KR101991926B1 - Composition for improving skin moisturization comprising royal jelly - Google Patents
Composition for improving skin moisturization comprising royal jelly Download PDFInfo
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- KR101991926B1 KR101991926B1 KR1020140125598A KR20140125598A KR101991926B1 KR 101991926 B1 KR101991926 B1 KR 101991926B1 KR 1020140125598 A KR1020140125598 A KR 1020140125598A KR 20140125598 A KR20140125598 A KR 20140125598A KR 101991926 B1 KR101991926 B1 KR 101991926B1
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- Prior art keywords
- royal jelly
- skin
- weeks
- protease
- food
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L21/00—Marmalades, jams, jellies or the like; Products from apiculture; Preparation or treatment thereof
- A23L21/20—Products from apiculture, e.g. royal jelly or pollen; Substitutes therefor
- A23L21/25—Honey; Honey substitutes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/40—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by drying or kilning; Subsequent reconstitution
- A23L3/44—Freeze-drying
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/20—Ingredients acting on or related to the structure
- A23V2200/23—Humectant
Abstract
The present invention provides an oral food composition and a cosmetic composition for enhancing skin moisturizing, comprising a freeze-dried royal jelly, or a freeze-dried royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient. . In the present invention, when the royal jelly is ingested in humans, by confirming the skin moisturizing enhancement effect, the composition according to the present invention can be usefully used as an oral health food or cosmetic composition having an excellent moisturizing enhancement effect without allergies or side effects.
Description
The present invention relates to oral food compositions and cosmetic compositions for improving skin moisturizing, including a freeze-dried royal jelly, or a freeze-dried royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient. .
With the development of medicine, increased income and improved quality of life, there is a growing interest in improving health and maintaining beauty, and there is a growing social demand for food ingredients that can be safely consumed for a long time.
In order to contribute to the health of the people and contribute to the national economic development, the health functional food law was enacted in 2003, and as of 2010, the health functional food market in Korea exceeded 1 trillion won, and the individual certified health functional food material market was more than 15 times ( Compared to 2006).
However, royal jelly, which has been treated as a precious medicine for a long time and has various efficacy reports, has been excluded from health food general notification materials due to the lack of scientific efficacy verification, which is contrary to the activation of the functional food industry.
In addition, domestic royal jelly production is greatly shrunk due to the import of cheap Chinese royal jelly due to the full-fledged opening of agriculture such as FTA, and is facing difficulties in converting high value-added industries such as systematization of production and lack of product diversification.
Royal jelly is widely recognized as a nourishing tonic, with reports on the prevention of hyperlipidemia and atherosclerosis, fatigue, relieving liver and kidney toxicity, anti-aging, antioxidant, immune enhancement, antidiabetic, anti-cancer, anti-inflammatory, neuroprotective. However, on the other hand, allergy-induced allergy-induced specific protein of royal jelly has been reported. In addition, the scientific evidence supporting the positive efficacy of royal jelly is limited in vitro, and there are no reports on skin efficacy in humans.
In addition, the cosmetic composition for improving the skin condition is composed of a formulation that is applied to most of the skin, there was a cumbersome and sticky inconvenience when applied to the skin. In addition, there is a problem in that the portability is lowered and the efficacy of lowering the body absorption rate when applied to the skin.
Therefore, there is a need for the development of oral royal jelly excellent in improving the absorption of the body while improving the skin moisturizing effect while reducing allergy induction.
Accordingly, the present inventors have been found to be effective in improving skin moisturizing and antioxidant activity by ingesting royal jelly and allergy-induced protein-treated royal jelly by protein and carbohydrate degrading enzymes for healthy men and women who are in their 30s and older as skin aging progresses. The present invention was completed by convincing the possibility of developing into a functional dietary material for skin health. In addition, we analyzed the changes in sebaceous membrane lipids including ceramide, which is a biomarker of moisturizing, and free amino acid, which is a major natural moisturizing factor, to confirm the mechanism for enhancing skin moisturizing. Newly announced.
An object of the present invention is to provide a food composition for promoting skin moisturizing.
Still another object of the present invention is to provide a cosmetic composition for improving skin moisture.
In order to solve the above problems, the present invention is an oral food for improving skin moisturizing, including a freeze-dried royal jelly, or a freeze-dried royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient. To provide a composition.
In addition, the present invention provides an oral cosmetic composition for improving skin moisturizing, comprising a freeze-dried royal jelly, or a freeze-dried royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient.
Royal jelly contained in the composition of the present invention is orally ingested in an amount of 400mg to 700mg / day (day).
Royal jelly included in the composition of the present invention is included in 30 to 50% by weight based on the total weight of the composition.
