KR102538762B1 - Method for producing a composition containing fermented ephedra - Google Patents

Abstract

A method for producing a composition containing fermented ephedra and a composition prepared thereby are disclosed.
Among these, the manufacturing method comprises the steps of drying and powdering domestic ephedra; After adding a 10% weight ratio of ephedra powder to distilled water, reflux cooling extraction at 100 ° C. for 24 hours; Using filter paper to remove the ephedra residue from the cooled extract and obtaining only pure solution; After adding the ephedra solution and 10 g of TSB medium powder to 1 L of distilled water, sterilization; Primary inoculation of Arthrobacter globiformis (ATCC 8010) or Pseudomonas putida (ATCC 12633) seed solution cultured in TSA medium at 25° C. at 1% (v/v) to 10% (v/v); After the first inoculation, fermenting at 25 ° C. at 100 rpm for 10 days to obtain a first-stage fermentation product; The culture medium containing MRS medium, which is a lactic acid bacteria medium, was mixed with the first-stage fermentation product at a ratio of 1:1 (v/v), and the mixture was heated to 37°C in a medium containing MRS medium powder at a ratio of 1:10 (w/v). Lactobacillus plantarum (ATCC 14917) seed solution cultured for 3 days Secondary inoculation with 1% (v/v) to 5% (v/v); After the second inoculation, fermenting at 37 ° C. at 120 rpm for 5 days to obtain a two-stage fermentation product; Obtaining a lysate by ultrasonically disrupting the second-stage fermented product at room temperature at 20 kHz to 120 kHz for 30 minutes to 4 hours; and preparing a fermented powder by sequentially filtering under reduced pressure, concentrating under reduced pressure, and freeze-drying the lysate.

Description

Method for producing a composition containing fermented ephedra and a composition prepared thereby {Method for producing a composition containing fermented ephedra}

It relates to a method for producing a composition containing fermented ephedra and a composition prepared thereby.

Ephedra is an evergreen shrub distributed in northern China and Mongolia, with a length of 30 cm to 70 cm and a width of about 1 m, and is well known as a herbal medicine. Known benefits include improvement of fever, asthma, headache and obesity.

Conventionally, obesity improvement effect of fermented ephedra lactic acid bacteria (J Korean Med Rehabil, 19(4); 37-57, 2009), fermented product manufacturing method using ephedra nano extract (domestic patent 10-0965961), ephedra nanoparticle method Papers and patent documents such as (Korean Patent No. 10-1065443) and a cosmetic composition containing ephedra root extract (Korean Patent No. 10-0562348) are known.

However, ephedra has many limitations in its use as a material for food, medicine or cosmetics due to the strong toxicity of ephedrine. Therefore, it is necessary to develop an ephedra processing method that can improve the functionality while lowering the content of ephedrine.

One aspect is 1. Drying and powdering domestic ephedra; 2. After adding ephedra powder in a weight ratio of 10% to distilled water, reflux cooling and extraction at 100 ° C. for 24 hours; 3. Removing ephedra residues from the cooled extract using filter paper and obtaining only a pure solution; 4. After adding the ephedra solution and 10 g of TSB medium powder to 1 L of distilled water, sterilization; 5. Primary inoculation of Arthrobacter globiformis (ATCC 8010) or Pseudomonas putida (ATCC 12633) seed solution cultured in TSA medium at 25° C. at 1% (v/v) to 10% (v/v); 6. After the first inoculation, fermenting at 25 ° C. at 100 rpm for 10 days to obtain a first-stage fermentation product; 7. Mix the culture medium containing the MRS medium, which is a lactic acid bacteria medium, with the first stage fermentation at a ratio of 1:1 (v/v), Lactobacillus plantarum (ATCC 14917) seed solution cultured for 3 days in the medium Secondary inoculation with 1% (v/v) to 5% (v/v); 8. After the second inoculation, fermenting at 37° C. at 120 rpm for 5 days to obtain a two-stage fermentation product; 9. Obtaining a homogenate by ultrasonically disrupting the two-stage fermentation product at room temperature at 20 kHz to 120 kHz for 30 minutes to 4 hours; And 10. Step of preparing a powder fermented product by sequentially filtering under reduced pressure, concentration under reduced pressure, freeze-drying the lysate; provides a method for producing a composition containing fermented ephedra.
Preferably, the ephedra may include ephedra above ground, underground or whole, ephedra fermented product.
Another aspect provides a composition comprising the fermentation product obtained by the above method.

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It provides a composition for whitening, antioxidant, anti-inflammatory, anti-aging or skin elasticity enhancement containing fermented ephedra.

