TWI780841B - An extract of sarcodia and uses thereof - Google Patents

Abstract

The present invention provides a use of a composition for anti-inflammatory and skin whitening, wherein the composition comprises an extract of sarcodia.

Description

A kind of sea fungus extract and its use

The present invention relates to a novel use of a sea fungus extract, in particular to the use of the sea fungus extract for preparing anti-inflammatory and skin whitening compositions.

Sea fungus is mainly distributed in the Indian Ocean and the Western Pacific. Sea fungus is a large marine heterotroph (heterotroph) algae. Because of its high content of micronutrients, it is also called longevity vegetable in Japan, and sea fungus is called the first sea vegetable in Europe and America. At present, sea fungus is mostly used for direct consumption or food additives. Although sea fungus has a long history of consumption, there is little research on its efficacy.

With the recent vigorous development of medical cosmetology, the cosmetics and skin care products market has grown rapidly. The factors that attract consumers to buy cosmetic and skin care products are novelty, durability and effectiveness. Cosmeceuticals are a bridge between physicians and cosmetic scientists with chemical backgrounds. After in vitro or in vivo experiments, cosmeceuticals are more valuable for specific purposes, or have better effects than traditional cosmetics. In recent years, the term cosmeceuticals (cosmeceuticals) has appeared in a large number of advertisements, which is defined between general cosmetics (cosmetics) and pharmaceuticals (pharmaceuticals); compared with general cosmetics, the bioactive ingredients contained in cosmeceuticals are more clearly defined Mechanism of action and activity, providing drug-like advantages.

Therefore, the industrial applicability of skin care products is very high, especially for modern people to skin care products high demand. Therefore, there is no relevant application of sea fungus active extracts on the skin at present, so there is still much room for research and development of sea fungus.

The present invention is to dry the sea fungus, and then extract it with alcohol to obtain an alcohol extract; then soak the alcohol extract in water to suspend it to obtain an aqueous extract; and then use ethyl acetate partitioning the aqueous extract to form an ethyl acetate layer and an aqueous layer, then collecting the aqueous layer; and partitioning the resulting aqueous layer with n-butanol to form an n-butanol alcohol and an aqueous layer, and then collect the aqueous layer to obtain a sea fungus extract EH.

In the whitening experiment, the sea fungus extract EH can reduce the expression of melanin in cells and zebrafish. This result shows that the sea fungus extract EH can inhibit the expression of melanin to achieve whitening effect.

In the anti-inflammatory experiment, the sea fungus extract EH can inhibit the expression of inflammatory factors cyclooxygenase-2 (cyclooxygenase-2, COX-2) and inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS). This result shows that the sea fungus extract EH can inhibit the expression of inflammatory factors to achieve anti-inflammatory effect.

In the experiment of atopic dermatitis, the sea fungus extract EH can improve the symptoms of atopic dermatitis in mice, such as erythema, edema, peeling and dryness. At the same time, the sea fungus extract EH can reduce the expression level of IgE raised by atopic dermatitis. In addition, because atopic dermatitis can cause spleen and lymph node enlargement, the addition of the sea fungus extract EH can improve the spleen and lymph node enlargement. This result shows that the sea fungus extract EH can treat atopic dermatitis.

The term "a" or "an" herein is used to describe elements and components of the present invention. This term is only for convenience of description and to give the basic concept of the present invention. This statement should be read to include one or at least one, and the singular also includes the plural unless it is clearly stated otherwise. When used in conjunction with the word "comprising" in the claims, the word "a" may mean one or more than one.

The term "or" used herein in claims means "and/or" unless it is expressly intended to mean only the other option, or unless the other options are mutually exclusive.

The invention provides a preparation method of sea fungus extract, which comprises the following steps: (a) extracting sea fungus with an alcohol solvent to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol (b) using an organic solution or supercritical extraction to partition and extract the sea fungus alcohol extract to form an organic solvent layer and an aqueous layer, and then collect the aqueous layer, wherein the organic solution is an organic solvent Or a mixture of water and an organic solvent, the organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, chloroform or Carbon tetrachloride; and (c) distributive extraction of the aqueous layer obtained in step (b) with a n-butanol to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the Sea fungus extract.

In a specific embodiment, the sea fungus is Sarcodia ceylanica .

In another specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In a specific embodiment, the organic solvent is ethyl acetate. According to the present invention, since the sea fungus extract is obtained from the aqueous layer, the sea fungus extract is a sea fungus extract Fungus Water Extract.

In a specific embodiment, the method includes a step (a1), before the step (a), wherein the step (a1) includes drying the sea fungus. In a preferred embodiment, the sea fungus is dried at a temperature of 20-70°C. In a more preferred embodiment, the sea fungus is dried at a temperature of 40-60°C. In another specific embodiment, the sea fungus is dried at 50°C.

In a specific embodiment, the drying time of the sea fungus is 12-48 hours. In a preferred embodiment, the drying time of the sea fungus is 16-32 hours. In a preferred embodiment, the drying time of the sea fungus is 20-24 hours.

According to the present invention, after the sea fungus is dried, it is filtered through a sieve with a certain mesh size and then extracted with ethanol. In a specific embodiment, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 30-70 mesh sieve. In a preferred embodiment, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 40-60 mesh sieve. In a more preferred embodiment, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 50-mesh sieve.

