KR19990039366A - Composition for inhibiting cholesterol lowering and cholesterol ester transfer protein containing jujube extract - Google Patents

Abstract

The present invention relates to a cholesterol ester transfer protein (CETP) activity inhibitor composition comprising a jujube extract, the composition of the present invention includes a pharmaceutical and food composition. Since the composition of the present invention inhibits CETP activity in the animal body and reduces blood cholesterol, it can be usefully used as a preventive and therapeutic agent for various cardiovascular diseases or as a component of a health food.

Description

Composition for inhibiting cholesterol lowering and cholesterol ester transfer protein containing jujube extract

The present invention relates to a composition for lowering cholesterol and a composition for inhibiting cholesterol ester transfer protein (CETP) activity including jujube extract.

Coronary cardiovascular disease currently accounts for more than 30% of all deaths, and in developed countries nearly 40%. On the other hand, in Korea, as the economic level develops, the cause of westernization, lack of exercise, overwork, etc. is increasing, and the mortality rate of heart disease due to excessive weight gain and blood cholesterol is increasing. It is reaching%. High blood cholesterol is known to cause atherosclerosis (Ross. R., Nature, 362, 801-809 (1993)). In particular, the amount of low-density lipoprotein (LDL) involved in the transport of cholesterol in the blood is proportional to the induction of atherosclerosis. In particular, the oxidized LDL is known to have a strong effect of atherosclerosis (SG Young and S. Parthasarathy, West J. Med. , 160, 153-164 (1994). Reducing the amount of cholesterol in the blood is a way to reduce excessive food intake of fat, and to continue to exercise. However, for people who have already had hyperlipidemia, it may be best to eat foods that contain special active ingredients. Currently, the mechanisms that regulate the amount of cholesterol in the blood are known to be very complex. Cholesterol ester transfer protein (CETP) is known to convert blood cholesterol from high density lipoprotein (HDL) to low density lipoprotein. It has been reported that lowering CETP activity may reduce the amount of LDL-cholesterol. (L. Lagrost, Biochemical et. Biophysica Acta, 1215, 209-236 (1994)).

The inventors of the present invention are looking for ingredients that are safe, non-toxic, and inhibiting CETP activity, which can be used to prevent cardiovascular diseases by reducing blood cholesterol, from jujube, a folk medicinal material traditionally used as a tonic in Korea. Extraction of components that reduce cholesterol in the blood, and also confirmed that they show CETP inhibitory activity to complete the present invention.

It is an object of the present invention to provide a composition for lowering blood cholesterol, which contains a jujube extract.

Another object of the present invention to provide a composition for inhibiting CETP activity, containing jujube extract.

The present invention provides a pharmaceutical composition for lowering cholesterol in blood comprising jujube extract with an pharmaceutically acceptable carrier as an active ingredient, and a food composition for lowering cholesterol in blood comprising jujube extract.

In addition, the present invention provides a pharmaceutical composition for inhibiting CETP comprising a CETP inhibitory substance in jujube extract as an active ingredient with a pharmaceutically acceptable carrier, and a food composition for inhibiting CETP comprising jujube extract.

The method of extracting the active ingredient from the jujube is as follows. That is, after washing the jujube with water, 2 to 5 L of 70 to 95% of ethanol or water is added per kg of jujube, or 1 to 24 hours at 50 ° C to 60 ° C for ethanol and 70 to 70 ° C for water. The active substance may be extracted by heating at 121 ° C. for 30 minutes to 12 hours. In general, when extracting the jujube using water or ethanol, the composition of the extract may vary depending on the reaction time, reaction temperature, alcohol concentration, jujube maturity.

Jujube extract obtained as described above reduces the blood cholesterol of rats, inhibits CETP activity. Toxicity studies studied to date have shown that jujube extract has little toxicity when administered orally to mice at a dose of 1,000 mg / kg. In addition, jujube extract does not show any side effects on the function of organs, including liver.

