KR20210127616A - Method for manufacturing a red algae microfibrillated cellulose and use thereof - Google Patents

Abstract

The present invention relates to a method for manufacturing red algae microfibrillated cellulose (MFC) and a use thereof. The red algae microfibrillated cellulose (MFC) manufactured by the manufacturing method of the present invention has effects of suppressing skin damage and inflammation by ultraviolet rays by having excellent effects of inhibiting activity of cyclooxygenase-2 (Cox-2), suppressing MAPKs phosphorylation, and suppressing expression of TNF-α. The method comprises a first step of removing agar from red algae, and a second step of blending the red algae.

Description

Method for manufacturing a red algae microfibrillated cellulose and use thereof

The present invention relates to a method for producing red algae microfibrillated cellulose (MFC) and its use, and more particularly, to a method for producing red algae microfibrillated cellulose (MFC) and prevention and treatment of skin cell damage or inflammation thereof. Or it relates to a composition for improvement.

Skin aging can be divided into intrinsic aging and extrinsic aging according to factors. It is known that intrinsic aging is caused by changes in the physiological functions of the skin epidermis and dermis according to age, and extrinsic aging is caused by changes in the physiological functions of the skin caused by environments such as air pollution, UV exposure, and stress. Among these mechanisms of aging, oxidative stress induced by UV increases free radicals in the body, increases the activity of MMP-1 that decomposes collagen, and reduces hyaluronic acid. Degradation causes damage to the epidermis and dermis of the skin due to increased activity of hyaluronidase.

In our daily life, we are always exposed to sunlight, and in particular, cell damage and skin cancer caused by UV-B exposure are unavoidable factors in our daily life. As a result, studies on the mechanism of inhibition of skin cell damage caused by UV exposure have been actively conducted, and studies on cosmetics, health functional foods, and pharmaceuticals showing biochemical efficacy and effects are being actively conducted. With the development of the research level and the increase in technology, materials with practical efficacy and effects are the main research subjects.

Red algae are composed of agar and cellulose, and form tetraspores, monospores, and bispores by asexual reproduction. It is divided into an annular shape, a cross shape, or a triangular pyramid shape. Sexual reproduction is accomplished by single-phase gametophytes, which form sperm and oviparous phases, respectively, and the carpospores produced as a result of their fertilization. Because it represents the morphology, it is regarded as one of the main distinguishing traits of the taxa. Therefore, the life history of red algae is based on the so-called Polysiphonia-type generational alternation, in which three generations of carposporophytes are alternately repeated by the union of spores, gametes, and gametes. It has also been found that this generational alternation has a slightly modified peculiar shape depending on the taxa, and is used as another distinguishing trait for the taxa. Red algae are largely divided into the protozoan subclass (Bangiophycidae; Protoflorideophycidae), to which seaweed and purple hair, and the like, and Florideophycidae, to which the algae, and the red algae belong. The former includes Goniotrichales and Bangiales , the latter include Nemaliales, Gelidiales, Cryptonemiales, Gigartinales, Rhodymeniales, and Ceramiales.

Meanwhile, along with the shortage of land resources, interest in marine resources, a new area, increased, and research and development of functional materials using seaweed began in earnest. Compared to wood, which is a terrestrial plant, seaweed has a softer tissue, finer fibers, and can be cultured in a much larger space than on land without special management, so it has a lot of biomass. The growth rate is also faster than that of trees, does not require a separate soil for cultivation, and the pulping process is simple due to the easy extraction of fibers, and the production facilities are inexpensive. In addition, studies are underway to use this seaweed pulp to improve skin hydration and adsorb harmful substances. However, there is no disclosure of the method for producing red algae fibrillar cellulose of the present invention and its use for inhibiting skin damage or inflammation.

Sousa, A. M. M. et al., Agar extraction from integrated multitrophic aquacultured Gracilaria vermiculophylla: Evaluation of a microwave-assisted process using response surface methodology, Bioresource Technology, 2010, 101(9), p.3258-3267.

