KR101779671B1 - Natural riposome comprising saccharomyces genus yeast extract, its preparation process, and food or pharmaceutical composition comprising it - Google Patents

Abstract

The present invention includes a first step of mixing at least one natural emulsifier with a natural-derived solvent and ultrasonication, a second step of adding a saccharomyces sp. Yeast extract as a capturing substance to the product of the first step and ultrasonication A liposome constituent substance containing a natural preservative, and a natural liposome containing a Saccharomyces cerevisiae extract as a capturing substance, a process for producing the same, and a process for producing the same, Thereby providing a food or pharmaceutical composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a natural liposome containing a saccharomyces cerevisiae extract, a method for producing the same, and a food or pharmaceutical composition containing the same. BACKGROUND ART [0002] Natural liposomes containing saccharomyces sp.

The present invention relates to a natural liposome containing a yeast extract of Saccharomyces sp., A method for producing the same, and a food or a pharmaceutical composition containing the same. More specifically, the present invention relates to a natural liposome which provides antioxidant, And a food or pharmaceutical composition containing the same.

As the eating habits of modern people gradually become westernized and the stress increases, there is a growing interest in antioxidants that can prevent the oxidative stress of the human body. As body activity increases, the body's oxygen consumption increases, resulting in increased free radical production due to oxidative stress [1].

There is a proper antioxidant system in the body that can treat active oxygen under physiological conditions. The antioxidant system is largely composed of enzymatic antioxidant groups including glutathione peroxidase (GPX) and catalase (CAT), and glutathione, which are highly intracellular antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase , Non-enzymatic antioxidant groups including vitamin C, vitamin E, carotenoids, and uric acid [2].

Glutathione (γ-glutamyl-cysteinyl-glycine (GSH)) acts not only on various enzymatic or nonenzymatic detoxification mechanisms, but also on free radical scavenging activity, And xenobiotics. ≪ / RTI > In addition, the kidneys, liver, lungs, small intestine and many other organisms that can be exposed to water can protect the body.

However, glutathione (GSH) has a low cellular uptake rate, is unstable in a neutral or alkaline environment, and has low bioavailability. This is because the thiol groups of glutathione (GSH) are easily oxidized to GSSG under oxidative stress. Therefore, there is an urgent need to develop a technique for preventing oxidation of glutathione (GSH).

To solve this problem, many studies have been conducted to protect glutathione from enzymatic damage. For example, nanoparticles containing glutathione, encapsulation, chitosan coating, and packaging films have been developed. Japanese Laid-Open Patent Publication No. 2013-0137561 discloses a method for producing a food packaging film having improved glutathione stability. However, liposomes made of natural substances, foods containing them or pharmaceutical compositions are not known at all.

Particularly, liposomes are biocompatible because they are composed of phospholipid, which is a main component of a vital cell membrane, and can store desired substances (drugs, nutrients, etc.) in a bilayer structure, so that unstable and difficult- There are advantages. However, in spite of these advantages, liposomes are unstable in formulation, have a low collection efficiency, and also have a problem that solvents and preservatives used in the production of liposomes cause skin irritation.

Patent Publication No. 2013-0137561

1. Alessio, H. M., & Goldfarb, A. H. (1998). Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. Journal of Applied. 2. Jeong, C. H., Choi, S. G., & Heo, H. J. (2008). Analysis of nutritional compositions and antioxidative activities of korean commercial blueberry and raspberry. Journal of the Korean Society of Food Science and Nutrition, 37 (11), 1375-1381. Physiology, 64, 1333-1336.

Disclosure of the Invention An object of the present invention is to provide a natural liposome which is easy to absorb in the body while alleviating oxidative stress, and an antioxidant food or pharmaceutical composition containing the same.

One embodiment of the present invention includes a first step of mixing at least one natural emulsifier with a natural-derived solvent and ultrasonication, adding a yeast extract of Saccharomyces genus as a capturing substance to the product of the first step, Wherein the natural liposome comprises at least one natural emulsifier, a natural-derived solvent, a liposome constituent material containing a natural preservative, and a natural liposome comprising Saccharomyces cerevisiae extract as a capturing agent, to provide.

Another embodiment of the present invention provides a food composition or a pharmaceutical composition containing the natural liposome as an active ingredient.

In another embodiment of the present invention, there is provided a method for producing a microorganism, comprising the steps of mixing at least one natural emulsifier with a natural solvent, and then ultrasonically treating the mixture at 50 to 80 ° C .; adding a Saccharomyces cerevisiae extract to the product of the first step And a third step of adding a distilled water heated to 40 to 60 ° C. to the product of the second step and a natural preservative and ultrasonically treating the product to form a liposome. to provide.

The natural liposome containing the Saccharomyces cerevisiae extract according to the present invention can increase the glutathione absorption capacity in the intestines upon oral ingestion. In addition, the food or pharmaceutical composition containing the natural liposome according to the present invention may exhibit excellent antioxidative and fatigue recovery effects.

