KR101566638B1 - A bark of Dioscorea batatas Decne. improving effect of cholesterol in blood, and health functional food comprising the same - Google Patents

Abstract

The present invention provides a health functional food comprising powder or extracts of the bark of Dioscorea batatas Decne., as an active ingredient. According to the present invention, a health functional food composition comprising powder or extracts of the bark of Dioscorea batatas Decne., as an active ingredient is capable of increasing antioxidant enzyme activity and inhibiting expression of an inflammation-related gene. In addition, according to the present invention, a health functional food composition comprising powder or extracts of the bark of Dioscorea batatas Decne., as an active ingredient is capable of improving blood cholesterol. The present invention is capable of improving general health by ingesting as a health functional food through a diet in daily life.

Description

[0001] The present invention relates to a method for producing cholesterol - lowering agents, improving effect of cholesterol in blood, and health functional food comprising the same}

Described herein is a health functional food composition comprising a peel of a maize.

Cholesterol (cholesterol) is an essential body component found in animal tissues. It is concentrated in the brain or nerve tissue and is a major component of the cell membrane with phospholipids. Cholesterol is also a component of lipoproteins, low density lipoprotein (LDL), high density lipoprotein (HDL) and very low density lipoprotein (VLDL). Among them, LDL and HDL are cholesterol-based lipoproteins. LDL transports cholesterol from the liver to tissues, whereas HDL plays a role in reversing the cholesterol of tissues to the liver. Thus, high levels of LDL in blood promote heart disease or atherosclerosis, while high levels of HDL inhibit the development of these diseases.

If the cholesterol metabolism is abnormal, the total cholesterol as well as the lipoprotein components such as LDL cholesterol and HDL cholesterol are changed quantitatively. When cholesterol accumulates, hypercholesterolemia occurs, which increases the total cholesterol level in blood plasma, which is the component of blood. If the total cholesterol level is not high, the LDL cholesterol is high or if the amount of HDL cholesterol involved in cholesterol reverse transportation is low, It can be a cause. The oxidation of LDL cholesterol in blood by reactive oxygen species is also involved in the development of cardiovascular diseases such as arteriosclerosis.

In order to improve hypercholesterolemia, there is a method of taking a cholesterol synthesis inhibitor. For example, drugs such as the fibrin acid derivative system that lower the 3-hydroxy-3-methylglutaryl coenzyme A, the main regulator of cholesterol synthesis, , Liver and kidney function are deteriorated.

Therefore, it is necessary to develop a natural product that can inhibit the synthesis of cholesterol in the blood through diet in daily life through the use of natural products which are safe to the human body and have no side effects.

Dioscorea batatas Decne.) has written a lot from the example Dios Correa (Dioscorea) in the perennial roots stem plants, the abundant intake of experience as a raw material for the food and not toxic to the medicine when diabetes, tuberculosis and physical weakness. It is said that the marjoram is said to be from the one side, and the marjoram is dried or steamed after removing the jjaji of the root of the marjoram. As described above, the processed product using the conventional hemp has been produced by peeling the bark of the hemp, and research on the characteristics of the hemp husk as a byproduct is insufficient.

Korean Registered Patent No. 10-1072926 (June 10, 2011)

The present invention aims to provide a health functional food composition which effectively inhibits cholesterol synthesis and exhibits antioxidant and anti-inflammatory effects by containing a natural raw material as an active ingredient.

On the other hand, other unspecified purposes of the present invention will be further considered within the scope of the following detailed description and easily deduced from the effects thereof.

In order to achieve the above object, the implementation of the invention do (Dioscorea batatas Decne.) peel powder or extract as an active ingredient.

The composition according to the present invention can increase antioxidant enzyme activity and inhibit the expression of an inflammation-related gene by containing a powder or extract of peel as an active ingredient. The peel, which is an active ingredient of the present invention, can inhibit colon cancer precancerous lesions before the oncogenesis by increasing the antioxidant enzyme activity by expressing the antioxidant enzyme gene of the colon mucosa, and suppresses the expression of the inflammatory mediator gene of the colon mucosa It can increase the antioxidant activity. In addition, the composition according to the present invention can prevent the occurrence of cardiovascular diseases such as hypercholesterolemia and arteriosclerosis by inhibiting cholesterol synthesis in the blood by including powder or extract of peel as an active ingredient.

Thus, the present invention can provide a healthier effect of enhanced health without adverse effects on the human body by providing the skin of a horse, which has been abandoned as a by-product of conventional horses, as a health functional food through dieting in daily life.

Hereinafter, the present invention will be described in detail.

Embodiments of the present invention are described in Dioscorea batatas Decne.) peel powder or extract as an active ingredient.

Active ingredient according to the embodiment of the invention the shell do DIOS in the hemp shells Correa (Dioscorea) in the perennial plant rhizomes, t (Dioscorea batatas Decaisne) or yams ( Dioscorea japonica Thunberg). The root of the edible horse root contains a large amount of milky white or yellowish brown sticky viscous substance and contains other phenanthrene derivative, saponin, allantoin, batatansin, choline and the like. The viscous substance is composed of glucomannan and protein occupying more than 80% of mannose.

The composition of the horse meat is as shown in Tables 1 to 3 according to the National Standard Food Ingredient List (content per 100 g of edible portion) of the agricultural product information system. As shown in Table 1 below, the discard rate is about 20%, and this discard rate includes the bark of the horse. The results of analysis of the general components of the crust are shown in Table 4 below.

division ingredient content unit General component energy 95 (kcal) General component moisture 73.5 (g) General component protein 5.1 (g) General component Lipid 0.7 (g) General component Ash 1.4 (g) General component carbohydrate 19.6 (g) General component Discard rate 20 (%)

division ingredient content unit Dietary Fiber Total dietary fiber 2 (g) Dietary Fiber Soluble fiber 0.6 (g) Dietary Fiber Insoluble fiber 1.4 (g) Minerals calcium 27 (mg) Minerals sign 53 (mg) Minerals iron 0.2 (mg) Minerals magnesium 17 (mg) Minerals zinc 0.3 (mg) Minerals cobalt 3 (㎍) Minerals Copper 0.1 (mg) vitamin Vitamin B1 0.1 (mg) vitamin Vitamin B2 0.02 (mg) vitamin Niacin 0.4 (mg) vitamin Vitamin C 9 (mg)

division ingredient content unit vitamin Vitamin B6 0.28 (mg) vitamin Pantothenic acid 0.45 (mg) vitamin Folic acid 24 (㎍) vitamin Vitamin E 0.4 (mg) amino acid Isoleucine 54 (mg) amino acid Leucine 81 (mg) amino acid Lysine 65 (mg) amino acid Methionine 23 (mg) amino acid Cysteine 21 (mg) amino acid Phenylalanine 67 (mg) amino acid Tyrosine 35 (mg) amino acid Threonine 59 (mg) amino acid Tryptophan 23 (mg) amino acid Balin 76 (mg) amino acid Histidine 38 (mg) amino acid Arginine 190 (mg) amino acid Alanine 110 (mg) amino acid Aspartic acid 160 (mg) amino acid Glutamic acid 350 (mg) amino acid Glycine 58 (mg) amino acid Proline 45 (mg) amino acid Serine 160 (mg)

Analysis item Analysis Standard error rate (% RSD) calorie 325.74 kcal - carbohydrate 70.7 g / 100 g - protein 11.34 g / 100 g 0.17% Fat 0.92 g / 100 g 4.02% moisture 8.60% 1.12% Ash 8.45% 0.90% Crude fiber 5.33 g / 100 g 1.63%

As described above, since the peel, which is a by-product of the production and processing of hemp, shows a difference in the composition of ingredients such as calorie and protein content as compared with the whole portion of hemp, the present invention includes the above-mentioned peel powder or extract as an effective ingredient, It is possible to provide a health functional food composition having enhanced improvement ability.