The ephedra is a generic term for plants of the genus Ephedra , and may be chomahuang, target ephedra, and heavy ephedra.

The ephedra may be the above-ground part, the underground part or the whole of ephedra, for example, stems, roots, leaves, flowers, fruits, and the like. In particular, the aerial part of ephedra is rich in alkaloids.

The fermented product means the product of fermenting the ephedra. In a narrow sense, the fermentation refers to a process of decomposing organic matter that obtains energy without using oxygen, and in a broad sense, refers to a process of obtaining useful substances for humans using microorganisms or fungi. For example, the ephedra fermented product may contain substances beneficial to humans produced through various enzymatic reactions by culturing with bacteria in a medium containing ephedra to be fermented.

The ephedra may be the raw material of the ephedra plant or dried it.

The ephedra may be an ephedra extract. The extract is obtained by physically extracting raw materials or using a solvent, and may include a juice, a concentrate, and the like in which specific components are not removed or separated. The extract may include a crude extract, a fraction obtained by additionally fractionating the crude extract, a dried product, a purified product, and the like.

The ephedra extract may be obtained by using an extraction method such as shaking extraction, hot water extraction, hot water extraction, cold needle extraction, ultrasonic extraction, immersion extraction of ephedra.

The ephedra extract may be obtained by using a weight ratio of 1% to 90%, 2% to 50%, 5% to 30% or 7% to 20% of the dried ephedra compared to the extraction solvent (the weight ratio is also w / w) Mark). The solvent may be selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol and butylene glycol. The alcohol having 1 to 4 carbon atoms includes methanol, ethanol, n-propanol, iso-propanol, n-butanol, tert-butanol, and the like.

For example, the ephedra extract may be obtained using hot water extraction at 70 ° C to 150 ° C, 80 ° C to 120 ° C or 90 ° C to 110 ° C. The ephedra extract may be obtained by using hot water extraction for 1 hour to 100 hours, 2 hours to 70 hours, 3 hours to 50 hours, 4 hours to 40 hours, 5 hours to 30 hours or 6 hours to 24 hours . When extracted under the above temperature and time conditions, it is possible to obtain a stable ephedra extract with high yield.

The ephedra fermented product may be produced by a manufacturing method comprising the step of fermenting ephedra in one or more steps to obtain a fermented product.

If the ephedra is the raw material of ephedra, the raw material of ephedra may be added at a weight ratio of 1% to 90% compared to the fermentation medium. When the ephedra is dried ephedra, the dried ephedra powder may be added at a weight ratio of 1% to 90% compared to the fermentation medium. Alternatively, when the ephedra is an ephedra extract, the ephedra extract may be added at a volume ratio of 10% to 90% compared to the fermentation medium. Under the above ratio conditions, it is possible to obtain an ephedra fermented product having excellent types and contents of active ingredients.

The ephedra fermented product may include a fermented product obtained by sequentially fermenting ephedra with Arthrobacter genus, Pseudomonas genus or a mixed strain thereof, and lactic acid bacteria, yeast or a mixed strain thereof. That is, the ephedra fermented product is fermented by culturing ephedra with Arthrobacter genus, Pseudomonas genus or a mixed strain thereof to obtain a first-stage fermentation product, and culturing the first-stage fermentation product with lactic acid bacteria, yeast or a mixed strain thereof to ferment It may be produced by a manufacturing method comprising the step of obtaining a two-stage fermentation product.

In the step of obtaining the first-stage fermentation product, the fermentation strain is A. agilis, A. bambusae, A. enclensis, A. flavus, A. gandavensis, A. globiformis, A. ginggonis, A. halodurans, A. ramosus, A. roseus, A. russicus and A. sanguinis . The fermentation strain may be selected from the group consisting of P. aeruginosa group, P. chlororaphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, and P. syringae group of the genus Pseudomonas.

The fermented ephedra may be prepared by a manufacturing method comprising fermenting ephedra to obtain a first-stage fermentation product and fermenting at a relatively high temperature compared to the first-stage fermentation product to obtain a second-stage fermentation product.

The step of obtaining the first-stage fermented product may be to ferment ephedra at 10 ° C to 50 ° C, 15 ° C to 40 ° C or 20 ° C to 30 ° C. The step of obtaining the first-stage fermentation product may be to ferment ephedra for 1 to 30 days, 2 to 25 days, 5 to 20 days, 7 to 15 days, or 8 to 12 days.