1 shows a process for producing a natural liposome according to an embodiment of the present invention.
Fig. 2 shows the results of the analysis of the glutathione content according to the kind of yeast and the treatment method.
3A, 3B, and 3C are electron micrographs of the extract of Comparative Example 1, the liposome of Example 1, and the liposome of Comparative Example 2, respectively.
Figs. 4A, 4B, and 4C show particle distributions of the extract of Comparative Example 1, the liposome of Example 1, and the liposome of Comparative Example 2, respectively, by a laser particle size analyzer.
Fig. 5 shows the results of the skin absorption test of the extract of Comparative Example 1, the liposome of Example 1, and the liposome of Comparative Example 2. Fig.
6 shows the cytotoxicity test results of the extract of Comparative Example 1 and the liposome of Example 1. Fig.
Figure 7 shows the cell survival rate during oxidative damage of each sample.
Figure 8 shows the intracellular SOD activity of each sample.
Figure 9 shows the Rat spleen index of each sample.
Figure 10 shows erythrocyte SOD activity for Rat of each sample.
Figure 11 shows erythrocyte catalase activity for Rat of each sample.
Figure 12 shows the intrathecal glutathione content for Rat of each sample.
Figure 13 shows the content of liver glutathione hydrolase relative to Rat of each sample.
Figure 14 shows the intrahepatic lipid peroxide content for Rat of each sample.
15 shows a liver tissue photograph of Rat of each sample.
Figure 16 shows the AST content in the Rat serum of each sample.
Figure 17 shows the ALT content in the Rat serum of each sample.
Figure 18 shows the CPK content in Rat serum of each sample.
Figure 19 shows the LDH content in Rat serum of each sample.
Figure 20 shows the antioxidant content in Rat serum of each sample.
Figure 21 shows the antioxidative effect of each sample in Rat serum.

Hereinafter, natural liposomes containing Saccharomyces cerevisiae extract according to the present invention will be described in detail with reference to the accompanying drawings.

However, these descriptions are provided only to illustrate the present invention, and the scope of the present invention is not limited by these exemplary explanations.

1. Natural Emulsifier

Natural emulsifiers are substances that constitute the lipid bilayer and emulsifiers derived from natural materials. The natural emulsifiers include phospholipids and fatty acids. The phospholipids contained in the natural emulsifiers are natural phospholipids derived from natural origin, and fatty acids are natural fatty acids derived from natural sources.

Examples of the phospholipid include phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or their hydrogenation products (for example, phosphatidylserine, phosphatidylglycerol, , Hydrogenated phophatidylcholine (derived from soybean), etc.) may be used. Hydrogenerated phosphatidylcholine, which is preferably excellent in oxidation stability, may be used.

Examples of the fatty acid include at least one natural fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid (derived from soybean) Can be used.

The natural emulsifier may comprise from 70 to 90% by weight of natural phospholipids and from 1 to 5% by weight of natural fatty acids based on the weight of the total natural emulsifier. Other solvent may be contained in the remaining amount. As the solvent, it is preferable to use a naturally derived solvent, and it is more preferable to use distilled water.

Natural emulsifiers can be used in combination of two or more kinds. For example, two kinds of natural emulsifiers or three kinds of natural emulsifiers may be mixed and used. When two natural emulsifiers are mixed and used, for example, the first natural emulsifier may be selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, palmitic acid, stearic acid stearic acid, oleic acid, linoleic acid and linolenic acid (derived from soybean), and the second natural emulsifier may be hydrogenated phosphatidylcholine (derived from soybean) . In addition, when three natural emulsifiers are mixed and used, for example, in addition to the first natural emulsifier and the second natural emulsifier, a third natural emulsifier such as cetearylolivate, sorbitanolivate- Olive) can be used.

2. Collecting material

Saccharomyces cerevisiae may be used as a trapping substance that can be trapped in the phospholipid layer of the liposome, for example, Saccharomyces cerevisiae cerevisiae ), Saccharomyces ellipsoideus ), Saccharomyces ( Saccharomyces carlsbergenis ) may be used. Preferably, Saccharomyces cerevisiae can be used, most preferably Saccharomyces cerevisiae cerevisiae MAB Y1 (KCTC 11386BP) may be used.

As the capturing material, it is preferable to use the yeast extract. It is particularly preferable to use an ultrasonicated yeast extract.

In general, yeast extract contains a large amount of nucleic acid and protein, which are tastes, and is widely used as a natural flavor ingredient. The tripeptide glutathione (L-glutamyl-L-cysteinylglycine) among the tastes of the yeast extract provides taste persistence. Glutathione present in yeast is a reduced form that is present in the cells of microorganisms, animals and plants, and has antioxidative effects against oxidative damage, and provides various pharmacological effects such as improvement of liver disease, treatment of diabetes, and anticancer activity Also known.