The health functional food composition according to embodiments of the present invention may include a powder or peel extract of peel. The particle size range of the peel powder may be 80 to 200 mesh, and more specifically 100 to 130 mesh. As an embodiment, the peel extract may be extracted with a solvent selected from the group consisting of water, an organic solvent and a mixed solvent thereof. The organic solvent may specifically include a solvent selected from the group consisting of C ₁ to C ₄ alcohols, dichloromethane, ethyl acetate, hexane, and mixed solvents thereof. More specifically, the C ₁ to C ₄ alcohols may include methanol, ethanol, and butanol.

In addition, the health functional food composition according to embodiments of the present invention may contain 1 to 30% by weight of powder or extract of peel based on the total weight of the composition, and more preferably 5 to 20% By weight.

RBCs are highly sensitive to reactive oxygen species (ROS) because fatty acids that constitute the blood erythrocyte membrane are highly unsaturated and have high oxygen content in the red blood cells as well as high iron content in hemoglobin molecules . Therefore, the effect of red blood cells on the red blood cells depends on the efficiency of the antioxidant system in red blood cells. The erythrocytes are rich in antioxidant enzymes such as glutathione (GSH), superoxid dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) Alpha-tocopherol as an antioxidant is also very abundant. However, when the production of active oxygen increases due to azoxymethane (AOM), etc., and the antioxidant system of red blood cells does not work properly, cells including red blood cells are damaged by oxidative stress. The superoxide anion is the only radical that can leak into the plasma through the red blood cell membrane through the anion channel of the red blood cell membrane. If the activity of superoxide dismutase (SOD) is not increased when the production of superoxide anion is increased due to azoxymethane or the like, the superoxide anion leaks into the plasma, and the plasma component is also changed.

Accordingly, embodiments of the present invention can provide a health functional food composition for antioxidation by containing the above-mentioned peel powder or extract as an active ingredient. Specifically, the peel powder or extract may contain at least one of Glutathione (GSH), Superoxid dismutase (SOD), Glutathione peroxidase (GPx) and Catalase (CAT) Antioxidant enzyme activity can be increased. Also, in one embodiment, the peel powder or extract may increase gene expression of NRF2 (Nuclear factor (erythroid derived 2) -like 2), which acts as a transcription factor for glutathione synthetase (GCLC) and antioxidative mechanisms have. The superoxide dismutase (SOD) can convert a highly reactive and highly toxic superoxide radical into hydrogen peroxide (H ₂ O ₂ ), and the hydrogen peroxide produced is converted to glutathione peroxidase (GPx) or catalase (CAT) Which protects cells from toxic effects by reactive oxygen species. In addition, GPx acts to protect cells from reactive oxygen species by converting hydrogen peroxide into water by using glutathione (GSH) to prevent hydrogen peroxide generated in the living body from being converted into toxic hydroxy radicals. Furthermore, in mitochondria, GPx activity together with GSH plays a major role in eliminating hydrogen peroxide produced in mitochondria than CAT. Glutathione S-transferase (GST) is an enzyme that conjugates and removes lipid peroxides with GSH. The activity of GST depends on the supply of GSH. That is, as described above, the present invention includes the powder or extract of peel as an active ingredient to increase the activity of the antioxidant enzyme, thereby inhibiting reactive oxygen species and protecting the cells against the toxicity of endogenous and exogenous compounds such as chemical carcinogens can do. In addition, the peel powder or extract according to embodiments of the present invention can increase the antioxidant activity against oxidative stress induced by AOM through gene expression and activity regulation of antioxidant enzyme.

In addition, embodiments of the present invention provide a health functional food composition for anti-inflammation by containing the above-mentioned peel powder or extract as an active ingredient. The powder or extract of the peel may be selected from the group consisting of NF-κB, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha -1 < / RTI > < RTI ID = 0.0 > β). ≪ / RTI >

Specifically, the NF-κB is a promoter of oncogenesis that regulates inflammation through overexpression of inflammatory mediator genes, and activated NF-κB can induce the transcription of pro-inflammatory mediators. The NF-kB is an important regulator of the first rapidly responding cellular response to deleterious cell stimulation. The known inducers of NF-κB activity are very diverse and include ROS, TNF-α, IL-1β, and LPS. Expression of NF-κB promotes cell proliferation whereas inhibition of NF-κB activity inhibits cell proliferation. Therefore, the present invention can suppress or inhibit inflammation by inhibiting the expression of NF-κB by including a powder or extract of peel. In addition, the TNF-a is one of the important inflammatory cytokines, mainly produced by mononuclear cells and macrophages. It is secreted in early stages of acute and chronic inflammatory diseases and causes a series of inflammatory reactions such as secretion of other cytokines including IL-1, IL-6, and IL-8. During the inflammatory reaction, the expression of TNF-α is dependent on the activity of NF-κB, and the cells exposed to TNF-α activate NF-κB and are activated by COX-2, LOX-2, cell adhesion molecules, inflammatory cytokines, Lt; RTI ID = 0.0 > iNOS < / RTI > In other words, suppression of the expression of TNF-a can inhibit the activation of NF-kB and improve or prevent inflammation. On the other hand, the enzyme responsible for the synthesis of prostaglandin (PG _S) is present the two homozygous protein (isoform), which are COX-1 and COX-2. Although COX-1 is continuously expressed in many tissues, the expression of COX-2 is regulated by mitogen, tumor promoters, cytokines and growth factors. That is, a diet that can inhibit NF-κB can inhibit the expression of COX-2. Nitric oxide synthase (NOS) releases gaseous free radical nitrogen oxides (NO) during the formation of L-citrulline in L-arginine. Overexpression of iNOS mediators and NO is associated with inflammation and tumorigenesis. iNOS is overexpressed in carcinogenesis as a human colon tumor or colon carcinogen, and the expression of COX-2 and iNOS is regulated by the transcription factor NF-κB. Therefore, the present invention can exhibit anti-inflammatory activity by inhibiting the expression of iNOS and COX-2 by including a powder or extract of peel.

Embodiments of the present invention provide a health functional food composition for improving cholesterol in blood by containing the above-mentioned peel powder or extract as an active ingredient. The peel powder or extract inhibits the expression of the gene of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is a cholesterol synthesizing enzyme, and inhibits the synthesis of cholesterol in the blood. Liver is a central organs that regulate cholesterol metabolism. Cholesterol is one of the constituents of mammalian cell membranes. It is a steroid hormone and is one of the lipid components essential for normal functioning of the body. However, when the total cholesterol content in the blood is increased, the oxidation of LDL-cholesterol is induced, and the macrophages that absorb the lipolytic enzyme form foam cells and settle on the blood vessel wall, thereby causing cardiovascular diseases caused by atherosclerosis. HDL-cholesterol lowers cholesterol levels in the blood by removing cholesterol accumulated in peripheral tissues or vascular walls and transporting it to the liver to increase excretion into bile acids. The present invention can reduce blood triglyceride content and total cholesterol content and reduce total cholesterol / high density lipoprotein (TC / HDL) by including peel powder or extract.