Obtaining the second-stage fermentation product may be fermenting the first-stage fermentation product at 20 ° C to 70 ° C, 25 ° C to 50 ° C, 30 ° C to 45 ° C, or 35 ° C to 40 ° C. The step of obtaining the two-stage fermentation product may be fermentation for 0.5 days to 20 days, 1 day to 15 days, 1.5 days to 10 days, or 2 days to 5 days. While the ephedrine content is significantly reduced within the above range, a fermented product having excellent radical scavenging ability, collagen generating ability, tyrosinase activity inhibiting ability, and nitroxide production inhibiting ability can be obtained.

In the step of obtaining the two-stage fermentation product, the fermentation strain is at least one selected from the group consisting of Acetobacter , Aspergilus , Bacillus , Bifidobacterium , Lactobacillus , Lactococcus , Leuconostoc , Rhodopseudomonas , Saccharomyces , and Streptococcus It may be fermented by culturing with a strain.

The strain of the genus Acetobacter may be one selected from the group consisting of A. xylinum, A. orleanensis, A. papayae, A. syzygii and A. thailandicus . The strain of the genus Aspergillus may be one selected from the group consisting of A. niger, A. acidus, A. caesiellus, A. ficuum, A. jaipurensis, A. nakazawae, A. saccari and A. vitricola . The strain of the genus Bacillus may be selected from the group consisting of B. stearothermophilus, B. subtilis and B. amyloliquefaciens . The Bifidobacterium genus strain may be selected from the group consisting of B. animalis, B. bifidum, B. adolescentis and B. longum . The strains of the genus Lactobacillus include L. acidophilus, L. brevis, L. bulgaricus, L. casei, L. delbrueckii, L. paracasei, L. plantarum, L. rhamnosus HK9, L. rhamnosus, L. diacetylactis and L. lactis . It may be selected from the group consisting of The strain of the genus Leuconostoc may be selected from the group consisting of L. mesenteroides, L. carnosum, L. inhae and L. garlicum . The strain of the genus Lactococcus may be selected from the group consisting of L. garvieae , L. diacetylactis and L. lactis . The strain of the genus Rhodopseudomonas may be selected from the group consisting of R. palustris, R. faecalis, R. julia, R. palustris and R. rhenobacensis . The strain of the genus Saccharomyces may be one selected from the group consisting of S. cerevisiae, S. exiguus, S. bayanus, S. ellipsoideus, S. eubayanus, S. pastorianus, S. florentinus and S. fragilis . The strain of the genus Streptococcus may be one selected from the group consisting of S. thermophilus, S. zooepidemicus, S. sanguinis, S. salivarius and S. mitis .

In the step of obtaining the first-stage fermentation product, the corresponding bacteria consume and grow the ephedrine present in ephedra to maximize the ephedrine content in the first-stage fermentation product, and in the step of obtaining the second-stage fermentation product, lactic acid bacteria or yeast Beneficial bacteria can consume and grow useful ingredients present in the first-stage fermented product to maximize the type and content of active ingredients.

The step of obtaining the fermented product is, after inducing the strain to grow for a certain period of time, the obtained cells, the substances secreted by the cells while growing, and the culture solution stock solution in which the nutrients necessary for growth are mixed, centrifugation or filtering out impurities Obtained through the process, it may be a cell body, a stock solution, a supernatant, or a mixture thereof.

The composition may include a lysate of fermentation. That is, the manufacturing method may further include the step of obtaining an ephedra lysate by sonicating the obtained fermented product. The step of obtaining the ephedra lysate may be to sonicate the fermented product under conditions of 10 kHz to 250 kHz, 15 kHz to 160 kHz or 20 kHz to 120 kHz. The step of obtaining the ephedra lysate may be ultrasonic treatment for 10 minutes to 20 hours, 20 minutes to 10 hours, or 30 minutes to 6 hours. Obtaining the ephedra lysate may be ultrasonic treatment at room temperature. The ultrasonic treatment can elute not only fermented products but also useful components in cells by disrupting lactic acid bacteria, yeast bacteria, and the like.

The composition may include a concentrate and/or a dried product of fermentation. That is, the manufacturing method may further include the step of concentrating and/or drying. The concentrating step may be vacuum concentration, evaporation concentration, vacuum concentration, or the like. The drying step may be freeze drying, hot air drying, natural drying, vacuum drying, spray drying, and the like.

The whitening refers to a method of increasing the brightness of skin whose brightness is reduced due to an excess of pigments such as melanin or maintaining the brightness of the skin at a certain level, skin with increased brightness formed by the method, etc. will be. Melanin is a pigment component widely distributed in living things such as skin, hair, and pupils. It is synthesized in melanosomes in melanocytes in the human epidermal layer. Melanin can be biosynthesized through oxidation and polymerization reactions that convert it into 3,4-dihydroxy-phenylalanine (DOPA) or dopaquinone. Since excessive synthesis of melanin causes pigmentation such as melasma, freckles, and age spots, measurement of tyrosinase activity can be an important indicator of skin whitening effect. The ephedra fermented product may have a preventive and improving effect on skin whitening and pigmentation through tyrosinase inhibitory activity.