It is an object of the present invention to provide a method for producing red algae microfibrillated cellulose (MFC).

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, another object of the present invention is to provide a cosmetic composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, another object of the present invention is to provide a health food and health functional food composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the red algae microfibrillated cellulose prepared by the above method.

Another object of the present invention is to provide a method for confirming fibrillation of red algae cellulose.

In order to achieve the above object, the present invention is a step of pre-treating red algae with a microwave sample decomposing device (microwave) to remove agar and physically blending the pre-treated red algae (red algae). It provides a method for producing red algae microfibrillated cellulose comprising the step.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, the present invention provides a cosmetic composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, the present invention provides a health food and health functional food composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising red algae microfibrillated cellulose prepared by the above method.

In addition, the present invention provides a method for confirming fibrillation of red algae cellulose, comprising the step of determining whether the fibers of the cellulose are fibrillated.

Red algae fibrillated cellulose (MFC) prepared according to the production method of the present invention is cyclooxygenase-2 (cyclooxygenase-2, Cox-2) activity inhibitory effect, MAPKs phosphorylation inhibitory effect, TNF-α expression inhibitory effect It is effective in inhibiting skin damage and inflammation caused by UV rays.

1 is a diagram showing the contents of glucan and galactan after microwave pre-treatment of red algae agar agar.
FIG. 2 is a diagram showing structural changes according to the microwave pretreatment temperature of red algae agaric agar.
Figure 3 is a diagram showing the water retention (water retention value) according to the physical treatment (blending) time of red algae cellulose.
Figure 4 is a diagram showing the results of the precipitation test according to the physical treatment (blending) time of red algae cellulose.
5 is a diagram showing the binding force of cellulose and Carbohydrate binding module protein according to the physical treatment (blending) time of red algae cellulose.
6 is a view of observing the fibers of red algae cellulose that vary depending on the presence or absence of physical treatment (blending).
7 is a diagram showing the COX-2 inhibitory activity of red algae microfibrillated cellulose.
8 is a diagram showing the MAPK phosphorylation inhibitory activity of red algae microfibrillated cellulose.
9 is a diagram showing the TNF-α expression inhibitory activity of red algae microfibrillated cellulose.

Hereinafter, the present invention will be described in more detail.

The present invention includes a first step of removing agar by pre-treating red algae by microwave decomposition, and a second step of blending red algae from which the agar is removed. It provides a method for producing red algae microfibrous cellulose (microfibrillated cellulose).

In addition, in the first step, the red algae microfibrillated cellulose manufacturing method is a solvent such that the content of red algae is 5 to 15% (w/v), preferably 8 to 12% (w/v). Step 1-1 of mixing with; and pre-treating the mixture at 110 to 190 ° C., preferably at 160 to 190 ° C., for 5 to 15 minutes, preferably for 8 to 12 minutes by microwave decomposition to obtain agar from red algae. It may include; removing and 1-2 steps of extracting cellulose (cellulose).

In addition, in step 1-2, when the agar is not sufficiently removed when the pretreatment is performed at a temperature of 110 ° C. or lower, and when the pretreatment is performed at a temperature of 190 ° C. or higher, decomposition of glucan and by-products are generated, so the mixture is determined in the present invention It is preferable to pre-treat under true conditions, but is not limited thereto.

In addition, in the second step, the cellulose is blended at 32000 to 42000 RPM, preferably 35000 to 39000 RPM for 40 minutes or more, preferably 80 minutes or more, more preferably 150 to 170 minutes. It may be to fibrillation.

The term “red algae” used in the present invention refers to seaweed that has red or purple color because it contains red algae and blue algae in addition to chlorophyll, and includes all seaweeds classified as red algae taxonomically. For example, seaweed (Porphyra tenera), agaric agar (Gelidium amansii), grasshopper (Gloiopeltis tenax), sagebrush (Gracilaria verrucosa), chaff (Chondrus ocellatus Holmes), gambling (Pachymeniopsis elliptica), silkworm (Grateloupia filicina), (Ceramium kondoi) and may include any one or more species from the group consisting of Hypnea charoides, preferably a seaweed of the family Gelidiaceae, more preferably a seaweed of the genus Gelidium, most preferably may be agar agar (Gelidium amansii).

