KR20050069410A - Composition comprising phytosterol, hesperidin and rutin for preventing and treating arteriosclerosis, hyperlipidemia and liver disease - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of hyperlipidemia, arteriosclerosis and liver disease comprising phytosterols, hesperidin and rutin, the composition of the present invention not only lowers total cholesterol, LDL-cholesterol and triglycerides in the body but also cholesterol in the body By inhibiting the enzyme activity that mediates accumulation, it can be usefully used for the prevention and treatment of hyperlipidemia, arteriosclerosis and liver disease.

Description

Composition comprising phytosterol, hesperidin and rutin for preventing and treating arteriosclerosis, hyperlipidemia and liver disease, including phytosterol, hesperidin and rutin

The present invention relates to a composition for preventing and treating hyperlipidemia, atherosclerosis and liver disease, including phytosterol, hesperidin and rutin, and more specifically, lowering triglyceride levels in blood, including phytosterol and flavonoid components hesperidin, rutin, The present invention relates to a composition for preventing and treating hyperlipidemia, arteriosclerosis, and liver disease having an arteriosclerosis index improving function through an effect of increasing HDL-cholesterol concentration.

Hyperlipidemia, a rapidly increasing trend, refers to a condition in which cholesterol or triglyceride levels are increased in blood, and in itself is not a disease state that shows clinical findings but also causes atherosclerosis, and further, myocardial infarction and cerebral infarction. It is the first risk factor. A large epidemiological study (Framingham, USA) found that hyperlipidemia, particularly hypercholesterolemia, correlated very positively with the incidence of ischemic heart disease. These correlations vary with age, and the increase in serum cholesterol levels before age 40 is more closely associated with the incidence of ischemic heart disease than after age 40. According to the 4 year follow-up study of 40,000 people, reducing blood cholesterol levels by 10% first reduces mortality from heart disease by 20% and secondly reduces the incidence of myocardial infarction by 17%. Has been reported. As a result, the treatment of hypercholesterolemia is the most efficient way to prevent and treat coronary heart disease.

Resin that binds bile acids (e.g. cholestyramine, colesripol, etc.), HMG-CoA reductase inhibitors that are major enzymes in cholesterol biosynthesis processes (e.g., lovastatin, fluvastatin, Simvastatin, pravastatin] and the like have been used in the clinic, there is a risk of side effects when long-term use. In particular, statin drugs, which are inhibitors of HMG-CoA reductase, are known to cause liver damage and myopathy.

Therefore, the treatment of hypercholesterolemia may be more effective with dietary supplements than drugs with side effects, given that long-term administration is necessary, especially below 220 mg / dl in blood cholesterol levels where drug treatment is not recommended. For patients with semi-hyperlipidemia, it is absolutely necessary to prevent the management using health foods.

At present, countries are focusing on the synthesis of various health functional foods and hyperlipidemia therapeutics to be used with diets to solve the above-mentioned problems of adult diseases, among which phytosterol is applied to various health functional foods. Phytosterols are commonly found in fruits, vegetables, nuts, cereals, corn, legumes, etc., and their efficacy began in the 1950s. The structure of phytosterols has been reported to be very similar to cholesterol, and research is now focused on the fact that phytosterols inhibit the absorption of cholesterol in the intestine. Phytosterol has been recognized as the US General GRAS (Generally Recognized As Safe) as a very safe substance for humans, and its stability has been recognized in Europe. In addition, FDA's scientific findings indicate that phytosterol esters have a cholesterol-lowering effect on 13g daily, and are recommended by the National Institutes of Health (NIH). Therefore, in the United States, Europe and Japan, a lot of phytosterol-enhanced products have been developed and marketed, attracting attention not only to patients with high cholesterol, but also to those concerned about health, so that medical food experts such as doctors, pharmacists, nutritionists, etc. First, the recommended food choice is recommended.

Flavonoids, on the other hand, are water soluble as pale yellow or yellow pigment compounds of plants, and widely distributed in all organs of plants. There are about 4,000 species in nature as natural products, and are known to exhibit a wide range of physiological and pharmacological effects. Among them, green tea polyphenol lowers blood LDL-cholesterol (low density lipoprotein-cholesterol), while HDL-cholesterol (high density lipoprotein-cholesterol) significantly increases, and it has been reported to have a cholesterol lowering effect in experimental animals.

Citrus peel extract or hesperidin or naringin purified therefrom is known to inhibit the activity of 3-Hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase to lower blood cholesterol levels (Korean Patent Application No. 1996- 0045735, 1997-0059427 and 1999-0051434). In addition, a material obtained by synthesizing beta-sitosterol, one of the components of phytosterol and pectin, which is a polysaccharide under acidic conditions, was used for cholesterol lowering. (WO 95/00158 and Korean Patent Application No. 2000-0067634) and a composition using lecithin, protein, oil, and the like have been developed for the purpose of increasing the absorption of phytosterol. (US Pat. Nos. 6,063,776, 6,113,972 and 6,267,963) However, the above prior arts exhibit only the conventionally known effects of bioflavonoids, polysaccharides and phytosterols, namely cholesterol lowering alone effects, which do not significantly detract from their scope.