Lyophilized royal jelly contained in the composition of the present invention in an oral intake for 4 to 6 weeks in an amount of 400mg to 700mg / day, lyophilized form treated with protease and non-starch polysaccharide degrading enzyme Royal jelly can be taken orally for 4 to 12 weeks in an amount of 400 mg to 700 mg / day.
The royal jelly of the lyophilized form included in the composition of the present invention includes the freeze-dried raw royal jelly with 60 to 70% moisture.
Lyophilized royal jelly treated with the protease and non-starch polysaccharide degrading enzyme contained in the composition of the present invention is treated with raw royal jelly diluted with water, and the starch polysaccharide degrading enzyme is treated at 45 ° C. Enzymatically reacting at a temperature of about 55 ° C .; And inactivating the enzymatically reacted royal jelly, followed by lyophilization.
In the present invention, the protease is derived from a proline type endo protease derived from Aspergillus niger , pepsin derived from pig, and Aspergillus oryzae . Exo-type proteolytic enzymes.
In the present invention, the non-starch polysaccharide degrading enzyme is a pectinase derived from Aspergillus niger , cellulase, hemicellulase, and beta-glucosidase (β-glucosidase). ).
Food in the food composition of the present invention includes a health functional food.
Royal jelly used in the present invention includes domestic royal jelly.
Lyophilized royal jelly used in the present invention has an effect of increasing the sebum membrane lipid content of the skin to promote skin moisturization and to increase epidermal thickness.
Lyophilized royal jelly treated with the protease and non-starch polysaccharide degrading enzyme used in the present invention has the effect of promoting the moisturizing of the skin through the change in the content of lactic acid and free amino acids.
In the present invention, when the royal jelly is ingested in humans, the skin moisturizing enhancement effect is shown, the skin sebaceous lipid content is changed, it was confirmed that the epidermal thickness is increased. Therefore, the composition according to the present invention can be usefully used as an oral health food or cosmetic composition having an excellent moisturizing enhancement effect without allergies or side effects. In addition, ingestion of the lyophilized royal jelly (RJ1), which is an active ingredient of the composition, promptly promotes moisturization at 5 weeks, while the degree of moisturizing is moderate, while ingestion of enzyme-treated lyophilized royal jelly (RJ2) is used. Moisture-promoting effect is not rapid, but 10-week long-term intake, the effect of the moisturizing enhancement is greater than the RJ1 intake can be developed RJ1 and RJ2 as a functional food material for the purpose of short-term or long-term moisturizing enhancement have.
FIG. 1 shows the histological appearance of the skin through H & E staining (A) and Messon trichrome staining (B) in each experimental group. Subjects were aged 30-59 years after placebo (placebo group, placebo group), lyophilized royal jelly (RJ1 group) and enzyme-treated lyophilized royal jelly (RJ2 group) for 10 weeks. Skin samples were taken from the inside of the arm and stained with H & E (A) and mesone trichrome (B).
Figure 2 is a total glass extracted by using a skin patch (Skin Patch) after 30-59 years old subjects to placebo (placebo group, placebo group) and enzyme-treated lyophilized royal jelly (RJ2 group) for 10 weeks. Amino acid content was measured with L-amino acid quantitation colorimetric / fluorometric kit.
The present invention is a food or cosmetic composition for skin moisturizing enhancement comprising a royal jelly in lyophilized form, or a lyophilized royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient, which is included in the composition. Royal jelly relates to a composition that is orally ingested in an amount of 400 mg to 700 mg / day.
In the present invention, the lyophilized royal jelly or lyophilized royal jelly treated with protease and non-starch polysaccharide degrading enzyme was ingested in a healthy adult having dry skin, and the skin moisturizing enhancement effect was confirmed.
In the past, royal jelly contained allergen-producing proteins, so it was limited to be used for diet, and the efficacy of royal jelly was studied only through animal models. The study is insufficient. Furthermore, the inventors of the present invention, for the first time to confirm that the royal jelly with allergen-induced protein is removed in an oral dosage form, a significant skin moisturizing enhancement effect when dietary intake to people with dry skin.
In the present invention, the dosage and administration of the royal jelly of the lyophilized form or the royal jelly of the lyophilized royal jelly treated with protease and non-starch polysaccharide degrading enzyme has the best mechanism and efficacy of enhancing skin moisturizing efficacy. By confirming the period, it was confirmed that royal jelly can be safely used as a food or cosmetic composition.
Specifically, royal jelly included in the composition is preferably included in 30 to 50% by weight based on the total weight of the composition. More preferably, it is included in 35 to 45% by weight. In one embodiment of the present invention was prepared oral soft capsules having a skin moisturizing effect, specifically lyophilized royal jelly, or lyophilized royal jelly treated with protease and non-starch polysaccharide degrading enzyme 41.6 It was to be included by weight.