In another specific embodiment, the sea fungus is extracted with an alcohol solvent at room temperature. In a preferred embodiment, in step (a), the sea fungus is soaked and extracted three times with an alcohol solvent, and the soaking solutions combined three times are filtered and concentrated under reduced pressure to obtain the sea fungus alcohol extract.

In a specific embodiment, in step (b), use the organic solution to the The sea fungus alcohol extract is partitioned and extracted to form an organic solvent layer and an aqueous layer, and then the aqueous layer is collected. In a preferred embodiment, in step (b), the organic solution is used to carry out distribution extraction of the sea fungus alcohol extract, and the operation of distribution extraction is repeated three times, so as to form an organic solvent layer and a Aqueous layer, then collect this aqueous layer.

In another specific embodiment, the step (b) further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain a sea fungus Aqueous solution of fungus. In a preferred embodiment, the step (b1) comprises soaking the maria eucalyptus extract in water, so as to suspend the marinum fungus alcohol extract in the water. After performing step (b1), in the original step (b), the organic solvent will be used to distribute and extract the sea fungus aqueous solution obtained in step (b1), without the need to mix the water with the organic solvent solution, so as to form an organic solvent layer and an aqueous layer, and then collect the aqueous layer.

In a specific embodiment, in step (c), the aqueous layer obtained in step (b) is subjected to distribution extraction with the n-butanol, and the distribution extraction operation is repeated three times, so as to form the n-butanol layer and The aqueous layer is then collected to obtain the sea fungus extract.

After step (b1) is performed, in the original step (b), the supercritical extraction will be used to distribute and extract the sea fungus aqueous solution obtained in step (b1). In a specific embodiment, in step (b), the supercritical extraction is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then collect the aqueous layer. In another embodiment, the supercritical extraction is CO ₂ supercritical extraction. According to the present invention, the process of _CO2 supercritical extraction and separation of nutritional components of natural products mainly uses supercritical carbon dioxide fluid to extract nutritional components from natural products under high pressure and low temperature conditions. In a specific embodiment, the CO ₂ supercritical extraction condition is CO ₂ : the flow rate ratio of 99% ethanol solution=10 mL/min: 1 mL/min; the critical pressure range is 150-400 bar (bar); and the critical temperature The range is 20-60°C. In a preferred embodiment, the CO ₂ supercritical extraction conditions are CO ₂ : the flow rate ratio of 95% ethanol solution=10mL/min: 1mL/min; the critical pressure is 250 bars (bar); and the critical temperature is 40 ℃. After CO ₂ supercritical extraction, it will also be divided into the raw material (aqueous layer) and the extracted material (organic solvent layer) left in the CO ₂ supercritical extraction reaction tank; therefore, the CO ₂ supercritical extraction reaction The aqueous layer in the tank was extracted with n-butanol to obtain the sea fungus extract.

According to the present invention, in step (c), the separated n-butanol layer and the aqueous layer are extracted with n-butanol, wherein after the aqueous layer is collected, it is first filtered with a C18 glass column to remove excess salt class, and then dried to obtain the sea fungus extract. In a specific embodiment, the method includes a step (d), after the step (c), wherein the step (d) includes filtering the aqueous layer obtained in the step (c), and drying after filtering, to obtain the sea fungus extract. In a preferred embodiment, the method includes a step (d), after the step (c), wherein the step (d) includes filtering the aqueous layer obtained in the step (c) with a C18 glass column , filtered and then dried to obtain the sea fungus extract.

In another embodiment, the ingredients of the sea fungus extract do not contain polysaccharides.

The present invention also provides a use of sea fungus extract for preparing a skin whitening composition, wherein the preparation method of the sea fungus extract includes the following steps: (a) extracting a sea fungus with an alcohol solvent to obtain a sea fungus Sea fungus alcoholic extract, wherein the alcoholic solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the aqueous layer to obtain a sea fungus aqueous layer extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, The machine solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then The aqueous layer was recovered.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (c), which, after the step (b), comprises distributing the aqueous layer obtained in the step (b) with n-butanol extraction to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the sea fungus extract.

In a specific embodiment, the sea fungus is Sarcodia ceylanica .

In another specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In a specific embodiment, the organic solvent is ethyl acetate.

According to the present invention, the sea fungus extract can inhibit melanin production or reduce black to achieve the effect of skin whitening. In a specific embodiment, the sea fungus extract inhibits melanin production or reduces melanin. In a preferred embodiment, the sea fungus extract is used to prepare a composition for inhibiting melanin production or reducing melanin. In a more preferred embodiment, the sea fungus extract is used to prepare medicines or cosmetics for inhibiting melanin production or reducing melanin.

In a specific embodiment, the composition includes a cosmetic material composition, a food additive or a pharmaceutical composition. In a preferred embodiment, the composition includes a cosmetic composition, a food or a medicine. Therefore, the composition can be used to prepare cosmetics, food or medicine. In another specific embodiment, the sea fungus extract is used to prepare medicines or cosmetics for skin whitening.

In an exemplary embodiment, the composition may be a cosmetic composition. In addition to the sea fungus extract, the cosmetic composition may contain functional additives and ingredients usually contained in cosmetic compositions. The functional supplement may include ingredients selected from water-soluble vitamins, oil-soluble vitamins, polypeptides, polysaccharides, sphingolipids, and seaweed extracts. In addition, oils, fats, wetting agents, emollients, surfactants, organic or inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, antioxidants, plant extracts , pH control agent (pH control agent), alcohol, coloring agent, fragrance, blood circulation enhancer, cooling agent, antiperspirant or pure water, etc.

