KR102587264B1 - Method for manufacturing a red algae microfibrillated cellulose and use thereof - Google Patents

Abstract

The present invention relates to a method for producing red algae microfibrillated cellulose (MFC) and its use. Red algae microfibrillated cellulose (MFC) prepared according to the production method of the present invention is cyclooxygenase-2 ( It has an excellent effect of inhibiting cyclooxygenase-2, Cox-2) activity, inhibiting phosphorylation of MAPKs, and inhibiting TNF-α expression, thereby suppressing skin damage and inflammation caused by ultraviolet rays.

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 specifically, to a method for producing red algae microfibrillated cellulose (MFC) and the prevention and treatment of skin cell damage or inflammation. Or it relates to a composition for improvement.

Skin aging can be divided into intrinsic aging and extrinsic aging depending on the factors. Intrinsic aging is caused by changes in the physiological functions of the skin's epidermis and dermis with age, while extrinsic aging is known to be caused by changes in the physiological functions of the skin that occur from environments such as air pollution, exposure to ultraviolet rays, and stress. Among these aging mechanisms, oxidative stress induced by UV increases free radicals in the body, increases the activity of MMP-1, which decomposes collagen, and hyaluronic acid. Decomposition causes damage to the skin epidermis and dermis due to increased activity of hyaluronidase.

In our daily lives, 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 lives. Accordingly, research on the mechanism for suppressing skin cell damage due to UV exposure has been actively conducted, and research is being actively conducted on cosmetics, health functional foods, and pharmaceuticals that demonstrate biochemical efficacy and effectiveness. With the development of research level and increase in technology, materials with practical efficacy and effectiveness are the main research targets.

Red algae are composed of agar and cellulose, and reproduce asexually to form tetraspores, monospores, and heterospores. These are formed through meiosis in double-phase sporophytes. In particular, tetraspores depend on the type. It is divided into a ring shape, a cross shape, or a triangular pyramid shape. Sexual reproduction is accomplished by single-phase gametophytes forming sperm and fruiting stage, respectively, and carpospores produced as a result of their fertilization. The process of forming carpospores from fertilization is a unique process that varies depending on the taxon of red algae. Because it represents the shape, it is considered one of the main identifying characteristics of a taxon. Therefore, the life cycle of red algae is based on the so-called polysiphonia-type alternation of generations, in which three generations of carposporophytes, created by the combination of spores, gametophytes, and gametes, are repeated alternately. This kind of generational alternation has also been found to have a unique appearance that is slightly modified depending on the taxonomic group, so it is used as another identification trait for the taxonomic group. Red algae are broadly divided into the native red algae (Bangiophycidae; Protoflorideophycidae), which includes seaweed and purple algae, and the true red algae (Florideophycidae), which includes agar agar, agar, etc. The former includes orders such as Goniotrichales and Bangiales. , the latter includes Nemaliales, Gelidiales, Cryptonemiales, Gigartinales, Rhodymeniales, and Ceramiales.

Meanwhile, along with the shortage of land resources, interest in marine resources, a new area, has increased, and research and development of functional materials using seaweed has begun in earnest. Compared to wood, which is a land plant, seaweed has a softer texture, has finer fibers, and can be cultured without special management in a much larger space than on land, so it has the advantage of having a much larger biomass. The growth rate is also faster than that of trees, no separate soil is required for cultivation, the extraction of fiber is easy, the pulping process is simple, and production facilities are inexpensive, so research has been conducted on the development of pulp using seaweed. Research is also being conducted to improve skin moisturization and adsorb harmful substances using this seaweed pulp. However, the method for producing red algae microfibrous cellulose of the present invention and its use for suppressing skin damage or inflammation have not been disclosed.

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.

The purpose of the present invention is to provide a method for producing red algae microfibrillated cellulose (MFC).

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

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

In addition, another object of the present invention is to provide a method for confirming fibrilization of red algae cellulose.

In order to achieve the above object, the present invention includes the steps of pretreating red algae with a microwave sample decomposition device to remove agar and physically blending the pretreated red algae. A method for producing red algae microfibrillated cellulose comprising the following steps is provided.