In the present invention, (a) jujube was extracted with acetone and then filtered, (b) the filtrate was concentrated under reduced pressure, hexane was added to separate the water layer and the hexane layer, and (c) ethyl acetate in the water layer containing the active substance. To separate the water layer and the ethyl acetate layer, (d) collecting the ethyl acetate layer, and then concentrated to dryness under reduced pressure, and (e) purifying the concentrate by reverse phase chromatography. A method of separating is provided. In step (a), the jujube is soaked in water, and then 1 to 3 L of acetone is added per kg of jujube and stirred at room temperature for 4 to 24 hours. In the reversed phase chromatography of step (e), the eluate is preferably water, water: methanol mixed solvent and acetonitrile, and the mixing ratio of water: methanol mixed solvent is 80:20 (v / v) to 20. It is desirable to change gradually to: 80 (v / v). CETP inhibitory active substances can be obtained from the acetonitrile fraction.

CETP activity inhibitors obtained from jujube in accordance with the method of the present invention inhibits CETP activity by 48% at a concentration of 0.05 mg / ml.

In order to reduce blood cholesterol or inhibit the activity of CETP, jujube extract, concentrate or dry powder thereof may be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a blood cholesterol lowering agent or a CETP activity inhibitor composition. The pharmaceutical composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents and the like, and by conventional methods tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, solutions Or in dosage unit forms or as multi-dose pharmaceutical preparations, such as preparations for parenteral administration.

Blood cholesterol lowering agent or CETP inhibitor composition containing jujube extract of the present invention as an active ingredient can be parenterally or orally administered as desired, 1 to 50 g of jujube extract per kg of body weight per day, preferably 5 The amount of from 10 g may be administered in one to several portions. In the case of a CETP inhibitory active substance isolated from jujube according to the method of the present invention, an amount of 0.1 to 5 g, preferably 0.5 to 1 g of active substance per kg of body weight per day is administered. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Jujube extract of the present invention may also be added to food for the purpose of lowering blood cholesterol or inhibiting CETP activity. Foods to which the jujube extract of the present invention can be added for the development of dietary supplements include, for example, various foods, meats, beverages, chocolates, snacks, snacks, pizzas, ramen noodles, other noodles, gums, ice creams, Alcoholic beverages, vitamin complexes, and health supplements.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following Examples are only for illustrating the present invention, and the scope of the present invention is not limited to these.

Example 1 Preparation of Jujube Extract

1) Ethanol Extract

15 L of 95% ethanol was added to 5 kg of jujube and extracted twice in a water bath at 50-60 ° C. for 3 hours. The extracted ethanol solution was filtered and the filtrate was concentrated under reduced pressure to obtain 2 kg of ethanol extract.

2) water extraction

15 L of water was added to 5 kg of jujube, and the mixture was boiled at 100 ° C. for 2 hours, filtered to remove solids, and the filtrate was concentrated under reduced pressure to obtain 3 kg of jujube extract.

Example 2: Effect of Jujube Extract on Plasma Cholesterol Concentration in Rats

30 Sprague-Dawley rats of 3 weeks of age were purchased from the Korean Experimental Animal Center in Negative Chungcheongbuk-do and were bred for 1 week in experimental animal feed, and at 4 weeks of age (90-110 g), Divided into three groups by randomized block design. Three groups of rats each had three different high cholesterol diets, namely 1% cholesterol (control) in normal diet (AIN-76 experimental animal diet); 1% cholesterol and 1.2% jujube ethanol extract (jujube ethanol extract group); And the diet containing 1% cholesterol and 1.5% jujube water extract (jujube water extract group), respectively. The composition of the diets ingested in the three groups is shown in Table 1 below.