The yeast extract may be a supernatant obtained by disrupting cells or cells by ultrasonication, or a mixture thereof may be used at a certain ratio. It is more preferred to use a supernatant. When the yeast extract is produced through ultrasonic disruption, it is preferable that the antioxidant component is prevented from destruction, the extract can be obtained in a short time, and the process is relatively simple. The content of the capturing material is 1 to 50% by weight based on the weight of the whole natural liposome.

3. Solvent

Examples of the solvent include natural solvents such as distilled water, glycerin from natural origin, butylene glycol from natural origin, propylene glycol from natural origin, natural ethanol, natural propanediol, natural glycerin, fermented alcohol, , Natural-derived glycerin, natural ethanol, and more preferably distilled water. The content of the solvent is the residual amount of the emulsifier, the trapping agent, and the preservative.

4. Preservatives

Preservatives may be any natural preservatives that can be used as food preservatives. For example, natural extracts can be used. Preferably, grapefruit extract, citrus extract may be used. It is distinguished from benzoic acid, sorbic acid, propionic acid, dehydroacetic acid, paraoxybenzoic acid and the like which are commonly used preservatives in that they are natural extracts. The preservative may comprise from 0.1 to 5% by weight, based on the total weight of the natural liposome.

5. Preparation of liposome

The liposome according to the present invention can be produced using ultrasonic waves. The specific manufacturing method is as follows.

One or more natural emulsifiers are mixed with a part of a natural solvent and homogenized by ultrasonic disruption at 50 to 80 degrees Celsius using an ultrasonic liquid processor. When the natural emulsifier is homogenized, it is mixed with ultrasonic wave after addition of the capturing material. Homogenization of the emulsifier and the collecting material can be enhanced and the particle size can be controlled by ultrasonic disintegration in the step of homogenizing the natural emulsifier and the step of adding the collecting material, unlike the prior art using the homogenizer and the like. When the emulsifier and the collecting material are completely homogenized, distilled water and natural extract (preservative) heated to 40 to 60 degrees Celsius are added and mixed with ultrasonic waves to form liposome type particles. Thereafter, the temperature is gradually lowered and ultrasonication is finally performed for 5 minutes to make the particles smaller and uniform so that a natural liposome is finally produced. The manufacturing process is shown in Fig.

The natural liposome according to an exemplary embodiment of the present invention is distinguished from conventional synthetic liposomes in that it does not contain a synthetic component, including natural emulsifiers, natural solvents, natural preservatives, and naturally occurring components.

Further, the liposome according to the present invention can improve the absorption rate by increasing the transmissivity in the body, and can effectively function as a food composition or a pharmaceutical composition. The liposome according to the present invention can be used in the form of beverage, health functional food, and the like.

<Selection of raw yeast - Analysis of glutathione content according to treatment method>

The contents of glutathione in microorganisms were checked and the contents of glutathione were analyzed according to the treatment method to select appropriate raw materials. For this purpose, Saccharomyces cerevisiae cerevisiae MAB Y1 (KCTC 11386BP) was cultured at 30 占 폚 in a medium containing glucose 3%, yeast extract 2%, NaCl 0.06%, and taurine 0.06%. The cultured Y1 broth was centrifuged at 12,000 rpm for 15 minutes to remove the supernatant, and the cells were recovered. The same amount of distilled water was added to the recovered cells, which was treated with an ultrasonic shredder at 10 Amp for 1 minute. The mixture was centrifuged at 12,000 rpm for 15 minutes and separated into pellet and supernatant. The pellet was removed and the supernatant was recovered.

0.6 ml of DTNB (5'-dithiobis (2-nitrobenzoic acid)) color development reagent was added to 0.5 ml of the microbial crush supernatant and diluted with the same amount of distilled water. After reacting for 15 minutes, absorbance was measured at 412 nm. The glutathione content was calculated from the standard calibration curves of glutathione.

In order to select an appropriate yeast extract, in addition to the glutathione content of the supernatant of the microorganism, the glutathione content of the cells, the glutathione content of the ultrasonic disruption pellet, the pellet / supernatant when disrupted using ethanol instead of ultrasonic disruption Glutathione content in the culture medium, glutathione content in the culture medium, and glutathione content in the yeast fermentation powder were measured. The results are shown in FIG.

As a result, as shown in FIG. 2, the content of glutathione in supernatant of supersonic wave was the highest at 1.29 mg / ml. Thus, supernatant supernatant was used for the preparation of Y1 extract.

< Example  1>

<Preparation of Y1 extract>

Mrs. serenity in a busy Saccharomyces (Saccharomyces cerevisiae) MAB Y1 (the resulting rice wine yeast), were cultured for 72 hours in accordance with the culture conditions shown in Table 1 below in a medium containing glucose (Glucose), salt (NaCl), Taurine.