The antioxidant refers to an action of suppressing oxidation. In the human body, prooxidants and antioxidants are in balance, but they are imbalanced by various factors, and when oxidation is promoted, oxidative Oxidative stress can be induced, leading to potential cell damage and disease. Reactive oxygen species (ROS), which is a direct cause of such oxidative stress, are unstable and highly reactive, easily reacting with various biomaterials, and attacking macromolecules in the body to cause irreversible damage to cells and tissues, mutations, It can cause cytotoxicity and carcinogenesis. Reactive nitrogen species (RNS) such as NO, HNO ₂ , and ONOO- are produced in large quantities due to immune responses of macrophages, neutrophils, and other immune cells during inflammatory reactions, and ROS are also produced at this time. In addition, the antioxidant refers to a function of inhibiting oxidation of cells caused by highly reactive free radicals or reactive oxygen species according to cellular metabolism or oxidative stress caused by ultraviolet rays, and removing free radicals or reactive oxygen species. This may include reducing damage to cells caused by this. The ephedra fermented product may have a preventive and ameliorative effect on diseases caused by active oxygen species or active nitrogen species through antioxidant action, aging of the human body, including the skin, and inflammatory diseases such as atopic dermatitis.

The anti-inflammatory means an action of suppressing inflammation, and the inflammatory response may be for enhancing and reducing damage of the repair system in vivo. Reactive oxygen species and reactive nitrogen species are deeply related to the inflammatory response. During the inflammatory process, a large amount of proinflammatory cytokines, nitric oxide (NO), and the like can be produced. The ephedra fermented product may have a preventive and ameliorative effect on various inflammatory diseases, including inflammatory skin diseases, through anti-inflammatory action.

The anti-aging refers to an action of alleviating the degree of deterioration of the ability of cells, bodies, etc. with increasing age. Collagen is a natural protein and is a component of skin and connective tissue, and is present in skin, tendons, ligaments, corneas, cartilage, bones, blood vessels, and intestines. It can be seen that the ephedra fermented product promotes collagen synthesis and at the same time inhibits the synthesis of collagen degrading enzymes to have a skin and connective tissue regeneration effect.

The skin elasticity promotion is to alleviate the degree of skin sagging or sagging or to restore elasticity, and elastic fibers composed of elastin exist together with collagen fibers called collagen, which means a state in which elastin and collagen are sufficiently present. The enhancement of skin elasticity is accompanied by an effect of reducing wrinkles, and the improvement of wrinkles means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles already formed. The ephedra fermented product may have an effect of improving skin sagging and wrinkles as it promotes collagen synthesis and at the same time inhibits collagen degrading enzyme synthesis to promote skin elasticity.

Another aspect provides a cosmetic composition for whitening, antioxidant, anti-inflammatory, anti-aging or skin elasticity enhancement containing the ephedra composition as an active ingredient. As can be seen from the examples below, the ephedrine fermented product has excellent radical scavenging ability, collagen generating ability, tyrosinase activity inhibiting ability, and nitroxide production inhibiting ability, while the ephedrine content is significantly reduced, and is suitable as a functional cosmetic composition.

The ephedra fermented product is as described above.

The cosmetic composition is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath additive, sunscreen cream, sun oil, suspension, emulsion, It may be selected from the group consisting of paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray, but is not limited thereto.

The cosmetic composition may further include one or more cosmetically acceptable carriers formulated in general cosmetics, for example, oil, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, pigments, It may be formulated with preservatives, flavorings, and the like.

Cosmetically acceptable carriers included in the cosmetic composition vary depending on the formulation of the cosmetic composition. When the formulation of the cosmetic composition is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. It may contain. When the formulation of the cosmetic composition is a powder or spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, and the like. When the formulation of the cosmetic composition is a solution or emulsion, the carrier component may include a solvent, a solubilizing agent, an emulsifying agent, and the like, and for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic ester, polyethylene glycol, sorbate Fatty acid esters of bitan may be used, but are not limited thereto. When the formulation of the cosmetic composition is a suspension, the carrier component is a liquid diluent such as water, ethanol, or propylene glycol, a suspension agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; It may include crystalline cellulose, aluminum metahydroxide, bentonite, agar, tracanth, and the like. When the formulation of the cosmetic composition is a soap, the carrier component includes an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, aliphatic alcohol, vegetable oil, glycerol, sugar, etc. it could be

Another aspect provides a pharmaceutical composition for whitening, antioxidant, anti-inflammatory, anti-aging or skin elasticity enhancement containing the ephedra composition as an active ingredient.

