KR102401597B1 - Protectant against neuronal cell death by oxidative stress comprising l-serine and the root extract of angelica gigas as an effective component - Google Patents

Abstract

The present invention relates to a composition for inhibiting neuronal cell death containing L-serine and Angelica asiatica extract as active ingredients. It relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases exhibiting neuronal apoptosis inhibitory activity, and to a health functional food prepared by adding a component for improving sensory function to the active ingredient. L-serine and Angelica basil extract as active ingredients of the composition for inhibiting neuronal cell death of the present invention have excellent efficacy in inhibiting cell death due to oxidative stress of nerve cells, as demonstrated through the examples of the present specification In addition, it exhibits excellent neuronal cell death inhibitory effect while minimizing the amount of expensive Angelica asiatica extract powder used. In addition, by mixing L-serine, which is difficult to ingest due to its low functionality alone, and sub-materials of a sweetener, an acidulant, and a flavoring agent to an angelica powder, a predetermined mixture is prepared to prepare a mixed powder having excellent functionality. The composition for inhibiting neuronal cell death of the present invention has no hemolytic activity against human red blood cells, has excellent thermal stability, and does not show loss of neuronal apoptosis inhibitory effect even under acidic conditions of pH 2 It is a very useful invention to use.

Description

Composition for inhibiting nerve cell death comprising L-serine and Angelica keis extract as active ingredients

The present invention relates to a composition for inhibiting neuronal cell death containing L-serine and Angelica asiatica extract as active ingredients. It relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases exhibiting neuronal apoptosis inhibitory activity, and to a health functional food prepared by adding a component for improving sensory function to the active ingredient.

Neuronal cell death in humans occurs naturally during development or is observed in brain-related central nervous system diseases. The death of nerve cells during development is essential as a normal process, but extensive and sudden abnormal nerve cell death is closely related to neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease, stroke, and traumatic brain injury. it is known However, this abnormal neuronal death mechanism is still not well known, oxidative stress (oxidative stress), calcium homeostasis abnormal, mitochondrial dysfunction, increased production of reactive oxygen species, excitatory neurotoxicity , caspase activation, trophic factor depletion, etc. are known to be involved (Nature Reviews Molecular Cell Biology, 1: 120-130, 2000; Neurotoxicology and Teratology, 24: 675-682, 2002). Therefore, apoptosis inhibitors with various mechanisms of action have been predicted to be available as therapeutic agents for related brain diseases, and antioxidants, ion chelators, free radical scavengers, neurotrophic factors, and excitability to protect neurons from actual apoptosis Excitatory amino acid antagonists, bioenergic supplements, agents that block protein aggregation/accumulation, and immuno-suppressants have been identified as potential neuroprotective agents (Neurotoxicology) and Teratology, 24, 675-682, 2002).

L-serine (L-serine) is an allowable additive that has [standards and standards for each item] set in the Food Additives Ordinance of Korea, and is also registered as a cosmetic material for hair and skin conditioning agents (EINECS No: 200-274-3) , is an amino acid that is safe for use. L-serine is biosynthesis of amino acids such as glycine and cysteine, biosynthesis of purine and pyrimidine, which are DNA precursors, biosynthesis of phosphatidylserine, a cell membrane phospholipid, and sphingomyeline, cerebrosides in the brain And it is also importantly involved in the biosynthesis of D-serine (D-serine). Therefore, the concentration of L-serine in the cell is the growth regulation according to cell division and proliferation, response to oxidative stress (Pompella et al., 2003. Biochem. Pharmacol. 66, 1499-1503), reactive oxygen species (reactive oxygen) radicals, ROS) may also contribute significantly to the defense mechanism that protects cells from damage (Aoyama et al., 2008. J. Pharmacol.Sci. 108, 227-238; Zhou et al., 2017, Mol Nutr. Food). Res. 61, https://doi.org/10.1002/mnfr.201700262). Recently, Korean Patent Registration No. 10-1952443 discloses the inhibitory effect of serine and its derivatives on neuronal cell death.

