to treat dermatological disorders and skin lesions; however, their efficacy is incomplete, and the drugs themselves can cause skin reactions, especially with long-term use. Therefore, currently available drugs not only have incomplete effects, but also cause cosmetic problems.

[0013] Therefore, the goal of the present invention is to provide a novel pharmaceutical for treating dermatological disorders and / or skin lesions, which is effective and well tolerated by the body and also has a favorable cosmetic effect. Summary of the Invention

[0014] The above goals have been achieved by the botanical composition of the present invention as set forth in the claims appended hereto.

[0015] In another aspect, the present invention relates to a dermatological formulation comprising the botanical composition and a suitable pharmaceutically acceptable excipient.

[0016] The botanical compositions and dermatological preparations can be advantageously used as therapeutic agents.

[0017] In particular, the botanical compositions and dermatological preparations can be advantageously used as therapeutic agents for the treatment of skin lesions.

[0018] The botanical compositions and dermatological preparations can be used in both human and veterinary medicine.

[0019] The botanical compositions and dermatological preparations of the present invention may also have cosmetic applications. For the purposes of the present invention, the dermatological diseases are: ichthyosis, sebum hypersecretion, microbial infections of the skin and / or dermis, dermatophytosis, acne, psoriasis, seborrheic dermatitis, rosacea, dandruff, alopecia, allergic skin diseases, urticaria, scleroderma, contact dermatitis, atopic dermatitis, chronic actinic dermatitis, photodermatosis; for the purposes of the present invention, the tissue lesions are macula, papules, vesicles, bullae, pustules, cysts, erosions, abrasions, rashes, ulcers, rough skin, sores, pressure ulcers, telangiectasias, scales, erythema, crusts, lichenification, abrasions, indurations, cuts, lacerations, diabetic lesions and ulcers, or burns, potentially including the dermis and also including lesions on the skin. Optionally, the dermatological diseases and / or skin lesions include abnormalities and / or lesions of the skin and / or dermis resulting from oxidation of the skin.

[0020] In a further aspect, the present invention relates to an ophthalmic product comprising the botanical composition and an ophthalmologically acceptable excipient.

[0021] The features and advantages of the present invention will become more apparent from the following detailed description, illustrative examples, and accompanying drawings. [Brief explanation of the drawings]

[0022] [Figure 1]The amounts of IL-6 (FIG. 1A) and IL-8 (FIG. 1B) are shown for Example 12. The absorbance measured at 450 nm is directly proportional to the amount of interleukin produced in the tissue. The values ​​are expressed as mean ± standard deviation. Statistical data processing was performed using Student's t-test. Values ​​were considered significant if p<0.05. NC = untreated and unstimulated control tissue; SDS = tissue treated with sodium dodecyl sulfate to stimulate cytokine production in order to mimic an inflammatory state; TS = tissue treated with the test sample. *p<0.05 for SDS; **p<0.01 for SDS. [Figure 2] The results of the protein assay performed in Example 13 are shown. The absorbance values ​​measured at 650 nm are directly proportional to the amount of protein produced by the cells. The values ​​are expressed as mean ± standard deviation. Statistical data processing was performed using Student's t-test. p<0.05. *p<0.05 vs. NC; **p<0.01 vs. NC. [Figure 3] The results of the wound healing test performed in Example 14 are shown. Figures 3A and 3B show the amount of IL-6 and IL-8, respectively; the absorbance measured at 450 nm is directly proportional to the amount of interleukin produced by the cells. The values ​​are expressed as mean ± standard deviation. Statistical data processing was performed using Student's t-test. p<0.05. NC = untreated control cells. *p<0.05 for NC; **p<0.01 for NC. Figures 3C-G show the morphological evaluation of the cell monolayer; Figure 3C = cut monolayer; Figure 3D = negative control (untreated cells); Figures 3E-G = test of samples at concentrations of 0.2 mg / ml, 0.1 mg / ml, and 0.05 mg / ml, respectively. Magnification 10x; dotted lines indicate the original wound margins. [Figure 4] Quantification of PCNA immunofluorescence assay in Example 15 is shown. The values ​​are expressed as mean ± standard deviation. Statistical data processing was performed by Student's t-test. p<0.05. NC = untreated control tissue; TS = test sample; *p<0.05 for NC; **p<0.01 for NC. [Figure 5]5 shows the results of the cell viability test of human fibroblasts and keratinocytes in Example 16. The test samples were the plant-based composition of the present invention (FIG. 5A), a serum formulation containing the same (FIG. 5B), and a commercially available serum (FIG. 5C). [Figure 6] Results after two months of application for severe psoriasis are shown. [Figure 7] The results of superficial necrosis after 15 days of application are shown. [Figure 8] Results are shown for hematomas resulting from dog bites after 3 days of application. [Figure 9] Results after 3 days of application are shown for a severe hematoma resulting from a motorcycle accident. [Figure 10] Shown are results after 10 days of known treatment for eye trauma (A); results after 3 days of treatment with the botanical composition of the present invention (B); and results after 1 week of treatment with the botanical composition of the present invention (C). [Figure 11] 1 shows the results of morphological observation of HaCaT cells (A); and fibroblasts (B) treated for 24 hours with the plant-based composition of the present invention as shown in Example 18. DETAILED DESCRIPTION OF THE INVENTION