External condition Time (Hr) rpm pH Temperature (° C) Condition 72 100 6.00 30 Nutritional condition Carbon source Nitrogen source salt amino acid Source Glucose Yeast extract NaCl Taurine density 3% 2% 0.06% 0.06%

The cultured microbial culture broth (Y1 culture broth) was centrifuged to collect cells, and 30 g of distilled water was added to 3 g of the recovered microbial cells, followed by sonication. It was then centrifuged at 3,000 rpm for 10 minutes to obtain a shredded pellet and a supernatant. Here, the pellet was removed and the supernatant was recovered, and this supernatant was used as a MAB Y1 extract (Y1 extract) in Saccharomyces cerevisiae as a capturing substance. Ultrasonic processing was carried out at 10 Amp for 10 minutes.

< Sakaromisses Serenity busy (Saccharomyces cerevisiae ) Manufacture of Natural Liposomes Containing MAB Y1>

(3% by weight of palmitic acid, 1% by weight of stearic acid, 3% by weight of oleic acid, and 7% by weight of linoleic acid) based on 100% by weight of the total amount of natural emulsifier (phosphatidylcholine 67% by weight, lysophosphatidylcholine 8% by weight, phosphatidylethanolamine 8% %, Linolenic acid 3 wt%) and distilled water were mixed and then homogenized by ultrasonic disruption at 65 ° C using an ultrasonic disrupter (Ultrasonic Liquid Processor, QSONICA, USA). Then, Y1 extract, which is a capturing material, was added to the homogenized natural emulsifier, followed by ultrasonic disruption and mixing. When the natural emulsifier and the capturing material were completely homogenized, distilled water heated to 50 ° C and a natural preservative were added, and the mixture was ultrasonically disrupted to form liposome type preliminary particles. Thereafter, the temperature was gradually lowered and ultrasonically disrupted for 5 minutes to make the particles smaller and uniform, thereby preparing a natural liposome (Y1 CPS) containing the Y1 extract. The above manufacturing process is shown in Fig.

&Lt; Example 2 >

A natural liposome was prepared in the same manner as in Example 1, except that the content of Y1 extract was 20% by weight and distilled water was used as the remaining amount.

&Lt; Example 3 >

A natural liposome was prepared in the same manner as in Example 1, except that the content of Y1 extract was 10% by weight and distilled water was used as the remaining amount. Table 2 shows the raw materials and their contents used in Examples 1 to 3 above.

Raw material name Content (% by weight) Example 1 Example 2 Example 3 Natural emulsifier One One One Grapefruit extract One One One menstruum
(Emulsification process) Distilled water (D. I. WATER) 30 30 30 Solvent (homogenization process) Distilled water (D. I. WATER) Balance Balance Balance Capture material Sakaromisses Serebijie
( Saccharomyces cerevisiae ) MAB Y1 30 20 10

&Lt; Comparative Example 1 &

In Comparative Example 1, 30% by weight of the Y1 extract used as the capturing material instead of the natural liposome of Example 1 and the remaining amount of distilled water were mixed and used.

&Lt; Comparative Example 2 &

In Comparative Example 2, a conventional synthetic liposome containing Y1 extract, lecithin, cholesterol, glycerin, and potassium sorbate was prepared.

Distilled water (30% by weight) was used as a solvent. Phospholipid (lecithin-1%), synthetic cholesterol (0.2% by weight) and synthetic glycerin (1% by weight) were mixed and heated to 65 占 폚. When the phospholipid was completely dissolved, the same amount of Y1 extract as in Example 1 was added and homogenized with a homogenizer. The remaining water was mixed with an antiseptic agent (potassium sorbate 0.1%) to form liposome particles, and the particles were homogenized through ultrasonic treatment.

<Experimental Example 1> Analysis of morphology and size of liposome by electron microscope

The shape and size of the Y1 extract of Comparative Example 1, Y1 natural liposome (Y1 CPS) of Example 1 and Y1 synthetic liposome of Comparative Example 2 were analyzed using a scanning electron microscope (Scanning Electron microscope S-1700, Hitachi) . The results are shown in Figs. 3A, 3B and 3C, respectively.

EXPERIMENTAL EXAMPLE 2 Analysis of Size of Liposome by Laser Particle Size Analyzer

The particle size of the sample was measured and the particle size distribution was analyzed using a laser particle size analyzer (Electrophoretic Light Scattering Spectrophotometer ELS-8000, Otsuka). The average particle sizes of the Y1 extract of Comparative Example 1, the Y1 natural liposome of Example 1 (Y1 CPS) and the Y1 synthetic liposome of Comparative Example 2 are shown in Table 3 below.

Sample Average particle size (nm) Comparative Example 1 125.0 Example 1 116.5 Comparative Example 2 601.9

4A, 4B and 4C show particle distributions of the Y1 extract of Comparative Example 1, Y1 natural liposome of Example 1 (Y1 CPS) and Y1 synthetic liposome of Comparative Example 2, respectively.