Angelica gigas Nakai is the dried root of Angelica gigas Nakai, a perennial herb belonging to the Umbrella family. Angelica is mainly used to prevent and treat anemia, and is known to be effective in various gynecological diseases. In particular, in Dongui Bogam, about the efficacy of Angelica 'Eohyeol (瘀血), use a hard knot on the head side, and use fine roots (止痛) when the goal is to stop bleeding (止血) or to stop pain (止痛). Use the head to relieve eohyeol, and use fine roots to stop bleeding. When the whole is used, it relieves eohyeol on the one hand and stops the blood on the other hand, so this is called hwahyeol (和血). In addition, Angelica has been used in oriental medicine to prevent diseases and improve cognitive function as basic medicines such as Gyeongokgo, Gongjinjin, and Chongmyeongtang. In Korea, as health functional food raw materials that can help improve cognitive function due to aging, "Angi Gui Extract Complex" (Daewoong Bio Co., Ltd., No. 2011-3) and "Angi Angelica Extract Powder" (Nutragen, No. 2014) -44) is designated and used. The "Angi Gui extract complex" and "Angi angelica extract powder" are limited in combination with anticoagulants or hypoglycemic agents.

On the other hand, in the academic literature related to Angelica larvae, the ischemic brain damage inhibitory effect and neuroprotective effect (Yeoni Jeon et al., 2003, Journal of the Korean Herbal Society, 18: 25-35), and memory improvement activity (Kang So-young, 2003, Seoul) University doctoral thesis), diabetes improvement effect (Park Man-joong et al., 2009. Journal of the Korean Society for Food and Nutrition, 22: 246-251), antioxidant effect (Kang Soon-ah et al. 2004, Journal of the Korean Society for Food and Nutrition, 33: 1112-1118), caspase-3 Cancer cell killing effect through activation (Kyungwook Park et al., 2007. Journal of the Korean Society for Food Distribution and Storage, 14: 655-661), inhibition of cancer cell growth, inhibition of mutagenesis and inhibitory activity of angiotensin converting enzyme (Munseon Ham et al., 1996. Korean Society of Applied Microbiology and Biotechnology) 24: 624-629), partial improvement of mouse fat metabolism and alcoholic hepatotoxicity (Oh Seok-Hong et al. 1999, Korean Journal of Agricultural Chemistry 42: 29-33), etc. have been reported. A study on the food science application of Angelica, such as analysis method (Kang Young-gu et al., 2003. Journal of Herbal Medicine 34: 201-205) and food preparation using Angelica extract (Sang-geun Jang et al., 2008. Journal of the Korean Society for Dietary and Lifestyle Culture, 23: 479-488) is also in progress. In addition, the platelet aggregation inhibitory activity of courmarin-based compounds of Angelica keiskei has been reported (Yongwook Lee et al., 2003, Arch, Pharm. Res 26: 723-726).

In addition, as patents related to Angelica perilla, Republic of Korea Patent Registration No. 10-0829122 (Registration date: May 6, 2008) "A composition for preventing lung cancer using an Angelica asiatica extract or a coumarin-based compound isolated therefrom as an active ingredient", Republic of Korea Patent No. 10-0746294 (Registration date: July 30, 2007) There is "a pharmaceutical composition for dendritic cell activation containing polysaccharide derived from Angelica basil as an active ingredient", such as gold and silver flowers, hyssop, Seokgok, Hwanggi, Angelica An antithrombotic composition according to platelet aggregation inhibition is known for an antithrombotic agent (Republic of Korea Patent No. 10-0784339, registration date: December 4, 2007) containing 9 kinds of mixed herbal extracts as active ingredients. On the other hand, Republic of Korea Patent No. 10-0775741 (Registration date: November 5, 2007), a method for preparing an Angelica asiatica extract containing high-purity decursin through a carbohydrate removal process from an ethanol extract of Angelica keis, and a method for preparing an Angelica keis extract and its composition and Korean Patent No. 10-0749233 (registration date: March 7, 2007) for a method of obtaining decursinol in a high concentration roll by heating and extracting after adding an alkalizing agent to the ethanol extract of Angelica oleracea. In addition, Republic of Korea Patent No. 10-0252194 discloses an immune enhancer containing the pectinyl polysaccharide of Angelica keiskei as an active ingredient, and Korean Patent Publication No. 10-2005-0121324 discloses a treatment for dysuria and edema using decursin or decursinol angelate as an active ingredient, In Korean Patent Publication No. 10-2006-0078343 and Patent Registration No. 10-0187881, anticancer composition containing decursin or decursinol angelate as an active ingredient is known. In particular, regarding the cognitive function improvement and neuroprotective action of Angelica keiskei, the composition containing nodakenin having neuroprotective activity in Korean Patent Registration No. 10-0886948, and the hydroxycinnamic acid derivative of Angelica in Patent Registration No. 10-0336182 A composition for preventing and treating dementia containing as an active ingredient, and an Angelica asiatica extract having a neuroprotective action and a pharmaceutical preparation containing the same are known in Korean Patent Publication No. 10-2002-0078850.