[0023] The botanical composition of the present invention contains natural ingredients, which may include multiple plant extract complexes.

[0024] In particular, the botanical composition of the present invention comprises the following components, each component being present in a concentration range by weight indicated in brackets (w / w, based on the total weight of the botanical composition): Tremella Fuciformis polysaccharides (10-20% w / w) Vitis Vinifera leaf extract (5-15%) Astragalus Membranaceus root extract (10-30%) Medicago Sativa extract (8-15%) Fucus Vesiculosus extract (4-15%) Spirulia Platensis powder (4-8%) Beta Vulgaris root extract (8-15%), and Polygonum Cuspidatum extract (5-30%) Preferably, the components of the botanical composition of the present invention are in the following concentration ranges by weight (w / w): Tremella Fuciformis polysaccharides (12-16%) Vitis Vinifera leaf extract (8-13%) Astragalus Membranaceus root extract (15-25%) Medicago Sativa extract (8-13%) Fucus Vesiculosus extract (8-15%) Spirulia Platensis powder (4-6%) Beta Vulgaris root extract (8-13%), and Polygonum Cuspidatum extract (15-25%)

[0025] For the preparation of extracts, unless otherwise specified, the above-ground parts of the plant are used; i.e., stems, leaves, flowers, or mixtures thereof. These parts can be used fresh or after drying under controlled conditions. The plant parts used for extraction are contacted with a suitable extraction solvent using traditional extraction methods (such as immersion or percolation) or more complex techniques (e.g., ultrasonic, microwave, pressure, or supercritical fluid extraction). Examples of extractive solvents that can be used include alcohols with up to four carbon atoms, including diols, triols, aldehydes, ketones, organic esters, chlorinated compounds, and mixtures thereof. If miscible, such solvents can also be used in mixtures with water.

[0026] Preferred solvents include methanol, ethanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, acetone, ethyl acetate and mixtures thereof, either alone or mixed with water.

[0027] After separating the extracted plant material, the extract can be used as is or after replacing it with one or more extraction solvents suitable for human use (such as glycerin or glycol if not used in the extraction phase), and preferably the extraction solvent is removed to obtain a dry extract, preferably by evaporation under reduced pressure and low temperature and spraying.

[0028] The extract may also be subjected to further purification steps to remove potential contaminants (such as fat-soluble pesticides), impurities (such as chlorophyll), or to increase the concentration of secondary metabolites.

[0029] Tremella fuciformis polysaccharide (CAS number: 778577-37-0) is extracted from the edible fruiting body of the Chinese Silver Ear mushroom. This polysaccharide contains glucuronic acid and N-acetylglucosamine. Tremella fuciformis extract can be obtained from Tremella fruiting bodies by solvent extraction. Vitis Vinifera leaf extract (CAS number: 84929-27-1) is an extract of red grape (Vitis) leaves.

[0030] Astragalus Membranaceus root extract (CAS number: 94166-93-5) is extracted from the dried roots of the medicinal herb Astragalus Membranaceus. Astragalus extract can be obtained through the following extraction process: Drying, grinding, screening and titration of individual plant extract complex components of Astragalus.

[0031] Medicago sativa extract (CAS number: 84082-36-0) is a whole plant extract of Medicago sativa (alfalfa), which contains folic acid and optionally one or more of oligosaccharides, vitamins B5e and Q10 coenzymes.

[0032] Fucus Vesiculosus Extract (CAS number: 84696-13-9) is an extract of the dried thallus of the bladderwrack alga Fucus.

[0033] Spirulia Platensis powder (CAS number: 223751-80-2) is a powder preferably obtained from the dried precipitate of the whole thallus of Arthrospira platensis (also known as Spirulina platensis (Spirulina)).