<Experimental Example 3> Measurement of collection rate

2 ml of the resultant Y1 natural liposomal suspension was taken and a 0.45 쨉 m to 25 mm syringe filter (GVS, USA) was attached to a 10 ml syringe to remove non-captured liposomal substances. The removed portion was supplemented with distilled water, and the absorbance was measured at the maximum absorption wavelength of the Y1 extract. The collection rate of the collected Y1 extract was confirmed by substituting the following equation.

Entrapment efficiency (%) = Y _e / Y _i x 100 (%)

Y _e : the concentration of the entrapped substance (encapsulated concentration)

Y _i : initial concentration of the whole substance

The collection rates are shown in Table 4 below.

Sample Collection rate (%) Example 1 98.11 Comparative Example 2 89.02

As a result of measuring the percentages of the natural liposome of Example 1 and the synthetic liposome of Comparative Example 2, the percentages of the Y1 natural liposomes (98.11%) of Example 1 were higher than those of the synthetic liposome of Comparative Example 2 (89.02%).

<Experimental Example 4> Skin absorption using Franz Diffusion cell

In order to examine the effect of the prepared Y1 natural liposome on skin permeation enhancement, a skin permeation experiment was conducted using an in vitro permeation experiment (Franz Diffusion cell). In the experiment, chicken skin tissue was used to remove hair. The absorption of the same amount of Y1 extract, Y1 natural liposome and Y1 synthetic liposome was compared. The chicken skin was fixed between the donor and receptor phases, and 1 ml of sample was added to the donor of the prepared Franz diffusion cell, and the temperature was maintained at about 36.5 ° C in a constant temperature water bath. At the time of 0 hour, 1 hour, 3 hours and 6 hours, 0.25 ml of receptor phase was recovered and the same amount of distilled water was supplied.

Fig. 5 shows the results of comparing the skin absorption tests of the Y1 extract of Comparative Example 1, the Y1 natural liposome of Example 1, and the Y1 synthetic liposome of Comparative Example 2. As shown in FIG. 5, the absorption power of the Y1 extract (Y1) of Comparative Example 1 was the lowest and the absorption power of the Y1 natural liposome (Y1 CPS) of Example 1 was the highest.

 [Cell Experiment]

<Experimental Example 5> Cytotoxicity using MTT

Fibroblast cells (1 × 10 ⁵ ) were dispensed into 48 wells. After 24 hours, the cells were replaced with fresh medium, and the samples were treated for 0.05 to 1% concentration for 24 hours. Subsequently, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (0.1 mg / ml) was added to each well and incubated for 3 hours. Insoluble formazan formed was dissolved in 0.04 N HCl / isopropanol And absorbance was measured at 570 nm through an ELISA reader.

As shown in FIG. 6, the cytotoxicity of the Y1 natural liposome (Y1 CPS) of Example 1 was lower than that of the Y1 extract of Comparative Example 1 at a concentration of 0.25% Was higher.

<Experimental Example 6> Recovery effect of oxidative damage

1 × 10 ⁵ keratinocyte HaCaT cells were plated in 48 wells, replaced with fresh medium after 24 hours, and treated for 24 hours. To induce oxidative stress, 500 uM H2O2 was treated for 4 hours and then 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (0.1 mg / After incubation, insoluble formazan formed was dissolved in 0.04 N HCl / isopropanol and the absorbance at 570 nm was measured by ELISA reader.

The keratinocytes were treated with hydrogen peroxide to induce oxidative stress, and the test material was treated to confirm cell viability. The results are shown in FIG.

As shown in FIG. 7, the cell survival rate of the group treated with hydrogen peroxide was only 77.9% of the cell survival rate of the group not treated with hydrogen peroxide. On the other hand, when the Y1 natural liposome of Example 1 was treated, the cell viability increased to 85.7%.

<Experimental Example 7> Intracellular SOD activity

Peroxidase (SOD) is an enzyme that catalyzes the conversion of excess oxidized ions to oxygen and hydrogen peroxide. The peroxidase-eliminating enzyme is known to act as an antioxidant defense mechanism in almost all cells exposed to oxygen, and is a typical antioxidant that inhibits active oxygen.

Dermal cells in 48 wells Human dermal fibroblast (HDF) cells were dispensed at 1 × 10 ⁵ cells, replaced with fresh medium after 24 hours, and treated for 24 hours. Then, the cells were collected and cell lysate was used for the experiment.

SOD activity was measured by measuring the absorbance at 420 nm using a UV / VIS spectrophotometer (UV-2401PC, Shimadzu) as a method for measuring the amount of inhibition of the auto-oxidation of pyrogallol. The activity of enzyme was calculated by converting the color development of pyrogallol into% SOD activity based on normal cells not treated with H2O2. The results are shown in Fig.