In order to solve the problems of the prior art, an object of the present invention is to provide a food composition for preventing and treating hyperlipidemia, arteriosclerosis and liver disease, including phytosterol, hesperidin and rutin.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating hyperlipidemia, arteriosclerosis, and liver disease, including phytosterol, hesperidin, and rutin.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of hyperlipidemia, arteriosclerosis liver disease, including phytosterol, hesperidin or hesperidin derivative, rutin or rutin derivative.

"Phytosterol" is a general term for sterol substances derived from all kinds of plants, and is mainly separated and purified from distillation components resulting from the final deodorization of vegetable oils such as soybean oil, jade oil and rapeseed oil under high temperature vacuum. In this case, an organic solvent is used alone or a supercritical device is used together with the organic solvent. These phytosterols are mainly composed of beta-sitosterol, stigmasterol, kempsterol and brassicasterol and the like. Phytosterols are structurally similar to cholesterol, and the reduced form of internal double bonds replaced by hydrogen is called phytostanol. The phytostanol is obtained mainly from by-products in the production of pulp used in paper mills, and its in vivo functionality has been reported to be nearly the same as phytosterols. Thus, in the present invention, phytostanol may be used instead of phytosterol and each single sterol material may be used.

Hesperidin, a flavonoid component, is obtained mainly from citrus rinds, and is licensed as a soluble vitamin P in Korea, and is generally known to strengthen capillaries, inhibit blood pressure increase, and prevent natural color fading by ultraviolet rays. Methyl Hesperidin is a form in which the hydroxyl group of the third carbon of the second benzene ring constituting the hesperidin is substituted with a methyl group and is known to be the same as hesperidin in terms of its functionality.

As used herein, the term “hesperidine derivative” includes methyl-hesperidine, hesperidin, neohesperidine and it is most preferred to use methyl-hesperidine when considering the solubility of the compound in the body.

"Routine (quercetin-3beta-d-rutinoside)" is also a flavonoid, which is present in a wide range of plants that we consume daily, such as buckwheat, parsley, asparagus, tomatoes, and citrus fruits. It is a pigment component. To increase the water solubility of rutin, alpha-G-routine is a form of binding sugars to the side chain of the lutinoside (glucose and rhamnose-bound disaccharides) using glucose transferase. It is known to be almost identical to the routine of.

As used herein, the term "rutin derivative" refers to the conversion of a side chain by enzymatic conversion of rutin, which includes alpha-G-routine. In the composition of the present invention, rutin and rutin derivatives can be used interchangeably, and it is most preferable to use alpha-G-routine in consideration of solubility in the body.

The composition of the present invention may further include flavonoid strains naringin, naringenin, neohesperidin, quercetin, catechin and the like, in addition to methyl-hesperidin and alpha-G-routine.

In the composition of the present invention, the compounding ratio of phytosterol, hesperidin or hesperidin derivative, rutin or rutin derivative alpha-G-routine is preferably 1: 0.4 to 1: 0.4 to 1, and more preferably 1: 0.4: It mixes in the weight ratio of 0.4.

The composition of the present invention is a combination of phytosterols, hesperidin, and rutin than each single component alone, blood triglycerides, total cholesterol and low-density lipoprotein (LDL) -cholesterol concentration, liver tissue triglyceride concentration At the same time, it can effectively reduce the arteriosclerosis indicators and improve the synergistic effect, hepatic tissue ACAT (acyl-CoA: cholesterol acyltransferase) activity is inhibited by reducing cholesterol accumulation in the tissue.

The composition according to the present invention may be a pharmaceutical composition or a food composition, the term food composition in the present invention is intended to include food, food additives, beverages and beverage additives and the like.

In addition to the above components, the composition may be prepared as a medicament by adding one or more pharmaceutically acceptable excipients in an amount of 1 to 99.9 parts by weight based on the total parts by weight. Excipients include, but are not limited to, magnesium stearate, cellulose, starch, amino acids, and the like, and any suitable agent known in the art may be used.

The composition is prepared in the form of warnings, granules, powders, syrups, liquids, liquid extracts, emulsions, suspensions, dips, tablets, injections, capsules, creams, troches, pastas, etc. Can be used orally. The dosage of the composition is not particularly limited, but may be used, for example, 2-3 grams per day. The dosage is not limited thereto, and it is preferable to be applied differently according to the age, sex, condition, absorbency of the active ingredient in the body, inactivation rate and excretion rate, disease type, and the concomitant drug.

Unless otherwise specified in the present invention,% means% by weight.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention is not limited to the scope of the invention by these examples.

Example 1 Investigation of the Effects of the Compositions of the Invention on Blood Glucose, Blood Lipid Levels, Atherosclerosis Indicators, Hepatic Function Indices, Hepatic Cholesterol Metabolizing Enzymes, Plasma and Hepatic Lipid Peroxide Levels

Experimental Example 1-1: Animal Administration

An experimental diet was conducted to evaluate the functionality of the composition of the present invention comprising phytosterols, methyl-hesperidine and alpha-G-routine.