In addition, according to one embodiment of the present invention, the powder or extract of mar peel contained in the health functional food composition can be used as a preventive agent against aberrant crypt foci (ACF) and mucin depleted it is possible to inhibit the formation of foci. The present invention can inhibit colon cancer precancerous lesions by increasing antioxidant enzyme activity. The reactive oxygen species (ROS) is an important precursor of carcinogenesis. In the condition that the level of reactive oxygen species is unbalanced with the antioxidant (oxidative stress condition), proliferation, inflammation or cell It can lead to death. Tumor cells use reactive oxygen species to stimulate proliferation, invasion, migration and angiogenesis, and the treatment of carcinogens or tumor promoters in the colon reduces the levels of antioxidant enzymes. Decreases in antioxidant enzymes can further increase oxidative stress and can lead to increased oxidation of DNA bases, mutagenesis and induction of carcinogenesis. In one embodiment, the peel powder or extract according to the present invention can inhibit the expression of the inflammatory mediator gene to inhibit the colon cancer precursor lesion. Active oxygen species, an important precursor of the carcinogenesis, play an important role in various signal transduction pathways, for example, as a messenger involved in the activation of NF-κB. The colorectal cancer precursor lesion may be one induced by azoxymethane (AOM). The cytotoxicity of the above azoxymethane (AOM) is considered to be a strong carcinogen to the large intestine, mediating oxidative stress. In addition, upon carcinogenesis, the production of reactive oxygen species in tumor cells increases and the ability to remove reactive oxygen species decreases. The ACF (Aberrant crypt foci) is a very small lesion considered to be the first precancerous lesion of colorectal cancer occurring before adenomatous development, and the generation of multiple ACF increases with time, and thus it is a predictor of tumor. Thus, inhibition of the formation of ACF in the colon means that it has potential chemical protective properties of colon tumors. In addition, precancerous lesions (MDF) that do not secrete mucin indicate the ability to control precancerous lesions in the large intestine.

The health functional food composition according to the embodiments of the present invention has a liquid or solid form, and the formulation is not particularly limited. For example, it can be formulated into tablets, capsules, soft capsules, pills, granules, beverages (drink), caramels, bars, chocolates or confections. The health functional food composition of each formulation may be formulated by those skilled in the art without difficulty, depending on the purpose of formulation or use, in addition to the active ingredient. For example, the health functional food composition according to one embodiment of the present invention may contain excipients, saccharides, flavors, pigments, oils, proteins and the like as needed.

Hereinafter, the present invention will be described with reference to the following Production Examples and Experimental Examples. The Examples and Experimental Examples are intended to further illustrate the present invention and are not intended to limit the scope of the present invention. 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 scope of the invention.

[Preparation Example 1] Preparation of peel powder

I bought the peel (Taesan Farm, Ohsanri, North and South of Andong) produced in Andong, Gyeongbuk. Twenty kilograms of the dried peel were ground by Pin Mill (grinder) of Gyeongbuk Biotechnology Research Institute and crushed at 120 mesh to obtain peel powder.

[Preparation Example 2] Preparation of Diet

(5Y, 10Y and 20Y) as one example of the present invention as a dietary test for evaluating the efficacy of the peel powder as an active ingredient of the present invention prepared in Preparation Example 1, (NC) and a positive control group, a silymarin-containing diet (0.5S), were prepared according to conventional methods (unit: g / kg diet) with the following composition.

Constituent Dietary Group NC 5Y 10Y 20Y 0.5S Starch 550 508 467 383 545 Sucrose 100 100 100 100 100 casein 200 194 189 177 200 Cone oil 50 50 50 50 50 cellulose 50 47 45 39 50 Mineral Mixture ¹⁾ 35 35 35 35 35 Vitamin mixture ²⁾ 10 10 10 10 10 DL-methionine 3 3 3 3 3 Colin Vita Trace 2 2 2 2 2 Peel (Preparation Example 1) - 50 100 200 - Silymarin - - - - 5

1) Mineral mixture (g / kg): CaCO ₃ 292.9, CaHPO ₄ -2H ₂ O 4.3, KH ₂ PO ₄ 43.1, NaCl 250.6, MgSO ₄ .7H ₂ O 99.8, Fe (C ₆ H ₅ O ₇ ) ₂ O 6.23, CuSO ₄ .5H ₂ O 1.56, MnSO ₄ .H ₂ O 1.21, (NH ₂ ) ₆ MO ₇ O ₂₄ .4H ₂ O 0.025, Na ₂ SeO ₃ .5H ₂ O 0.015, ZnCl ₂ 0.005); 2) Vitamin mixture (mg / kg): Vitamin D ₃ 58.2,? -Tocopherol-acetate 1200.0, retinol-acetate 93.2, vitamin K ₃ Biotin 1.0, folic acid 2.0, inositol 1176.0, calcium pantothenate 235.0, riboflavin 59.0, nicotinic acid 294.0, sucrose 96257.017, thiamine-HCl 59.0, vitamin B ₁₂ 0.2, vitamin C 588.0, pyridoxine-HCl 29.0,

[Experimental Example 1] Analysis of biochemical liver function index change in blood

Animal Breeding and Diet Benefits

F344 male rats were purchased from BioLink prior to the experiment and were sufficiently accommodated for 1 week under solid feed and standard feeding conditions (temperature 23 ± 1 ℃ and humidity 50 ± 5%). (AOM, A5Y, A10Y, A20Y, A0.5S) were divided into 5 groups (NC, 5Y, 10Y, 20Y and 0.5S) Respectively.

The NC, 5Y, 10Y, 20Y and 0.5S were the dietary groups prepared in Preparation Example 2, NC was the AIN-76 general formula, 5Y was the 5% peanut containing diet, 10Y was the 10% 20Y is a dietary group containing 20% parboil and 0.5S is a dietary group fed with 0.5% silymarin. The AOM, A5Y, 10Y, A20Y, and A0.5S were groups fed with the azo-methane (AOM) reagent and the diets prepared in Preparation Example 2, After AOM injection, A10Y and A10Y after AOM injection, A10Y and A20Y after AOM injection, and A20Y and A0.5S after AOM injection, respectively.

After two weeks, the mice were injected with azoxymethane (AOM) reagent at a dose of 15 mg / kg subcutaneously twice (once / week) to induce colorectal cancer. After 8 weeks, The diets were fed with the samples.

Sampling

After 8 weeks, the animals were fasted for 12 hours and then anesthetized with CO ₂ gas. The blood was collected through the abdominal aorta and centrifuged (3,000 rpm, 20 minutes) to separate plasma and erythrocytes and stored at -70 ° C. Liver organs were harvested and rinsed with physiological saline. Water was removed and weighed. The whole colon tissue was rinsed with PBS buffer and cut longitudinally. The cut colon was placed between two sheets of filter paper in a Petri dish, flattened and fixed in 4% formaldehyde in PBS (4% formaldehyde in PBS) for 24 hours (4 ° C). Some of the large intestine mucosa and liver tissues were stored at -70 ° C after RNAlater ^™ treatment and used as samples for gene expression measurement.

Liver sample fraction

The remaining liver was separated into cytosol, microsomes and mitochondria and stored at -70 ° C for enzyme activity measurement. The liver tissues were homogenized with 0.25 M sucrose solution of 4 times the weight of the liver tissue and centrifuged at 4 x 10 x g for 10 min at 1,000 x g. The supernatant was centrifuged at 10,000 xg for 20 min at 4 ° C Separate the supernatant and the mitochondria by centrifugation. The post-mitochondrial fraction was centrifuged at 105,000 xg for 1 hour at 4 ° C, and the microsomes and supernatants (Cytosol) were stored at -70 ° C for enzyme activity measurement.