[0034] Beta vulgaris root extract (CAS No.: 89957-89-1 / 89957-90-4) is an extract of the root of sugar beet (Beta vulgaris L); it can be obtained by drying, extraction, and titration of Beta vulgaris powder or by solvent extraction. Polygonum cuspidatum extract (CAS No.: 501-36-0) can be obtained by drying and titration of powder or by solvent extraction.

[0035] The Vitis Vinifera leaf extract and the Polygonum Cuspidatum extract preferably contain resveratrol.

[0036] Preferably, the botanical composition according to the present invention is formulated as a dermatological preparation (also referred to herein as a "dermatological product"), more preferably as a dermatological preparation intended for topical (external) use. The botanical composition is preferably present in the pharmaceutical preparation at a concentration of 1 to 5% by weight relative to the weight of the dermatological preparation; more preferably, the botanical composition is present at a concentration of 2 to 4% by weight relative to the weight of the dermatological preparation, most preferably at a concentration of about 3% by weight.

[0037] The present invention is also directed to a dermatological formulation comprising the above-described botanical composition, preferably at a concentration of 1-5% by weight, more preferably 2-4% by weight, and most preferably about 3% by weight, based on the weight of the dermatological formulation, and suitable pharmaceutically acceptable excipients, including rheological additives, buffers, antimicrobials, antioxidants, antiisothermal agents, antistatic agents, absorbers, UV absorbers, astringents, chelating agents, skin protectants, preservatives, coating agents, denaturants, depigmenting agents, emulsifiers, film-forming agents, gelling agents, colloidal agents, humectants, hydrotropic agents, binders, emollients, leveling agents, opacifiers, plasticizers, propellants, skin protectants, reducing agents, cooling agents, sebum restoration agents, solvents, stabilizers, emulsion stabilizers, toning agents, humectants, bulking agents, or combinations thereof. Preferably, the pharmaceutically acceptable excipient comprises or consists of one or more of a rheological additive, a buffering agent, an antimicrobial agent, an antioxidant, an emulsifier, a wetting agent, a hydrotropic agent, a lubricating agent, and a solvent.

[0038] Dermatological formulations according to the present invention are preferably aqueous formulations, more preferably aqueous formulations comprising at least one alcoholic solvent, at least one antimicrobial agent, at least one smoothing agent, at least one rheological additive, at least one antioxidant, and / or at least one humectant. Particularly preferred pharmaceutically acceptable excipients present in the dermatological formulations of the present invention include: methoxymethylbutanol as the alcoholic solvent; carbomer as a thickening agent; propylene glycol, polyethylene glycol (PEG) compounds (such as PEG-6, PEG-32, and PEG-4, including PEG-4 proline linolenate and PEG-4 proline linoleate). Preferably, the dermatological formulations of the present invention further comprise one or more of vitamins and / or provitamins (such as vitamin A, vitamin D, vitamin E, and panthenol), preferably colloidal vitamins (such as colloidal vitamin D and colloidal vitamin A), inorganic elements with antimicrobial activity (such as silver, copper, and zinc), and other antimicrobial compounds (such as phenoxyethanol and 2-bromo-2-nitropropane-1,3-diol). Optionally, the dermatological preparation may further comprise glycosaminoglycans, such as chondroitin, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronic acid, and mixtures thereof.

[0039] The dermatological formulations may also contain antioxidants and / or antimicrobial agents as active ingredients.The botanical compositions of the present invention as described above have been shown to exert surprising and unexpected regenerative, repairing, anti-inflammatory, and cicatrizing effects on a variety of tissue lesions.

[0040] Therefore, the botanical compositions and dermatological formulations comprising them disclosed in the present invention are suitable for medical use. In particular, the botanical compositions and dermatological formulations comprising them of the present invention are disclosed with respect to their medical use in the treatment of dermatological diseases and skin lesions.

[0041] In a more particular aspect, the botanical compositions of the present invention and dermatological formulations containing them are disclosed for their medical use in the treatment of psoriasis.

[0042] Furthermore, the present invention also relates to the cosmetic use of said botanical compositions and dermatological preparations containing them.

[0043] In particular, the present invention discloses the cosmetic use of said botanical compositions and dermatological formulations containing them in the treatment of dermatological diseases and / or skin lesions.