The animals used in the experiment were Sprague-Dawley male rats (4 weeks old, 100 ± 10 grams), which were adapted to the laboratory environment with solid feed for 5 days, followed by randomized complete block design. Each group was divided into groups and fed for 5 weeks. The breeding environment of the breeding room was maintained at a temperature of 24 ° C., a humidity of 55 ± 5%, and a photoperiod of 12 hours daily. Dietary intake was measured between 11 am and 12 pm daily and body weight once a week.

Phytosterol was purchased from a commercially available product (Generol 122N, Cognis, Germany) and used as a flavonoid (Hesperidin Methyl Chalcone, Chong Qing Economic & Technological Development, China) and alpha-G-rutin (alpha-G). -Routine PS, Oriental Party, Japan).

Detailed compositions of the eight experimental diets used in this experiment are shown in Table 1. The normal control diet (ND) was prepared according to the experimental composition of AIN-93 (Philip et al., J Nutr 123 (11): 1939-1951, 1993), and the normal diet (ND) for the high cholesterol control diet (HCD). 1% (wt / wt) cholesterol was mixed and the high cholesterol / methyl-hesperidin diet (HCD + H) or the high cholesterol / plant sterols diet (HCD + P) was 0.1% methyl-hesperidin or 0.25% phytosterol was added. High Cholesterol / Phytosterol + Lecithin Addition The diet (HCD + PL) was the same as HCD + P, but 0.15% lecithin was added. A diet group that prescribed 0.25% phytosterol, 0.1% methyl-hesperidine, and 0.1% alpha-G-routine in a high cholesterol diet was named HCD + FA, and 0.15% lecithin was added to the HCD + FA diet group. One diet group was named HCD + FB. In the experimental diet group, the total amount of the diet was adjusted by excluding corn starch by the amount of the added substances, tap water in the normal diet group, and tap water in which 0.5% ethanol was added in the high cholesterol diet group (the remaining 6 groups). Free meals with diet.

Dietary composition for comparing the functionalities between the composition and a single component (Unit:% diet) Ingredient Diet Group ND 1) HCD HCD + H HCD + P HCD + PL HCD + FA HCD + FB Corn Starch (Samyang Genex) 39.749 38.749 38.649 38.499 38.349 38.299 38.149 Alpha-Starch (Samyang Genex) 13.2 13.2 13.2 13.2 13.2 13.2 13.2 Sugar (Samyang Corporation) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Casein (Scerma, 59290, France) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 L-Cystine (Sigma, USA) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Soybean Oil (CJ, Korea) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Mineral mixtures 2) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 Vitamin mixtures 3) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Cellulose Powder (Borac, Korea) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Choline bitartrate (Sigma, USA) 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Tert-butylhydroquinone (Sigma) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Cholesterol (Nippon, Japan) - 1.0 1.0 1.0 1.0 1.0 1.0 Dietary Additives Methyl-hesperidine - - 0.1 - - 0.1 0.1 Phytosterol - - - 0.25 0.25 0.25 0.25 Lecithin - - - - 0.15 - 0.15 Alpha-G-Routine - - - - - 0.1 0.1 Total (%) 100 100 100 100 100 100 100

1) According to AIN-93 diet

2) AIN-93 mineral mixture (AIN-93G-MX; ICN Biomedical, USA)

3) AIN-93 Vitamin Mixture (AIN-93-VX; ICN Biomedical, USA)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + phytosterols), HCD + PL (high cholesterol diet) Phytosterol + Lecithin), HCD + FA (High Cholesterol + Plant Sterol + Methylhesperidin + Rutin), HCD + FB (High Cholesterol + Phytosterol + Methyl-Hesperidin + Rutin + Lecithin)

The animals bred for 5 weeks were fasted for 16 hours, and then opened in anesthesia with ethyl ether, and blood was collected from the abdominal aorta. Blood was collected using a tube treated with heparin, and the collected blood was centrifuged at 1000 × g and 4 ° C. for 15 minutes to separate plasma and frozen at −80 ° C. until biochemical analysis. Liver was extracted immediately after blood collection and washed with 0.9% sodium chloride solution, and weighed. After snap-freeze in liquid nitrogen, it was stored at -80 ℃ until lipid and enzyme activity analysis.

Statistical analysis of all data was performed using the Statistical Analysis System (SAS version 8.01, SAS Institute Inc, Cary, NC) PC package. Significant differences according to the experimental diet were verified by performing ANOVA (one way analysis of variance), and when significance was observed at p <0.05 level, the significance of the difference between the mean values of each experimental group was found in Duncan's multiple range test. Evaluated using

Experimental Example 1-2: Measurement of Fasting Glucose Levels According to Diet