Composition of Experimental Diet (%) Dietary Ingredients Control Jujube Ethanol Extract Group Jujube Water Extract Group Casein 20 20 20 D, L-methionine 0.3 0.3 0.3 Corn starch 15 15 15 Sucrose 49 47.8 47.5 Cellulose powder 5 5 5 Mineral mixtures 3.5 3.5 3.5 Vitamin mixtures One One One Lee Joo Seok Choline 0.2 0.2 0.2 Corn oil 5 5 5 cholesterol One One One Jujube extract - 1.2 1.5 gun 100 100 100

All diets corresponded to a high cholesterol diet (1%, wt / wt) and the experimental diet contained 1.2-1.5% jujube extract. Cellulose, mineral mixtures and vitamin mixture components in the AIN-76 dietary composition of rats were purchased from TEKLAD premier, Madison, Wisconsin.

All dietary rats were given free diet with water for 6 weeks. The dietary intake was recorded daily and weighed every 7 days. After the animal breeding was completed, the data on the breeding journals were analyzed.

Example 3 Determination of Total Cholesterol, HDL-Cholesterol and Neutral Lipid Contents in Experimental Animals

The effect of jujube extract on the blood cholesterol and triglyceride content in rats was investigated as follows.

Blood samples were taken from three dietary rats, and plasma HDL fractions were isolated from the HDL-cholesterol reagent (Sigma Chemical Co. Cat. No. 352-3) including dextran-sulfate. Total cholesterol and HDL-cholesterol concentrations were measured by enzymatic coloration using Sigma's Total Cholesterol Kit (Cat. No. 352-100) (Allain et al., Clin. Chem., 20, 470-475 (1974)). It was. Neutral lipid concentrations were measured using Sigma Diagnostic Kit (Cat. No. 339-50) (Bucolo, G. and David, H., Clin. Chem., 19, 476-482 (1973)). The results are shown in Table 2 below, plasma total cholesterol concentration was reduced by 28% compared to the control group in the 1.2% jujube ethanol extract administration group, 30% compared to the control group in the 1.5% jujube water extract administration group.

Effect of Jujube Extract on Blood Lipid Levels in Experimental Animals Dietary group lipid content Control Jujube Ethanol Extract Group Jujube Water Extract Group Total cholesterol (mg / dl) 142 103 100 HDL cholesterol (mg / dl) 31 25 26 22 25 26 Triglycerides (mg / dl) 96 61 70

Example 4 CETP Inhibitor Extraction from Jujube

(Step 1) Partial purification of cholesteryl ester transfer protein (CETP) from human plasma

Cholesteryl ester transfer protein (CETP) was partially purified from fresh human plasma purchased from Red Cross blood sources as follows. After adding MnCl ₂ (final concentration 0.2 M) and a 10% solution of Na-dextran sulfate (final concentration 1.2%) to 920 ml (5 packs) of human plasma, 10,000 xg (Sorvall RC5C, GS-3 rotor, 8,000 rpm) Centrifugation for 30 minutes) to remove the supernatant. BaCl ₂ was added to the supernatant to a final concentration of 70 mM, followed by secondary centrifugation at 10,000 × g for 30 minutes to obtain lipoprotein deficient plasma (LPDP) from which Na- residues were removed. To this lipoprotein deficient plasma was added solid NaCl to a solution concentration of 4M, which was then added to 200 ml of phenyl-Sepharose CL-4B and stirred at 4 ° C. for 1 hour to adsorb the protein to the resin. The protein-adsorbed resin was washed with 200 ml of 4M NaCl solution using a fritted-glass funnel, followed by 500 ml of 50 mM Tris-HCl / 150 mM NaCl (pH 7.4) buffer A. After washing, the glass column was filled (2.5 × 50 cm). The buffer A was washed so that the absorbance at 280 nm was 0.3 or less, and CETP was eluted using H ₂ O containing 0.02% NaN ₃ . 0.5 M Na-acetate buffer was added to the CETP elution fraction in a volume of 1/9 fraction to adjust the pH of the eluate to 4.5 and then centrifuged at 10,000 xg for 30 minutes to remove the resulting precipitate. CETP was partially purified from the supernatant using a CM-cellulose ion exchange column. The supernatant was applied to a CM-cellulose column (1.6 × 20 cm) equilibrated with 50 mM Na-acetate (pH 4.5, 1 mM Na ₂ EDTA, 10 mM β-mercaptoethanol) buffer B, and the buffer B and 90 After washing with Buffer B containing mM NaCl, eluting and fractionating with increasing NaCl concentration from 90 mM to 400 mM. The active fractions obtained by CETP analysis were dialyzed with Buffer A containing 1 mM Na ₂ EDTA, 10 mM β-mercaptoethanol, 0.02% NaN ₃ , and the low protein using bovine serum albumin as the standard protein. The protein was quantitated according to the method of (Lowry) and the like, diluted to a protein concentration of 0.13 ~ 0.15 mg / ml and fractionated in small portions and used while storing in a -80 ℃ freezer.