In the blood  Biochemical liver function index change analysis

There are many enzymes in hepatocytes, but only a small amount in the blood. However, when the cell membrane is damaged, the enzyme in the hepatocyte leaks into the blood, thereby increasing the activity of the enzyme in the blood. Therefore, the activity of plasma GOT (Glutamate-oxalacetate transaminase), GPT (Glutamate-pyruvate) and γ-GT (γ-glutamyl transferase) increases in liver diseases such as hepatitis, fatty liver and liver cirrhosis. GPT is mainly present in the cytoplasm of hepatocytes, and sensitively reflects the degree of necrosis when severe hepatocellular necrosis such as acute hepatitis is severe. When GOT is present in the cytoplasm of the mitochondria and the rest is in the cytoplasm, GOT is increased more than GPT.

In order to analyze the liver function improvement ability of the peeled diet, the azoxymethane (AOM) reagent was subcutaneously injected twice (once / week) at a concentration of 15 mg / kg, and after 8 weeks A5Y, 10Y, A20Y, and A0.5S, which were fed diets containing NC, 5Y, 10Y, 20Y and 0.5S, respectively.

Specifically, the activity of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), which indicates the liver function index, was measured using a kit (Asan Pharmaceutical Co., Ltd.). 1 ml of GOT and GPT substrate solution was heated in a 37 ° C water bath for 5 minutes and 0.2 ml of each of the AOM, A5Y, 10Y, A20Y, and A0.5S plasma was added. The GOT was maintained for 60 minutes in a 37 ° C water bath, Lt; / RTI > 1 ml of colorimetric solution was added and allowed to stand at room temperature for 20 minutes. Then, 10 ml of 0.4 N NaOH solution was added and the absorbance was measured at 505 nm. GOT and GPT standards (2 mM pyruvate) were developed by the same method as above, and the absorbance was measured, and then the concentration of the sample was calculated by inserting it into a standard curve. Data were analyzed using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 16), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

Dietary Group GPT (U / ml) GOT (U / ml) AOM 66.75 ± 17.25 ^b 83.13 + - 4.37 ^b A5Y 58.06 ± 12.43 ^ab 78.50 ± 6.21 ^ab A10Y 53.53 +/- 14.40 ^a 75.86 +/- 8.23 ^a A20Y 56.19 ± 16.75 ^ab 76.06 +/- 10.77 ^a A0.5S 51.94 ± 12.39 ^a 76.13 + - 10.36 ^a

As a result, as shown in Table 6, GPT and GOT were significantly lower in the A10Y group and A0.5S group, both of which were fed the peel diet and the positive control group, respectively, as compared with the AOM group. That is, according to the embodiment of the present invention, the activity of blood enzymes such as GOT and GPT in hepatocytes damaged by azomethymethane (AOM) when the peel-containing diet is fed reduces the damage of hepatocytes .

Lipid changes in blood

Liver is a central organs controlling cholesterol metabolism. Cholesterol is one of the constituents of mammalian cell membranes and is one of the lipid components essential for normal functioning of the body as a steroid hormone. However, when the total cholesterol content in the blood increases, the oxidation of LDL-cholesterol is induced and the macrophages Forms foam cells and deposits them in the blood vessel walls, which leads to cardiovascular diseases caused by atherosclerosis. HDL-cholesterol lowers cholesterol levels in the blood by removing cholesterol accumulated in peripheral tissues or vascular walls and transporting it to the liver to increase excretion into bile acids. Thus, elevated serum concentrations of HDL-cholesterol indicate improved hyperlipemia.

Subsequently, the above azoxymethane (AOM) reagent was subcutaneously injected twice (once / week) at a concentration of 15 mg / kg, NC, 5Y, 10Y, 20Y and 0.5S The changes in biochemical liver function index of AOM, A5Y, 10Y, A20Y and A0.5S fed diets were analyzed. Specifically, plasma triglyceride concentration, plasma total cholesterol concentration, and plasma HDL-cholesterol concentration as lipid of blood were analyzed as follows.

First, plasma triglyceride (TG) concentration was measured by a kit for measuring triglycerides by enzymatic method (Asan Pharmaceutical, Korea). Add 0.02 ml of plasma and 3 ml of enzyme reagent, mix well and incubate at 37 ° C for 10 min. Measure the absorbance at 550 nm with the reagent blank as a control. Add the absorbance of the sample to the following equation to determine the concentration of triglyceride Respectively.

The measurement of plasma cholesterol (TC) concentration was measured by a kit for measuring cholesterol by an enzymatic method (Asan Pharmaceutical, Korea). Add 0.02 ml of plasma and 3 ml of enzyme reagent, mix well and incubate for 5 minutes at 37 ° C. Measure the absorbance at 500 nm with the reagent blank as a control. Add the absorbance value of the sample to the following equation to determine the total cholesterol concentration Respectively.

Plasma HDL-cholesterol concentration was measured by a kit for measuring cholesterol by an enzymatic method (Asan Pharmaceutical, Korea). 0.2 ml of plasma and 0.2 ml of separation reagent were reacted at room temperature for 10 minutes and centrifuged at 3,000 rpm for 10 minutes. The supernatant was mixed with 1.0 ml of the supernatant and 3 ml of the enzyme reagent, and the mixture was incubated at 37 ° C for 5 minutes. After absorbance at 500 nm was measured, the absorbance value of the sample was substituted into the following equation to calculate the plasma HDL-cholesterol concentration.

Data were analyzed using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 16), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

group TG (mg / dL) TC (mg / dL) HDL (mg / dL) TC / HDL AOM 118.1 ± 30.5 ^c 82.2 ± 10.8 ^b 38.3 ± 5.6 ^ab 2.2 ± 0.3 ^c A5Y 84.1 ± 30.8 ^b 83.3 ± 12.9 ^b 40.8 ± 8.0 ^ab 2.1 ± 0.3 ^bc A10Y 79.6 ± 28.6 ^ab 73.5 ± 16.7 ^ab 39.6 ± 6.7 ^ab 1.9 ± 0.4 ^ab A20Y 60.9 ± 24.2 ^a 65.6 ± 10.6 ^a 36.3 ± 4.7 ^a 1.8 ± 0.3 ^a A0.5S 71.8 ± 25.0 ^ab 79.9 ± 13.9 ^b 42.2 ± 5.5 ^b 1.9 ± 0.3 ^ab

(ns: not significant)

As shown in Table 7, total cholesterol and TC / HDL were significantly decreased in A10Y, A20Y, and A0.5S fed with peeled diet compared with AOM, and triglyceride (TG) A10Y, A20Y and A0.5S, respectively. A20Y was significantly lower than A0.5S group. According to one embodiment of the present invention, when the diet containing the peel is fed, the peel reduces the blood cholesterol metabolism and improves the hyperlipemia. In particular, in the case of A20Y containing 20% It means that it has improvement ability.

[Experimental Example 2] Enzyme activity assay

In order to confirm the antioxidant ability of the parasite as an active ingredient of the present invention, the enzyme activity was analyzed as follows.