Fasting blood glucose of rats dieted in Experimental Example 1-1 was measured at 505 nm using a commercial kit (Glucose-E kit, Yeongdong Pharm.) Used for the enzyme method. The fasting blood glucose level of the high cholesterol diet control group (HCD) in combination with the high cholesterol diet was 141 ± 8.2mg / ml, which was not significantly different from the 159 ± 4.49mg / ml of the ND diet. There was no significant change in blood glucose levels in the experimental group (HCD + H) added with methyl-hesperidin alone (HCD + H) to HCD or the experimental group (HCD + P) added to HCD alone. Breeding with addition of + lecithin (HCD + PL), phytosterol + methyl-hesperidin + alpha-G-routine (HCD + FA) or phytosterol + methyl-hesperidine + alpha-G-routine + lecithin (HCD + FB) The blood glucose level of the group was 119 ± 6.68mg / dl, 112 ± 2.48mg / dl or 116 ± 5.36mg / dl, respectively, which was significantly lower than the HCD group (p <0.05). It was found that the highest glycemic control effect in the experimental group (HCD + FA) added phytosterol, methyl- hesperidin and alpha-G-routine (Table 2).

The results indicate that the single components added to the high cholesterol diet did not have a glycemic control effect in itself, but had a hypoglycemic effect when the composition was ingested. Phytosterols, methyl-hesperidin and alpha- It was found that the highest glycemic control effect in the experimental group (HCD + FA) added G-routine (Table 2).

Blood Sugar Levels According to Diet Diet Group ND HCD HCD + H HCD + P HCD + PL HCD + FA HCD + FB Blood sugar (mg / dl) 159 ± 4.5a 141 ± 8.2a 141 ± 5.7a 148 ± 7.2a 119 ± 6.7b 112 ± 2.5b 116 ± 5.4b

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + plantsterol), HCD + PL (high cholesterol diet) + Plant sterol + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (high cholesterol diet + plant sterol +

Methylhesperidin + rutin + lecithin)

Experimental Example 1-3: Change in Serum Lipid Concentration According to Diet

Plasma triglycerides (Triglycerides kit) and total cholesterol concentrations (Cholesterol E kit, Yeongdong Pharmaceutical) were measured using a commercial kit, respectively. The HDL-cholesterol concentration was measured using a commercial (HDL-cholesterol kit, Yeongdong Pharmaceutical Co., Ltd.). It was. VLDL + LDL-cholesterol concentration of plasma was determined by subtracting HDL-cholesterol concentration from plasma total cholesterol concentration.

The plasma lipid concentrations of animals bred for five weeks on the experimental diet are shown in Table 3. No significant difference in plasma triglyceride levels was observed between the high cholesterol control group (HCD) and the normal diet group. Methyl-hesperidine (HCD + H), phytosterol (HCD + P), phytosterol + lecithin (HCD + PL) or methyl-hesperidine + phytosterol + alpha-G-routin + lecithin (HCD + FB) Plasma triglyceride concentration of the group added was lower than that of the high cholesterol control group, but there was no significant difference. However, the plasma triglyceride concentrations of the group added with phytosterol + methyl-hesperidin + alpha-G-routine (HCD + FA) to the high cholesterol control group (14.4 ± 0.78 mg / dl) were higher than the high cholesterol control group (20.4 ± 1.29 mg / dl). 29% significantly lower than) (p <0.05). In other words, when methyl-hesperidine and alpha-G-routine were added to phytosterols, plasma triglyceride concentrations tended to decrease compared to phytosterols, whereas phytosterols alone were added to phytosterols. Plasma triglyceride concentrations tended to increase (P <0.05).

Plasma total cholesterol concentration of the high cholesterol control group (125.8 ± 8.99mg / dl) was 47% higher than that of the normal diet group (85.8 ± 4.98mg / dl) (p <0.05). As a result of adding methyl-hesperidine alone to the high cholesterol control group, there was no significant change in the plasma total cholesterol concentration, and phytosterol (HCD + P), phytosterol + lecithin (HCD + PL), phytosterol + methyl Hesperidin + alpha-G-routine (HCD + FA) or phytosterol + methyl-hesperidin + alpha-G-routin + lecithin (HCD + FB) resulted in a total plasma concentration of 82.4 ± 6.73 mg / dl, respectively. 103.4 ± 13.85 mg / dl, 81.5 ± 5.73 mg / dl or 92.7 ± 5.49 mg / dl, which was significantly lower than the high cholesterol control group (p <0.05). In particular, the diet group (HCD + FA) added with phytosterol, methyl-hesperidine and alpha-G-routine was the most effective with a 35% decrease in total plasma cholesterol concentration.

The plasma high density lipoprotein-cholesterol concentration of the high cholesterol control group (HCD) with cholesterol and alcohol intake was 24.8 ± 2.51mg / dl, which is 53% higher than the normal diet (53.1 ± 3.03 mg / dl). Low (p <0.05). The HDL-cholesterol concentration of the group added with phytosterol, methyl-hesperidine and alpha-G-routine (HCD + FA) to the high cholesterol control group showed the highest value. This is a 37% significant increase (p <0.05) compared to the group added only phytosterol (HCD + P).