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

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

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

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

Microfibrillated cellulose (MFC) from red algae prepared according to the production method of the present invention has the effect of inhibiting cyclooxygenase-2 (Cox-2) activity, inhibiting phosphorylation of MAPKs, and inhibiting TNF-α expression. It is effective in suppressing skin damage and inflammation caused by ultraviolet rays.

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

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

The present invention includes a first step of removing agar by pretreating red algae using a microwave digestion method, and a second step of blending the red algae from which the agar has been removed. A method for producing microfibrillated cellulose from red algae is provided.

In addition, in the first step, the method for producing red algae microfibrillated cellulose involves adding a solvent so 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 the mixture is pretreated by microwave digestion at 110 to 190 ° C., preferably 160 to 190 ° C. for 5 to 15 minutes, preferably 8 to 12 minutes to produce agar from red algae. It may include steps 1-2 of removing and extracting cellulose.

In addition, in steps 1-2, if pretreated at a temperature of 110 ℃ or lower, the agar is not sufficiently removed, and if pretreated at a temperature of 190 ℃ or higher, glucan decomposition and by-products are generated, so the mixture is as defined in the present invention. It is preferable to pre-treat under normal conditions, but it is not limited thereto.

In addition, in the second step, the cellulose is blended at 32,000 to 42,000 RPM, preferably 35,000 to 39,000 RPM for 40 minutes or more, preferably 80 minutes or more, more preferably 150 to 170 minutes. It may be fibrillation.

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

In addition, the red algae is 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 measuring the galactan content to confirm whether the agar has been removed.

The term “galactan” used in the present invention refers to a polysaccharide containing D-galactose residues as a constituent sugar. In the present invention, it was used to confirm the D-galactose content of red algae.

The term “microwave decomposition” used in the present invention is a pretreatment method that removes organic substances and interfering substances in the sample by adding distilled water to the sample and heating it. Polar molecules or ions have a dipole moment in the microwave region using a microwave device. The sample is heated using the principle of increasing temperature 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 digestion method.

In addition, the blending refers to a physical treatment, and through homogenization of the sample, fibers having a long and intact fibrous structure of cellulose (see FIG. 6A) are changed from thin threads or ribbons to broad sheet-like fibers. This may mean transforming the fibers into fibers of various sizes (see FIG. 6B) to fibrillate the fibers.

The present invention provides a pharmaceutical composition for preventing or treating skin damage or inflammatory diseases containing red algae microfibrillated cellulose prepared by the above method.

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

The term “damage” used in the present invention includes death of human skin cells caused by ultraviolet rays, skin cell DNA damage, increased reactive oxygen species (ROS), increased lipid peroxidation, etc. Symptoms include erythema, This can include sunburn, pigmentation, photoaging, and skin cancer. In addition, “improvement” of damage refers to all actions that alleviate the damage to skin cells caused by ultraviolet rays or reduce the severity of symptoms.

In addition, 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, It may be selected from the group consisting of burns, dermatitis, periodontitis, and gingivitis, but is not limited thereto.

Additionally, the red algae microfibrous cellulose may inhibit cyclooxygenase-2 (Cox-2) activity.

The term “cyclooxygenase-2 (Cox-2)” used in the present invention is also called prostaglandin-endoperoxide synthase 2 and refers to prostaglandin, a substance that causes inflammation and pain. (prostaglandin, PGs) refers to an enzyme that promotes the formation. The prostaglandin is known to be deeply involved in the development of cancer by promoting inflammatory reactions such as pain and fever, immune reactions, and angiogenesis. Blocking the activity of the enzyme can reduce the inflammatory reaction or tumor growth. there is.

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

Additionally, the red algae microfibrous cellulose may inhibit the expression of TNF-α.

In addition, the pharmaceutical composition for preventing or treating skin damage is a skin external preparation that has a skin protection effect from ultraviolet rays and may include creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, or cataplasma agents. It can be prepared and used as a pharmaceutical composition in the form of an external skin preparation, but is not limited thereto.