The preparation of the cosmetic composition is not particularly limited, and may be appropriately selected according to the purpose. For example, it can be prepared to be selected from the group consisting of one or more of the following preparations: facial cleanser (skin lotion), skin softener (softener), skin toner (toner), astringent (astringent), lotion, emulsion ( milk lotion), moisturizing lotion, nourishing lotion, massage cream, nourishing cream, Moisturizing cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion ( cleansing lotion), cleansing cream, body lotion and body cleanser, but not limited thereto.

The present invention further provides a method for whitening skin by inhibiting melanin production or reducing melanin, which comprises administering an effective dose of a composition to a skin of an individual, wherein the composition comprises a sea fungus extract, wherein the sea fungus extract The preparation method of the substance comprises the following steps: (a) extracting a sea fungus with an alcoholic solvent to obtain an alcoholic extract of the sea fungus, wherein the alcoholic solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the aqueous layer to obtain a sea fungus aqueous layer extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, The machine solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), use the organic solvent to carry out the sea fungus aqueous solution obtained in step (b1) Partition extraction to form an organic solvent layer and an aqueous layer, followed by recovery of the aqueous layer.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (c), which, after the step (b), comprises distributing the aqueous layer obtained in the step (c) with n-butanol extraction to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the sea fungus extract.

In a specific embodiment, the sea fungus is Sarcodia ceylanica .

In another specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In a specific embodiment, the organic solvent is ethyl acetate.

In another embodiment, the individual is an animal, preferably a mammal, more preferably a human.

According to the present invention, the composition is administered on the skin of the individual to inhibit or reduce the melanin in the skin area, so as to whiten the skin in the area. In a specific embodiment, the composition is prepared in the form of an emulsion, a lotion, an essence, a sunscreen, a barrier cream or a mask.

The term "effective dose" herein refers to a therapeutic dose which prevents, reduces, arrests or reverses the development of a symptom in a subject under specified conditions, or partially or completely alleviates the symptoms that were present in the subject when the treatment was started. symptoms. Those skilled in the art can also readily determine the appropriate dosage regimen for administering the sea fungus extract to an individual. For example, the sea fungus extract can be administered to the individual once or twice. When a dosage regimen includes multiple administrations, it can be understood that the effective dosage of the sea fungus extract administered to the individual can include all dosages Total amount of product administered during administration.

In terms of skin whitening, in a specific embodiment, the effective dosage range of the sea fungus extract is 5 μg/ml to 500 μg/ml. In a preferred embodiment, the effective dosage range of the sea fungus extract is 20 μg/ml to 200 μg/ml. In a more preferred embodiment, the effective dosage range of the sea fungus extract is 50 μg/ml to 100 μg/ml.

The present invention also provides a composition for the preparation of anti-inflammatory drugs, wherein the composition contains a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcoholic solvent to A sea fungus is extracted to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the aqueous layer to obtain a sea fungus aqueous layer extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, The machine solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), use the organic solvent to carry out the sea fungus aqueous solution obtained in step (b1) Partition extraction to form an organic solvent layer and an aqueous layer, followed by recovery of the aqueous layer.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (c), which, after the step (b), comprises distributing the aqueous layer obtained in the step (b) with n-butanol extraction to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the sea fungus extract.

In a specific embodiment, the sea fungus is Sarcodia ceylanica .

As used herein, the term "anti-inflammatory" includes, but is not limited to, treating or managing inflammatory conditions.

In one embodiment, the anti-inflammatory comprises inhibiting an inflammatory factor. In a preferred embodiment, the inflammatory factors include cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In a more preferred embodiment, the sea fungus extract inhibits inflammation caused by cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).

Therefore, the sea fungus extract can inhibit inflammation, and can be applied to treat atopic dermatitis. In one embodiment, the sea fungus extract treats atopic dermatitis through anti-inflammation. In a preferred embodiment, the drug treats atopic dermatitis through anti-inflammation.

As used herein, the term "treating" refers to alleviating symptoms or complications; delaying the progression of a disease, disorder or condition; alleviating or alleviating symptoms and complications; and/or curing or eliminating the disease, disorder or condition.

As used herein, the term "allergic dermatitis" refers to a general term for skin diseases caused by allergic reactions, characterized by chronic itching and eruptions on the face, neck, elbows and/or knees. At once Examples of allergic dermatitis include contact dermatitis and atopic dermatitis. "Contact dermatitis" refers to an eczematous inflammatory disease caused by the contact of foreign antigens with the skin, such as allergic contact dermatitis, photocontact dermatitis, systemic contact dermatitis and Contact hives. Furthermore, examples of antigens include metal allergens (cobalt, nickel, etc.), plant allergens (sumac, primrose, etc.), and food allergens (mango, ginkgo, etc.). "Atopic dermatitis (AD)" refers to a skin disease in which many patients have atopic tendencies. Symmetrical generalized eczema characterized by repeated exacerbations and remissions, for example, diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Besnier prurigo, acute Infantile eczema, flexural eczema, extremities childhood eczema, childhood atopic eczema, childhood eczema sicca, childhood eczema, adult atopic dermatitis, endogenous eczema, childhood dermatitis and chronic childhood eczema rash.