As described above, although L-serine and Angelica are known to have a neuroprotective action, Angelica is very expensive, so it is difficult in terms of economics to use it at a concentration showing a neuroprotective effect, and further , L-serine and Angelica have very low organoleptic properties, so there was a problem that they could not be taken as food as they are.

KR 10-1952443 B KR 10-0886948 B

Soyoung Kang, 2003, Seoul National University PhD thesis "Neuroprotective and cognition-enhancing coumarins of Angelica gigas" Kim Pil-seok, 2017, Pusan National University Master's thesis "Strengthening effect of angiogenesis through complex prescription of mesenchymal stem cells and Angelica asiatica extract in stroke"

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to exhibit an excellent neuronal cell death inhibitory effect by a synergistic effect with L-serine and Angelica keis as active ingredients. To provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases that can minimize the use of expensive Angelica keis, and to provide a health functional food with improved sensuality that can be conveniently taken while maintaining the pharmacological effect of the active ingredient will do

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising L-serine and Angelica asiatica extract as active ingredients.

The neurodegenerative disease is preferably amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

In addition, the present invention provides a health functional food for preventing or improving degenerative neurological diseases comprising L-serine and Angelica keis extract as active ingredients.

The neurodegenerative disease is preferably amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

The health functional food is L-serine 61 to 70% by weight, Angelica asiatica extract powder 16 to 25% by weight, brewer's yeast powder 4 to 5% by weight, xylitol 4 to 5% by weight, pineapple flavor powder 2.0 to 2.5% by weight, milk flavor It is preferable to include 2.0 to 2.5% by weight of powder and 2.0 to 2.5% by weight of stevia powder.

L-serine and Angelica basil extract as active ingredients of the composition for inhibiting neuronal cell death of the present invention have excellent efficacy in inhibiting cell death due to oxidative stress of nerve cells, as demonstrated through the examples of the present specification In addition, it exhibits excellent neuronal cell death inhibitory effect while minimizing the amount of expensive Angelica asiatica extract powder used. In addition, by mixing L-serine, which is difficult to ingest due to its low functionality alone, and sub-materials of a sweetener, an acidulant, and a flavoring agent to an angelica powder, a predetermined mixture is prepared to prepare a mixed powder having excellent functionality. The composition for inhibiting neuronal cell death of the present invention has no hemolytic activity against human red blood cells, has excellent thermal stability, and does not show loss of neuronal apoptosis inhibitory effect even under acidic conditions of pH 2 It is a very useful invention to use.

1 shows (1) the composition powder of the present invention, (2) L-serine and (3) Angelica basil powder.
2 is a graph showing the neuronal cell death inhibitory activity of the composition of the present invention.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention developed a composition for inhibiting neuronal cell death that is excellent in safety, functionality, sensibility, portability, accessibility and economic feasibility. The mixture was selected. In addition, the functionality of the powder was improved based on the results of mixing in various composition ratios, and the mixing ratio of L-serine: Angelica asiatica extract is 2.0:1 to 5.0:1, preferably, 2.44:1 to 4.38:1, more preferably was confirmed to have the best functionality in the case of 2.5:1 to 3.8:1.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases comprising L-serine and Angelica basil extract as active ingredients.

The neurodegenerative disease is preferably amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

In addition, the present invention provides a health functional food for preventing or improving degenerative neurological diseases comprising L-serine and Angelica keis extract as active ingredients.