In addition, the red algae are composed of agar and cellulose, and the agar is D-galactose and 3,6-anhydro-alpha-L- It may be composed of lactose (3,6-anhydro-α-L-lactose).

In addition, the manufacturing method may further include the step of determining whether agar is removed by measuring the galactan content.

As used herein, the term “galactan” refers to a polysaccharide having a D-galactose residue as a constituent sugar. In the present invention, it was used to confirm the D-galactose content of red algae.

The term “microwave decomposition method” used in the present invention is one of the pretreatment methods for removing organic substances and interfering substances in the sample by adding distilled water to the sample and heating it. The sample is heated using the principle that the temperature rises by causing (dipole moment) and ionic conductance. In the present invention, red algae nanocellulose is prepared by removing agar from red algae and extracting cellulose using a microwave decomposition method.

In addition, the blending (blending) means physical treatment, from the form of a fine thread or ribbon to a fiber having a long and intact fibrous structure of cellulose (see FIG. 6A) through homogenization of a sample to a fiber such as a broad sheet It may mean to deform into fibers of various sizes (see FIG. 6B ) to make the fibers fibrillated.

The present invention provides a pharmaceutical composition for the prevention or treatment of skin damage or inflammatory diseases comprising red algae microfibrillated cellulose prepared by the above method.

In addition, the skin damage may be caused by ultraviolet rays, but is not limited thereto.

The term “damage” as used in the present invention includes apoptosis of human skin cells by ultraviolet rays, DNA damage of skin cells, an increase in reactive oxygen species (ROS), an increase in lipid peroxidation, and the like, and the symptoms include erythema, It may include sunburn, pigmentation, photoaging, skin cancer, and the like. In addition, "improvement" of damage refers to any action of alleviating the damage state of the skin cells by the ultraviolet rays or reducing the degree of symptoms.

In addition, the inflammatory disease is arthritis, gout, hepatitis, asthma, obesity, keratitis, gastritis, enteritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, arteriosclerosis, sepsis, It may be selected from the group consisting of burns, dermatitis, periodontitis and gingivitis, but is not limited thereto.

In addition, the red algae fibrillar cellulose may inhibit cyclooxygenase-2 (Cox-2) activity.

As used herein, the term “cyclooxygenase-2 (Cox-2)” is also called prostaglandin-endoperoxide synthase 2, and prostaglandin, a substance that causes inflammation and pain. (prostaglandin, PGs) means an enzyme that promotes the formation. The prostaglandins are known to be deeply involved in cancer development, such as promoting inflammatory responses such as pain and fever, immune responses, and angiogenesis. have.

In addition, the red algae fibrillar cellulose may inhibit the phosphorylation level of JNK and p38 mitogen-activated protein kinase (MAPK).

In addition, the red algae fibrillar cellulose may inhibit the expression of TNF-α.

In addition, the pharmaceutical composition for preventing or treating skin damage is an external preparation for skin having an effect of protecting the skin from ultraviolet rays, such as cream, gel, patch, spray, ointment, warning agent, lotion, liniment agent, pasta agent or cataplasma agent. It can be prepared and used as a pharmaceutical composition in the form of an external preparation for skin, but is not limited thereto.

The "prevention" means any action that reduces the frequency or degree of occurrence of a pathological phenomenon. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the symptoms of skin disease or inflammatory disease in the individual are reduced compared to the case where the composition is not used.

The "treatment" refers to any action that clinically intervenes to alter the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, decrease all direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the disease progression rate. alleviating or temporarily ameliorating the condition, or improving the prognosis.

The composition according to the present invention may contain a pharmaceutically effective amount of red algae fibrillar cellulose alone or may contain one or more pharmaceutically acceptable carriers, excipients or diluents. In the above, the pharmaceutically effective amount refers to an amount sufficient to prevent, improve and treat symptoms of immune diseases. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.