In addition, the lyophilized royal jelly contained in the composition of the present invention is preferably ingested orally for 4 to 6 weeks in an amount of 400 mg to 700 mg / day, and treated with protease and non-starch polysaccharide degrading enzyme. One lyophilized royal jelly is preferably taken orally for 4 to 12 weeks in an amount of 400 mg to 700 mg / day.
In the present invention, in the case of freeze-dried royal jelly, after about 5 weeks, the moisturizing enhancement effect appeared quickly, and after the ingestion, it was confirmed that the degree of moisturizing enhancement was moderate, and in the case of long-term intake of the freeze-dried royal jelly after enzymatic treatment It was confirmed that the effect of the moisturizing enhancement was greater than that of the royal jelly that was only freeze-dried. Through this, it is possible to utilize the active ingredient of the composition of the present invention for the purpose of promoting short-term or long-term moisturizing.
Lyophilized royal jelly contained in the composition of the present invention as an active ingredient can be obtained by lyophilizing the raw royal jelly with 60 to 70% moisture. Preferably it is lyophilized to a level of 64 to 69%.
Lyophilized royal jelly treated with the protease and non-starch polysaccharide degrading enzyme included in the composition of the present invention is treated with raw royal jelly diluted with water, and the non-starch polysaccharide degrading enzyme. Enzymatic reaction at 45 ° C. to 55 ° C. conditions; And inactivating the enzymatically reacted royal jelly and then lyophilizing.
Specifically, proline-type endo type protease derived from raw royal jelly, Aspergillus niger diluted in water, pepsin derived from pig, and Aspergillus oryzae Exo-type proteolytic enzymes derived from c) and pectinase, cellulase, hemicellulase, and beta-glucosidase derived from Aspergillus niger . treating the non-starch polysaccharide degrading enzyme including (β-glucosidase) with an enzyme reaction at about 50 ° C .; It can be prepared including the step of inactivating the enzymatically reacted royal jelly after lyophilization.
After the deactivation of the enzymatically reacted royal jelly in the manufacturing method may further include a mesh filtration step for removing foreign matters, it may be lyophilized.
In the present invention, the skin moisturizing enhancement means that the moisture content of the skin is increased.
In the present invention, when orally ingested lyophilized royal jelly or enzyme-treated lyophilized royal jelly, general blood components including indicators such as hepatotoxicity, blood sugar, immunity, etc. are all determined in the normal range, the composition of the present invention Can be applied to human body without side effects.
In addition, lyophilized royal jelly was found to significantly increase the content of skin sebaceous membrane lipids, in particular cholesterol, free fatty acids, ceramides, thereby improving the moisturizing effect of the skin. In addition, it was confirmed that the effect of promoting the thickness of the epidermis, which is known to decrease as skin aging progresses.
In addition, the royal jelly lyophilized after the enzyme treatment enhances the content of vitamin C in the blood, it was confirmed that the natural moisturizing factor, in particular, the change in the content of free amino acid to improve the moisturizing. In other words, the royal jelly lyophilized after the enzyme treatment can enhance the moisturizing of the skin without side effects by increasing the content of the natural moisturizing factor without changing the lipid.
The food composition may include other foods or ingredients other than lyophilized royal jelly, or lyophilized royal jelly treated with protease and non-starch polysaccharide degrading enzyme as an active ingredient. The mixing amount of the active ingredient can be appropriately determined depending on the intended use.
The oral food composition of the present invention may be prepared by various methods known in the food or pharmaceutical arts, and may be ingested by itself or mixed with a food acceptable carrier, excipient, diluent or the like in any food form to be ingested orally. Can also be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the extract of the present invention, for example, starch, calcium carbonate, sucrose (Sucrose), lactose (Lactose) or gelatin can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various carriers, excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. Can be.
Preferably, the food composition of the present invention may be made of any one type selected from powder, granule, capsule, ring or liquid.
There is no particular limitation on the type of food of the present invention. Examples of foods to which the royal jelly can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, teas, drinks , Alcoholic beverages and vitamin complexes, etc., and may include all foods in a conventional sense, and include foods used as feed for animals.
In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonic acid used in carbonated beverages. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. In addition, the food may also be prepared in the form of tablets, granules, powders, capsules, liquid solutions and pills according to known production methods. There is no particular limitation on other components except including royal jelly according to the present invention as an active ingredient, and may include various conventional flavoring agents or natural carbohydrates as additional ingredients.
The food composition of the present invention includes a dietary supplement.
In the present invention, a health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition which has added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to fully express the gymnastics function on recovery. The dietary supplement may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of dietary supplements.
In one embodiment of the present invention, oral capsules were prepared, including 41.6% royal jelly, 56.4% soybean oil, 1% lead, 1% soy lecithin, and confirmed the effect of enhancing moisturizing.