[0044] In the present invention, the dermatological disease is ichthyosis, sebum hypersecretion, microbial infection, dermatophytosis, acne, psoriasis, seborrheic dermatitis, rosacea, dandruff, alopecia, allergic skin disease, urticaria, scleroderma, contact dermatitis, atopic dermatitis, chronic actinic dermatitis, and photodermatopathy.Preferably, the dermatological disease is ichthyosis, more preferably psoriasis.In the present invention, the tissue lesion is macules, papules, vesicles, bullae, pustules, cysts, erosions, abrasions, rashes, ulcers, rough skin, sores, decubitus ulcers, telangiectasias, scales, erythema, crusts, lichenification, abrasions, indurations, cuts, lacerations, diabetic lesions, and ulcers, or burns.

[0045] Formulation of the botanical composition with certain excipients may further improve one or more of the above-described dermatological benefits of the botanical composition, thereby providing a formulation that is particularly suitable for treating certain dermatological disorders or tissue lesions.

[0046] In a particularly preferred embodiment, the dermatological formulation of the present invention comprises the botanical composition together with a mixture of methoxymethylbutanol, carbomer, propylene glycol, PEG-4 proline linoleate, PEG-4 proline linoleate, colloidal silver, phenoxyethanol and 2-bromo-2-nitropropane-1,3-diol.

[0047] A preferred formulation with further improved anti-inflammatory activity comprises the botanical composition together with vitamin D and one or more of silver, iron, copper, phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol; preferably, the vitamins, silver, and / or iron are present in the form of a colloidal compound.

[0048] A preferred formulation with further improved antioxidant, antibacterial, anti-inflammatory, and soothing activity comprises the botanical composition together with vitamin A and / or panthenol, and further comprises one or more of silver, zinc, phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol; preferably, the vitamin A and / or silver are present in the form of a colloidal compound.

[0049] A preferred formulation having further improved antioxidant, antibacterial, anti-inflammatory, and anti-acne activity comprises the botanical composition, together with sulfur, hyaluronic acid, and one or more of silver (preferably colloidal silver), phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol.

[0050] Preferred formulations with further improved antioxidant, antibacterial, cicatrizing, regenerating, restoring, and antipsoriatic activity comprise the botanical composition, together with vitamin E and / or panthenol, and further comprising one or more of silver (preferably colloidal silver), copper, phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol. Particularly preferred are the following dermatological formulations 1 to 4:

[0051] Formulation 1 is particularly suitable for use as an antioxidant, antibacterial, anti-inflammatory and soothing formulation, preferably in the form of a serum.

[0052] [Table 1]

[0053] Formulation 2 is particularly suitable for use as an antioxidant, anti-aging and antibacterial formulation, preferably in the form of a serum. [Table 2]

[0054] Formulation 3 is particularly suitable for use as an antioxidant, antibacterial, anti-inflammatory and anti-acne formulation, preferably in serum form. [Table 3]

[0055] Formulation 4 is particularly suitable for use as an antioxidant, antibacterial, cicatrizing, regenerating, restorative and antipsoriatic formulation, preferably in serum form. [Table 4]

[0056] The dermatological preparation of the present invention can be administered by topical (external) route.The dermatological preparation containing the plant composition can be in the form of ointment, lotion, cream, emulsion, paste, gel, aqueous solution, colloid, spray, patch, serum, impregnated gauze, dressing, mouthwash, or combination thereof.Particularly preferred is the preparation in the form of serum.

[0057] Preferably, the dermatological preparation is administered topically (externally) to the skin, 1 to 4 times daily, in a suitable amount, such as the amount that can be absorbed when the preparation is applied to the skin and massaged in.

[0058] All of the dermatological formulations may be prepared by methods known in the pharmaceutical art.

[0059] In a further aspect, the present invention relates to an ophthalmic product comprising the botanical composition and an ophthalmologically acceptable excipient.

[0060] The ophthalmic product can be a tear substitute, an eyewash, a suspension, an eye spray, a foam, a liquid-impregnated wipe, a spray patch, or a combination thereof.

[0061] The term "ophthalmologically acceptable excipient" refers to a compound or mixture suitable for use in administering a composition to the external surface of the eye. For example, such excipients generally should not cause adverse reactions in users or significantly inhibit the action of the active ingredient at the ocular surface. Suitable excipients include antioxidants, gelling agents, sequestering agents, binders, lubricants, thickeners, tonicity adjusting agents, film-forming agents, and mixtures thereof.