In addition, the composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, may be at least one selected from the group consisting of propylene glycol and liquid paraffin, but It is not limited, and all conventional carriers, excipients or diluents may be used. The ingredients may be added independently or in combination to the active ingredient, red algae fibrillar cellulose.

In addition, solid preparations for oral administration may include tablet pills, powders, granules, capsules, etc., and these solid preparations include one or more compounds and at least one excipient, for example, starch, calcium carbonate, sucrose. Alternatively, it may be prepared by mixing lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

In addition, the pharmaceutical composition of the present invention is a group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have any one formulation selected from As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

In addition, the effective dose of the red algae fibrillar cellulose of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health status and disease level, and is generally about 0.001-100 mg/kg/day. and preferably 0.01-35 mg/kg/day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg/day, preferably 0.7-2500 mg/day, and once a day at regular time intervals according to the judgment of a doctor or pharmacist It may be administered in several divided doses.

The present invention provides a cosmetic composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, since the cosmetic composition for preventing or improving skin damage of the present invention contains the red algae fibrillar cellulose described above, the content overlapping with the red algae fibrillar cellulose of the present invention is excessive in the present specification by the description of the overlapping content. In order to avoid complexity, the description thereof is omitted.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, thickener, A chelating agent, a colorant, a preservative, a fragrance, etc. may be appropriately mixed, but the present invention is not limited thereto.

Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, softening lotion, nutritional lotion, various creams, essences, packs, foundations, and cleansing, face wash, soap, treatment, and serum. etc.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing essence, pack, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder, loose powder, and eye shadow.

In the cosmetic composition containing red algae fibrillar cellulose of the present invention, the active substance of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, to the cosmetic composition usually contained therein.

When the red algae fibrillar cellulose of the present invention is used as an external preparation for skin, additionally, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, Surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in skin care. In addition, the ingredients may be introduced in an amount generally used in the field of dermatology.

The present invention provides a health food and health functional food composition for preventing or improving skin damage comprising red algae microfibrillated cellulose prepared by the above method.

In addition, since the health food and health functional food composition for preventing or improving skin damage of the present invention contains the above-mentioned red algae fibrillar cellulose, the content overlapping with the red algae fibrillar cellulose of the present invention is in the description of the overlapping content. In order to avoid excessive complexity of the present specification, the description thereof is omitted.

The food composition of the present invention may be formulated in various forms, such as tablets, pills, granules, capsules, liquid preparations, and beverages, and added to food. There are no special restrictions on the type of food. Examples of foods to which the red algae microfibrous cellulose of the present invention can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream. There are products, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and processed dairy products, and includes both health food and health functional food in the ordinary sense.

The health food and health functional food composition containing red algae microfibrous cellulose according to the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of red algae fibrillar cellulose may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the composition in health food and health functional food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for health maintenance or health control, the above amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health food and health functional food composition of the present invention is not particularly limited in other ingredients except for containing the red algae fibrillar cellulose of the present invention as an essential ingredient in the indicated ratio. It can contain as an ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 of the nutraceutical composition of the present invention.

In addition to the above, the health food and health functional food composition containing red algae fibrillar cellulose of the present invention are various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health food and health functional food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

These components may be used independently or in combination. Although the proportion of these additives is not so important, it is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the red algae fibrillar cellulose of the present invention.

The present invention provides a method for confirming fibrillation of red algae cellulose, comprising the step of determining whether fibrillation of the fibers of the cellulose is fibrillated.

The step of determining whether or not the fibrillation is to measure the water retention value of cellulose, to check the degree of sedimentation (sedimentation) to determine whether an assembly is formed, or to bind cellulose and Ct CBD3 protein It may be to check

The water retention value is related to the binding force of the fibers and the cellulose surface area, and as more fibers are defibrillated, water retention increases. Therefore, it can be determined whether the fibrillation of the cellulose according to the water retention value (water retention value).