The cosmetic composition of the present invention may include other ingredients that can give a synergistic effect of the main effect within the range that does not impair the main effect of the present invention, for example, fragrances, pigments, It may further include additives such as fungicides, antioxidants, preservatives, humectants, thickeners, inorganic salts, emulsifiers, synthetic polymers, etc., and the amount of the additives may be selected within a range that does not impair the object and effect of the present invention. Can be.
The ingredients can be formulated by a person skilled in the art according to the formulation or purpose of use without difficulty, preferably in order to have an excellent skin moisturizing effect, oral cosmetic composition, more preferably powder, granules, capsules or liquids It is made of any one type selected from the diet can be used for diet to enhance the skin moisturizing effect through ingestion.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.
Experimental Example 1: Study approval and target
This study was approved for the application of human body at Kyung Hee University Hospital in November 2013 (approval number: KMC-IRB-1331-02). Healthy men and women aged 30-59 years with dry skin and Fitzpatrick type Ⅲ and Ⅳ skin during the period of November 2013-January 2014 can fully understand and cooperate with the purpose and content of this study. A total of 52 subjects were recruited and selected. Exclusions were based on general exclusion criteria such as chronic diseases and drug users including hypertension, diabetes, arthritis, etc. In particular, skin diseases such as atopic dermatitis, psoriasis, vitamins, minerals, and lipid supplements Health functional food users were excluded.
Experimental Example 2: lyophilized form Royal jelly And Enzyme-treated Royal jelly secure
Lyophilized royal jelly and enzyme-treated lyophilized royal jelly used in this experiment were supplied by Samyang Genex Food Research Institute. Specifically, lyophilized royal jelly was quenched and lyophilized after mesh filtering the raw royal jelly to remove foreign substances, and the lyophilized royal jelly was enzymatically treated to the raw royal jelly of food additive standard. 3 kinds of proteolytic enzymes for food [Brewers Clarex, DSM Food Specialties: Proline type endoproteinase derived from Aspergillus niger , BC Pepsin 1: 3000 (Biocatalysts): Pepsin from pigs, Prozyme 2000P (Vision Biochem): exo-type protease derived from Aspergillus oryzae and non-specialized polysaccharides lyase [Tora between the (Cytolase) PCL5 (DSM Food Specialties ): aspergillus you ger the Pectinase dehydratase (pectinase), cellulase (cellulase), hemicellulase (hemicellulase) derived from a (Aspergillus niger), A beta-glucosidase (β-glucosidase)] by treating to deactivate the enzyme by heating after 12 hours the enzyme reaction at 50 ℃ condition. The inactivated enzymes were then filtered through a mesh to remove foreign substances and then rapidly frozen and lyophilized.
Experimental Example 3: Experimental group And intake method
Subjects who signed the agreement and were judged eligible to participate in the study were asked to consume the product provided for 10 weeks in a double-blind manner as directed. The state of ingestion was checked weekly to ensure that the plan of the trial was carried out correctly. When dose compliance was below 75%, the subjects were alerted for compliance, and when compliance was below 60%, non-compliance was classified and dropped. The specific experimental group was as follows.
3.1: Placebo group ( Placebo Group )
Placebo soft capsules 2 capsules (1 day) containing no royal jelly extract was taken for 10 weeks.
625mg / capsule / time x 2 times (morning and evening) = 1250mg / 1 day for 10 weeks
[Placebocapsule content: 41.6% corn starch, 56.4% soybean oil, 1% lead, 1% soy lecithin]
3.2: Lyophilization Royal Jelly ( RJ1 Group )
2 capsules / day of soft capsules containing lyophilized royal jelly were taken for 10 weeks.
625mg / capsule / time x 2 times (morning and evening) = 1250mg / 1 day for 10 weeks
[Freeze Dried Royal Jelly Soft Capsule Content: Freeze Dried Royal Jelly 41.6%, Soybean Oil 56.4%, Sacrose 1%, Soy Lecithin 1%]
3.3: Enzyme Freeze Drying Royal Jelly ( RJ2 Group )
Enzyme-treated lyophilized royal jelly 2 soft capsules / day was taken for 10 weeks.
625mg / capsule / time x 2 times (morning and evening) = 1250mg / 1 day for 10 weeks
[Enzyme-treated lyophilized royal jelly Soft capsules: Enzyme-treated lyophilized royal jelly 41.6%, soybean oil 56.4%, lead 1%, soy lecithin 1%]
Experimental Example 4: general questionnaire
Questionnaires related to the general health and skin health of the subjects were administered twice every 0 and 10 weeks.
4.1: Paperweight on rejection
Subjects were questioned about the rejection of ingestion twice every 5 and 10 weeks.