Of blood and liver tissue Glutathione ( GSH ) Content measurement

GSH content was determined by using GSH measurement kit (Cayman Chem. Co.). Plasma GSH was prepared by mixing 50 μl of 5-fold diluted plasma and 50 μl of detector reagent in assay wells in 96 wells, reacting at room temperature for 5 minutes, Fluorescence was measured with a fluorescent microplate reader (Molecular Devices Co.). The red blood cells were lysed with distilled water four times the volume of the red blood cells, centrifuged at 10,000 xg for 15 minutes at 4 ° C, and the supernatant was measured in the same manner as the plasma using the sample. Cytosol and mitochondrial fractions were obtained by centrifugation of hepatic tissues, and each was diluted with assay buffer and then measured by the same method. The GSH content was calculated as the fluorescence value of the standard solution, and the regression equation was obtained. The content was calculated using the following equation and corrected for protein concentration: plasma, liver cytosol and mitochondria in nmol / mg protein and red blood cells in hemoglobin concentration And corrected to nmol / mg Hb. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.). Erythrocyte concentrations were determined by the Cyanomethemoglobin method using a Drabkin's solution.

Of blood and liver tissue Superoxide Dismutase ( SOD ) Active measurement

Superoxide dismutase (SOD) activity was measured in blood (plasma and red blood cells), Cu / Zn SOD in liver cytosol and Mn SOD in liver mitochondria. The concentration of superoxide radicals produced by the reaction of xanthine oxidase and hypoxanthine was measured using a kit (Cayman Chem. Co.) using tetrazolium salt as a radical detector , And 1 unit of SOD was defined as the amount of enzyme needed to dismutate 50% of the superoxide radical. 10 μl of a sample diluted with the sample buffer, 200 μl of a radical detector, and 20 μl of xanthine oxidase (10 μl of sample, 190 μl of a radical detector, 10 μl of potassium cyanide and 20 μl of xanthine oxidase in the case of Mn SOD) After mixing in wells and shaking for 20 minutes, the absorbance was measured at 450 nm. The red blood cells were lysed with four times the volume of red blood cells, and the supernatant obtained by centrifugation at 10,000 xg for 15 minutes at 4 ° C was diluted with a sample buffer and used as a sample. SOD activity was calculated by calculating the linear rate (LR) as the absorbance of the standard solution and the sample, creating a standard curve, calculating the regression equation, and calculating the activity using the following equation (5). Enzyme activity was expressed as U / mg protein in plasma, liver cytosol and mitochondria, and U / mg Hb in erythrocytes corrected to hemoglobin concentration. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.). Erythrocyte concentrations were determined by the Cyanomethemoglobin method using a Drabkin's solution.

Of blood and liver tissue Catalase ( CAT ) activation

Catalase (CAT) activity was measured using a kit (Cayman Chem. Co.) using the peroxidation activity of CAT in blood (plasma and red blood cells) and hepatic tissue fractions (cytosol and mitochondria). That is, CAT reacts with methanol in the presence of a certain concentration of H ₂ O ₂ to form a formaldehyde, which is then developed with 4-amino-3-hydrogene-5-mercapto-1,2,3-triazole (Purpald) Of the CAT activity. 20 μl of the sample diluted with the sample buffer, 100 μl of assay buffer, 30 μl of methanol and 20 μl of H ₂ O ₂ were mixed in 96 wells and reacted at room temperature for 20 minutes. 30 μl of potassium hydroxide solution and 30 μl of coloring agent were added to each well. After 10 minutes of shaking, 10 μl of potassium periodate solution was added, and the mixture was further shaken for 5 minutes. Then, the absorbance was measured with a microplate reader at 540 nm. The red blood cells were lysed with distilled water four times the volume of red blood cells, and the supernatant obtained by centrifugation at 10,000 xg for 15 minutes at 4 ° C was diluted with a sample buffer and used as a sample. CAT activity was calculated as the absorbance value of the standard solution and the regression equation was obtained and the formaldehyde concentration was calculated using the following equation.

From the calculated formaldehyde concentration, the CAT activity was calculated by the following equation. The enzyme (CAT) activity was corrected by protein concentration, and the plasma was expressed in nmol / mg protein, the liver fraction in μmol / mg protein, and the erythrocyte corrected by hemoglobin concentration in μmol / mg Hb. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.). Erythrocyte concentrations were determined by the Cyanomethemoglobin method using a Drabkin's solution.

Of blood and liver tissue Glutathione  Peroxidase ( GPx ) activation

Glutathione peroxidase (GPx) activity was measured using a kit (Cayman Chem. Co.) in blood (plasma and red blood cells) and hepatic tissue fractions (cytosol and mitochondria). The principle of measurement is that hydroperoxide is reduced by GPx and oxidized glutathione (GSSG) is generated. GSSG is reduced to GSH by glutathione reductase (GR) and NADPH, and NADPH is oxidized to NADP ⁺ Of the absorbance was proportional to the activity of GPx. Mix 20 μl of sample diluted with sample buffer, 120 μl of assay buffer and 50 μl of Co-substrate mix (NADPH, GSH and GR) in 96 wells and add 20 μl of cumene hydroperoxide After addition, it was shaken thoroughly and the change of absorbance at 340 nm for 5 minutes was measured. The red blood cells were lysed with distilled water four times the volume of red blood cells, and the supernatant obtained by centrifugation at 10,000 xg for 15 minutes at 4 ° C was diluted with a sample buffer and used as a sample. A standard curve was calculated from the change in absorbance of the GPx control group by ΔAbs / min, and the GPx activity was calculated using the following equation (8).

One unit of GPx was defined as the amount of enzyme that oxidized 1 nmol of NADPH to NADP ⁺ for 1 minute at 25 ° C. Enzyme activity was expressed as nmol / min / mg protein in plasma, liver cytosol and mitochondria in nmol / min / mg protein and red blood cells in nmol / min / mg Hb corrected to hemoglobin concentration. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.). Erythrocyte concentrations were determined by the Cyanomethemoglobin method using a Drabkin's solution.

Glutathione -S-transferase ( GST ) activation

Glutathione S-transferase (GST) activity was measured using a kit (Sigma) in hepatic tissue fractions (cytosol and mitochondria). GST catalyzes the conjugation of a thiol group of glutathione to 1-chloro-2,4-dinitrobenzene (CDNB) to produce a GS-DNB conjugate. Since the amount of this reaction product is proportional to the GST activity, GS -DNB conjugate was measured at 340 nm and expressed as GST activity. After mixing 10 μl of the sample diluted with buffer and 190 μl of the substrate mixture (100 μl of 200 mM GSH, 100 μl of 100 mM CDNB and 9.8 ml of PBS) in 96 wells, the absorbance change at 340 nm was measured Respectively. Enzyme activity was calculated as nmol / min / mg protein by calibrating with the following formula. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.).

The effects of the peel on the antioxidant enzymes (GSH, SOD, GPx, and CAT) measured above are shown in the following table as enzyme activity in blood and enzyme activity in liver. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 8), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

The results of analyzing the enzyme activity changes in blood and liver tissues are shown in Table 8 below.