The degree of sedimentation (sedimentation) can be confirmed through a sedimentation test, and the visible deposited layer exists as individual fibers or is entangled or incompletely separated fibers that interact with other fibers to form an assembly and visually fibers that can be observed. Therefore, it is possible to determine whether the cellulose is microfibrillated according to the settling height of the fibers.

The Ct CBD3 protein is known to be attached to the crystalline form of cellulose including CBM1, CBM2a, CBM3, CBM5, and CBM10, which are surface-binding-carbohydrate-binding modules. As the fibrillation progresses, the surface area of the cellulose increases and thus the Ct CBD3 protein binding rate increases. Therefore, it is possible to determine whether cellulose is microfibrillated according to the degree of binding of Ct CBD3 protein.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to embody the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Removal of agar from red algae and extraction of cellulose

First, a sample was prepared by mixing 2 g of agar agar and 18 ml of distilled water so that the content of Gelidium amansii was 10% (w/v). Thereafter, the samples were pretreated at 120, 150 and 180° C. for 10 minutes using Microwave (MARS6 System, CEM Corporation, USA) Easy Prep ^{TM (see Table 1).}

Pretreatment temperature (℃) 120 150 180 Pretreatment time (min) 10 Content (% (w/v)) 10

<Example 2> Confirmation of whether agar (agar) is removed

In order to confirm whether the agar was removed, the glucan and galactan contents of the sample of Example 1 were measured. The glucan and galactan contents were measured as follows.

The method of Laboratory Analytical Procedure provided by the US Department of Energy's Renewable Energy Laboratory (Department of Energy, Nation Renewable Energy Laboratory) was referred to. The dried assay sample (dry content: 25 mg) was reacted with 250 μl of 72% (w/w) sulfuric acid at 30° C. for 1 hour, then transferred to a serum bottle, diluted with 7 mL of distilled water to 4%, and reacted at 121° C. for an additional hour. . After the reaction, the sample is centrifuged to separate the solid, and the supernatant is neutralized to pH 6-8 with calcium carbonate. In the neutralized sample, glucose and galactose contents were confirmed through HPLC analysis. The glucan content means glucose, that is, the cellulose content, and the galactan content means galactose, that is, the agar content. The results are shown in Figure 1 and Table 2.

1 is a graph showing the contents of glucan and galactan after microwave pretreatment of red algae agaric, and FIG. 2 is a structure according to microwave pretreatment temperature of red algae agaric. This is a graph showing the change.

Glucan % (w/v) Galactan % (w/v) control 13.9±0.7 45.2±4.6 120 ℃ 20.5±1.1 42.0±1.8 150 ℃ 27.9±2.4 23.9±1.9 180 ℃ 40.2±3.1 1.8±0.1

As shown in FIGS. 1 and 2, the control sample, which was not pretreated, had the highest galactan content because agar was not removed, and the glucose content increased according to the pretreatment temperature and galactose ), it was confirmed that the content was decreased. In addition, as shown in FIG. 2 , in the control group (untreated) ^{, the peaks of 1315 cm -1} and 1371 cm ^-1 did not exist, and the higher the pretreatment temperature, the higher the cellulose content at the peak of ^{1110 cm -1 indicating the cellulose content.} was shown. In particular, it was confirmed that the sample pretreated at 180 ° C. had a galactan content of 1.8%, and most of the agar was removed. Therefore, it can be seen that the sample is cellulose extracted from agar agar ( Gelidium amansii ). From the examples below, only samples pretreated at 180 °C were used.

<Example 3> Preparation of microfibrillated cellulose

The sample pretreated at 180° C. of Example 1, that is, cellulose extracted from agar agar (Gelidium amansii ) was blended for 40, 80, 120 and 160 minutes, respectively, at 37000 RPM using a Vitamix TNC5200 blender (vitamix, japan). (blending) to prepare microfibrillated cellulose.

<Example 4> Check whether cellulose is microfibrillated

4-1. Determination of water retention value

In order to check whether the cellulose is microfibrillated, the water retention value of each of the samples of Example 3, that is, microfibrillated cellulose, was measured.