4.2: Blood Analysis
Blood samples were collected twice a week from 0 and 10 weeks and requested to analyze general health status (liver toxicity, blood glucose, lipids, etc.) at Kyunghee University Hospital.
Experimental Example 5: Probe skin condition (moisturizing, Oil , PH) measurement
Moisturize, oil and pH the back side of neck at
Experimental Example 6: Autofluroscence (AF) Used skin Antioxidant activity Skin Antioxidant Measurement
Skin antioxidant activity was measured in a non-invasive way using an Ecoskin Fluoroscence Video Dermascope autofluorescence instrument. At excitation (408 nm) and recording (530 nm: G wavelength) on the outer and inner side of the forearm, which are UV and non-exposed areas at 0 and 10 weeks. The autofluorescence of the skin was measured to analyze the intensity (G peak) of the autofluorescence and the change in the pattern (total area of the G spectrum).
Experimental Example 7: skin sensitivity measurement ( UVB Erythema index measurement after irradiation)
At
Experimental Example 8: Determination of Antioxidant Vitamins, Lipid Peroxides and Antioxidant-related Enzyme Activities in Blood
At 0 and 10 weeks, 1 mL of blood was collected in a test tube treated with heparin, an anticoagulant, in a fasting state, and centrifuged at 3000C for 4 minutes to obtain plasma. Vitamin C and vitamin E concentrations in plasma were analyzed using High Performance Liquid Chromatography (HPLC) along with the column used in the previous study and the mobile phase required for each separation.
5% metaphosphoric acid was added at a ratio of 1: 4 (v / v) to the plasma obtained for vitamin C concentration measurement. Vitamin C concentrations were analyzed using high performance liquid chromatography (HPLC). The C18 ODS column (250 × 4.60 mm; particle size 5 μm; Beckman) was used, and the mobile phase was 0.05 M sodium phosphate, 0.05 M sodium acetate, A mixture of methanol and H 2 O containing 189 μM dodecyltrimethyl ammonium chloride, 3.66 μM tetraoctylammonium bromide (30:70, v / v, pH 4.8) Was carried out at a rate of 1.0 mL / min and quantified using a stoichiometric detector.
50 μl of internal standard α-tocopherol acetate (50 μg / ml) dissolved in acetonitrile in 200 μl of plasma for measurement of vitamin E concentration and chloroform: methanol ( 2: 1) 750 [mu] l was added and mixed well throughout using a vortex mixer. The lower layer obtained after centrifugation at 6000 rpm for 7 minutes was taken, dried over nitrogen gas, and dissolved in methanol: methanol: dichloromethane = 85:15 (v / v) and measured by HPLC. That is, using a Micro Bondapak C-18 column (300 × 3.90mm; particle size 5μm) and the mobile phase is 1.5ml / acetonitrile: methanol mixture (75:25, v / v) Progress at min speed and quantified using a UV detector (280 nm).
Plasma lipid peroxides (malonedialdehyde) obtained at fasting states at
Malondialdehyde (MDA) produced during the oxidation of blood lipids reacted with blood MDA and thiobarbituric acid (TBA) to develop the product and quantify it at 532 nm to determine the degree of lipid oxidation. 1 ml of plasma is mixed with 0.1 ml of 8.1% sodium dodecyl sulfate, 0.5 ml of 0.8% thiobarbituric acid (TBA) dissolved in 20% sodium acetate (pH 3.5), and 0.15 ml of distilled water at 95 ° C. After heating for 1 hour, the mixture was cooled, and 2.5 ml of n-butanol / pyridine (15: 1, v / v) and 0.5 ml of distilled water were added and mixed. After centrifugation at 3.000 rpm for 10 minutes, the upper layer was taken, and the absorbance at 532 nm was measured using KCl as a control.
In order to measure SOD activity, the plasma of which red blood cell concentration was measured was washed with physiological saline and then hemolyzed by adding 1.5-fold distilled water. Dilute 20 µl of the lysate with 3 ml of Drabkins solution, and leave for 10 minutes. Add 0.5 ml of hemolysate, add 3.5 ml of cold distilled water, 1.0 ml of ethanol, and 0.6 ml of chloroform. And 3000 rpm, centrifuged for 2 minutes. The obtained supernatant was divided into various concentrations, left at 25 ° C. for 10 minutes, 20 ml pyrogallol was added, and pyrogallol oxide was produced at 320 nm.
Catalase activity was measured by adding 50 mM phosphate buffer (pH 7) and hydrogen peroxide to hydrogenated erythrocytes, and then reducing the amount of hydrogen peroxide at 240 nm for 30 minutes.
GSH-Px activity was reduced by adding glutathione, glutathione reductase and NADPH to hemolyzed erythrocytes, and leaving it at 37 ° C for 10 minutes before reacting with t-butylhydroperoxide. NADPH concentration was measured for 90 seconds at 340 nm to determine the antioxidant level of GSH-Px.