In a specific embodiment, the allergic dermatitis comprises a contact dermatitis or an atopic dermatitis. In a preferred embodiment, the allergic dermatitis comprises atopic dermatitis. In a more preferred embodiment, the atopic dermatitis includes diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Bernier prurigo, acute infantile eczema, flexural eczema , Children's eczema of extremities, children's atopic eczema, children's sicca eczema, children's eczema, adults' atopic dermatitis, endogenous eczema, children's dermatitis or chronic children's eczema.

In another embodiment, the medicament inhibits the symptoms of atopic dermatitis including erythema, edema, peeling or dry skin.

In a specific embodiment, the drug reduces the increase of IgE expression level caused by the atopic dermatitis.

In another embodiment, the drug improves the atopic dermatitis A condition in which the spleen or lymph nodes are enlarged.

In a specific embodiment, the medicament further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is determined by the particular combination of administration and the particular method of administering the composition. The term "carrier" as used herein includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, penetration and absorption delaying agents such as antibacterial and antifungal agents, buffers, carrier solutions, suspensions or colloid etc. Such media and agents for pharmaceutically active substances are well known in the art. Unless any conventional media or agents are incompatible with the active ingredients, their combination for treatment needs to be considered. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce allergic or similar adverse reactions when administered to a subject. The preparation of aqueous compositions with proteins as active substances is well known in the art. Usually, the composition is prepared as a liquid solution, tablet, capsule or suspension injection; it can also be prepared as a solid form that can be dissolved or suspended for injection. In another embodiment, the pharmaceutically acceptable carrier comprises a dermatologically acceptable vehicle. "Dermatologically acceptable medium" refers to biologically appropriate substances, such as salts, esters and/or amides. This means that the substance, when used together with selected active ingredients, does not cause undesired biological effects when administered to the individual. In addition, a substance that does not adversely interact with any of the components of the pharmaceutical composition in which it is contained. Likewise, "dermatologically acceptable salts" or "dermatologically acceptable esters" herein are biologically acceptable salts or esters.

According to the present invention, the formulation of the composition (comprising the sea fungus extract) and the pharmaceutically acceptable carrier may be via sterile aqueous solution or dispersion, aqueous suspension, oil emulsion, water in oil-in-emulsion, specific point emulsions, long stay emulsions, viscous emulsions, microemulsions Liquids, nanoemulsions, liposomes, microparticles, microspheres, nanospheres, nanoparticles, micromercury and several sustained-release natural or synthetic polymers. The pharmaceutically acceptable carrier and the sea fungus extract can also be configured into aerosol, tablet, pill, capsule, sterile powder, suppository, lotion, cream, ointment, paste, gel, hydrogel , or other formulations that can be used for composition delivery.

In addition, compositions referred to in the present invention include compositions for topical and regional application. The term "topical" as used herein refers to the use of the composition described herein incorporated in a suitable pharmaceutical carrier and applied to a site of skin, such as an affected area of atopic dermatitis, for local action. Accordingly, such topical compositions include those pharmaceutical forms in which the compound is applied externally through direct contact with the skin surface to be treated. Conventional pharmaceutical forms for this purpose include ointments, lotions, creams, shampoos, lotions, pastes, gels, sprays, aerosols, etc., and may be presented as patches or dips depending on the part of the body to be treated. Applied in the form of an ulcer dressing. The term "ointment" includes formulations (including creams) having oily bases, water-soluble bases and emulsion-type bases such as petrolatum, lanolin, polyethylene glycols and mixtures of these combinations.

The medicament of the present invention may further comprise a pharmaceutically acceptable carrier, which can be administered to an individual through many different routes in the treatment mode known in the field related to the present invention. In some embodiments, the composition (comprising the sea fungus extract) and the pharmaceutically acceptable carrier are administered externally, intravenously, intramuscularly, subcutaneously, topically, orally or inhaled. The drug will be delivered to the target through the digestive and circulatory system.

In another embodiment, the individual is an animal, preferably a mammal, more preferably a human.

In terms of anti-inflammation, in a specific embodiment, the effective dose of the sea fungus extract ranges from 1 mg/kg body weight to 200 mg/kg body weight. In a preferred embodiment Among them, the effective dosage range of the sea fungus extract is 5 mg/kg body weight to 100 mg/kg body weight. In a more preferred embodiment, the effective dose of the sea fungus extract ranges from 10 mg/kg body weight to 40 mg/kg body weight.

In terms of treating atopic dermatitis, in a specific embodiment, the effective dose of the sea fungus extract ranges from 0.05 to 20 mg/ml. In a preferred embodiment, the effective dosage range of the sea fungus extract is 0.1 to 10 mg/ml. In a more preferred embodiment, the effective dosage range of the sea fungus extract is 0.5 to 2 mg/ml.

Figure 1 is a flow chart of the extraction of sea fungus.

Figure 2 is the effect of sea fungus extract EH on the content of melanin in cells.

Figure 3 is the effect of sea fungus extract EH on zebrafish body pigment. Zebrafish embryos were given sea fungus extract EH 9 hours after fertilization. After soaking for 48 hours, the larvae were taken out to take pictures and analyzed. 5.4mg/mL arbutin and 30.4μg/mL PTU were used as the positive control group. The belly of the fish was circled by image quantitative software, and the expression of melanin production in each group of zebrafish was analyzed. The results showed that EH (100 μg/mL) could inhibit the production of melanin in zebrafish and had whitening activity. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference (P<0.05) compared with the control group are represented by *.