[0062] It is to be understood that all preferred and advantageous aspects identified for the botanical composition should be considered as equally preferred and advantageous for the dermatological formulation, its uses, and vice versa. It is to be understood that all combinations of preferred aspects of the botanical composition of the present invention, and such combinations of the dermatological formulations, and such combinations of uses thereof, should be considered to be disclosed herein, as set forth above.

[0063] The following are examples of the present invention provided for illustrative purposes.

[0064] Example Example 1 The botanical composition is prepared by mixing the following ingredients: [Table 5]

[0065] Example 2 The botanical composition is prepared by mixing the following ingredients: [Table 6]

[0066] Example 3 The botanical composition is prepared by mixing the following ingredients: [Table 7]

[0067] Example 4 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 2% weight to formulation weight (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, propylene glycol, vitamin D, and 2-bromo-2-nitropropane-1,3-diol.

[0068] Example 5 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 3% weight to formulation weight (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, propylene glycol, panthenol, zinc, and 2-bromo-2-nitropropane-1,3-diol.

[0069] Example 6 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 1.5% weight to weight of formulation (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, propylene glycol, sulfur, zinc hyaluronate, silver, phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol.

[0070] Example 7 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 2.5% weight to weight of formulation (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, propylene glycol, hyaluronic acid, and 2-bromo-2-nitropropane-1,3-diol.

[0071] Example 8 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 3% weight to formulation weight (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, panthenol, vitamin E, phenoxyethanol, and 2-bromo-2-nitropropane-1,3-diol.

[0072] Example 9 Dermatological formulations were prepared using the botanical composition of Example 2 at a final concentration of 3% weight to formulation weight (w / w) by mixing with water together with the following excipients: Alcoholic solvent, carbomer, PEG compound, panthenol, colloidal silver, copper, and 2-bromo-2-nitropropane-1,3-diol.

[0073] Example 10 The following dermatological formulations containing the botanical composition of Example 1 were prepared for testing. [Table 8] [Table 9] [Table 10] [Table 11]

[0074] Example 11 The products tested in Example 10 were evaluated for their antioxidant activity by measuring their inhibitory capacity to inhibit free radical (ROS) formation in cell cultures of human keratinocytes.

[0075] An appropriate number of human keratinocytes (Huker) were seeded in 96-well plates and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2). Upon reaching a semi-confluent monolayer, the cells were treated with serially diluted concentrations (1:2 dilutions starting from 1.0 mg / ml) of the test product and positive control and incubated overnight (18-22 hours). The following day, the cells were washed with phosphate-buffered saline (PBS) and then incubated in DCFDA solution for 20 minutes under standard culture conditions. DCFDA was then removed, and the cells were washed twice with PBS before being exposed to UVA radiation for 2, 4, 6, 8, and 10 minutes to stimulate ROS production. The UVA range was 315-400 nm, with an irradiance of 1.7 mW / cm. 2 The treatment was carried out while ensuring constant irradiation of 1000 μg / ml. Untreated cells represent the negative control (NC).

[0076] At the end of each irradiation period, fluorescence was read using a fluorometer set to excitation at 485 nm and emission at 530 nm. A substance with known antioxidant activity was used as a positive control (PC). To confirm that there was no significant decrease in cell viability under the experimental conditions, NRU assays were also performed before and after UV irradiation. The results of the NRU assay performed before UV irradiation indicated that the test concentrations (0.0156–1.0 mg / ml) did not inhibit cell viability. Therefore, ROS quantification was performed using these concentrations.

[0077] All formulations tested showed antioxidant activity (significant % reduction in ROS) after UV exposure.

[0078] The formulations of the present invention are therefore suitable for attenuating and delaying the process of skin ageing caused by oxidative stress.