The ephedra fermented product is as described above.

As can be seen from the following examples, the ephedrine fermented product has excellent radical scavenging ability, collagen generating ability, tyrosinase activity inhibiting ability, and nitroxide production inhibiting ability, while the ephedrine content is significantly reduced, pigmentation disease, active nitrogen or active oxygen It is suitable as a pharmaceutical composition for preventing, improving, and treating diseases or inflammatory diseases, aging, and skin damage caused by .

The pigmentation disease is freckles, senile spots, liver spots, melasma, brown or black spots, solar pigment spots, cyanic melasma, gravidic chloasma, melasma in women taking oral contraceptives, scratches And it may include hyperpigmentation, phototoxic reaction, etc. after inflammation due to wounds including burns or dermatitis.

Diseases caused by the active nitrogen or active oxygen include arteriosclerosis, Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases including amyotrophic sclerosis and Huntington's disease, myocardial infarction, angina pectoris, coronary artery disease, and ischemic heart disease It may include cardiovascular diseases, ischemic brain diseases including stroke, diabetes, digestive system diseases including gastritis and gastric cancer, cancer, leukemia, aging, rheumatoid arthritis, hepatitis, atopic dermatitis, aging, and the like.

The inflammatory disease is a general term for diseases with inflammation as a main lesion, and includes allergic asthma, allergic rhinitis, allergic mucositis, allergic diseases including urticaria and anaphylax, systemic sclerosis, skin Myopathy, including dermatomyositis and inclusion body myositis, arthritis, atopic dermatitis, psoriasis, asthma, multiple sclerosis, viral infections, sepsis, polychondritis, scleroderma, eczema, gout, periodontal disease, Behcet's syndrome , edema, vasculitis, Kawasaki disease, diabetic retinitis, autoimmune pancreatitis, vasculitis, glomerulonephritis, acute and chronic bronchitis, and the like.

The aging means that the ability of cells, bodies, etc. deteriorates with increasing age.

The skin damage may include wounds including scratches and burns, or skin necrosis after inflammation due to dermatitis.

The pharmaceutical composition may be a single agent or a combination agent that further includes a pharmaceutical composition known to have similar effects.

The pharmaceutical composition may be formulated for pharmaceutical unit administration by adding a pharmaceutically acceptable carrier, excipient or diluent. The "pharmaceutically acceptable" means that it does not significantly stimulate an organism and does not inhibit the biological activity and properties of the active substance administered, and the administration means introducing the pharmaceutical composition into a subject by any suitable method.

The composition may be an oral or parenteral formulation. The composition may be prepared using diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like. The solid preparation may further include excipients such as starch, calcium carbonate, sucrose or lactose, and gelatin, lubricants such as magnesium stearate, and talc. Liquid preparations for oral administration may include suspensions, emulsions, syrups, and the like.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending solvents may further include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. The suppository may further contain witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogelatin, and the like as a base.

The pharmaceutical composition varies according to the patient's age, weight, general health condition, sex and diet, administration time, administration frequency, administration route and secretion rate of the composition, treatment period, various factors including concurrently used drugs, etc., depending on the purpose of administration. can be applied

Another aspect provides a method for producing a fermented ephedra product comprising the step of fermenting ephedra in one or more steps to obtain a fermented product. The manufacturing method is a step of culturing and fermenting ephedra with Arthrobacter genus, Pseudomonas genus or a mixed strain thereof to obtain a first-stage fermentation product, and culturing the first-stage fermentation product with lactic acid bacteria, yeast bacteria, or a mixed strain thereof and fermenting the two-stage fermentation It may include the step of obtaining water. In addition, the manufacturing method may further include the step of obtaining an ephedra lysate by sonicating the obtained fermented product. The step of obtaining the ephedra, ephedra extract, fermented ephedra, and ephedra lysates are as described above.

The ephedra fermented product has excellent efficacy such as whitening, antioxidant, anti-inflammatory, anti-aging and elasticity enhancement, while the content of ephedrine having conventional toxicity is low, and can be applied to cosmetic compositions and pharmaceutical compositions.