The plasma VLDL + LDL-cholesterol concentration of the high cholesterol control group (HCD) was 101.1 ± 3.81 mg / dl, which was 209% higher than the normal diet (32.7 ± 2.53 mg / dl) (p <0.05). However, the addition of phytosterol, methyl-hesperidine and alpha-G-routine (HCD + FA) to the high cholesterol control group resulted in 50.7 ± 7.06 mg / dl, with the lowest plasma VLDL + LDL-cholesterol concentration of 50%. The decrease was found to be (p <0.05). Therefore, phytosterol, methyl-hesperidine and alpha-G-routine had the best effect of lowering plasma VLDL + LDL-cholesterol concentration in rats fed the high cholesterol diet, which was similar to the result observed at the total cholesterol concentration.

Changes in Serum Lipid Concentration According to Diet (Unit: mg / dl) Item Dietary Group ND HCD HCD + H HCD + P HCD + PL HCD + FA HCD + FB Triglyceride 19.5 ab ± 1.24 20.4 ab ± 1.29 18.4 abc ± 1.51 16.0 bc ± 1.34 19.3 ab ± 1.83 14.4 c ± 0.78 16.5 bc ± 0.86 Total cholesterol 85.8 c ± 4.98 125.8 ab ± 8.99 133.9 a ± 11.07 82.4 c ± 6.73 103.4 bc ± 13.85 81.5 c ± 5.73 92.7 c ± 5.49 HDL-cholesterol 53.1 a ± 3.03 24.8 bc ± 2.51 27.9 bc ± 2.31 22.5 c ± 2.86 27.8 bc ± 2.07 30.9 b ± 2.84 27.4 bc ± 1.90 VLDL + LDL-Cholesterol 1) 32.7 cd ± 2.53 101.1 a ± 3.81 106.0 a ± 12.12 60.0 bc ± 7.87 75.6 b ± 13.91 50.7 bcd ± 7.06 65.4 b ± 4.02

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + plantsterol), HCD + PL (high cholesterol diet) + Plant sterol + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (high cholesterol diet + plant sterol +

Methylhesperidin + rutin + lecithin)

1) VLDL + LDL-Cholesterol = Total Cholesterol-HDL-Cholesterol

Experimental Example 1-4: Investigation of the change of arteriosclerosis index according to diet

Table 4 shows the results of evaluating atherosclerotic indicators (atherogenic indices) of experimental animals. The high cholesterol control group (HCD, 0.80 ± 0.09), which combined cholesterol and alcohol intake, had a 116% higher ratio of plasma triglycerides to HDL-cholesterol concentration than the normal diet (ND, 0.37 ± 0.03) (p < 0.05). Phytosterol + methyl-hesperidin + alpha-G-routine (HCD + FA) was fed to the high cholesterol control group, and the ratio (0.50 ± 0.06) was significantly decreased by 38% (p <0.05).

Calculation of the ratio of malignant VLDL and LDL-cholesterol concentrations (total plasma total cholesterol minus HDL-cholesterol concentration) to HDL-cholesterol concentration showed that VLDL and LDL malignant to HDL-cholesterol concentration for high cholesterol control group The ratio of cholesterol concentration was 4.28 ± 0.51, which was 590% higher than that of the normal diet group (ND, 0.62 ± 0.04), indicating that the risk of atherosclerosis was significantly increased by high cholesterol diet and alcohol intake (p <0.05). Phytosterol + methyl-hesperidin + alpha-G-routine (HCD + FA, 1.52 ± 0.26) was added to the high cholesterol control group, resulting in malignant VLDL and LDL for HDL-cholesterol concentration compared to the high cholesterol control group (HCD). -Cholesterol concentration ratio decreased by 64% (p <0.05). It was found that the composition of the present invention has a very good anti-arteriosclerosis effect compared to the case of adding methyl-hesperidine and phytosterol alone to the high cholesterol control group.

Changes in Atherosclerosis Indicators According to Diet Diet group item Triglyceride / HDL-cholesterol 1) (Total Cholesterol-HDL-Cholesterol) / HDL-Cholesterol 2) ND 0.37 ± 0.03 d 0.62 ± 0.04 d HCD 0.80 ± 0.09 a 4.28 ± 0.51 a HCD + H 0.70 ± 0.08 ab 4.08 ± 0.62 a HCD + P 0.77 ± 0.08 a 3.17 ± 0.75 ab HCD + PL 0.63 ± 0.08 abc 2.89 ± 0.64 ab HCD + FA 0.50 ± 0.06 bcd 1.52 ± 0.26 cd HCD + FB 0.63 ± 0.06 abc 2.42 ± 0.13 bc

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + plantsterol), HCD + PL (high cholesterol diet) + Plant sterol + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (high cholesterol diet + plant sterol +

Methylhesperidin + rutin + lecithin)

1) Triglyceride (mg / dl) / HDL-cholesterol (mg / dl)

2) [Total Cholesterol (mg / dl) -HDL-Total Cholesterol (mg / dl)] / HDL-Cholesterol

Experimental Example 1-5: Investigation of changes in activity of liver function indicator enzyme according to diet

When hepatocytes are destroyed, enzymes such as GOT (glutamate oxaloacetate transeferase) and GPT (glutamate pyruvate transferase), which are contained in high concentrations, are released into the blood, which increases the activity of these enzymes in plasma. It is used as an indicator that reflects the extent of disease damage, such as hepatitis, cirrhosis and necrosis. Plasma GOT and GPT activities of rats fed the experimental diet for 5 weeks are shown in Table 7. Plasma GOT activity of the high cholesterol control group (HCD) was 90.6 ± 2.49U / L, which was 25% higher than that of the normal diet (ND, 72.4 ± 5.07U / L) (p <0.05). Plasma GOT activity in rats fed with the addition of hesperidin and alpha-G-routine (HCD + FA) tended to decrease compared to the HCD group.