Experimental Example 9: Sebaceous membrane Lipid analysis
Each subject stayed in a space where temperature (22-24 ° C) and humidity (55-60%) were maintained for 30 minutes, and then tape-strip the inside of the subject's forearm (Tape 1601, Skin lipids and proteins were collected twice every 0 and 10 weeks using Handeight Enterprise, Wickford, essex, UK). The tape-strip was immersed in a Polch's solution (Chloroform / Methanol 2: 1, v / v) for 2 hours, lipid was extracted using sonication, and the same amount of 0.1M KCl solution was used. After the addition, centrifugation was performed at 3000 rpm for 5 minutes. A portion of the separated upper layer was protein quantified using albumin as standard. Separate lower layer is N 2 After drying with gas and dissolving in 100 μl of Folch solution, various lipids in the stratum corneum were fractionated by using the solvent used in the pre-test in High Preformance Thin Layer Chromatography (HPTLC). Expressed as μg protein.
Experimental Example 10 Analysis of sebum film total free amino acid content
After extracting amino acids from the skin using tape strips (22-mm D-SQUARE Tape; Cu-Derm Co., USA) on the fore arm, chloroform / Methanol (2 : 1, v / v)) solution was extracted using sonication for 2 hours. The amino acid collected from the tape was measured by using distilled water to separate the layers, and then using L-amino acid quantitation colorimetric / fluorometric kit (Biovision Co., USA). Take 5 μL of standard and extract for each concentration and dispense into 96 well plate, and then 45 μL of assay buffer and 50 μL of master reaction mix (assay 46 μL of buffer), 2 μL of probe (pprobe) and 2 μL of enzyme mix were added and mixed well. Afterwards, a 96 well plate was wrapped in foil to block light and reacted at 37 ° C. for 30 minutes, followed by ELISA reader (VICTOR ™ X3, PerkinElmer Co., Singapore) at 535/590 nm. Measured.
Experimental Example 11: Biopsy Tissue Acquisition and Histological Examination
At
Experimental Example 12: Statistical Analysis
Descriptive statistics of all data obtained from human trials were conducted using the SPSS 18.0 program (SPSS Inc., Chicago, IL, USA). Data from all subjects were performed as a two-sided test at the significance level of 0.05 using a paired T test of measurement results before and after ingestion (weeks 0-5 or 0-10 weeks). The number of final subjects in each group after the 10-week intake period is not the same (placebo group: n = 14, RJ1 group: n = 17, RJ2 group: 16). Absolute change in each measurement parameter. Significance between groups was determined using ANOVA and Tukey's honestly significance difference (HSD) for unequal sample size.
Example 1: Obtain test subjects
The first subjects selected based on the exclusion criteria of the human application test with the consent form were obtained: 19 placebo groups, 17 lyophilized royal jelly groups (RJ1 group), and lyophilized royal jelly groups (RJ2 group). A total of 52 people were 16 (see Table 1).
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SD
During ingestion period, 2 patients at 1-5 weeks, 3 patients at 6-10 weeks, and 5 patients were dropped out. The reason for this was subjective judgment of no efficacy of the food. The final subjects after ingestion were 47 in placebo group, 17 in RJ1 group and 16 in RJ2 group. The mean age of each group was in the early 40's, and there was no significant difference between the groups. The Body Mass Index (BMI) was 21-23 (kg / m 2 ), which was normal.
Example 2: Royal jelly General blood analysis and rejection before and after ingestion
All common blood components, including indices such as hepatotoxicity and blood glucose, in the placebo group and RJ1 and RJ2 groups before and after ingestion were measured in the normal range (see Table 2).
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SEM
3 Significance before and after ingestion (week 0) by student's paired t-test
At week 5 and
Example 3: Royal jelly Moisturizing the skin by ingestion, Oil And acidity changes
At
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SEM
3 disposed one won ANOVA (one way ANOVA) and the significance between groups by Tukey's honestly significance difference (HSD) for unequal sample size
4 Significance before and after ingestion (week 0) by student's paired t-test
As a control group, there were significant differences between the groups of skin moisturizing changes in RJ1 and RJ2 groups at week 5 and
The oil content of the skin did not change between groups. Healthy skin is slightly acidic and is known to increase acidity with disease development (Behne MJ, Barry NP, Hanson KM et al. J Invest Dermatol 120 (6): 99801006 (2003)). Intake of RJ1 and RJ2 groups significantly decreased acidity at
Example 4: Royal jelly Skin by ingestion Antioxidant activity change
Tryptophan, NADH, flavin, porphyrin, keratin, collagen, elastin, etc., present in the skin release autofluorescence in the skin (Portugal-Cohen M , Soroka Y, Frui-Zlotkin M et al. Exp Dermatol 20: 749-755 (2011)), and their analysis of autofluorescence differences is an indicator of the degree of oxidation and antioxidant activity of skin depending on the surrounding micro-hazardous environment. It can be used as. These substances in the skin can be monitored primarily in the green (G) visible light region at 530 nm wavelength. The sensitivity of self-fluorescence was measured by measuring the antioxidant capacity of the inner arm and the outer arm which are almost irradiated with UV radiation using Ecoskin self-fluorescence measuring device. Both G values) and area% of the G full spectrum showed no change between groups (see Table 4).