[0079] Example 12 The soothing activity of Formulation 1A of Example 10 was tested using a reconstituted human epidermis (RHE) model consisting of normal human keratinocytes cultured on an inert polycarbonate filter at the air / liquid interface. This is a model of a highly differentiated stratified epidermis, containing the main basal layer, the suprabasal layer, the spinous layer, and the granular layer, as well as a functional stratum corneum. This RHE model exhibits histological morphology, composition, and biochemical characteristics comparable to those of human in vivo tissue, making it highly representative and predictable for the efficacy of products used in vivo. Cytokine quantification was performed using three sets of tissues, each consisting of at least three tissues; one set of three tissues received no treatment (NC, negative control); a second set of three tissues was treated with sodium dodecyl sulfate (SDS) to stimulate interleukin production; and the third set of three tissues was treated with SDS and the test substance (TS) itself to determine whether the substance could reduce interleukin production (mitigation activity). After a 60-minute incubation, the tissues were washed to remove all treatment solution. After 24 hours, the medium was collected for interleukin quantification. The interleukin content of the medium was measured using ELISA (enzyme-linked immunosorbent assay), a plate assay technology designed for the detection and quantification of substances such as peptides, proteins, antibodies, and hormones. The color reaction is proportional to the amount of cytokine present in the medium. The results were read using a spectrophotometer at 450 nm. Interleukin concentrations were calculated by interpolating the OD values ​​on a standard interleukin curve. The percentages were calculated based on the unstimulated, untreated tissue (NC). As can be seen from Figure 1, the test product showed a significant decrease in IL-6 (protective activity 14.5% compared to the untreated tissue) and IL-8 (protective activity 10.8% compared to the untreated tissue).

[0080] These results demonstrate the palliative effect of the tested formulation and support its use in the treatment of dermatological disorders.

[0081] Example 13 The regenerative activity of Formulation 4A of Example 10 was tested by assessing its ability to induce protein production in cell cultures of human fibroblasts.

[0082] The above test was performed on human fibroblasts (NHDF) cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2). Cells were seeded and treated with the test products at concentrations of 0.2 mg / ml, 0.1 mg / ml, and 0.05 mg / ml; these concentrations were selected based on preliminary cytotoxicity tests (not shown). Some cells were maintained in culture without treatment (NC, negative control); others were treated with substances known to have regenerative activity (PC, positive control). After 24, 48, and 72 hours of contact, the cells were lysed and the cytoplasmic contents were collected; total protein was quantified from these cytoplasmic contents using the Lowry method. The absorbance value at 650 nm was used to calculate the content, and the OD of non-treated cells (NC) was considered as 100%.

[0083] As can be seen from Figure 2, the test product was found to significantly increase protein content after 24 and 48 hours of contact at test concentrations of 0.2 and 0.1 mg / ml, and after 24 hours of contact at test concentration of 0.05 mg / ml; this indicates a regenerative effect and supports its use for the treatment of skin lesions.

[0084] Example 14 The wound healing activity of Formulation 4A of Example 10 was tested by assessing the ability of the test product to accelerate tissue repair in a wound simulated by disrupting a monolayer of human fibroblast cells.

[0085] The above test was performed on human fibroblasts (NHDF) cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated under standard culture conditions (37°C, 5% CO2). Cells were seeded and maintained under standard culture conditions until a confluent monolayer was reached. At this point, we performed a wound-simulating procedure on the monolayer and treated the cells with the product at concentrations of 0.2 mg / ml, 0.1 mg / ml, and 0.05 mg / ml; these concentrations were selected based on the results of preliminary cytotoxicity tests (not shown). Untreated cells maintained in culture medium represented a negative control (NC). Some cells were treated with a positive control (PC), which is known to stimulate wound repair. The cells were observed under a light microscope to assess whether the wound edges approached, the disruption decreased, or the disruption closed. Bright-field images were acquired under a light microscope, and IL-1α assay was performed.

[0086] After photography, the culture medium in which the cells were incubated was collected and subjected to an IL assay. The IL content in the medium was quantified by ELISA (enzyme-linked immunofluorescence assay). Interleukin concentrations were calculated by interpolating OD values ​​on a standard interleukin curve. Percentages were calculated relative to untreated cells (NC). As can be seen in Figures 3A and 3B, the test products significantly reduced IL-6 and IL-8 at all test concentrations. As can be seen in Figures 3C–G, the approximation of the edges of the divided sections was observed after 24 hours of contact in cells treated with each test sample concentration. The results indicate that the test formulations can stimulate cell repair processes in human fibroblast cell cultures, suggesting scar-forming and regenerative activities that support their use in the treatment of skin lesions.

[0087] Example 15 The restorative activity of Formulation 4A of Example 10 was tested by assessing its ability to induce the expression of proliferating cell nuclear antigen (PCNA) in a reconstituted human epidermis (RHE) model. PCNA plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. Increased expression of this marker is indicative of the restorative activity of the test product.

[0088] The above test was performed using a reconstituted human epidermis (RHE) model. Tissues were treated with the test products for 1 and 4 hours. Untreated tissue represented a negative control (NC). At the end of the contact period, the tissues were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned with a microtome. The tissue sections were deparaffinized with a series of alcohol dilutions, washed with PBS, and then immunolabeled for PCNA. Nuclei were stained with Hoechst 33258. The preparations were mounted in a drop of Mowiol for fluorescence microscopy.