As shown in FIG. 8, it was confirmed that SOD activity was lowered to 20% by treatment with hydrogen peroxide in skin dermal cells. SOD activity was increased when the cells were treated with an antioxidant (Ascorbic acid (AA), BHT). SOD activity was increased to 51.652% by the Y1 natural liposome treatment of Example 1.

 [Animal experiment]

<Experimental Example 8> Oxidative stress-improving effect on t-BHT-induced Rat

Experimental animals and oxidative induction

SD Rat was used for the experiment and 4 week old male rats weighing 130g were received from BioLink (Chungcheongbuk - do, Korea) and underwent a treatment period of 4 weeks after the adaptation period of 1 week. During the test period, feed and water were freely taken, and the temperature of the breeding room (22 ± 2), relative humidity (55 ± 5%) and intensity were maintained for 12 hours. Oxidative stress was induced by injecting t-butyl hydroperoxide (t-BHT) 0.5 mmol / kg into the peritoneal cavity of the rats on the last day of the experiment.

<Experimental Example 9> Spleen index comparison of Rat causing oxidative stress

For immunological evaluation, the spleen was sacrificed at the end of the experiment and the weight was measured. The results are shown in the graph of FIG. The spleen index was expressed as the ratio of the spleen weight (mg) to the weight (g) of the mouse.

As shown in FIG. 9, the spleen index was 2.54 mg / g in the normal group which did not induce the oxidative stress but the spleen index increased to 2.74 mg / g in the group that caused the oxidative stress Respectively. In the case of treatment with the natural liposome (Y1 CPS) of Example 1, it was 2.35 mg / g, which was the most similar to that of the normal group. These results suggest that the natural liposome of Example 1 significantly inhibited the increase of T lymphocytes in the spleen.

<Experimental Example 10> Comparison of SOD activity of erythrocytes of Rat causing oxidative stress

After collecting blood of Rat causing oxidative stress, red blood cells were collected and used in the experiment. For pretreatment, add 0.9 ml of 10 mM Tris-1 mM EDTA buffer to 100 μl of red cell suspension, add 0.4 ml of Chloroform-Ethanol solution and mix for 2 minutes, then add 140ul of tertiary distilled water and centrifuge at 20,000g at 4 ℃ for 30 minutes The supernatant was used for the experiment.

SOD activity in erythrocytes was determined based on SOD activity in erythrocytes of normal group which did not induce oxidative stress. The results are shown in Fig.

As shown in FIG. 10, the SDO activity of erythrocytes in the control group treated with only t-BHT decreased to 43.7%. On the other hand, in the group administered with Y1 natural liposome of Example 1, 77.8% of the SOD activity showed the closest activity to the normal group which did not induce oxidative stress.

Experimental Example 11: Comparison of erythrocyte catalase activity of oxidative stress induced Rat

Catalase activity of erythrocytes recovered from blood of Rat causing oxidative stress was measured. Pretreatment was performed by adding 1.35 ml of 10 mM Tris-1 mM EDTA buffer to 150 μl of a red cell suspension, and then diluting the solution 1000 times with 0.01 M phosphate buffer (pH 7.4). Then 15ml tube to _{250mM KH 2 PO 4 -NaOH buffer (} pH 7.0) 300ul, into a 100% methanol 300ul, 0.27% H2O2 60ul was added 600ul of the sample was reacted at 20 ℃ 20 minutes. The reaction was terminated by the addition of 7.8 M KOH (300 ul), followed by addition of 600 ul of 34.2 mM Pulpald solution, followed by reaction at 20 ° C for 10 minutes. Finally, 300μl of 65.2mM Potassium periodate was added, developed and left at room temperature for 10 minutes, and absorbance was measured at 550nm. The results are shown in Fig.

Catalase activity was 122.77 nmol in the normal group that did not induce oxidative stress and 119.18 nmol in the control group that induced oxidative stress by t-BHT. Respectively.

In contrast, the catalase activity in the erythrocytes treated with the natural liposome (Y1 CPS) of Example 1 was 123.16 nmol, which was similar to that of the normal group, although it induced oxidative stress. That is, it was found that catalase activity did not decrease due to oxidative stress.

EXPERIMENTAL EXAMPLE 12 Comparison of Glutathione Content in Liver of Rat Induced by Oxidative Stress

To 0.2 ml of homogenate fraction of liver tissue, 0.3 ml of tertiary distilled water and 0.5 ml of 0.4% sulfosalicylic acid were added and mixed. After centrifugation, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) coloring reagent was added to 0.3 ml of supernatant And the absorbance at 412 nm was measured. The glutathione content was calculated from the standard calibration curves of glutathione and expressed in ml per mg of liver tissue. The results are shown in Fig.