Erythema index (Mj / ㎠)
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SEM
3 disposed one won ANOVA (one way ANOVA) and the significance between groups by Tukey's honestly significance difference (HSD) for unequal sample size
4 Significance before and after ingestion (week 0) by student's paired t-test
As shown in Table 4, there was no change in the erythema index, which is an indirect indicator of skin sensitivity and skin antioxidant activity.
Example 5: Royal jelly Blood by ingestion Antioxidant activity change
Antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and lipid peroxide (malonedialdehyde: MDA) increased when antioxidant activity decreased As a result of the content determination, there was no significant difference between groups of all these measurements (see Table 5).
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SEM
3 disposed one won ANOVA (one way ANOVA) and the significance between groups by Tukey's honestly significance difference (HSD) for unequal sample size
4 Significance before and after ingestion (week 0) by student's paired t-test
The blood concentration of vitamin E, a fat-soluble antioxidant vitamin, was increased in all groups, and there was no significant difference between the groups. On the other hand, the blood concentration of vitamin C, a water-soluble antioxidant vitamin, was significantly increased at
Example 6: Royal jelly By ingestion Sebaceous membrane Change in lipid content
Lipids in the sebaceous membrane, the outermost layer of the epidermis, are a mixture of triglyceride (TG), cholesterol (cholesterol (Chol)), free fatty acid (FFA), and ceramides (Cer). Among them, FFA and Chol are produced and secreted in both the epidermis and sebaceous glands, TG is produced in the sebaceous glands and Cer is produced only in the epidermis, respectively (Clarys P, Barel A. Clin dermatol 13: 307-321 (1995)).
After extraction using skin patch, the lipid changes of sebaceous membrane were identified through HPTLC. Total lipids were significantly increased only in RJ1 group, and there was no change in placebo and RJ2 groups before and after ingestion. 6).
(Μg / μg protein)
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SEM
3 disposed one won ANOVA (one way ANOVA) and the significance between groups by Tukey's honestly significance difference (HSD) for unequal sample size
4 Significance before and after ingestion (week 0) by student's paired t-test
There was no significant change in all sebaceous lipids (TG (triglycerides), Chol (cholesterol), FFA (free fatty acids) and Cer (ceramide)) in the placebo and RJ2 groups after the 10-week intake period. On the other hand, in the RJ1 group, Chol (p = 0.06, see Table 6), FFA (p = 0.001, see Table 6) and Cer (p = 0.000, see Table 6) of sebaceous membrane lipids significantly increased. p = 0.013, see Table 6) The Cer content (p = 0.000, see Table 6) was significant between the groups. Chol is a lipid that is produced and secreted from both the epidermis and sebaceous glands, whereas ceramide is a lipid produced and secreted only from the epidermis and the folding area inside the arm where biopsy is taken is not an area of sebaceous gland development. Increased skin moisturization is due to increased ceramide production and cholesterol, which are the major constituent lipids of the sebaceous membrane, which play a major role in maintaining the sebum membrane's lamella integrity.
On the other hand, despite the increase of vitamin C concentration in blood, which is suggested as an indirect indicator of moisturizing with skin moisturization and ceramide de novo production after RJ2 ingestion for 10 weeks, all sebaceous lipid content did not change. It suggests the possibility of causing changes in Natural Moisturizing Factor (NMF), another biomarker of skin moisturizing rather than lipids. Natural moisturizers are mixtures containing lactic acid, free fatty acids and free amino acids (Rawlings AV, Harding CR Dermatol Ther 17 (suppl 1): 43-48 (2004), Verdier-Svrain S, Bont F. J Cosmet Dermatol 6 ( 2): 75-82 (2007)), free fatty acids showed no change after RJ2 intake. Intake of RJ2 suggests that it may lead to a change in the content of natural moisturizing factors, such as free amino acids, rather than lipid components such as ceramides and free fatty acids, ultimately enhancing moisturization.