Changes in the Activity of Hepatic Function Marker Enzymes According to Diet Diet Group ND HCD HCD + H HCD + P HCD + PL HCD + FA HCD + FB GOT 1) 72.4 ± 5.1d 90.6 ± 2.5bc 101.9 ± 7.6ab 86.1 ± 2.0bcd 107.2 ± 6.0a 79.8 ± 2.2cd 101.1 ± 8.9ab GPT 2) 21.8 ± 0.9c 26.8 ± 1.3abc 27.8 ± 1.9abc 27.3 ± 1.5abc 30.8 ± 2.3ab 26.5 ± 1.3abc 33.3 ± 5.0a

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + plantsterol), HCD + PL (high cholesterol diet) + Plant sterol + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (high cholesterol diet + plant sterol +

Methylhesperidin + rutin + lecithin)

1) Glutamate oxaloacetate transferase, 2) Glutamate pyruvate transferase

Experimental Example 1-6: Changes in the Activity of Cholesterol Metabolizing Enzymes in Liver Tissues According to Diet

HMG-CoA reductase (3-Hydroxy-3-methylglutaryl-Coenzyme A reductase) is an important rate-determinant in the synthesis of cholesterol from HMG-CoA in liver tissue, and ACAT (acyl coenzyme A: cholesterol acyltransferase) It is an enzyme that catalyzes the process of converting free cholesterol into cholesterol esters and storing them in liver tissues when free cholesterol levels increase. After breeding for 5 weeks in an experimental diet, the activity of cholesterol metabolizing enzymes in liver tissues is shown in Table 6. The hepatic ACAT activity of the high cholesterol control group (HCD) was 154 ± 5.53 pmolemin-1 mg protein-1, which was not significantly different from the normal diet group (ND), but methyl-hesperidine alone was added to the high cholesterol control group. The results showed no significant change in liver ACAT activity, whereas phytosterol (HCD + P), phytosterol + lecithin (HCD + PL), phytosterol + methyl-hesperidine + alpha-G-routine (HCD + FA). ) Or hepatic ACAT activity in the group to which phytosterol + methyl-hesperidin + alpha-G-routin + lecithin (HCD + FB) was added was significantly lower (p <0.05) compared to the HCD group. Therefore, ingestion of the composition of the present invention in rats with a high cholesterol diet and alcohol intake did not significantly affect cholesterol biosynthesis, but significantly reduced the accumulation of cholesterol in tissues by inhibiting ACAT activity in liver tissues. It can be seen.

Changes in Cholesterol Metabolizing Enzymes in Hepatic Tissues According to Diet   Diet Group HMG-CoA reductase ACAT 1) (pmolemin-1mg protein-1) ND 117 ± 9.91 NS 157 ± 4.76 a HCD 116 ± 4.51 154 ± 5.53 ab HCD + H 119 ± 6.10 143 ± 3.90 bc HCD + P 113 ± 3.00 135 ± 3.38 cd HCD + PL 113 ± 5.53 136 ± 3.90 cd HCD + FA 109 ± 2.70 133 ± 3.43 cd HCD + FB 109 ± 6.39 126 ± 3.97 d

Values are mean ± standard deviation (n = 8)

The other alphabets beside the numbers indicate significant differences (p <0.05), no significant differences in NS ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet) + Methyl-hesperidine), HCD + P (high cholesterol diet + plant sterols), HCD + PL (high cholesterol diet + plant sterols + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (High Cholesterol Diet + Plant Sterol + Methylhesperidin + Rutin + Lecithin)

1) acyl coenzyme A: cholesterol acyltransferase

Experimental Example 1-7: Changes in Lipid Peroxide Levels in Plasma and Liver Tissues According to Diet

The concentration of thibarbituric acid reactive substances (TBARS) formed to evaluate lipid peroxide concentrations in plasma was analyzed. 0.4 ml of plasma was added to 0.8 ml of 15% trichloroacetic acid (w / v), 0.375% thibarbituric acid (w / v) and 0.25 N hydrochloric acid solution (TCA-TBA-HCl reagent), and then mixed well. Heated at 95 ° C. for 15 minutes and then cooled on ice for 3 minutes. After centrifugation at 2000 × g for 10 minutes, the supernatant

Colorimetric quantification was performed at 535 nm (DU530 Life Science UV / Vis Spectrophotometer, USA) to compare malondialdehyde standards.