(Step 2) Determination of CETP Activity

Cholesterol ester transfer protein (CETP) activity was measured using the [ ³ H] -CETP scintillation proximity assay as follows. [ ³ H] -cholesteryl esters are transported from HDL to LDL bound to biotin, which binds to avidin-coupled fluomicrospheres to induce aqueous ³ β in aqueous solution. Emitters emit energy (light) by being very close to the scintillation molecules, and by measuring the intensity of the light, they detect the activity of CETP.

All reagents were cooled with ice water before measuring CETP activity. 10 μl sample, 10 μl assay buffer (50 mM Hepes, 0.15 M NaCl, 0.1% (w / v) NaN ₃ , pH 7.4), 10 μl of [ ³ H] cholesteryl in 1 ml centrifuge tube The reaction was initiated by adding ester HDL, 10 μl Biotin LDL, followed by the addition of 10 μl CETP (isolated from human plasma, 1.3-1.5 μg). At this time, 10 μl of water was added instead of 10 μl of CETP, and control was reacted under the same conditions by adding water instead of 10 μl of analyte. After 4 hours of reaction at 37 ° C., 200 μl of SPA beads were added to stop the reaction and allowed to stand at room temperature for 1 hour to reach equilibrium. CETP activity was measured by the liquid scintillation counter of the reacted centrifuge tube to determine the amount of cholesteryl ester transferred from HDL to LDL in units of CPM (count per minute) The activity inhibition (%) was calculated accordingly.

(Step 3) Isolation of CETP Activity Inhibitor from Jujube

The seeds of jujube (4 kg) purchased from Yeongcheongnam-do, Korea were removed, the jujube pulp was divided into 4-6 pieces, and 1 liter of water was added for about 1 hour. 4 L of acetone was added to the soaked jujube and stirred for 24 hours, and then the liquid and solid portions were separated using a filter paper. After collecting the liquid phase and concentrated under reduced pressure, the same amount of hexane was added to separate the water layer and the hexane layer, and the separatory funnel separated the water layer and the hexane layer. The same process was repeated two more times and the hexane layer was removed to remove the fatty acid-based material contained in the hexane layer. The same amount of ethyl acetate was added to the water layer containing the active material, and the water and ethyl acetate layers were separated. The same process was repeated twice. The ethyl acetate layer containing the active material was collected and concentrated to dryness under reduced pressure to give 7.3 g of a tan material.

The next step is to adsorb yellow material on Licroprep RP-18 (Merck, Art No. 13900, mesh size 40-60 μm, 9.5 x 4.5 cm), wash the non-adsorbed material with water, and eluate As water, water: methanol (80:20 (v / v)), water: methanol (60:40 (v / v)), water: methanol (40:60 (v / v)), water: methanol (20 : 80 (v / v)) and acetonitrile were separated by reverse phase chromatography with 500 ml of each flowing sequentially, and the active material was obtained from the acetonitrile fraction (219 mg). As a result of measuring the CETP inhibitory activity of this active substance according to the method of (step 2) above, 0.05 mg / ml of the active substance solution inhibited the CETP activity by 48%.