[0089] After immunofluorescence reaction, images were acquired using Cell F software. ImageJ software was used for quantitative analysis of the images. The percentages listed were calculated based on the measured fluorescence values ​​and the percentage of PCNA compared to untreated tissue (NC). As can be seen from Figure 4, the immunofluorescence for PCNA increased after both 1 hour and 4 hours of contact (reaching +55.2% after 1 hour of contact; +114.9% after 4 hours of contact). The results obtained demonstrate that the test product has restorative activity on reconstructed human epidermis, which supports its use in the treatment of dermatological diseases and skin lesions.

[0090] Example 16 The safety of the botanical compositions of the present invention, either in their original form or formulated into serums for topical administration, was evaluated. These compounds were tested in vitro using a cell model consisting of two human cell lines (dermal fibroblasts and epidermal keratinocytes, both of which constitute skin tissue). After direct addition to the culture medium, both the botanical composition (Figure 5A) and serum (Figure 5B) were well tolerated in both human skin cell lines. Even at the highest doses added to the model, no reduction in cell viability beyond 20%, a threshold potentially indicative of cellular dysfunction, was observed. Compared to other commercially available topical serums that exhibit toxicity at concentrations above 10 mg / ml (Figure 5C), the serums tested in the present invention could be added to cells at twice the concentration (20 mg / ml) without reducing cell viability; thus, this clearly demonstrates a robust safety profile.

[0091] Example 17 Figures 6-10 herein show that the compositions of the present invention were surprisingly effective after conventional treatments had failed or were unsatisfactory. especially: Figure 6 shows the results of two months of application on severe psoriasis. Figure 7 shows the results of 15 days of application on superficial necrosis. Figure 8 shows the results of a 3-day application on a hematoma resulting from a dog bite. Figure 9 shows the results of a 3-day application on a severe hematoma resulting from a motorcycle accident. Figure 10 shows the results of 10 days of known treatment (A), 3 days of treatment with the botanical composition of the present invention (B), and 1 week of treatment with the botanical composition of the present invention for eye trauma.

[0092] Example 18 The botanical compositions of the present invention are suitable for treating psoriasis.

[0093] Methods and operating principles used Flow cytometry analysis of the proliferation marker bromodeoxyuridine (BrdU), in conjunction with propidium iodide (IP) labeling, was performed as a functional parameter to examine cellular distribution during each phase of the replicative cycle following treatment with a plant extract complex mixture. Following 24 hours of treatment with the plant extract complex, the thymine analog BrdU (20 μM) was added directly to cells in the culture medium for 1.5 hours to allow incorporation into DNA in actively proliferating cells. Cells were then fixed with 70% ethanol and stored at -20°C until further immunolabeling. After allowing cells to warm to room temperature, DNA was subjected to acid hydrolysis; DNA was denatured by treatment with 2N HCl followed by sodium tetraborate (NA2B4O7); cells were washed and resuspended in PBS + 0.2% Tween 20 and 1% albumin. After 30 minutes, labeling was initiated using anti-BrdU antibody (DaKo) as the primary antibody, followed by a 2-hour incubation. After appropriate washing, incubation with FITC-conjugated secondary antibody was performed for 30 minutes, followed by IP labeling using a PBS solution containing propidium iodide, Nonidet, and RNase A for 30 minutes at room temperature and then overnight at 4°C. Finally, the samples were analyzed using an Attune™ NxT Acoustic flow cytometer Focusing Cytometer (Invitrogen, US). In a bivariate analysis of BrdU relative to propidium iodide (IP) labeling, BrdU is, in fact, incorporated into DNA during replication, whereas IP is a DNA intercalator commonly used to label the nucleic acid content of cells.

[0094] result Morphological observation of HaCaT cells treated for 24 hours with two concentrations of a plant extract complex mixture, which was found to be noncytotoxic, revealed a decrease in cell proliferation, as evidenced by the absence of mitosis (observed in Figure 11A, which appears as small rings under phase-contrast microscopy) and, at the highest concentration, a morphological shift toward a more mesenchymal phenotype. More specifically, the proliferative potential of the cells was examined by flow cytometry in relation to their distribution in the cell cycle (see Figures 11A and 11B). The images show representative cytograms obtained from BrdU labeling as a function of cell distribution in the replicative cycle.