The term “prevention” refers to any action that reduces the frequency or severity of pathological phenomena. Prevention may be complete or partial. In this case, it may mean that the symptoms of skin disease or inflammatory disease in an individual are reduced compared to the case where the composition is not used.

The term “treatment” refers to any clinical intervention to change the natural process of the target or cell to be treated, and can be performed while the clinical pathology is progressing or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or It may include alleviating or temporarily relieving the condition or improving the prognosis.

The composition according to the present invention may contain a pharmaceutically effective amount of red algae microfibrillar cellulose alone or may include 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. In the above, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans.

Additionally, the composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, It may be one or more selected from the group consisting of polyvinyl pyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, and liquid paraffin. It is not limited, and all common carriers, excipients, or diluents can be used. The ingredients may be added independently or in combination to the active ingredient, red algae microfibrous cellulose.

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

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

In addition, the pharmaceutical composition of the present invention includes tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. It may have any one formulation selected from. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

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

The present invention provides a cosmetic composition for preventing or improving skin damage containing 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 microfibrous cellulose described above, any content that overlaps with the red algae microfibrous cellulose of the present invention is excessive in the present specification due to the description of the duplicated content. To avoid complexity, the description is omitted.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, Chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are 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, nourishing lotion, various creams, essences, packs, foundations, cleansing products, face washes, soaps, treatments, and serums. etc.

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

In the cosmetic composition containing red algae microfibrous 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, in a cosmetic composition that is usually contained.

When the red algae microfibrous cellulose of the present invention is used as an external skin agent, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, Surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or topical agents for the skin. may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in dermatology. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology.

The present invention provides a health food and health functional food composition for preventing or improving skin damage containing 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 includes the red algae microfibrous cellulose described above, any content that overlaps with the red algae microfibrous cellulose of the present invention described above is included in the description of the duplicated content. In order to avoid excessive complexity of this specification, the description is omitted.

The food composition of the present invention can 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, sausages, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, and dairy products including ice cream. Products, various soups, beverages, alcoholic beverages and vitamin complexes, dairy and milk products, etc., and include both health foods and health functional foods in the conventional sense.

The health food and health functional food composition containing red algae microfibrous cellulose according to the present invention can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of red algae microfibrous cellulose can be appropriately determined depending on the purpose of use (prevention or improvement). In general, the amount of the composition in health foods and health functional foods can be 0.1 to 90 parts by weight of the total weight of the food. However, in the case of long-term intake for the purpose of maintaining health or regulating health, the amount may be below 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 has no particular restrictions on other ingredients other than containing the red algae microfibrous cellulose of the present invention as an essential ingredient in the indicated ratio, and various flavoring agents or natural carbohydrates are added like ordinary beverages. It can be contained as an ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 of the health functional food composition of the present invention.

In addition to the above, the health food and health functional food composition containing red algae microfibrous cellulose of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, and thickening agents (cheese, Chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc. In addition, the health food and health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

These ingredients can be used independently or in combination. The ratio of these additives is not that important, but 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 red algae microfibrous cellulose of the present invention.

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

The step of determining whether fibrillation occurs is measuring the water retention value of cellulose, checking sedimentation to determine whether or not aggregates are formed, or binding between cellulose and Ct CBD3 protein. It may be to confirm.

The water retention value is related to the binding force of the fibers and the cellulose surface area, and as more fibers are separated (defibrillation), the water retention value increases. Therefore, it is possible to determine whether cellulose is fibrillated according to its water retention value.

The degree of sedimentation can be confirmed through a sedimentation test, and the visible sedimented layer exists as individual fibers or is tangled or incompletely separated fibers that interact with other fibers to form an assembly, making them visible. Refers to fibers that can be observed. Therefore, it is possible to determine whether cellulose is microfibrillated depending on the precipitation height of the fiber.

The Ct CBD3 protein is known to be attached to the crystallines of cellulose including surface-binding-carbohydrate-binding modules CBM1, CBM2a, CBM3, CBM5, and CBM10. As fibrillation progresses, the surface area of cellulose increases, so the Ct CBD3 protein binding rate increases. Therefore, the presence or absence of microfibrillation of cellulose can be determined depending on the degree of binding of the Ct CBD3 protein.