Figure 1 shows the cytotoxicity of fermented ephedra.
Figure 2 shows the 1,1-diphenyl-2-picrylhydrazyl (2,2-Dipheny-1-picrylhydrazyl: DPPH) free radical scavenging ability of ephedra fermentation.
Figure 3 is a diagram showing the collagen synthesis ability of fermented ephedra.
Figure 4 shows the matrix metalloproteinase (Matrix metalloproteinase: MMP-1) production inhibition rate of ephedra fermentation.
Figure 5 shows the tyrosinase enzyme activity inhibition rate of ephedra fermentation.
Figure 6 shows the ability to inhibit nitroxide production of macrophages of ephedra fermentation.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

manufacturing example. Ephedra multi-stage fermentation and obtaining fermented product

The entire aerial part of domestic ephedra was dried and prepared as a powder. After adding 10% ephedra powder by weight to distilled water, reflux cooling extraction was performed at 100 ° C. for 24 hours. Ephedra residues were removed from the extract using filter paper, and only a pure solution was obtained.

After adding the ephedra extract and 10 g of TSB medium powder to 1 L of distilled water, it was sterilized. Then, Arthrobacter globiformis (ATCC 8010) Alternatively, Pseudomonas putida (ATCC 12633) seed solution (cultured in TSA medium at 25°C) was inoculated at 1% (v/v) to 10% (v/v). After inoculation, fermentation was performed at 25° C. at 100 rpm for 10 days to obtain a single fermentation product.

The culture medium containing MRS medium, which is a lactic acid bacteria medium, was mixed with the first-stage fermentation product at a ratio of 1:1 (v/v), and L actobacillus plantarum (ATCC 14917) was added to the mixture. Seed culture (cultured for 3 days in a medium containing 1:10 (w/v) of MRS medium powder at 37 ° C) Inoculated at 1% (v/v) to 5% (v/v). After inoculation, a two-stage fermentation was obtained by fermentation at 37° C. and 120 rpm for 5 days.

Thereafter, the two-stage fermentation was ultrasonically disrupted at room temperature at 20 kHz to 120 kHz for 30 minutes to 4 hours to obtain a lysate. Subsequently, the lysate was sequentially filtered under reduced pressure, concentrated under reduced pressure, and freeze-dried to prepare a fermented powder.

Example 1. Confirmation of ephedrine content in fermented ephedra

The extract, the first-stage fermentation product and the second-stage fermentation product were concentrated under reduced pressure in a rotary vacuum evaporator at a volume ratio of 1/10 and freeze-dried to obtain a powder, and the concentration of ephedrine remaining in the powder was measured.

First, 20 mg of ephedrine standard was diluted in methanol to prepare a standard solution stock of 1000 mg/L. Then, the standard solution was diluted to prepare an ephedrine solution of 1 mg/L to 100 mg/L.

1 g of the powder of the extract, the first fermentation product and the second fermentation product was put into a 50 mL flask, followed by 30 mL of methanol:mobile phase A (7:3), followed by ultrasonic shaking extraction for about 30 minutes. It was then filtered with methanol:mobile phase A (7:3) and filtered.

The measurement conditions of the liquid chromatograph used in the analysis are that the column is C18 (4.6 mm × 250 mm, 5 μm) or equivalent, and two mobile phases are used at 40 ° C., mobile phase A: 0.5 mM sodium-1-hexane-sulfo A 0.1% phosphoric acid solution containing sodium-1-hexane sulfonate and mobile phase B: 95% acetonitrile were flowed through the column in the following manner. At this time, the speed of the mobile phase was 1.0 mL/min, the UV detector wavelength was set to 210 nm, and the PDA detector wavelength was set to 200 nm to 400 nm.

time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 30 50 50 42 0 100 50 90 10

Table 2 shows the results of comparing the ephedrine content present in ephedra extract, ephedra first-stage fermented product and ephedra second-stage fermented product. As shown in Table 2, it can be seen that the first-stage fermented product and the second-stage fermented product have a significantly lower content of ephedrine than the extract.

Comparison of ephedrine content in ephedra extract and ephedra 1st and 2nd fermented product extract 1 stage fermented product 2 stage fermented product unit 27.53 15.29 1.04 mg/g

Example 2. Cytotoxicity evaluation of fermented ephedra

Cytotoxicity of the fermented ephedra was confirmed by MTT (Thiazolyl Blue Tetrazolium Bromide) assay.

Human skin fibroblasts (CCD-986sk) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). The cells were inoculated into the plate and maintained while being cultured in an incubator at 37° C. and 5% CO ₂ while exchanging the medium every 2 days.

Cells were plated 1×10 ⁴ in a 48-well plate. Inoculated into cells/well, administered with each concentration (0.05%, 0.04%, 0.03%, 0.02%, 0.01% in weight ratio) of ephedra extract, ephedra 1st stage fermentation product and ephedra 2nd stage fermentation product, and cultured for 24 hours After that, cell viability was confirmed.