AOM A5Y A10Y A20Y A0.5S Plasma GSH (nmol / mg protein) 1.94 ± 0.23 ^ns 1.93 ± 0.24 ^ns 2.23 ± 0.61 ^ns 2.25 ± 0.09 ^ns 2.23 ± 0.29 ^ns SOD (U / mg protein) 2.99 ± 0.48 ^ns 3.26 ± 0.47 ^ns 2.71 ± 0.93 ^ns 2.79 ± 0.68 ^ns 3.43 ± 0.89 ^ns GPx (nmole / min / mg protein) 38.61 ± 3.11 ^a 38.89 ± 3.37 ^a 39.45 + 2.52 ^a 37.61 ± 1.75 ^ab 41.82 ± 1.78 ^b Erythrocyte GSH (nmol / mg Hb) 1.58 ± 0.27 ^a 2.52 ± 1.00 ^ab 4.15 ± 2.11 ^b 2.95 ± 1.78 ^ab 2.58 ± 1.48 ^ab SOD (U / mg Hb) 222.41 ± 63.09 ^b 134.52 ± 31.48 ^a 299.08 + - 47.53 ^c 219.98 +/- 42.71 ^b 170.70 ± 26.04 ^a GPx (nmole / min / mg Hb) 731.41 + - 76.88 ^ns 787.44 + 84.88 854.69 + - 71.76 829.96 ± 179.70 756.17 + - 152.03 CAT (nmole / min / mg Hb) 1865.37 ± 215.54 ^ns 1891.72 + - 444.97 1942.22 + 192.27 1938.11 ± 559.00 1992.60 ± 260.59 Liver Cytosol GSH (nmol / mg protein) 1.21 ± 0.44 ^ns 1.10 0.41 1.53 + - 0.66 1.34 ± 0.58 1.21 ± 0.69 Cu / Zn SOD (U / mg protein) 68.84 +/- 14.40 ^a 81.05 ± 10.71 ^b 94.12 ± 13.77 ^c 99.96 + - 6.61 ^c 92.35 + - 11.66 ^bc GPx (nmole / min / mg protein) 199.65 ± 43.93 ^a 243.17 ± 19.59 ^b 253.63 ± 19.20 ^b 272.68 ± 19.61 ^b 267.05 ± 24.75 ^b CAT (nmol / min / mg protein) 2131.53 ± 309.28 ^ns 2101.96 + 479.01 1790.18 ± 608.93 2205.18 ± 453.11 2051.34 + - 585.76 GST (nmole / min / mg protein) 98.91 +/- 10.70 ^a 105.99 ± 13.96 ^ab 104.53 ± 12.33 ^ab 115.80 ± 5.45 ^b 109.75 ± 16.82 ^ab Liver mitochondria
(Liver Mitochondria) GSH (nmol / mg protein) 1.89 ± 0.53 ^ns 3.07 ± 2.09 3.17 ± 1.42 3.84 ± 2.55 3.74 ± 1.93 Mn SOD (U / mg protein) 8.58 ± 2.17 ^b 8.60 ± 1.41 ^b 4.37 ± 0.83 ^a 5.98 ± 1.57 ^a 7.64 ± 1.85 ^b GPx (nmole / min / mg protein) 40.92 ± 16.44 ^ab 35.88 ± 12.50 ^a 47.60 ± 13.46 ^abc 62.68 ± 13.60 ^c 54.75 ± 17.93 ^bc CAT (nmol / min / mg protein) 1636.51 ± 402.02 ^a 1748.82 + - 616.98 ^a 2684.10 ± 692.91 ^bc 3122.47 + - 469.54 ^c 2505.56 + 514.44 ^b GST (nmole / min / mg protein) 52.70 + - 8.60 ^b 35.07 ± 8.94 ^a 54.93 + - 9.81 ^b 66.77 ± 16.71 ^b 64.73 ± 21.39 ^b

(ns: not significant.)

As shown in Table 8, enzymatic activities of antioxidant enzymes GSH and SOD in the blood were increased by the parasite diet. As a result, GSH contents in red blood cells were significantly increased in the A10YS group compared to AOM group due to consumption of parasite. SOD activity in red blood cells was significantly increased in the A10Y group due to consumption of the parasite. There was no change in GPx and CAT activity in plasma and red blood cells due to consumption of AOM group and parchment.

Changes in red blood cells have been detected after exposure to a number of human pathologic conditions or biohazard indicating oxidative stress. Erythrocytes are in constant contact with potentially damaging levels of oxygen, but under normal conditions, red blood cell metabolism and antioxidant defense systems can prevent damage. Wherein the antioxidant system comprises SOD, CAT, GPx, GST and GR and GSH. Oxidation of red blood cells is associated with membrane damage, methemoglobin formation, osmotic vulnerability and cell destruction, and oxidative stress of red blood cells is an indicator of RBC-related disease or overall oxidative stress. Accordingly, the above results indicate that the health functional food composition according to the present invention not only strengthens the oxidative stress protection system but also can remove free radicals by containing the powder or extract of peel as an active ingredient.

On the other hand, as shown in Table 8, the antioxidative enzymes SOD, GPx, CAT, and GST in the liver tissues caused by the consumption of AOM group and parchment were increased by the parasite diet. Specifically, the changes in GSH content in liver tissues due to consumption of AOM group and parchment were not changed, but the change of SOD activity due to consumption of parasite was measured by Cu / Zn SOD in liver cytosol In liver mitochondria, the Mn / SOD activity of liver cytosol was significantly increased compared to that of AOM due to the consumption of parasite, whereas the activity of Mn SOD in liver mitochondria was increased in the AOM group Compared with the A10Y group. It is considered that the radicals are cleaved due to the increase of Cu / Zn SOD activity in the cytosol. Changes in GPx activity due to ingestion of parasite were significantly increased in hepaticositol compared to AOM group. CAT activity was significantly increased in the hepatic mitochondria from the A10Y group, especially in the A20Y group than in the A0.5S group. GST activity due to the consumption of parasites in liver cytosol and mitochondria was significantly increased in the liver cytosol and mitochondria, respectively. Therefore, it can be seen that the health functional food composition according to the present invention exhibits antioxidant activity by containing a powder or extract of peel as an active ingredient.

[Experimental Example 3] Measurement of enzyme gene expression level

The expression of cholesterol synthase (liver), antioxidant enzyme (large intestine mucosa) and inflammation related gene expression (large intestine mucosa) by the peeled diet, which is an active ingredient of the present invention, were analyzed as follows.

First, total RNA was extracted from liver using RNeasy Mini Kit (Quiagen, Valencia, CA, USA). The extracted total RNA (0.1 μg) was analyzed using a One Step SYBR PrimeScript RT-PCR kit (TaKaRa) using an Exicycler3 system (Bioneer Co., Daejeon, Korea). The enzyme primers were custom-made on Bioneer Co. (Daejeon, Korea) and the primer sequences are shown in Table 9 below.

Target gene The primer sequence (5 'to 3') β-Actin F: CAC CAG TTC GCC ATG GAT
R: ATC ACA CCC TGG TGC CTA HMG CoA reductase F: GCG TGC AAA GAC AAT CCT GGA G
R: GTT AGA CCT TAG GAA CCC AAT G

Β-actin was used as an internal control. The degree of gene expression was calculated by the comparative Ct method. Obtaining the difference (ΔCt) of the (threshold cycle) value and the measured Ct β-actin, determined the difference (ΔΔCt) between control and experimental groups were then calculated the relative expression ratio (2 ^{-ΔΔ CT)} to the control group. The results are shown in Table 10 below. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 6), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

AOM A5Y A10Y A20Y A0.5S HMG-CoA ΔC _T 3.43 ± 0.21 ^a 4.10 ± 0.27 ^b 4.47 ± 0.29 ^b 4.52 ± 0.54 ^b 4.09 ± 0.44 ^b 2- ^{ΔΔ CT} 1.00 0.63 0.49 0.47 0.63

As a result, the expression level of HMG-CoA reductase, which is a cholesterol synthesizing enzyme of liver tissue, was significantly decreased as compared with that of AOM group due to consumption of march, and thus it can be confirmed that the present invention can inhibit cholesterol synthesis.