Figure 4 shows the effect of sea fungus extract EH on iNOS induced by lipopolysaccharide (LPS). Raw264.7 cells were treated with 10ng/mL lipopolysaccharide to induce inflammatory response, and added 20, 50, and 100 μg/mL sea fungus extract EH for 16 hours, and 10 μM Dicortisol (Dex) was used as a positive control group. The expression of the inflammatory factor iNOS was analyzed by Western blot method, and the results showed that EH can significantly inhibit iNOS. Data are presented as mean ± standard error of the mean (mean ± SEM). with LPS Significant difference between groups (P<0.05) is indicated by *.

Figure 5 shows the effect of sea fungus extract EH on COX-2 induced by lipopolysaccharide (LPS). Raw264.7 cells were treated with 10ng/mL lipopolysaccharide to induce inflammatory response, and added 20, 50, and 100 μg/mL sea fungus extract EH for 16 hours, and 10 μM Dicortisol (Dex) was used as a positive control group. The expression of the inflammatory factor COX-2 was analyzed by western blot method, and the results showed that EH could significantly inhibit COX-2. Data are presented as mean ± standard error of the mean (mean ± SEM). Compared with LPS group, those who achieved significant difference (P<0.05) are represented by *.

Fig. 6 is the effect of sea fungus extract EH on the skin appearance of mice with atopic dermatitis. Figure 6A shows the use of DNCB to induce atopic dermatitis. After the skin of mice showed symptoms of atopic dermatitis, the drug was applied daily; DNCB-induced atopic dermatitis was photographed on the 1st, 7th, and 12th days of treatment. Skin appearance of mice, scale bar = 1 cm. The results showed that compared with the AD group, both the EHL group (low dose group) and the EHH (high dose group) group had the effect of alleviating the symptoms of atopic dermatitis, and the edema, redness, dryness and crusting of the epidermis were all improved. Relieve. Fig. 6B shows the clinical dermatitis score of the relief of atopic dermatitis symptoms by sea fungus extract EH on the 12th day of treatment. EH relieved symptoms of atopic dermatitis evaluated by clinical dermatitis score. Four skin surface symptoms were used to assess the severity of dermatitis: erythema/hemorrhage, edema, desquamation/erosion, and dryness. Scores from 0 to 3 are given according to the severity of each symptom, with more scores being more severe. The total score of dermatitis is the sum of the above 4 scores, and the highest score is 12. The higher the score, the more severe the severity of the dermatitis. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Figure 7 shows the effect of sea fungus extract EH on IgE in serum. before animal sacrifice Blood was collected to detect IgE in serum. DNCB-induced atopic dermatitis caused IgE to rise, and after applying the sea fungus extract EH, IgE all decreased. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Figure 8 is the effect of sea fungus extract EH on the weight of enlarged spleen induced by DNCB. Spleen tissue was collected, photographed and weighed at the time of animal sacrifice. DNCB-induced atopic dermatitis caused enlargement of spleen tissue, and after applying EH extract of sea fungus, the weight of spleen decreased. Scale bar = 1 cm. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Fig. 9 is the effect of sea fungus extract EH on the weight of lymph node enlargement induced by DNCB. Lymph node tissues were collected, photographed and weighed when animals were sacrificed. DNCB-induced atopic dermatitis caused swelling of lymph node tissue, and after applying EH extract of sea fungus, the weight of lymph nodes decreased. Scale bar = 0.5 cm. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Extraction of cultured sea fungus

The present invention uses artificially cultured double-cracked sea fungus ( Sarcodia ceylanica ).

The sea fungus was dried at 50°C for 24 hours to obtain dried sea fungus. 10.8kg dried sea fungus is pulverized, filtered through a 50 mesh (mesh) sieve, and carried out at room temperature 95% ethanol (EtOH) was soaked and extracted three times, and the soaking solution combined three times was filtered and concentrated under reduced pressure to obtain 370.5 g of sea fungus alcohol crude extract. Add the above sea fungus crude extract to 1L of water to make it into a suspension; then add 1L of ethyl acetate (EtOAc) for distribution extraction, repeat the above distribution extraction three times to form an ethyl acetate layer and an aqueous layer, and transfer Except above-mentioned ethyl acetate layer, then collect this aqueous layer; Use 1L n-butanol (n-BuOH) this aqueous layer is carried out distribution extraction, repeat above-mentioned distribution extraction three times, in order to form a n-butanol layer and an aqueous layer, The n-butanol layer was removed, and the separated aqueous layer was filtered through a C18 glass column to remove excess salts, and then dried to obtain extract EH (150 g). The extraction process of the whole sea fungus is shown in Figure 1.

In addition, in the above extraction step, the alcohol solution can be replaced with methanol solution. At the same time, the extraction step with ethyl acetate can be replaced by other organic solvents, such as propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, Chloroform or carbon tetrachloride solvent for partition extraction or supercritical extraction (such as _CO2 supercritical extraction), and then extract with n-butanol can obtain the extract with the same composition as EH.

In addition, water can be mixed with ethyl acetate or other organic solvents to form an organic solution to directly extract the sea fungus alcohol crude extract, so there is no need to separate water and organic solvents into two steps for extraction. In addition, the sea fungus alcohol crude extract can also be directly extracted with ethyl acetate or other organic solvents, without the step of soaking in water first. The changes of the above steps can finally obtain the extract with the same composition as EH.