The neurodegenerative disease is preferably amyotrophic lateral sclerosis, Alzheimer's disease or Parkinson's disease.

The health functional food is L-serine 61 to 70% by weight, Angelica asiatica extract powder 16 to 25% by weight, brewer's yeast powder 4 to 5% by weight, xylitol 4 to 5% by weight, pineapple flavor powder 2.0 to 2.5% by weight, milk flavor It is preferable to include 2.0 to 2.5% by weight of powder and 2.0 to 2.5% by weight of stevia powder.

Hereinafter, efficacy experiments of the composition for inhibiting neuronal cell death of the present invention will be described in more detail.

After the composition of the present invention was prepared at various concentrations, and treated with hippocampal neuronal cell HT-22, the apoptosis inhibitory activity against oxidative stress induced by DMNQ (2,3-dimethoxy-1,4-napthoquinone) was evaluated by MTT. As a result of the assay, concentration-dependent neuronal apoptosis inhibitory activity was confirmed, which was confirmed to be a very strong apoptosis inhibitory activity compared to serine-only treatment.

In addition, after dissolving the composition of the present invention, as a result of evaluating various radical scavenging ability and reducing power at a concentration of 0.5 mg/ml, it was confirmed that it exhibited superior DPPH anion scavenging ability and ABTS cation scavenging ability and reducing power compared to serine, and excellent functionality Confirmed.

Therefore, the active ingredient of the present invention can be used as a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases through inhibition of neuronal cell death caused by oxidative stress.

In addition, the active ingredient of the present invention may be used as a pharmaceutical composition for preventing or treating diseases related to oxidative stress.

The diseases may be, for example, cancer, dementia, inflammation and cardiovascular disease, wherein the cardiovascular disease is myocardial infarction, chest pain, shortness of breath, loss of consciousness, stroke, angina, hypertension, headache, dyskinesia, sensory Abnormality, decreased visual acuity, numbness pain, etc. are mentioned.

The pharmaceutical composition of the present invention is formulated in various forms, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injection solutions, according to conventional methods for each purpose of use. It can be formulated and used, and it can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

The pharmaceutical composition may additionally include a carrier, excipient or diluent, and the like, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. and the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per body weight is administered daily or every other day or It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell. In the present invention, the "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as foods and beverages effective for preventing or improving neurodegenerative diseases related to inhibition of neuronal cell death due to oxidative stress.

In addition, the health functional food of the present invention can be used in various ways, such as antioxidant functional food and beverage.

The active ingredient of the present invention can be used for prevention or improvement of diseases related to oxidative stress. The diseases may be, for example, cancer, dementia, inflammation and cardiovascular disease, wherein the cardiovascular disease is myocardial infarction, chest pain, shortness of breath, loss of consciousness, stroke, angina, hypertension, headache, dyskinesia, sensory Abnormalities, blurred vision, chest pain, etc. are mentioned.

Foods containing the composition of the present invention, for example, various foods, beverages, gum, tea, vitamin complexes, health supplements, etc., powder, granules, tablets, capsules or beverages can be used in the form. The active composition of the present invention can generally be added in 0.01 to 15% by weight of the total food weight, and the health beverage composition can be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain, as an additional ingredient, in addition to containing the above compound as an essential ingredient in the indicated ratio, a food pharmaceutically acceptable food supplement additive, such as natural carbohydrate and various flavoring agents. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and common sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, thaumatin, rebaudioside A, glycyrrhizin, saccharin, aspartame, and the like may be used. The ratio of the flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice, and fruit for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The active ingredient of the present invention can be used as a functional cosmetic composition for antioxidant use. Effects according to the antioxidant activity, for example, may include pigmentation improvement, whitening effect, wrinkle improvement, skin elasticity improvement, skin aging prevention, and the like.

The active ingredient of the cosmetic composition of the present invention may be included in an amount of 0.01 to 50% by weight based on the total weight of the composition, preferably 0.1 to 20% by weight, and more preferably 1 to 10% by weight. In this content range, proper formulation stability can be ensured, and the desired antioxidant effect can be expected.

In addition to the active ingredient of the present invention, the composition of the present invention can be used by additionally including known natural extracts or other ingredients for the effects of antioxidant, skin aging prevention and skin regeneration promotion within the range that does not inhibit the effective activity of the present invention. have.