The water retention value (WRV) is the ratio of the percentage of water contained in the sample after centrifugation with force and time to the dry weight of the sample. Put a 70㎛ nylon cap on the 50 mL tube and cover the membrane filter with a 0.2㎛ pore size. The sample was filled on top and placed in a centrifuge. After centrifugation at 4 °C with a relative centrifugal force (RCF) of 900 g for 30 min, the samples were weighed. After that, it was dried in a dry oven at 80 °C for 24 hours and dried at 103 °C until all moisture was removed. Water retention was calculated using Equation 1 below. W _wet is the weight of the sample after centrifugation, and W _dry is the dry weight of the sample.

Figure 3 is a graph showing the water retention (water retention value) according to the physical treatment (blending) time of red algae cellulose. As a negative control group (0 min), the sample pretreated at 180° C. of Example 1 without blending treatment was used.

As shown in FIG. 3 , the experimental group had a significantly higher water retention value than the control group that did not perform blending, which is a physical pretreatment step. In particular, it can be seen that the sample blended for 80 minutes or more maintains high water retention. Fibrillation was confirmed through the physical pretreatment step of cellulose extracted from agar agar, and this result means that microfibrillated cellulose was formed from agar agar by blending.

4-2. sedimentation test

In order to confirm whether the cellulose is microfibrillated, a sedimentation test of each of the samples of Example 3, that is, microfibrillated cellulose, was performed.

Experiments were carried out at 0.0005 g/mL, 0.001 g/mL, 0.002 g/mL, and 0.004 g/mL. The degree of precipitation is a method that can confirm the dispersion stability of the MFC suspension. Red algae cellulose samples were prepared at a concentration of 0.05% 0.1% 0.2% 0.4% with a total volume of 10 mL, and then thoroughly mixed before the precipitation test. And it was left for 48 hours to allow the precipitate to settle. After the cellulose fibers were completely submerged, the height of the sediment was measured. The results are shown in FIG. 4 . The visible precipitated layer refers to fibers that can be visually observed as cellulose fibers are microfibrillated and the separated fibers interact with other fibers to form an assembly (see FIG. 6 ).

4 is a graph showing the results of the precipitation test according to the physical treatment (blending) time of red algae cellulose. Here, x-axis: Hs (height of sedimentation)/Ho (height before sedimentation), y-axis: means concentration.

As shown in FIG. 4 , it can be confirmed that the Hs/Ho value is significantly increased in the samples subjected to blending (40 minutes, 80 minutes, 120 minutes, and 160 minutes). Since there is a difference in the Hs/Ho values depending on the presence or absence of blending, it can be seen that the microfibrillation of the fibers is increased when the physical treatment is performed. In particular, it was found that the sample blended for 160 minutes among the samples of Example 3 had the highest Hs/Ho value. These results indicate that microfibrillated cellulose was formed from agar agar by blending.

In addition, the degree of suspension retention can also be confirmed through sedimentation test data. Suspension stability increases with higher Hs/Ho values. It can be seen that the suspension stability of the sample blended for 160 minutes is the highest, which means that it has superior stability than the positive control (Borregard MFC).

4-3. Ct CBD3 protein binding force measurement

Ct CBD3 protein binding force of each of the samples of Example 3, that is, microfibrillated cellulose, was measured to determine whether cellulose was microfibrillated.

Binding experiments were performed by adding 200 μg/mL of Ct CBD3 to the substrate for 1 mg of fibrillar cellulose. The substrate and protein were reacted with a total of 500 μL of 50 mM potassium phosphate buffer (pH 7.0) at 4 °C for 30 minutes. After reacting Ct CBD3 with the substrate, the supernatant was separated by centrifugation at 13,000 rpm and 4°C for 10 minutes. The amount of protein remaining in the supernatant without binding to the substrate through BCA was measured. The results are shown in FIG. 5 .

5 is a graph showing the binding force between cellulose and Ct CBD3 protein according to the physical treatment (blending) time of red algae cellulose.