The above CO ₂ supercritical extraction conditions are: flow rate ratio of CO ₂ : 95% ethanol solution = 10 mL/min: 1 mL/min; critical pressure is 250 bar; and critical temperature is 40°C. After the _CO2 supercritical extraction, it will also be divided into the raw material (aqueous layer) and the extracted material remaining in the _CO2 supercritical extraction reaction tank; therefore, the raw material in the _CO2 supercritical extraction reaction tank ( aqueous layer) was extracted with n-butanol to obtain the sea fungus extract EH.

At the same time, the present invention uses the phenol-sulfuric acid method to measure the polysaccharide content of EH, and the result shows that EH does not contain polysaccharide components. Therefore, the extraction method of soaking sea fungus in alcohol first will not dissolve the polysaccharide components, so the crude sea fungus alcohol extract has no polysaccharide components.

experimental method

(1) Evaluate the whitening efficacy of EH

(a) In vitro cell testing

B16-F10 cells were cultured in the holes of a ²⁴ -well culture plate at a density of 104 cells per well, and placed in a cell culture incubator. After 24 hours, the original cell culture medium was removed, and the extract containing sea fungus was added. The cell culture medium was put into the incubator for 48 hours. Using the action of trypsin, the cells were collected, washed with phosphate-buffered saline (PBS), and then added with 100 μl of 1N NaOH aqueous solution to react at 90° C. for one hour. Use an enzyme immunoassay analyzer to detect the absorbance at a wavelength of 405nm, and then analyze the data. Taking the data of the control group as 100%, compare the intensity of light absorption between the experimental groups to show the whitening effect of sea fungus extract EH, and use Arbutin and N-phenylthiourea (N- phenylthiourea, PTU) as a positive control group. α-Melanocyte-stimulating hormone (α-Melanocyte-stimulating hormone, α-MSH) is used to promote the production of melanin in cells.

(b) Analysis of whitening activity in living zebrafish

In the experiment, zebrafish (AB strain Danio rerio) aged over four months were used as the experimental species. They were raised in acrylic water tanks with filters and circulation systems, and the water temperature was controlled at 28.5°C. The light and dark periods were controlled at 14 and 10 hours, respectively.

Embryos taken 9 hours after fertilization of zebrafish were injected into a 96-well plate, with 3 embryos and 100 μL of Hank's buffer (0.04mM NaHCO ₃ , 1.3mM CaCl ₂ , 2mM MgSO ₄ ) in each well. The control group was added with 100 μL of 1% dimethylsulfoxide (DMSO); the positive control group and the experimental group were respectively added with 100 μL of 5.4 mg/mL arbutin (Arbutin, Sigma-Aldrich Co., MO, USA; Cat.No.# A4256), 30.4μg/mL N-phenylthiourea (N-phenylthiourea, PTU; Sigma-Aldrich Co., MO, USA; Cat.No.#P7629) and sea fungus extract with concentrations of 50, 100 and 200μg/mL EH (both dissolved in 1% DMSO) was placed in a temperature-controlled light incubator (light/dark cycle: 14/10 hours, 28.5°C) and incubated for 48 hours. 48 hours after the drug treatment (ie 57 hours after fertilization), the juvenile fish were anesthetized with the anesthetic tricaine (Tricaine, 168ppm, MS-222), placed in the groove of the concave slide, and filled with 2% methylcellulose (Methylcellulose). Cellulose, Sigma-Aldrich Co., MO, USA; Cat.No.#M0512) fixed the fish body, observed with a solid microscope (Z16 APO, Leica, Heerbrugg, Switzerland), with image acquisition system and software (idea SPOT & SPOT software VERSION 4.6, Diagnostic instruments Inc., USA), to obtain fish body images. The fish images were processed using image processing software (Image J; National Institute of Health, MD, USA), and the abdomen area of the fish was circled, respectively, and the melanin value of zebrafish in each group was quantitatively analyzed. Taking the melanin value of the control group as 100%, compare the melanin value of the experimental group to evaluate the whitening effect of sea fungus extract EH.

(2) Assess the anti-inflammatory activity of EH

In Vitro Cell Testing

5 x 10 ⁵ Raw264.7 cells were implanted in a 6 cm culture dish. After being attached, they were given lipopolysaccharide (LPS) (0.01 μg/mL; Sigma-Aldrich Co., MO, USA; Cat.No.# L2654). After 10 minutes, different concentrations of EH were added, and 10 μM Dexamethasone (Dex) (Sigma-Aldrich Co., MO, USA; Cat.No.#D4902) was used as a positive control group, and co-cultured for 16 hours. The anti-inflammatory activity of EH was evaluated by Western blot, and the pro-inflammatory related proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were analyzed ) relative performance. The group that added lipopolysaccharide (LPS) alone was taken as 100%, and finally β-actin was used as the internal control group.

(3) Evaluate the curative effect of EH on atopic dermatitis

The experimental animals used in the present invention are BALB/c strain mice. The mouse was anesthetized with 2.5% isoflurane (isoflurane), and the hair on the back of the mouse was shaved using a razor and hair removal cream, and a silicone ring with a diameter of 1 cm was used to mark (the area size was 78.5 mm ² ). Once a day, drop 26 μL of 2% 2,4-dinitrochlorobenzene (2,4-dinitirochlorobenzene, DNCB) (Sigma-Aldrich Co., MO, USA; Cat.No.#138630) within this range once a day, A total of 8 times to induce skin lesions.