[0095] In control and solvent-controlled cells, a characteristic distribution of BrdU relative to iron is observed in the Y-shaped region of actively proliferating cells; in particular, this population of cells is in the S phase. The distribution of cells in replicating cell cycles also clearly indicates a predominance of the S phase.

[0096] In the presence of the plant extract complex at concentrations of 0.1 mg / ml and 0.2 mg / ml, the cell population corresponding to S phase flattened, and the cells appeared arrested in G1; DNA profiles also showed a corresponding distribution. From these results, it is not possible to definitively answer whether the inhibitory response may be dose-dependent. Similar to what was found in HaCaT cells, morphological observations performed at 24 h after treatment also revealed a complete arrest in mitosis in fibroblasts. In contrast, this was completely evident in control cells. Flow cytometry analysis revealed a smaller population of BrdU-positive cells compared to HaCaT cells, consistent with a less proliferative cell type; G1 arrest was also observed after treatment with the plant extract complex at concentrations of 0.1 mg / ml and 0.2 mg / ml.

[0097] The distribution of cells in the replicative cycle reveals a G1 arrest and a marked reduction in cells in S phase.

[0098] conclusion Overall, it can be said that treatment of HaCaT cells and fibroblasts with the mixture at concentrations of 0.1 and 0.2 resulted in the induction of antiproliferative activity, which confirms the hypothesis of inhibition of replicative synthesis presented in the introduction of this study.

Claims

1. A botanical composition comprising: 10-20% (w / w) Tremella Fuciformis polysaccharides 5-15% (w / w) of Vitis Vinifera leaf extract 10-30% (w / w) Astragalus Membranaceus root extract 8-15% (w / w) extract of Medicago Sativa 4-15% (w / w) extract of Fucus Vesiculosus 4-8% (w / w) Spirulia Platensis powder 8-15% (w / w) of Beta Vulgaris root extract, and 5-30% (w / w) of an extract of Polygonum Cuspidatum Consists of Botanical composition.

2. 10. The botanical composition of claim 1 , 12-16% (w / w) Tremella Fuciformis polysaccharides 8-13% (w / w) of Vitis Vinifera leaf extract 15-25% (w / w) Astragalus Membranaceus root extract 8-13% (w / w) of an extract of Medicago Sativa 8-15% (w / w) extract of Fucus Vesiculosus 4-6% (w / w) Spirulia Platensis powder 8-13% (w / w) of Beta Vulgaris root extract, and 15-25% (w / w) extract of Polygonum Cuspidatum Consists of Botanical composition.

3. A formulation comprising the botanical composition of claim 1 or 2 and a pharmaceutically acceptable excipient.

4. 4. The formulation of claim 3, wherein the concentration of the botanical composition is 1-5% by weight, preferably 2-4% by weight, relative to the weight of the dermatological formulation.

5. 5. The formulation of claim 3 or 4, further comprising at least one vitamin selected from vitamin A, vitamin E, vitamin D, panthenol, and mixtures thereof, and / or at least one antimicrobial agent selected from silver, sulfur, copper, phenoxyethanol, 2-bromo-2-nitropropane-1,3-diol, and mixtures thereof.

6. The formulation according to any one of claims 3 to 5, further comprising at least one glycosaminoglycan, preferably hyaluronic acid.

7. A formulation according to any one of claims 3 to 6, comprising: in the form of an ointment, lotion, cream, emulsion, paste, gel, aqueous solution, colloid, spray, patch, serum, impregnated gauze, dressing, mouthwash, or a combination thereof, preferably in the form of a serum; formulation.

8. A formulation according to any one of claims 3 to 7, which is a dermatological formulation.

9. A botanical composition or formulation according to claim 1 or 2, or a formulation according to any one of claims 3 to 7, for use as a therapeutic agent.

10. A botanical composition according to claim 1 or 2 or a formulation according to any one of claims 3 to 8 for use as a therapeutic agent in the field of human or veterinary medicine.

11. A botanical composition according to claim 1 or 2 or a formulation according to any one of claims 3 to 8 for use as a therapeutic agent in the treatment of dermatological diseases and / or skin lesions.

12. 10. An ophthalmic product comprising the botanical composition of claim 1 or 2 and an ophthalmologically acceptable excipient, the ophthalmic product being a tear substitute, an eyewash, a suspension, an eye spray, a foam, a liquid-impregnated wipe, a spray patch, or a combination thereof.

13. Cosmetic use of the plant composition of claim 1 or 2 or the formulation of any one of claims 3 to 8.

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