Next, total RNA was extracted from liver and colon mucosa using RNeasy Mini Kit (Quiagen, Valencia, CA, USA). The extracted total RNA (0.1 μg) was analyzed using a One Step SYBR PrimeScript RT-PCR kit (TaKaRa) using an Exicycler3 system (Bioneer Co., Daejeon, Korea). The enzyme primer was custom-made on Bioneer Co. (Daejeon, Korea), and the primer base sequence is shown in Table 11 below.

Target gene The primer sequence (5 'to 3') β-Actin F: CAC CAG TTC GCC ATG GAT
R: ATC ACA CCC TGG TGC CTA Catalase F: AAG CTG GTT AAT GCG AAT GG
R: CAA GTT TTT GAT GCC CTG GT Cu / Zn SOD F: GTC GTC TCC TTG CTT TTT GC
R: TCT GCT CGA AGT GAA TGA CG Mn SOD F: CGT CAC CGA GGA GAA GTA CCA CGA
R: CAG CCT GAA CCT TGG ACT CCC ACA GPx 2 F: AAA CCG AAG CAT TTC CTCE
R: AGC ACC TAC CCC AGA CTT AGA NrF2 F: CGG CAT TTC ACT GAA CAC AA
R: AGC TCT TCC ATT TCC GAG TC GCLc F: ACC TGC ATT CCA GAG TTTCA
R: ACA GCA AGA GGA GAC TCA AG

(SOD), superoxide dismutase (GPX), glutathione peroxidase (CAT), catalase, NRF 2 (erythroid-derived 2) -like 2, GCLC: Glutamate cysteine ligase catalytic subunit

Β-actin was used as an internal control. The degree of gene expression was calculated by the comparative Ct method. Obtaining the difference (ΔCt) of the (threshold cycle) value and the measured Ct β-actin, determined the difference (ΔΔCt) between control and experimental groups were then calculated the relative expression ratio (2 ^{-ΔΔ CT)} to the control group. The results are shown in Table 12 below. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 6), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

Antioxidant enzyme AOM A5Y A10Y A20Y A0.5S NRF2 ΔC _T 5.15 + 0.49 ^c 1.98 + 0.11 ^a 1.86 ± 0.67 ^a 2.01 + 0.40 ^a 3.71 + - 0.46 ^b 2- ^{ΔΔ CT} 1.00 9.00 9.74 8.79 2.70 GCLC ΔC _T 3.32 ± 0.39 ^bc 3.01 ± 0.42 ^ab 2.70 ± 0.19 ^a 3.29 ± 0.62 ^bc 3.70 + - 0.30 ^c 2- ^{ΔΔ CT} 1.00 1.24 1.54 1.02 0.77 Cu / Zn SOD ΔC _T 1.80 ± 0.47 ^ns 1.93 + - 0.23 1.65 0.95 2.30 0.61 1.78 + - 0.31 2- ^{ΔΔ CT} 1.00 0.92 1.11 0.71 1.01 Mn SOD ΔC _T 4.22 ± 0.85 ^ab 4.07 ± 0.21 ^ab 3.96 ± 0.84 ^ab 4.62 ± 0.65 ^b 3.73 ± 0.44 ^a 2- ^{ΔΔ CT} 1.00 1.11 1.21 0.76 1.40 GPx 2 ΔC _T 6.05 ± 0.71 ^ns 5.36 ± 0.55 5.28 ± 1.20 5.52 + - 1.05 5.72 + - 0.56 2- ^{ΔΔ CT} 1.00 1.62 1.72 1.45 1.26 CAT ΔC _T 3.17 ± 0.64 ^b 2.70 ± 0.48 ^ab 3.44 ± 0.53 ^b 2.70 ± 0.72 ^ab 1.95 + 0.33 ^a 2- ^{ΔΔ CT} 1.00 1.39 0.83 1.38 2.32

(ns: not significant)

As a result, there was no change due to consumption of peel in Cu / Zn SOD, Mn SOD, CAT, and GPx2. However, GCLC, a glutathione synthetase, and NRF2, an antioxidant related factor, were significantly increased by a peel diet.

The expression of inflammatory mediator genes in the intestinal mucosa due to ingestion of falciparum was analyzed as follows.

First, total RNA was extracted from the colon mucosa using RNeasy Mini Kit (Quiagen, Valencia, CA, USA). The extracted total RNA (0.1 μg) was analyzed using a One Step SYBR PrimeScript RT-PCR kit (TaKaRa) using an Exicycler3 system (Bioneer Co., Daejeon, Korea). The enzyme primers were custom-made on Bioneer Co. (Daejeon, Korea) and the primer sequences are shown in Table 13 below.

Target gene The primer sequence (5 'to 3') β-Actin F: CAC CAG TTC GCC ATG GAT
R: ATC ACA CCC TGG TGC CTA iNOS F: GTG TTC CAC CAG GAG ATG TT
R: ACC GCT TTC ACC AAG ACT G COX-2 F: ACT ACG CCG AGA TTC CTG AC
R: CAA TGT TCC AGA CTC CCT TG NF-Kb F: GGA ACT GGG CAA ATG TTTCA
R: GGT ATG GGC CAT CTG TTGA Ik-Bα F: TAC TCC CCC TAC CAG CTT AC
R: GAC TCC GTG TCA TAG CTC TC IL-1? F: GCC TCA AGG GGA AGA ATC TAT
R: CAA ACC GCT TTT CCA TCT TCT TNF F: GAG CTC AAG CCC TGG TAT G
R: CGG ACT CCG TGA TGT CTA AG

(NF-κB), necrosis factor kappaB (COX), cyclooxygenase (iNOS), inducible nitric oxide synthase (IL), interleukin, TNF-α and tumor necrosis factor alpha.

Β-actin was used as an internal control. The degree of gene expression was calculated by the comparative Ct method. Obtaining the difference (ΔCt) of the (threshold cycle) value and the measured Ct β-actin, determined the difference (ΔΔCt) between control and experimental groups were then calculated the relative expression ratio (2 ^{-ΔΔ CT)} to the control group. The results are shown in Table 14 below. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 6), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

AOM A5Y A10Y A20Y A0.5S iNOS ΔC _T 9.84 ± 1.11 ^b 10.29 ± 0.62 ^b 9.73 + - 0.41 ^b 9.59 ± 0.88 ^b 8.07 ± 0.53 ^a 2- ^{ΔΔ CT} 1.00 0.73 1.07 1.19 3.39 COX-2 ΔC _T 5.45 ± 0.28 ^b 6.30 ± 0.52 ^c 5.27 ± 0.21 ^ab 5.67 ± 0.65 ^b 4.75 ± 0.50 ^a 2- ^{ΔΔ CT} 1.00 0.55 1.13 0.86 1.62 NF-κB ΔC _T 2.95 + - 0.38 ^a 5.20 0.35 ^c 4.58 ± 0.29 ^b 4.66 ± 0.49 ^b 8.14 ± 0.35 ^d 2- ^{ΔΔ CT} 1.00 0.21 0.32 0.31 0.03 IKBα ΔC _T 6.86 ± 0.42 ^c 2.59 ± 0.28 ^b 2.57 + - 0.36 ^b 2.61 ± 0.67 ^b 1.88 + 0.42 ^a 2- ^{ΔΔ CT} 1.00 19.36 19.54 19.05 31.69 TNF-a ΔC _T 0.41 + 0.36 ^a 5.87 ± 1.07 ^c 6.20 ± 1.43 ^c 6.01 ± 1.33 ^c 4.53 ± 0.26 ^b 2- ^{ΔΔ CT} 1.00 0.02 0.02 0.02 0.06 IL-1? ΔC _T 2.59 ± 0.11 ^a 7.77 ± 0.76 ^d 6.80 ± 1.00 ^c 8.22 ± 0.39 ^d 4.62 ± 0.32 ^b 2- ^{ΔΔ CT} 1.00 0.03 0.05 0.02 0.24

(ns: not significant)

As a result, COX-2, NFkB, TNF-α and IL-1β were significantly decreased, and IKBα was an inhibitor of NF-kB. This means that the present invention can inhibit inflammation by containing a powder or extract of peel as an active ingredient.