After the 7th day of DNCB induction, 100 μL of vehicle, sea fungus extract EH or crisaborole, a drug for treating atopic dermatitis, was applied every day. Experimental animals were grouped as follows (3 animals in each group): (1) Control group: no induction; (2) AD group: only given 2% DNCB; (3) ADV group: given 2% DNCB and vehicle (Vehicle); ( 4) EHL group: after DNCB induction, given 50 μg EH/100 μL 1% methylcellulose; (5) EHH group: after DNCB induction, administered 200 μg EH/100 μL 1% methylcellulose; and (6) Cri group: DNCB induced Afterwards, 25 μg crisborole/100 μL 1% acetone alcohol (acetone-EtOH) was administered. 5 minutes after applying DNCB, wait for the DNCB to dry before applying the medicine. Before inducing or applying the medicine, take pictures with a digital camera to record the changes of skin symptoms.

Animals were sacrificed on day 18, blood was collected and the back skin, spleen and lymph were collected knot tissue. Observe the size of the spleen and subiliac lymph nodes, and weigh them for comparison. Four skin surface symptoms were used to assess the severity of clinical dermatitis: erythema/hemorrhage, edema, excoriation/erosion, and dryness. According to the severity of each symptom, 0-3 points are given individually: 0 is asymptomatic; 1 is mild; 2 is moderate; 3 is severe. The total score of dermatitis is the sum of the above 4 scores, and the highest score is 12. Higher scores indicate more severe dermatitis.

Before sacrifice, the blood of the mice was collected into blood collection tubes (BD vacutainer-SST, NJ, USA) by means of cardiac blood collection. The blood was then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was stored in a -80°C refrigerator until further use. IgE concentrations in serum were measured according to the manufacturer's instructions (IgE-ELISA kit, Thermo Fisher Scientific Inc., Vienna, Austria).

If inflammation or immune-related diseases occur, the lymph nodes and spleen will swell, so when the mice are sacrificed, these two organs are collected for photography and weighed.

Statistical Analysis

All experimental data are presented as mean ± standard error of the mean (mean ± SEM). For data comparison among multiple groups, one-way analysis of variance (ANOVA) was used for statistical analysis of data, and the difference between multiple groups was compared according to Duncan's Method. When the P value is less than 0.05, it means that there is a significant difference between the groups.

result

(1) Whitening effect of EH

B16F10 cells were treated with 100mM α-MSH to induce melanin production, and given 10μg/ml EH solution or 100μM PTU to culture for 48 hours, then collected the cells, and treated them with 1N NaOH Dissolve the melanin, and finally read the absorbance value at a wavelength of 405nm. The more melanin, the higher the absorbance value. The results are shown in Figure 2, the PTU of the control group was 100±5.33% and the positive control group was 37.41±3.94%, and 10 μg/mL of EH could significantly inhibit cell melanin production (72.64±2.79).

The whitening activity of EH was evaluated by environmental drug administration. After 48 hours of drug administration to zebrafish embryos, the fish body pigments of the larvae were photographed, and the pigment spots were quantitatively analyzed. The results are shown in Figure 3, the control group was 100±6.06%, the arbutin and PTU in the positive control group significantly inhibited the fish pigment (9.86±1.40%, 3.08±0.62%), and the EH of 50, 100 and 200 μg/mL Can significantly inhibit fish body pigment (78.61±7.68%, 70.12±8.62%, 63.64±8.50%).

(2) Anti-inflammatory effect of EH

To detect whether EH has the effect of inhibiting inflammatory response, Raw264.7 cells were added LPS to induce inflammatory response, and added EH at a concentration of 20, 50 and 100 μg/mL to co-culture for 16 hours, and then the inflammatory proteins iNOS and COX were detected by Western blot method -2 performance amount. The results are shown in Figure 4. The expression level of iNOS was 1.04±0.15% in the control group without LPS-induced inflammatory response, while the expression level of iNOS in the LPS group was 100.00±10.74%. Using 10 μM Dex as the positive control group, the expression of iNOS was significantly decreased (14.78±0.92%), while 100 μg/mL EH could significantly inhibit the expression of iNOS (65.95±1.95%).

Figure 5 shows that the inflammatory reaction was not induced by LPS in the control group, and the expression level of COX-2 was 4.65±3.12%, while the expression level of COX-2 in the LPS group was 100.00±0.99%. With the concentration of 10μM Dicortisol (Dex) as the positive control group, it can significantly inhibit the expression of COX-2 (2.90±0.71%), while 20~100μg/mL EH can significantly inhibit the expression of COX-2, the expression of 68.55± 0.46%, 35.43±9.55% and 72.77±7.81%.

(3) Curative effect of EH on atopic dermatitis

DNCB was used to induce atopic dermatitis (AD) symptoms in mice, including erythema, edema, exfoliation, dryness and lichenification. After the mice showed obvious symptoms such as erythema, edema, desquamation and dryness, EH or crisaborole (Cri) was applied daily to test whether EH has the effect of treating AD. Observe and take pictures to record the skin appearance of the mice, as shown in Figure 6A, and quantify the therapeutic effect of EH with clinical dermatitis scores, as shown in Figure 6B.