As shown in FIG. 5 , there is a difference in the binding rate of Ct CBD3 protein depending on the presence or absence of blending, which is the physical pretreatment step, so it can be seen that the microfibrillation of the fibers is increased when the physical treatment is performed. In particular, the sample blended for 160 minutes had the highest Ct CBD3 protein binding capacity. Fibrillation was confirmed through the physical pretreatment step of cellulose extracted from agar agar, and this result means that microfibrillated cellulose was formed from agar agar by blending.

<Example 5> Cox-2 inhibitory activity of red algae fibrillar cellulose

The expression level of cyclooxygenase-2 (Cox-2) was measured to confirm the skin damage and inflammation inhibitory effect of the microfibrillated cellulose prepared in Example 3 above.

For in vitro Western blot analysis, HaCaT cells were seeded in a 6 cm dish at a ^{concentration of 3×10 5} cells/mL and cultured in an incubator at 37° C. and 5% CO _{2 conditions.} Thereafter, the concentration of the fibrillar cellulose of Example 3 was pre-treated with 50 and 100 μg/mL for 1 hour, followed by UVB (0.03 J/cm ² ) treatment and cultured for 4 hours. Then, the dish was placed on ice, washed twice with cold PBS, and then lysed with a cell lysis buffer (lysis buffer, Cell Signaling Technologies). Lysis buffer was recovered with a cell scraper, transferred to a 1.5 mL tube, and then vortexed on ice once every 10 minutes for a total of 3 times. After centrifugation in a centrifuge at 4 °C and 12,000 rpm for 15 minutes to obtain a supernatant, the protein was quantified using a DC Protein Assay Kit reader (Bio-Rad Inc.).

After separating the protein through electrophoresis by loading the protein mixture on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), polyvinylidene difluoride (PVDF) membrane (Millipore, Immobilon®-P transfer membrane, MA, USA) ) was transferred. After blocking with 5% skim milk at room temperature for 1 hour, each primary antibody was reacted at 4° C. overnight. After that, the membrane was washed three times once every 5 minutes by changing TBST (tris-buffered saline Tween-20), and a secondary antibody (horseradish peroxidase (HRP) labeled with HRP dissolved in skim milk) )-conjugated secondary antibody, Thermo Scientific) at room temperature for 1 hour. After washing the membrane, EzWestLumi plus was treated to measure protein expression and phosphorylation. Protein bands were quantified and visualized using a chemiluminescence detection kit (ATTO, Tokyo, Japan) and GeneGnome XRQ NPC (Syngene, Cambridge, UK), respectively. The results are shown in FIG. 7 .

7 shows the skin damage inhibitory effect of red algae microfibrillated cellulose.

As shown in FIG. 7 , it was confirmed that the expression level of Cox-2 was reduced in both cellulose and fibrillar cellulose extracted from agar agar compared to the control group (UVB+). In particular, it can be seen that the expression of Cox-2 was reduced in the fibrillar cellulose to a level similar to that of the control (UVB-). These results mean that the fibrillar cellulose of the present invention has the effect of inhibiting skin damage and inflammation.

<Example 6> UVB-induced phosphorylation inhibitory activity of MAPK family of red algae fibrillar cellulose

HaCaT (Human Adult Low Calcium High Temperature) skin cells were pretreated with the MFC of Example 3 at 0, 25, 50 and 100 μg/mL for 1 hour, respectively, stimulated with UVB irradiation, and harvested after 10 or 30 minutes. . Phosphorylation and expression levels of MAPKs (JNK1/2, p38 and ERK1/2) were detected by Western blot using specific antibodies. Western blot was performed in the same manner as in the experimental method described in Example 5 above. The results are shown in FIG. 8 .

As shown in FIG. 8 , the MFC of Example 3 was shown to inhibit UVB-induced phosphorylation of JNK1/2 and p38 in HaCaT cells. These results indicate that the fibrillar cellulose of the present invention has an effect of inhibiting skin damage and inflammation by inhibiting MAPK, which plays an important role in inflammation.