In addition, the composition may contain ingredients commonly added to cosmetic compositions, for example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics, such as fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives; It may be molded into a specific formulation including an adjuvant or carrier commonly used in the field of dermatology.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant- It may be formulated as containing cleansing, oil and spray, but is not limited thereto. More specifically, flexible lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, pact, lip gloss It can be prepared in formulations such as rose, lipstick, shadow, shampoo and conditioner.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.

Hereinafter, a specific method of the present invention will be described in more detail through examples. The following examples are only one preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example 1: Preparation and sensory evaluation of a composition for inhibiting neuronal cell death with excellent sensitivity containing L-serine and Angelica keis extract as active ingredients

In order to develop a composition for inhibiting neuronal cell death with excellent safety, functionality, functionality, portability, and accessibility, the sensory properties of the powder mixed with commercially available L-serine powder and Angelica keis extract in various composition ratios were evaluated several times. The most excellent organoleptic ratio was found between 2.5:1 and 3.8:1 of serine: Afterwards, through the repeated ratio change of the composition to increase the functionality, finally, 61 to 70% by weight of serine, 16 to 25% by weight of Angelica basil extract powder, 4 to 5% by weight of brewer's yeast powder, 4 to 5% by weight of xylitol , pineapple flavor powder 2.0 to 2.5% by weight, milk flavor powder 2.0 to 2.5% by weight, stevia powder 2.0 to 2.5% by weight of the final product recipe was confirmed.

After dissolving 2 g of the final powder product in 8 ml of sterile water, the pH was 5.4, the acidity was 0.14, and the Brix (water-soluble solid content) was 19, and most of the water-soluble solid content was L-serine. On the other hand, in the case of L-serine (20% aqueous solution), pH was 6.3, acidity was 0.26, and Brix (aqueous solids) was ~20.0. 0.02 and brix (water soluble solid content) were found to be 2.8. Therefore, it was confirmed that the sugar/acid ratio of the composition of the present invention was maintained at 13.6, which was the most excellent in functionality (Table 1).

[Table 1] Physicochemical properties of the composition of the present invention, L-serine, and Angelica keiskei

In addition, as a result of color difference analysis of the composition of the present invention, L-serine and Angelica basil powder, as shown in FIGS. 1 and 2, the composition of the present invention has an intermediate brightness between L-serine and Angelica keis (75.39±0.01). , redness (0.26±0.02), yellowness (14.70±0.03) and color difference (21.52±0.03) were shown. However, as a result of color difference analysis of 20% aqueous solution, the composition of the present invention had higher brightness (22.12±0.01), lower redness (-0.92±0.01), yellowness (-2.61±0.03) and higher than L-serine and Angelica keiskei. A color difference (70.40±0.01) was exhibited, indicating a color difference with high functionality. At this time, color difference analysis was measured using a Hunter Color Difference meter (Super color SP-80 Colormeter, Tokyo Denshoku Co., Japan), brightness (white 100 ~ 0 black), redness (red 100 ~ -80 green), Yellowness (yellow 70 to -80 black) was measured. At this time, the chromaticity of the standard white plate was set as L value of 92.44, a value of -0.06, and b value of 1.35. It was measured 3 times per sample and the average value was obtained and expressed. was calculated.

[Table 2] Color difference analysis of the composition of the present invention, L-serine, and Angelica keiskei powder

The composition of the present invention was packaged in a 2g powder stick and sensory evaluation was conducted, and the sensory evaluation was evaluated using a 7-point scale method for 10 food and nutrition experts and 40 consumers in their 20s by classifying them by taste, aroma and overall preference. and the results are shown in Table 3. In this case, as controls, L-serine and Angelica keiskei powder were used.