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

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

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

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

<Example 2> Confirmation of removal of agar

To check whether agar was removed, the glucan and galactan contents of the sample of Example 1 were measured. Glucan and galactan contents were measured by the following method.

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

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

Glucan % (w/v) Galactan % (w/v) control group 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 Figure 1 and Table 2, the control sample without pretreatment had the highest galactan content because the agar was not removed, and the glucose content increased with pretreatment temperature and galactose ) content was confirmed to be reduced. In addition, as shown in Figure 2, the control group (untreated) did not have peaks at 1315 cm ^-1 and 1371 cm ^-1 , and at the peak of 1110 cm ^-1 , which indicates cellulose content, the higher the pretreatment temperature, the higher the cellulose content. indicated. In particular, the sample pretreated at 180°C had a galactan content of 1.8%, confirming that most of the agar was removed. Therefore, it can be seen that the sample is cellulose extracted from Gelidium amansii . From the following examples, only samples pretreated at 180°C were used.

<Example 3> Preparation of microfibrillated cellulose

The sample pretreated at 180°C in Example 1, that is, cellulose extracted from Gelidium amansii, was blended at 37,000 RPM for 40, 80, 120, and 160 minutes, respectively, using a Vitamix TNC5200 blender (Vitamix, Japan) Microfibrillated cellulose was prepared by blending.

<Example 4> Confirmation of microfibrillation of cellulose

4-1. Measurement of water retention value

In order to confirm whether microfibrillation of cellulose occurred, the water retention value of each sample of Example 3, that is, microfibrillated cellulose, was measured.

Water retention value (WRV) is the ratio of the percentage of water contained in a sample after centrifugation with force and time to the dry weight of the sample. Insert a 70㎛ nylon cap into a 50 mL tube and cover it with a membrane filter with a pore size of 0.2 ㎛. The sample was filled on top and placed in a centrifuge. After centrifugation at 4 °C for 30 minutes at 900 g relative centrifugal force (RCF), the sample was weighed. Afterwards, 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 capacity 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 value according to the physical processing (blending) time of red algae cellulose. As a negative control (0 min), the sample pretreated at 180°C in Example 1 without blending was used.

As shown in Figure 3, the water retention value of the experimental group was significantly higher than that of the control group that did not undergo blending, a physical pretreatment step. In particular, it can be seen that samples blended for more than 80 minutes maintain high water retention. Microfibrilization of cellulose extracted from agar agar was confirmed through a physical pretreatment step, and this result means that microfibrillated cellulose was formed from agar agar by blending.

4-2. sedimentation test

In order to confirm whether microfibrillation of cellulose occurred, a sedimentation test was performed on each sample of Example 3, that is, microfibrillated cellulose.

The experiment was conducted at 0.0005 g/mL, 0.001 g/mL, 0.002 g/mL, and 0.004 g/mL. Sedimentation is a method to check the dispersion stability of MFC suspension. The total volume was 10 mL, and red algae cellulose samples were prepared at concentrations of 0.05%, 0.1%, 0.2%, and 0.4%, and then thoroughly mixed before the sedimentation test. Then, it was left for 48 hours to allow the sediment to settle. After the cellulose fibers had completely settled, the height of the sediment was measured. The results are shown in Figure 4. The visible deposited layer refers to fibers that can be visually observed by microfibrillation of cellulose fibers and the separated fibers interact with other fibers to form an assembly (see Figure 6).

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

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

In addition, the degree of suspension maintenance 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, meaning that it has superior stability than the positive control (Borregard MFC).

4-3. CT Measurement of CBD3 protein binding force

In order to confirm whether microfibrillation of cellulose occurred, the Ct CBD3 protein binding force of each sample of Example 3, that is, microfibrillated cellulose, was measured.

A binding experiment was performed by adding 200 μg/mL of Ct CBD3 to 1 mg of microfibrillar cellulose as a substrate. The substrate and protein were reacted in 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 without binding to the substrate was measured in the supernatant using BCA. The results are shown in Figure 5.