<Example 7> TNF-α inhibitory activity on UVB-induced phosphorylation of red algae fibrillar cellulose

HaCaT (Human Adult Low Calcium High Temperature) skin cells were pretreated with the MFC of Example 3 at 0, 50 and 100 μg/mL for 1 hour, respectively, stimulated with UVB irradiation, and harvested after 10 or 30 minutes. Total RNA was isolated from HaCaT cells using RNAiso Plus (TAKARA, China). The isolated total RNA was used for cDNA synthesis using ReverTra Ace® qPCR RT master mix (Toyobo, Japan). Then, RT-PCR was performed using SYBR® Green Realtime PCR Master Mix (Toyobo, Japan). The level of TNF-α mRNA was measured using CFX Real-Time PCR Detection Systems (Bio-Rad). Relative gene expression was corrected (normalized) with glyceraldehyde-3-phosphate dehydrogenase using the ^{2-ΔΔCT method to reduce inherent errors in the experiment.} The results are shown in FIG. 9 .

As shown in FIG. 9 , when the MFC of Example 3 was treated at 50 and 100 μg/mL concentrations after UVB irradiation, the expression of TNF-α mRNA compared to the TNF-α mRNA expression level of the positive control group (UVB irradiation) was approximately It has been shown to inhibit by 10 to 20%. These results mean that the fibrillar cellulose of the present invention suppresses the expression of TNF-α, which is closely related to inflammation, and has an effect of inhibiting skin damage and inflammation.

As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

Claims (15)

Red algae comprising a first step of removing agar by pre-treating red algae with a microwave decomposition method and a second step of blending red algae from which the agar (ager) is removed. Method for producing microfibrillated cellulose.
According to claim 1,
The red algae are agar agar ( Gelidium amansii ), grass agar ( Gloiopeltis tenax ), ragweed ( Gracilaria verrucosa ), chungdubal ( Chondrus ocellatus Holmes ), gambling ( Pachymeniopsis elliptica ), ginseng ( Grateloupia filicina kondoramium ), silkworm ( Grateloupia filicina ) ), a red algae microfibrillated cellulose (microfibrillated cellulose) manufacturing method comprising any one or more from the group consisting of ), Hypnea charoides and laver ( Porphyra tenera).
According to claim 1,
The first step is a method for producing red algae microfibrillated cellulose, characterized in that made at 110 to 190 ℃.
According to claim 1,
The second step is a method for producing red algae microfibrillated cellulose, characterized in that the fibers of the cellulose are fibrillated through the homogenization of the red algae sample.
According to claim 1,
The second step is a method for producing red algae microfibrillated cellulose, characterized in that the blending (blending) for at least 80 minutes.
A pharmaceutical composition for preventing or treating skin damage, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
7. The method of claim 6,
The red algae fibrillar cellulose is a pharmaceutical composition for preventing or treating skin damage, characterized in that it inhibits the activity of cyclooxygenase-2.
7. The method of claim 6,
The red algae fibrillar cellulose is a pharmaceutical composition for preventing or treating skin damage, characterized in that it inhibits the phosphorylation level of JNK and p38 MAPK (mitogen-activated protein kinase).
7. The method of claim 6,
The red algae fibrillar cellulose is a pharmaceutical composition for preventing or treating skin damage, characterized in that it inhibits the expression of TNF-α.
A cosmetic composition for preventing or improving skin damage, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
A health food composition for preventing or improving skin damage, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
A health functional food composition for preventing or improving skin damage, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
A pharmaceutical composition for preventing or treating inflammatory diseases, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
14. The method of claim 13,
The inflammatory diseases include arthritis, gout, hepatitis, asthma, obesity, keratitis, gastritis, enteritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, arteriosclerosis, sepsis, burns, A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it is selected from the group consisting of dermatitis, periodontitis and gingivitis.
Measuring the water retention value, sedimentation, or binding force with Ct CBD3 protein of red algae cellulose; and
Including; determining whether fibrillation of the fibers of the cellulose;
The water retention is calculated by Equation 1 below, a method for confirming fibrillation of red algae cellulose
[Equation 1]

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