[Table 3] Sensory evaluation of the final developed composition

As shown in Table 3, L-serine powder showed a value of 3.12 to 3.55 in taste, aroma, swallowing, and residual feeling in the mouth, and also showed 3.2 in overall preference, indicating a preference below normal (standard value of 3.5). it was In addition, due to its unique flavor, strong off-flavor and bitterness, chamomile powder showed values of 1.25 to 2.10 points in taste, aroma, swallowing, and residual feeling in the mouth. state was However, the final developed product showed a high value of 5.65 to 6.05 points in the evaluation of taste, aroma, swallowing, and residual feeling in the mouth, and a high preference of 5.86 points was confirmed in the overall preference. Therefore, it is judged that the composition of the present invention has a low degree of irritation due to low acidity and low sugar content, and has excellent flavor due to the addition of milk flavor and pineapple flavor, and has high sensibility, so that continuous intake is possible.

Example 2: Component analysis of the composition of the present invention

The content of total polyphenols, total flavonoids, total sugars and reducing sugars was measured by analyzing the composition of the composition. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1N NaOH was added again, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method, and total sugar was quantified by phenol-sulfuric acid method.

[Table 4] Components of the composition of the present invention

As a result, as shown in Table 4, the composition of the present invention had a total polyphenol content of 3.1 mg/g and a total flavonoid content of 0.2 mg/g, and the total sugar and reducing sugar content were 30.6 mg/g and 11.2, respectively. mg/g. Therefore, the composition of the present invention shows a low total sugar content, unlike the conventional Angelica product, so that it can be ingested even if you have a chronic disease such as diabetes.

Example 3: Evaluation of neuronal apoptosis inhibitory activity

The neuronal apoptosis inhibitory activity of L-serine and Angelica asiatica extract was evaluated using the hippocampal neuronal cell HT-22 cell line. After the sample was treated with the cell line, DMNQ (2,3-dimethoxy-1,4-napthoquinone) was treated. The degree of neuronal apoptosis in response to oxidative stress was measured by MTT analysis. First, the hippocampal neuronal cell HT-22 cell line was transplanted into 96-well plates at 1.2 × 10 ⁴ cells/well and cultured for 21 hours, then L-serine and Angelica asiatica extract powder of various concentrations were added and cultured 1 hour before. , then DMNQ (12.5 μM) was treated for 20 hours to induce cell death by oxidative stress. Finally, it was mixed with 50 μl of MTT (3-(4,5-dimethyl thiazolyl)2,5-diphenyl tetrazolium bromide) solution (1.1mg/ml) and incubated for 4 hours more. The formed formazan crystal was dissolved in 150 μl of DMSO solution, and the OD was measured at 540 nm using a plate reader. As a result, when only DMNQ was treated, the cell viability was 55% compared to that of the DMNQ untreated group, whereas when various concentrations of L-serine and Angelica keis extract powder were added at the same time, a significant apoptosis inhibitory effect was exhibited (Fig. 2). ). The survival rate of 66% when treated with the concentration combination of L-serine 0.085mg/ml + Angelica asiatica extract 0.015mg/ml, 70% when treated with the concentration combination of L-serine 0.21mg/ml + Angelica asiatica 0.04mg/ml Survival rate, 73% survival rate when treated with a concentration combination of L-serine 0.64mg/ml + Angelica keis extract 0.11mg/ml, L-serine 1.7mg/ml + concentration combination of Angelica asiatica 0.3mg/ml treatment 75 %, the apoptosis of hippocampal neuronal cells HT-22 cells decreased as the concentration of L-serine and Angelica keis extract increased. On the other hand, when L-serine was treated alone (0.1~0.75mg/ml), the neuron survival rate was 56~57%, similar to that of the untreated group. . However, when L-serine was treated at a high concentration of 2 mg/ml, the neuron survival rate was slightly increased to 67%, and it was found that L-serine had a weak apoptosis inhibitory effect when treated with a high concentration of L-serine. The above results suggest that the treatment of serine 1.7mg/ml + Angelica asiatica extract 0.3mg/ml can most effectively inhibit neuronal cell death (FIG. 2).