As shown in FIG. 12, the content of glutathione was 0.746 mg / ml in the normal group but 0.737 mg / ml in the control group, indicating that the content of glutathione decreased when oxidative stress was induced I could confirm. On the other hand, it was confirmed that the glutathione tends to increase in the extract (Y1) of Comparative Example 1, the natural liposome (Y1 CPS) of Example 1 and the synthetic liposome of Comparative Example 2 (Y1 synthetic liposome) The glutathione content of liposomes (Y1 CPS) was the highest at 0.772 mg / ml.

< Experimental Example  13> Oxidative  In the liver of stress-induced rats Glutathione  Comparison of hydrolytic enzyme content

Glutathione (GSH) -Px is an antioxidant enzyme with selenium, which is involved in the detoxification of peroxidized lipids and H2O2 using glutathione (glutathione (GSH)) present in the body as a substrate. In case of deficiency of iron, vitamin E and essential fatty acids It is reported that the activity is decreased and the activity is increased by oxidative stress.

To measure the amount of GSH-Px in liver tissue, 100ul of tissue fluid was added to 96 wells and 100ul of GPX buffer mixture (2mM NADPH, 4mM reduced glutathione, 25.6mU / ml glutathione reductase, 5mM EDTA, 50mM Tris, Respectively. 50 ul of GPx Substrate Mic (16 mM hydrogen peroxide) was added and the absorbance was measured at 340 nm for 10 minutes at intervals of 1 minute. The results are shown in Fig.

The GSH-px of 13.73 Unit / ml was measured in the normal group which did not induce oxidative stress and the GSH-px content of 26.47Unit / ml in the group which induced oxidation by t-BHT Showed that the content of GSH-px was increased when oxidative stress was induced. On the other hand, the Y1 natural liposome of Example 1 showed GSH-px of 15.13 Unit / ml, which was similar to that of the normal group.

< Experimental Example  14> Oxidative  Liver lipid peroxidation in stressed rats MDA ) Content comparison

Peroxide lipid content (MDA) of the homogenate fraction from liver tissue was measured by TBARS method. 2 ml of TBA (thiobarbituric acid) reagent was added to 1 ml of the fraction solution containing 1 mg of protein, followed by 30 minutes of reaction in boiling water, followed by cooling at room temperature, centrifugation at 3,000 rpm for 10 minutes, nm absorbance was measured. The results are shown in Fig.

As a result, the content of lipid peroxidation was 75.87 nmol / ml in the normal group and 77.16 nmol / ml in the control group causing oxidative stress. As a result, it was confirmed that the content of lipid peroxide in the Y1 natural liposome of Example 1 was remarkably low.

EXPERIMENTAL EXAMPLE 15 Histological Evaluation of Rat Induced Oxidative Stress

Liver tissues were examined to determine the effect of oxidative stress on the liver tissue of Rat. A portion of the liver tissue was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosi (H & E). The results are shown in Fig. Histologic observation showed that in the normal group without oxidative stress, liver tissue was constant, while in the control group injected with t-BHT, cell tissue was damaged and pores were formed. In the case of the group treated with the natural liposome (Y1 CPS) of Example 1, it was confirmed that the arrangement of the cells was constantly restored similar to that of the normal group.

<Experimental Example 16> Biochemical indicators of oxidative stress induced Rat serum

The AST, ALT, CPK total (S) and LDH contents in the serum were analyzed and the results are shown in FIGS. 16 to 19, respectively.

The levels of AST and ALT, the enzymes secreted by liver injury, were the lowest in the normal group without oxidative stress. On the other hand, in the group treated with t-BHT, it was confirmed that the value was increased. It was confirmed that the content of the enzyme decreased in the group treated with the natural liposome (Y1 CPS) of Example 1 (Figs. 16 and 17).

The contents of creatine kinase (CPK) and lactate dehydroxygenase (LDH) were analyzed to analyze serum fatigue substances. As a result of the analysis, it was confirmed that the CPK value of the CPK was increased in the control group that caused the oxidative stress when compared with the normal group, and that of the natural liposome (Y1 CPS) of Example 1 was 308 U / L. In addition, the LDH content was also found to be 245 U / L in the normal group and 393 U / L in the oxidation-induced control group. As a result, it was confirmed that the LDL value increased and the natural liposome Y1 CPS) treated group showed an LDH content of 228.33 U / L, which is similar to that of the normal group, even though oxidative stress was induced (FIGS. 18 and 19).

<Experimental Example 17> Antioxidant analysis of serum induced by oxidative stress

To determine the antioxidant activity in the serum, vitamin C content of the sera isolated from the blood was analyzed. The content of VitaminC was determined by adding 2 ml of 5% TCA (Trichloroacetic acid) to 0.5 ml of the sample, precipitating the protein, centrifuging and adding color development reagent (85% orthophosphoric acid 0.05 ml + 8% a, a'-Bipyridyl 0.05 ml + % aqueous ferric chloride 0.05ml) was added to the wells and incubated at 25 ° C for 1 hour to confirm formation of ferrous dipyridyl chromophore. Absorbance was measured at 525 nm using a UV spectrophotometer. The results are shown in Fig.