[Experimental Example 4] Histopathological changes of colon tissue

Colon cancer Pre-cancerous lesion ( Aberrant crypt foci , ACF ) Measure

ACF (Aberrant crypt foci) is a microscopic lesion considered as the first precancerous lesion of colorectal cancer occurring before adenomatous development. It is a predictor of the tumor because the production of multiple ACF increases with time. Therefore, the change of ACF number in the colon tissue was measured in order to analyze the ability to control the precancerous lesion of colon cancer by the peeled diet of the present invention.

First, in order to measure the ACF of the colon tissues, the colon tissues of the experimental animals were washed with PBS buffer and then cut longitudinally. The cut colon was placed between two sheets of filter paper in a Petri dish, flattened and fixed in 4% formaldehyde (4% formaldehyde in PBS) dissolved in PBS for 24 hours (4 ° C). In order to fix the tissue flat, the slide glass was placed on the filter paper. The fixed colon was stained with 1% methylene blue in PBS (PBS) for 10-20 minutes, rinsed with PBS, And the number of ACFs was counted by observing with an optical microscope (X40) with the mucous membrane facing upward. The ACF was distinguished from the stained tissue by dark blue staining. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 6), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

Table 15 shows the total number of pre-cancerous lesions (total ACF number / large intestine) and the multiplicity of ACF (total aberrant crypts / large intestine) produced in the colon in each group investigated according to the above method.

Mucin  Pre-cancerous lesion ( Mucin -depleted Foci ) Measure

In a recent study, Giovanna Caderni, A. Pietro Femia, and Piero Dolara (2003) showed that mucin depleted foci (MDF), which are not secreted as mucin secretion by pre-cancerous lesions, are likely to progress to cancer. Hereinafter, the change of the MDF number of the colon tissue by the parasite diet as the active ingredient of the present invention was measured.

First, the entire colon tissue is immersed in sterilized water for 5 minutes, and then dipped in a petri dish containing freshly prepared diamine solution. The Petri dish was wrapped with a foil and stained for 18 hours at room temperature in the state of blocking light. The colon was rinsed 3 times with sterile water and stained with 1% Alcian blue (in 3% acetic acid) for 30 minutes. Rinsed three times with 80% ethanol, rinsed with sterilized water and observed under a microscope (40X-mucosal side up) and analyzed as shown in Table 15 below. Data analysis was done using SPSS ver21 PC package. Results were expressed as mean ± standard deviation (n = 10), and one-way ANOVA was performed for each variable. Duncan's multiple range test was used for the post test and the significance was tested at α = 0.05 level.

In the colon mucosal tissue PGE ₂  Production amount

Prostaglandin E ₂ (ProstaGlandin-E ₂ , PGE ₂ ) is a signaling molecule of the prostaglandin system and is a factor directly affecting the inflammatory response. Hereinafter, as a part of the analysis of histopathological changes by the peeled diet of the present invention, PGE ₂ The amount of production was measured.

The amount of prostaglandin E ₂ (ProstaGlandin-E ₂ , PGE ₂ ) was measured by using a kit (Cayman Chem. Co.) using a monoclonal antibody of PGE _{2 in} the intestinal mucosa. That is, PGE _{2 for} the monoclonal antibody of PGE ₂ And PGE ₂ is based on competition between? che (acetylcholinesterase) conjugate (PGE ₂ tracer). The concentration of PGE ₂ tracer is always constant, but PGE ₂ concentration will depend on the sample, the amount of PGE ₂ tracer capable of binding with the monoclonal antibody of PGE ₂ and PGE ₂ concentration in the well (well) shows an inverse relationship. After adding 50 μl of EIA buffer to a 96-well plate, PGE ₂ 50 μl of EIA standard was added at a low concentration, and 50 μl of each sample was diluted to two concentrations with buffer, 50 μl of PGE ₂ tracer was added, and 50 μl of monoclonal antibody was added. After the film was well mixed, the film was reacted at 4 ° C for 18 hours. After washing 5 times, 200 μl of Ellman's reagent (containing substrate for Ache) was added and light was blocked for 60 to 90 minutes in an orbital shaker. The PGE ₂ content was calculated as the absorbance value of the standard solution, the regression equation was calculated, the PGE ₂ content (pg / ml) was calculated, and the final concentration was calculated by multiplying the dilution factor. Protein concentration was corrected to nmol / mg protein. Protein concentration was determined using the Pierce BCA ^TM protein assay kit (Pierce BCA ^TM protein assay kit, Thermo Sci.). This is shown in Table 15 (n = 6).

AOM A5Y A10Y A20Y A0.5S Total number of ACFs / colon 310.6 + - 82.0 ^bc 217.6 ± 63.7 ^a 396.7 ± 70.1 ^c 386.6 ± 110.7 ^c 246.4 ± 34.7 ^ab Total aberrant crypts / colon 715.1 + - 192.1 ^b 537.1 + 141.7 ^a 957.3 ± 150.8 ^c 975.9 ± 285.3 ^c 541.7 ± 91.1 ^a Total number of MDF / Large intestine 192.5 ± 31.9 ^b 194.4 ± 57.5 ^b 118.5 ± 20.9 ^a 161.0 ± 19.8 ^b 113.5 ± 12.9 ^a MDF distribution Proximal colon 88.1 ± 28.5 ^c 84.6 ± 23.5 ^c 50.4 ± 13.5 ^a 81.5 ± 10.6 ^bc 61.8 + 16.8 & lt ^; Middle colon 54.5 ± 14.3 ^bc 65.5 ± 31.7 ^c 36.5 ± 13.1 ^ab 58.3 ± 12.2 ^c 34.3 ± 14.7 ^a Distal colon 49.9 ± 11.5 ^c 44.3 + - 13.8 ^c 31.6 ± 6.1 ^b 21.3 + - 6.4 ^a 17.5 + 4.8 ^a PGE ₂ (ng / mg protein) 9.09 ± 2.85 ^b 5.11 ± 2.19 ^a 5.71 ± 1.77 ^a 6.05 ± 2.43 ^a 5.00 ± 1.89 ^a

(ns: not significant)

As shown in the above table, pre-cancerous lesions of A5Y and A0.5S were significantly decreased in the AOM group fed with the normal diet without parchment, and the positive control group, A5Y, respectively. For the AOM group fed with the normal diet without parchmenting, it was confirmed that the MDF number was significantly decreased in A10Y and A0.5S. In addition, the PGE ₂ counts of A5Y, A10Y and A20Y were significantly decreased in the AOM group fed with the normal diet without parchment.

The scope of protection of the present invention is not limited to the description and the expression of the embodiments explicitly described in the foregoing. It is again to be understood that the present invention is not limited by the modifications or substitutions that are obvious to those skilled in the art.

Claims (10)

Dioscorea batatas Decne. A health functional food composition for improving blood cholesterol, comprising as an active ingredient a powder of a crust obtained by crushing the crust to a particle size of 80 to 200 mesh. The health functional food composition according to claim 1, wherein the composition comprises 1 to 30% by weight of a powder of peel based on the total weight of the composition.
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