The control group had a dry skin surface due to the lack of hair covering, so the score was 0.75 ± 0.25. The skin surface of AD group and ADV group formed a thick layer of scab due to keratinocyte hyperplasia. As the number of days increases, after part of the scab peels off, it can be observed that the skin around the induction is dry, and the skin has wrinkles; also because of the inflammation symptoms, the skin has erythema and edema. The keratinocytes are still proliferating, so there is still a layer of scab on the skin surface. The clinical dermatitis scores of these two groups were significantly increased compared with the control group, which were 9.17±0.40 and 9.50±0.56, respectively. With the passage of days, after the scab peeled off in the EH and crisborole group, although dryness, erythema and swelling could still be observed, the degree of keratinocyte hyperplasia seemed to be milder than that in the AD group. Therefore, before animal sacrifice, EHL The dermatitis scores of the EHH and EHH groups also decreased significantly (4.50±0.50 and 3.67±0.67). The dermatitis score of the Cri group was 4.80±0.37, which was statistically significant compared with the AD group. The above results show that EH has the effect of relieving AD symptoms.

IgE is one of the indicators for clinical diagnosis of atopic dermatitis. Before the mice were sacrificed, the serum was collected to detect the IgE content, and the results are shown in Figure 7. The IgE content in serum of the control group was 1.71±0.14μg/mL. The IgE concentration of AD group and ADV group increased significantly (68.89±4.12 and 68.95±2.60μg/mL). The IgE content of EHL and EHH group decreased significantly to 52.32±3.41μg/mL and 46.31±3.81μg/mL. But the serum IgE content of Cri group decreased to 65.61±1.74μg/mL. Applying EH can reduce the IgE in the serum that rises due to atopic dermatitis.

If inflammation or immune-related diseases occur, the lymph nodes and spleen will swell, so when the mice are sacrificed, these two organs are collected for photography and weighed. The results of the spleen weight are shown in Fig. 8, the spleen weight of the control group was 91.08±3.61 mg. The spleens of the AD group and the ADV group were significantly enlarged, which was 2.5 times that of the control group (234.46±6.56 and 221.14±13.32mg). Spleen weights also decreased significantly in EHL and EHH groups (161.23±9.97 and 171.07±5.58 mg). In Cri group, the spleen weight decreased significantly to 167.46±10.61mg.

The results of the total weight of the lymph nodes are shown in Figure 9, the total weight of the two lymph nodes in the control group was 4.46±0.29 mg. The lymph nodes in the AD group and the ADV group were significantly enlarged, which was three times that of the control group (12.07±0.33 and 11.61±0.54mg). Lymph node weights were also significantly decreased in EHL and EHH groups (9.06±0.54 and 9.13±0.75 mg). The weight of lymph nodes in Cri group decreased significantly to 9.49±0.81mg. The results showed that the swelling of lymph nodes and spleen due to inflammation or immune reaction caused by atopic dermatitis, application of EH can inhibit the aforementioned phenomenon.

The invention suitably described can be practiced subject to provisos or limitations not specifically disclosed herein. The terms that have been used to describe are not limiting. There is no difference in expression or description using these terms and any equivalents otherwise, but it should be recognized that rights within the invention may be modified. Therefore, while the present invention has described embodiments and others, the disclosure herein may be modified and changed by those skilled in the art, and such modifications and changes are considered to be within the scope of the present invention.

Claims (10)

A use of sea fungus extract for preparing a skin whitening composition, wherein the preparation method of the sea fungus extract comprises the following steps: (a) extracting a sea fungus with an alcohol solvent to obtain a sea fungus alcohol extract, wherein the alcoholic solvent is methanol or ethanol; (b) partitioning and extracting the sea fungus alcoholic extract with an organic solution to form an organic solvent layer and an aqueous layer, wherein the organic solution is a Mixture of water and organic solvent, the organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, chloroform or tetrachloride and (c) distributive extraction of the aqueous layer obtained in step (b) with a n-butanol to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the sea fungus extraction things. The use as described in Claim 1, wherein the sea fungus extract inhibits melanin production or reduces melanin. The use as claimed in item 1, wherein the composition comprises a cosmetic composition, a food or a medicine. A composition for the preparation of anti-inflammatory drugs, wherein the composition contains a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) treating a sea fungus with an alcoholic solvent extraction to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol; (b) extracting the sea fungus alcohol with an organic solution The extract is distributed and extracted to form an organic solvent layer and an aqueous layer, wherein the organic solution is a mixture of water and an organic solvent, and the organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane , heptane, octane, nonane, methyl ether, ether, dichloromethane, chloroform or carbon tetrachloride; and (c) carry out partition extraction to the obtained aqueous layer of step (b) with n-butanol, so that to form a n-butanol layer and an aqueous layer, and then collect the aqueous layer to obtain the sea fungus extract. The use as claimed in claim 4, wherein the anti-inflammation includes inhibiting an inflammatory factor. The use as described in claim 5, wherein the inflammatory factor comprises cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The use as described in claim 4, wherein the drug treats atopic dermatitis through anti-inflammation. The use as claimed in item 7, wherein the allergic dermatitis comprises atopic dermatitis. The use as described in any one of claim 1 or 4, wherein the alcoholic solvent is ethanol. The use as described in any one of claim 1 or 4, wherein the organic solvent is ethyl acetate.

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