Example 7: Sebaceous membrane total free amino acid content analysis
(nmol / μg protein)
1 RJ2: Enzyme treated lyophilized royal jelly intake group
2 Mean ± SEM
3 disposed one won ANOVA (one way ANOVA) and the significance between groups by Tukey's honestly significance difference (HSD) for unequal sample size
4 Significance before and after ingestion (week 0) by student's paired t-test
The total free amino acid content extracted using the Skin Patch was measured using L-amino acid quantitation colorimetric / fluorometric kit. The total free amino acid content was little changed. On the other hand, the total free amino acid content of the RJ2 group was significantly increased (p = 0.029, Table 7 and Figure 2), and as a result showed a significant difference between the groups (p = 0.042, Table 7). Total free amino acids, along with ceramides as the skin's main natural moisturizing factor, are the major biomarkers of skin moisturization. Total free amino acids significantly increased by RJ2 intake means that the increase in moisturization by RJ2 ingestion was due to an increase in the content of free amino acids, as well as the results of content of ceramide and free fatty acids that did not change before and after RJ2 intake.
Example 8: Royal jelly Histological changes due to ingestion
The thickness of the epidermis varies by body part (75-150 μm), but this decreases with aging. In addition, skin aging is known to cause the production and secretion of collagen, a collagen protein, in the dermis. The thickness of the stratum corneum, the outermost layer of the epidermis measured after H & E staining in the biopsy tissues obtained from volunteers in each group did not change, but the thickness of the epidermis was significantly increased compared to the placebo group (Fig. 1 and Table 8). On the other hand, the density of collagen fibers in the dermis, which was examined by Masson trichrome staining, was increased in the RJ2 group.
1 RJ1: lyophilized royal jelly group, RJ2: enzyme-treated lyophilized royal jelly group
2 Mean ± SD
* p <0.05, significance with placebo group by student's unpaired t-test
Although the above description has been made based on the embodiments, these are merely examples and are not intended to limit the present invention. Those skilled in the art to which the present invention pertains may not have been exemplified above without departing from the essential characteristics of the present embodiments. It will be appreciated that many variations and applications are possible. For example, each component specifically shown in the embodiment can be modified. And differences relating to such modifications and applications will have to be construed as being included in the scope of the invention defined in the appended claims.
Claims (13)
The protease is a proline type endo protease derived from Aspergillus niger , pepsin derived from pig, and Aspergillus oryzae Exo type protease,
The non-starch polysaccharide degrading enzyme is a pectinase derived from Aspergillus niger , cellulase, hemicellulase, and beta-glucosidase. sign,
Food composition for promoting skin moisturization by increasing the free amino acid content of the skin.
Treating the raw royal jelly in water diluted with proteolytic enzymes and non-starch polysaccharide degrading enzymes at 45 ° C. to 55 ° C .; And
It is a royal jelly prepared by inactivating the enzyme-activated royal jelly and then lyophilized food composition for skin moisturizing.
As a method of manufacturing a food composition for enhancing skin moisturizing by increasing the free amino acid content of the skin, including the step of inactivating the enzyme-reacted royal jelly after lyophilization,
The protease is a proline type endo protease derived from Aspergillus niger , pepsin derived from pig, and Aspergillus oryzae Exo type protease,
The non-starch polysaccharide degrading enzyme is a pectinase derived from Aspergillus niger , cellulase, hemicellulase, and beta-glucosidase. sign,
Method for producing a food composition for promoting skin moisturizing by increasing the free amino acid content of the skin.
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| JP6450882B1 (en) * | 2018-06-11 | 2019-01-09 | アピ株式会社 | Moisture transpiration control composition |
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| JP2009029772A (en) * | 2007-07-05 | 2009-02-12 | Api Co Ltd | Enzyme-treated royal jelly, and antioxidant, moisturizer, skin fibroblast proliferation promoter, hypotensive, fatigue-relieving agent and calcium absorption promoter, each containing the same |
| JP2013221017A (en) | 2012-04-17 | 2013-10-28 | Api Co Ltd | Epidermal cell activator, cosmetic including the same, skin care external preparation and anti-wrinkle cosmetic |
| KR101418067B1 (en) | 2012-12-20 | 2014-07-14 | 주식회사 삼양제넥스 | Preparation method of hypoallergenic royal jelly by enzyme treatment |
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| JP2002112715A (en) * | 2000-10-03 | 2002-04-16 | Api Co Ltd | Low-allergenized royal jelly and method for producing the same |
| JP2009029772A (en) * | 2007-07-05 | 2009-02-12 | Api Co Ltd | Enzyme-treated royal jelly, and antioxidant, moisturizer, skin fibroblast proliferation promoter, hypotensive, fatigue-relieving agent and calcium absorption promoter, each containing the same |
| JP2013221017A (en) | 2012-04-17 | 2013-10-28 | Api Co Ltd | Epidermal cell activator, cosmetic including the same, skin care external preparation and anti-wrinkle cosmetic |
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