In the normal group, the content of 34.66 ug / ml was measured, and in the control group, the concentration was decreased to 22.23 ug / ml. On the other hand, in the case of the natural liposome (Y1 CPS) of Example 1, 31.98 ug / ml was recovered to a level similar to that of the normal group.

<Experimental Example 18> Antioxidant activity of serum of Rat causing oxidative stress

 DPPH scavenging ability was confirmed to confirm antioxidative capacity in serum. The DPPH solution was prepared by dissolving 16 mg of DPPH in 100 ml of ethanol, mixing 100 ml of distilled water, and filtering through a filter paper. 0.5 ml of the sample solution was mixed with 2.5 ml of DPPH solution, and reacted at room temperature for 30 minutes. Absorbance was measured at 528 mm on a spectrophotometer (HITACHIU-2990). The DPPH free radical scavenging activity was expressed as a percentage (%) of absorbance difference between the sample and the non-additive. The results are shown in Fig.

Free radical scavenging activity in serum was 71.32% in normal group and 67.55% in control group, which caused oxidative stress. In the group treated with the sample other than the natural liposome (Y1 CPS) of Example 1, the control value was similar to that of the control, and the radical scavenging ability was not restored. On the other hand, in the group treated with the natural liposome (Y1 CPS) of Example 1, the radical scavenging ability was increased up to 70.21% and the oxidative stress was restored to a level similar to that of the normal group.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. The present invention is not limited to the drawings.

Claims (19)

A first step of mixing at least one kind of natural emulsifier with a natural solvent and ultrasonication at 50 to 80 ° C, a second step of adding a saccharomyces cerevisiae extract as a capturing substance to the product of the first step and ultrasonication 1. A natural liposome formed by the method comprising:
One or more natural emulsifiers, a natural derived solvent, a liposome constituent material containing a natural preservative,
A saccharomyces cerevisiae extract as a capturing substance,
Wherein the natural derived solvent is at least one selected from the group consisting of distilled water, butylene glycol, propylene glycol, propanediol, glycerin, ethanol, and fermented alcohol. The method according to claim 1,
The Saccharomyces cerevisiae extract is a natural liposome that is a yeast extract of Saccharomyces cerevisiae . The method according to claim 1,
The Saccharomyces cerevisiae extract is a natural liposome which is a yeast extract of Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP). The method according to claim 1,
The Saccharomyces cerevisiae extract is a natural liposome, which is an ultrasonically pulverized yeast extract. The method according to claim 1,
Wherein the natural emulsifier comprises at least one natural phospholipid selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and their hydrogenation products. The method according to claim 1,
Wherein the natural emulsifier comprises at least one naturally occurring fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. delete The method according to claim 1,
The natural preservative is a natural liposome, which is a natural-derived extract containing grapefruit or citrus extract. The method according to claim 1,
The solvent is distilled water. The method according to claim 1,
Wherein said natural emulsifier comprises a first natural emulsifier containing at least one fatty acid and at least one phospholipid and a second natural emulsifier containing at least one phospholipid. 11. The method of claim 10,
Wherein the second natural emulsifier is at least one emulsifier selected from the group consisting of sorbitan olivate and cetearyl olivate. The method according to claim 1,
Wherein the natural liposome comprises 2 to 9% by weight of a natural emulsifier based on the total weight of the natural liposome, 1 to 5% by weight of a natural preservative, 0.01 to 5% by weight of an extract of Saccharomyces cerevisiae and 0.01 to 5% by weight of a solvent. The method according to claim 1,
Natural liposomes for antioxidants. A food composition comprising the natural liposome of claim 1 as an active ingredient. delete A first step of mixing at least one natural emulsifier with a natural solvent and ultrasonication at 50 to 80 캜,
A second step of adding a Saccharomyces cerevisiae yeast extract to the product of the first step,
Adding a distilled water warmed at 40 to 60 ° C to the product of the second step and a natural preservative and sonicating to form a liposome;
13. The method according to any one of claims 1 to 6, 8 to 13, wherein the natural liposome is a liposome. 17. The method of claim 16,
The fourth step of performing ultrasonic processing while gradually lowering the temperature after the third step
&Lt; / RTI &gt; 17. The method of claim 16,
Prior to the second step,
Saccharomyces cerevisiae, a makgeolli-derived yeast, cerevisiae ) MAB Y1,
The culture solution is centrifuged to collect the cells and subjected to ultrasonic treatment to obtain saccharomyces yeast extract
&Lt; / RTI &gt; 19. The method of claim 18,
After the ultrasonic treatment, centrifuging the ultrasonic treated cells again, removing the pellet, and recovering the supernatant
&Lt; / RTI &gt;

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