To measure the lipid peroxide concentration of liver tissue, 0.2 ml of 8.1% sodium dodecyl sulfate (SDS) and 0.6 ml of distilled water were added to 0.2 ml of the homogenate, followed by reaction at room temperature for 5 minutes. 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% TBA (thiobarbituric acid) solution were added thereto, and the mixture was heated in a 95 ° C shaker bath for 60 minutes. The mixture was cooled on ice for 5 minutes, and the reaction was terminated. Then, 1 ml of distilled water and 5 ml of n-butanol: pyridine (15: 1, v / v) mixture were added and mixed well. The absorbance of the supernatant obtained by centrifugation (1000 × g, 20 ° C., 15 min) was colorimetrically determined at 532 nm in comparison with malondialdehyde standard solution.

Lipid peroxide concentrations in plasma and liver tissues of rats bred in the experimental diet for 5 weeks are shown in Table 7. Plasma lipid peroxide concentrations in the high cholesterol control group (HCD, 1.32 ± 0.05umol / L) were not different from those of the normal diet group (ND), but methyl-hesperidine (HCD + H) or phytosterol (HCD +) diets in the high cholesterol diet (HCD). The addition of P) alone significantly increased lipid peroxide concentrations by 38% or 36% (p <0.05). Therefore, the addition of methyl-hesperidine or phytosterol alone to the high cholesterol diet promotes lipid peroxidation of plasma, but the composition of the present invention has an effect of reducing the lipid peroxidation increased by a single component.

The hepatic lipid peroxide concentration of the high cholesterol diet group (HCD) did not change significantly with the normal diet group. Similar to the results observed in plasma, lipid peroxide concentrations of liver tissues increased by ingestion of methyl-hesperidine or phytosterol alone tended to decrease by supplementing these components in combination (p <0.05).

Changes in Lipid Peroxide Levels in Plasma and Hepatic Tissues According to Diet   Diet group item plasma liver (Μmol MDA 1) / L) (nmol MDA / g liver) ND 1.42 ± 0.06 bc 36.6 ± 0.61 d HCD 1.32 ± 0.05 cd 35.4 ± 1.69 d HCD + H 1.82 ± 0.13 a 41.5 ± 1.56 cd HCD + P 1.80 ± 0.12 a 51.1 ± 3.41 a HCD + PL 1.27 ± 0.10 cd 46.4 ± 2.72 abc HCD + FA 1.35 ± 0.09 bcd 34.0 ± 1.69 bc HCD + FB 1.61 ± 0.11 ab 48.7 ± 2.37 ab

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet, HCD + H (high cholesterol diet + methyl-hesperidine), HCD + P (high cholesterol diet + plantsterol), HCD + PL (high cholesterol diet) + Plant sterol + lecithin), HCD + FA (high cholesterol + phytosterol + methylhesperidin + rutin), HCD + FB (high cholesterol diet + plant sterol + methylhesperidin + rutin + lecithin)

1) MDA: malondialdehyde

Example 2 Derivation of Optimal Formulation of Phytosterol, Methyl-Hesperidin, and Alpha-G-Routine in the Improvement of Blood Lipid Metabolism

In a composition comprising phytosterol, methyl-hesperidine, and alpha-G-routine, Sprague-Dawley male rats (4 weeks old) at different addition ratios are determined to determine an optimal blending ratio of the components. , 100 ± 10 grams) for 5 weeks. Compounding ratios of the main components are shown in Table 8, and the experimental conditions were carried out in the same manner as in Example 1.

Diet composition with different compounding ratios (unit:% diet) Ingredient Diet Group ND 1) HCD HCD + FA1 HCD + FA2 HCD + FA3 HCD + FA4 HCD + FA5 Corn Starch (Samyang Genex) 39.749 38.749 38.449 38.149 38.249 38.299 37.749 Alpha-Starch (Samyang Genex) 13.2 13.2 13.2 13.2 13.2 13.2 13.2 Sugar (Samyang Corporation) 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Casein (Scerma, 59290, France) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 L-Cystine (Sigma, USA) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Soybean Oil (CJ, Korea) 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Mineral mixtures 2) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 Vitamin mixtures 3) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Cellulose Powder (Borac, Korea) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Choline bitartrate (Sigma, USA) 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Tert-butylhydroquinone (Sigma) 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Cholesterol (Nippon, Japan) - 1.0 1.0 1.0 1.0 1.0 1.0 Dietary Additives Methyl-hesperidine - - 0.1 0.1 0.2 0.1 0.3 Alpha-G-Routine - - 0.1 0.2 0.1 0.1 0.3 Phytosterol - - 0.1 0.3 0.2 0.25 0.4 Total (%) 100 100 100 100 100 100 100

1) According to AIN-93 diet, 2) AIN-93 mineral mixture (AIN-93G-MX; ICN Biomedical, USA), 3) AIN-93 vitamin mixture (AIN-93-VX; ICN Biomedical, USA)

ND normal diet, HCD high cholesterol diet

HCD + FA1 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Phytosterol = 1: 1: 1)

HCD + FA2 (high cholesterol diet, methyl-hesperidin: routine: plant sterol = 1: 2: 3)

HCD + FA3 (high cholesterol diet, methyl-hesperidin: routine: plant sterol = 2: 1: 2)

HCD + FA4 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Phytosterol = 1: 1: 2.5)

HCD + FA5 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Plant Sterol = 3: 3: 4)

Plasma lipid concentrations according to the blending ratios are shown in Table 9, where the compounding ratio of methyl-hesperidine, alpha-G-routine and phytosterol was 1 to 2.5 (FA4) in the high cholesterol control diet compared to other formulations. Reduction of fat, total cholesterol and malignant cholesterol was better, and HDL-cholesterol, which has a good effect on lipid metabolism in the body, was also higher than other combination ratios.