As shown in Figure 1, the ephedra fermented product-administered group had significantly lower toxicity than the ephedra extract-treated group. did In addition, it was confirmed that the cell viability was about 93% even at the highest concentration of 0.05%, and the toxicity was low. That is, it can be seen that the ephedra fermented product has little cytotoxicity. On the other hand, ephedra extract showed relatively toxicity with cell viability less than 30% at all dose concentrations.

Example 3. Confirmation of antioxidant activity of fermented ephedra

Antioxidant activity of fermented ephedra was confirmed by DPPH assay.

Trolox was used as a positive control, and 0.2 A mM DPPH solution was added and reacted at room temperature for 30 minutes. After the reaction was completed, the absorbance was measured at 520 nm to confirm the DPPH radical scavenging ability according to the concentration according to the following formula. The free radical scavenging activity was expressed as a percentage compared to the negative control to which no sample was added.

Free radical scavenging activity (%)=[1-(A-B)/C] x 100

A: absorbance of sample group

B: absorbance of blank

C: negative control absorbance

As a result, as shown in FIG. 2, the ephedra fermented product administration group had significantly higher free radical scavenging activity than the ephedra extract administration group. In particular, the group administered with 0.125% of ephedra two-stage fermentation showed more than 95% free radical scavenging activity, and the groups administered with 0.125% and 0.25% of ephedra two-stage fermentation showed free radical scavenging activity equivalent to that of the positive control group.

Example 4. Confirmation of elasticity enhancement of fermented ephedra

Example 4-1. Confirmation of collagen synthesis ability of fermented ephedra

The collagen synthesis ability of ephedra fermentation was confirmed by PICP EIA (Procollagen Type I C-peptide (PIP) EIA) assay.

Fibroblasts were 1 × 10 ⁴ per well in a 48-well plate. After inoculation into cells/well, ephedra extract, ephedra 1st stage fermented product and 2nd stage fermentation product were administered by concentration (weight ratio 0.05%, 0.04%, 0.03%, 0.02% and 0.01%), cultured for 24 hours, and culture medium was collected to measure the amount of collagen synthesis in the cell culture medium (PICP EIA Takara biomedical Co. kit). As a positive control group, 0.1 ng/mL of TGF-beta 1 (TGF-β 1) was administered.

100 μl of the antibody-PpD conjugate solution was put into each well of the plate, and then 20 μl of the sample and standard solutions were added and reacted at 37° C. for 3 hours. After the reaction was completed, the reaction solution of each well was removed and washed 4 times with washing buffer (PBS: phosphate buffer saline). 100 μl of the substrate solution was added and reacted at room temperature for 15 minutes. Subsequently, after stopping the reaction by adding 100 μl of 1N sulfuric acid (H ₂ SO ₄ ), the absorbance was measured at 450 nm with an ELISA reader.

As a result, as shown in Figure 3, the ephedra fermentation product showed a collagen synthesis ability of 60% or more. In particular, in the ephedra two-stage fermented product administration group, collagen synthesis was significantly increased by 20% or more compared to the negative control group and equal to that of the positive control group.

Previously, the ephedra fermentation product administration group was found to have very low cytotoxicity compared to the ephedra extract administration group.

Example 4-2. Confirmation of collagenase inhibitory ability of fermented ephedra

Collagen degrading enzyme inhibitory ability of ephedra fermentation was confirmed to the extent of MMP-1 production.

Fibroblasts were 1 × 10 ⁴ per well in a 48-well plate. After inoculation into cells/well, ephedra extract, ephedra 1st stage fermented product and 2nd stage fermentation product were administered by concentration (weight ratio 0.05%, 0.04%, 0.03%, 0.02% and 0.01%), cultured for 24 hours, and culture medium were collected to measure the amount of collagenase MMP-1 in the cell culture medium (Human MMP-1 ELISA Abcam kit). As a positive control group, 0.1 ng/mL of TGF-beta 1 (TGF-β 1) was administered.

100 μl of the antibody-PpD conjugate solution was put into each well of the plate, and then 50 μl of the sample and standard solutions were added and reacted at 37° C. for 3 hours. After the reaction was completed, the reaction solution in each well was removed and washed 4 times with washing buffer. 100 μl of the substrate solution was added and reacted at room temperature for 15 minutes. Subsequently, after stopping the reaction by adding 100 μl of 1N sulfuric acid (H ₂ SO ₄ ), the absorbance was measured at 450 nm with an ELISA reader.

As a result, as shown in Figure 4, the ephedra fermented product administration group significantly reduced MMP-1 production. The ephedra 1st fermentation product administration group inhibited MMP-1 production equally or more than the positive control group, and in particular, the ephedra 2nd fermentation product administration group inhibited MMP-1 production by more than 40%.