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

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

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

To confirm the effect of the microfibrillated cellulose prepared in Example 3 on suppressing skin damage and inflammation caused by ultraviolet rays, the expression level of cyclooxygenase-2 (Cox-2) was measured.

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

The protein mixture was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), the proteins were separated through electrophoresis, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Immobilon®-P transfer membrane, MA, USA). ) was transferred to. After blocking with 5% skim milk at room temperature for 1 hour, each primary antibody was reacted at 4°C overnight. Afterwards, the membrane was washed a total of 3 times with TBST (tris-buffered saline Tween-20) once every 5 minutes, and a secondary antibody labeled with HRP dissolved in skim milk was incubated with horseradish peroxidase (HRP). )-conjugated secondary antibody, Thermo Scientific) for 1 hour at room temperature. After washing the membrane, it was treated with EzWestLumi plus to measure protein expression and phosphorylation levels. Protein bands were quantified and visualized using chemiluminescence detection kit (ATTO, Tokyo, Japan) and GeneGnome XRQ NPC (Syngene, Cambridge, UK), respectively. The results are shown in Figure 7.

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

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

<Example 6> UVB-induced phosphorylation inhibitory activity of the MAPK family of red algae microfibrillar 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, 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 the experimental method described in Example 5 above. The results are shown in Figure 8.

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

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

HaCaT (Human Adult low Calcium High Temperature) skin cells were pretreated with 0, 50, and 100 μg/mL of the MFC of Example 3 for 1 hour, 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 to synthesize cDNA using ReverTra Ace® qPCR RT master mix (Toyobo, Japan). Afterwards, 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 normalized with glyceraldehyde-3-phosphate dehydrogenase using the 2 ^-ΔΔCT method to reduce inherent errors occurring in the experiment. The results are shown in Figure 9.

As shown in Figure 9, when the MFC of Example 3 was treated at a concentration of 50 and 100 μg/mL after UVB irradiation, the expression of TNF-α mRNA was about It was found to be suppressed by 10 to 20%. These results mean that the microfibrous cellulose of the present invention has an effect of suppressing skin damage and inflammation by suppressing the expression of TNF-α, which is closely related to inflammation.

As discussed above, specific embodiments of the present invention have been described in detail, but 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, thereby creating other regressive inventions. However, other embodiments that are included within the scope of the present invention can be easily proposed. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as

Claims (15)

Red algae, comprising a first step of removing agar by pretreating red algae by microwave decomposition and a second step of blending the red algae from which the agar has been removed. Method for producing microfibrillated cellulose.
According to paragraph 1,
The red algae include Gelidium amansii , Gloiopeltis tenax , Gracilaria verrucosa , Chondrus ocellatus Holmes, Pachymeniopsis elliptica , Grateloupia filicina , and Ceramium kondoi . ), a method of producing red algae microfibrillated cellulose, comprising at least one species from the group consisting of Hypnea charoides and Porphyra tenera .
According to paragraph 1,
A method for producing red algae microfibrillated cellulose, characterized in that the first step is carried out at 110 to 190 ° C.
According to paragraph 1,
The second step is a method of producing red algae microfibrillated cellulose, characterized in that the cellulose fibers are fibrillated through homogenization of the red algae sample.
According to paragraph 1,
The second step is a method of producing red algae microfibrillated cellulose, characterized in that blending is performed for more than 80 minutes.
A pharmaceutical composition for preventing or treating skin diseases caused by ultraviolet rays, comprising red algae microfibrillated cellulose prepared by the method of any one of claims 1 to 5.
According to clause 6,
A pharmaceutical composition for preventing or treating skin diseases caused by ultraviolet rays, wherein the red algae microfibrous cellulose inhibits the activity of cyclooxygenase-2.
According to clause 6,
The red algae microfibrous cellulose is a pharmaceutical composition for preventing or treating skin diseases caused by ultraviolet rays, characterized in that it inhibits the phosphorylation level of JNK and p38 MAPK (mitogen-activated protein kinase).
According to clause 6,
A pharmaceutical composition for preventing or treating skin diseases caused by ultraviolet rays, wherein the red algae microfibrous cellulose 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.
According to clause 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.
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