 Comparison of Lipid Metabolism Improvement Effect According to the Composition Ratio of the Composition (Unit: mg / dl) Item Dietary Group ND HCD HCD + FA1 HCD + FA2 HCD + FA3 HCD + FA4 HCD + FA5 Triglyceride 19.5 ab ± 1.24 20.4 ab ± 1.29 18.7 abc ± 1.28 16.5 bc ± 1.14 17.3 bc ± 1.63 14.4 c ± 0.78 16.9 bc ± 0.76 Total cholesterol 85.8 c ± 4.98 125.8 ab ± 8.99 110.9 a ± 10.07 82.9 c ± 6.93 89.4 bc 12.65 81.5 c ± 5.73 88.7 c ± 5.25 HDL-cholesterol 53.1 a ± 3.03 24.8 bc ± 2.51 27.1 bc ± 2.38 21.5 c ± 2.84 27.7 bc ± 2.04 30.9 b ± 2.84 27.1 bc ± 1.80 VLDL + LDL-Cholesterol 1) 32.7 cd ± 2.53 101.1 a ± 3.81 96.0 a ± 11.17 60.3 bc ± 6.42 65.6 b ± 8.90 50.7 bcd ± 7.06 63.2 b ± 3.92

Values are mean ± standard deviation (n = 8)

Meaning that there is a significant difference in the other alphabets beside the figure (p <0.05)

ND normal diet, HCD high cholesterol diet

HCD + FA1 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Phytosterol = 1: 1: 1)

HCD + FA2 (high cholesterol diet, methyl-hesperidin: routine: plant sterol = 1: 2: 3)

HCD + FA3 (high cholesterol diet, methyl-hesperidin: routine: plant sterol = 2: 1: 2)

HCD + FA4 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Phytosterol = 1: 1: 2.5)

HCD + FA5 (High Cholesterol Diet, Methyl-Hesperidin: Routine: Plant Sterol = 3: 3: 4)

1) VLDL + LDL-Cholesterol = Total Cholesterol-HDL-Cholesterol

Example 3 Preparation of Health Food Compositions Containing Methyl-Hesperidin, Alpha-G-Routine and Phytosterols

Based on the above experimental results, a health food composition including the following ingredients was prepared.

Phytosterol 224 mg

Methyl-hesperidine 88 mg

Alpha-G-Routine 88 mg

Cellulose 56 mg

L-valine 20 mg

Magnesium Stearate 4 mg

480 mg total

The ingredients were mixed well and filled into hard gelatin or cellulose capsules to a total of 480 mg per capsule. By ingesting five capsules per day, the risk of atherosclerosis could be reduced by effectively lowering cholesterol and triglycerides in the body without side effects.

As mentioned above, the composition comprising phytosterol, methyl-hesperidine and alpha-G-routine of the present invention was able to significantly lower blood cholesterol and triglycerides simultaneously compared to the addition of a single component in a high cholesterol diet. . In particular, the present invention inhibits the enzymatic activity of mediating cholesterol accumulation in the body in a high cholesterol diet compared to a single component, and also has an effect of significantly improving arteriosclerosis index, which can be widely used for health functional food materials and pharmaceutical uses.

Claims (6)

Phytosterol, hesperidin or hesperidin derivative, rutin or a composition for preventing and treating hyperlipidemia, arteriosclerosis and liver disease, characterized in that it comprises a rutin derivative. 2. The composition for preventing and treating hyperlipidemia, atherosclerosis and liver disease according to claim 1, wherein the hesperidin derivative is methyl-hesperidine. The composition for preventing and treating hyperlipidemia, atherosclerosis and liver disease according to claim 1, wherein the rutin derivative is alpha-G-routine. The composition for preventing and treating hyperlipidemia, arteriosclerosis and liver disease according to claim 1, wherein the compounding ratio of phytosterol, hesperidin or hesperidin derivative, rutin or rutin derivative is 1: 0.4 to 1: 0.4 to 1 weight ratio. The method for preventing and treating hyperlipidemia, arteriosclerosis, and liver disease according to claim 1, further comprising any one or more selected from the group of neohesperidin, naringin, quercetin, and catechin. The method of claim 1, wherein the phytosterol is a plant sterol extracted from the deodorizing distillate or by-products of pulp processing of liquid fats containing beta-sitosterol, stigmasterol, camphorsterol, brassicasterol and stanol forms thereof. Hyperlipidemia, arteriosclerosis and liver disease prevention and treatment composition characterized in.