Example 5. Confirmation of whitening ability of fermented ephedra

The whitening ability of the fermented ephedra was confirmed by the degree of tyrosinase activity inhibition.

First, mushroom-derived tyrosinase (sigma aldrich) was dissolved in 0.1 M phosphate buffer at 2000 units/mL and cryopreserved, and the substrate was L-3,4-dihydroxylphenyl alanine (L-3,4-dihydroxylphenyl alanine: L -DOPA) was prepared and used immediately before analysis.

Tyrosinase was added at 78 units/mL to a 1/15M phosphate buffer solution and preincubated at 25 ° C for 5 minutes, and each of the ephedra extract, ephedra first-stage fermentation product and second-stage fermentation product was prepared by concentration (weight ratio 0.05%) , 0.04%, 0.03%, 0.02% and 0.01%), L-DOPA was added. Arbutin was administered as a positive control group. Absorbance was measured at 475 nm for 10 minutes, and the degree of tyrosinase inhibition was confirmed by the following formula.

Tyrosinase inhibition (%) = [(A-B)-(C-D)/(A-B)] x 100

A: Absorbance at 475 nm of the control without sample

B: Absorbance at 475 nm of sample and control without enzyme

C: Absorbance at 475 nm of the experimental group containing the sample and enzyme

D: Absorbance at 475 nm of control without enzyme

As a result, as shown in Figure 5, the ephedra fermented product administration group showed a similar level of tyrosinase inhibition as the positive control group and was significantly higher than the ephedra extract administration group.

Example 6. Confirmation of improvement in inflammation of ephedra fermented product

The effect of enhancing skin immunity of fermented ephedra was confirmed by the degree of inhibition of NO production in macrophages.

Mouse macrophages (RAW 264.7) were cultured at 1.5×10 ⁵ per well in a 96-well plate. Cells/mL were inoculated and incubated for 24 hours, then divided into groups treated with 1 μg/mL of Lipopolysaccharide (LPS) (marked +) and groups not treated (marked -), 200 μl of each concentration (0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1mg/mL) of ephedra extract, ephedra first-stage fermentation product and second-stage fermentation product to the ride treatment group were added and incubated for another 24 hours. 50 μl of the culture supernatant was transferred to a new 96-well plate, then 50 μl of Griess reagent was added and reacted at 25 ° C for 20 minutes, and then the absorbance was measured at 540 nm and the amount of NO production was quantified using a quantification method using NaNO ₂ .

As a result, as shown in Figure 6, the ephedra fermented product administration group significantly reduced NO production compared to the ephedra extract administration group. In particular, in the ephedra two-stage fermented product administration group, NO production was further inhibited to 10% or less.

So far, we have looked at the preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. In particular, fermented products using various types of bacteria that can be used to obtain fermented products have the same function and are not excluded from the subject of this embodiment. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (5)

1. Drying and powdering domestic ephedra;
2. After adding ephedra powder in a weight ratio of 10% to distilled water, reflux cooling and extraction at 100 ° C. for 24 hours;
3. Removing ephedra residues from the cooled extract using filter paper and obtaining only a pure solution;
4. After adding the ephedra solution and 10 g of TSB medium powder to 1 L of distilled water, sterilization;
5. Primary inoculation of Arthrobacter globiformis (ATCC 8010) or Pseudomonas putida (ATCC 12633) seed solution cultured in TSA medium at 25° C. at 1% (v/v) to 10% (v/v);
6. After the first inoculation, fermenting at 25 ° C. at 100 rpm for 10 days to obtain a first-stage fermentation product;
7. Mix the culture medium containing the MRS medium, which is a lactic acid bacteria medium, with the first stage fermentation at a ratio of 1:1 (v/v), Lactobacillus plantarum (ATCC 14917) seed solution cultured for 3 days in the medium Secondary inoculation with 1% (v/v) to 5% (v/v);
8. After the second inoculation, fermenting at 37° C. at 120 rpm for 5 days to obtain a two-stage fermentation product;
9. Obtaining a homogenate by ultrasonically disrupting the two-stage fermented product at room temperature at 20 kHz to 120 kHz for 30 minutes to 4 hours; and
10. Step of preparing a powder fermented product by sequentially filtering under reduced pressure, concentrating under reduced pressure, and freeze-drying the lysate; The method of claim 1,
The ephedra is a composition manufacturing method comprising the ephedra aboveground, underground or whole, ephedra fermented product. A composition comprising the fermented rice yellow product obtained by the method of claim 1 or 2. delete delete

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