Example 4: Evaluation of antioxidant activity of the composition of the present invention

The antioxidant activity of the composition of the present invention was evaluated, and the sample was dissolved in DMSO (dimethylsulfoxide) and diluted to an appropriate concentration to obtain DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging ability, ABTS[2,2-azobis ( 3-ethylbenzo thiazoline-6-sulfonate)] was used to measure cation radical scavenging ability, nitrite scavenging ability and reducing power. First, in the case of DPPH scavenging ability, 380 μl of a 2 x 10 ^-4 M DPPH solution dissolved in 99.5% ethanol was added to 20 μl of a sample diluted to various concentrations, mixed, and reacted at 37° C. for 30 minutes, followed by a microplate reader (Asys) at 516 nm. The absorbance was measured using Hitech, Expert96, Asys Co., Austria). The DPPH radical scavenging ability was expressed as a percentage of the sample-added group and non-additive group. In the case of ABTS scavenging activity, 5ml of 7mM ABTS (Sigma Co., USA) and 88ml of 140mM potassium persulfate were mixed, and light was blocked at room temperature for 16 hours to form ABTS cations. Then, this solution was mixed with ethanol so that the absorbance value at 414nm was 1.5. diluted with After mixing 190 ml of the prepared diluted solution and 10 ml of samples prepared at various concentrations, reacting at room temperature for 6 minutes, absorbance was measured at 734 nm, and the ABTS radical scavenging ability was calculated by the following equation.

On the other hand, in the case of measuring nitrite scavenging ability, the sample solution was added to a nitrite solution (1 mM), and 0.1N HCl was added thereto to adjust the pH to 1.2, and after reacting at 37°C for 1 hour, Griess reagent (Sigma Co., USA) was added and mixed. Thereafter, after standing at room temperature for 15 minutes, the absorbance was measured at 520 nm to measure the amount of remaining nitrite. The nitrite scavenging ability (%) was calculated by the following formula.

In the case of evaluation of the reducing power of the composition, it was measured by modifying the method of Oyaizu et al. (J. Life Sci. 21: 576-583, 2011). 2.5ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5ml of 10% potassium ferricyanide were added to 2.5ml of sample dissolved in ethanol, and after reacting at 50℃ for 20 minutes, 2.5ml of 10% trichloroacetic acid was added to terminate the reaction and centrifuged at 4,000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was diluted two-fold with distilled water, mixed with a freshly prepared 0.1% ferric chloride solution at a ratio of 5:1 (v/v), and absorbance was measured at 700 nm to evaluate. In the antioxidant experiment, vitamin C (Sigma Co., USA) was used as a control, and DMSO was used as a solvent control. Each activity evaluation was expressed as the mean and deviation of each repeated experiment 3 times (Table 5).

[Table 5] Antioxidant activity of the composition of the present invention

As a result, as shown in Table 5, the composition of the present invention exhibited good DPPH scavenging ability, ABTS scavenging ability and reducing power, and these radical scavenging ability and reducing power will contribute to inhibition of oxidative stress in nerve cells and alleviation of other oxidative stress. is judged

Example 5: Evaluation of the human hemolytic activity of the composition of the present invention

The main components of the composition of the present invention, L-serine and Angelica asiatica extract, have been registered as food raw materials and have been used for food and medicine since the past, and it is judged that there is no specific toxicity. In addition, added sweeteners, acidulants, flavoring agents, etc. are all listed as food additives, so it is judged that stability is secured.

In order to evaluate the acute toxicity of the composition of the present invention, human hemolytic activity was evaluated, and the results are shown in Table 7. At this time, the hemolytic activity was evaluated according to the existing report (Korean J. Microbiol. Biotechnol. 42: 285-292, 2014). Simply, 100 μl of human red blood cells washed three times with PBS was added to a 96-well microplate and various concentrations were 100 μl of the sample solution was added, followed by reaction at 37° C. for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. . DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 6] Human erythrocyte hemolytic activity of the composition of the present invention

First, it was confirmed that DMSO and distilled water used as controls did not have red blood cell hemolytic activity, and Triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. In the case of empoteracin ratio, which is used as an antifungal and anticancer agent, a strong hemolytic activity of 85.3% was shown even at a concentration of 0.025 mg/ml. On the other hand, hemolytic activity did not appear even when the composition of the present invention and L-serine were treated at 10 mg/ml. Therefore, it is judged that the composition of the present invention will not show a separate problem of causing acute toxicity.

Claims (5)

delete delete delete A functional cosmetic composition for preventing skin aging by antioxidant activity comprising 61 to 70% by weight of L-serine and 16 to 25% by weight of Angelica basil extract powder as active ingredients. delete

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