Example 5: Oral Toxicity Test of Jujube Extract

Specific pathogens free of 7-8 weeks of age, 6 females weighing 25-29 g and 6 males weighing 34-38 g, temperature 22 ± 1 ° C, humidity 55 ± 5%, illumination 12L / 12D was bred in the animal room. Mice were allowed to acclimate for about a week before being used for the experiment. Feed for experimental animals (Jeil Jedang Co., Ltd., mice and rats) and drinking water were supplied after sterilization and free ingestion.

The jujube extract obtained in Example 1 was prepared at a concentration of 100 mg / ml using 0.5% Tween 80 as a solvent, followed by oral administration of 0.2 ml per 20 g of mouse body weight. Samples were administered orally once and observed for side effects or lethality for 10 days after administration. That is, on the day of dosing, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least 1 pm, 4 hours, 8 hours, 12 hours, and the next day until the 10th day of administration. In addition, the animals were killed and dissected at 10 days of administration, and the internal organs were visually examined. The change in body weight was measured at the interval of 1 day from the day of administration to observe the weight loss phenomenon of the animal by hesperidin.

The purpose of this experiment was to provide information on available doses in general pharmacology and drug efficacy tests and to derive basic data on toxicity by identifying the degree of acute toxicity in the oral route of jujube extract. Got.

In case of acute oral toxicity, no lethal animals were observed for 10 days at the dose of 1,000 mg / kg jujube extract. An autopsy of the surviving animals was performed 10 days later, and there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after the administration.

In conclusion, the jujube extract was not toxic upon oral administration at the above concentrations.

The following formulation examples are merely illustrative of the formulation of the present invention and do not limit the scope of the invention.

My example

Hard gelatin capsules were prepared using the following ingredients:

Amount (mg / capsule)

Active ingredient (jujube extract) 1,000

Dry starch 160

Magnesium Stearate 40

Total 1,200 mg

Since the composition of the present invention containing the jujube extract reduces blood cholesterol in the animal body, it can be usefully used as a prophylactic and therapeutic agent for various cardiovascular diseases. In addition, since the active substance of the present invention extracted from jujube reduces the blood cholesterol by inhibiting CETP activity, it can be usefully used as a preventive and therapeutic agent for various cardiovascular diseases or as a component of health foods.

Claims (12)

A plasma cholesterol lowering agent composition comprising an effective amount of jujube ethanol extract as an active ingredient with a pharmaceutically acceptable carrier. The method of claim 1, A composition wherein the effective amount of jujube ethanol extract is 1 to 50 g / kg body weight / day. A plasma cholesterol lowering agent composition comprising an effective amount of jujube extract as an active ingredient with a pharmaceutically acceptable carrier. The method of claim 3, wherein A composition having an effective amount of jujube water extract of 1 to 50 g / kg body weight / day. A food composition for lowering plasma cholesterol, containing an effective amount of jujube ethanol extract as an active ingredient. A food composition for lowering plasma cholesterol, containing an effective amount of jujube water extract as an active ingredient. (a) Jujube was extracted with acetone and filtered. (b) The filtrate was concentrated under reduced pressure, hexane was added to separate the water layer and the hexane layer. Separating the ethyl acetate layer, (d) collecting the ethyl acetate layer and then concentrated to dryness under reduced pressure, and (e) purifying the concentrate by reverse phase chromatography; CETP) A method for separating activity inhibitors. CETP activity inhibitory substance isolated by the method of Claim 7. A CETP activity inhibitor composition comprising, as an active ingredient, an effective amount of the CETP activity inhibitory substance of claim 8 together with a pharmaceutically acceptable carrier. The method of claim 9, The effective amount of the CETP activity inhibitory substance is 0.1 to 5 g / kg body weight / day. A food composition for inhibiting CETP activity, containing an effective amount of the CETP activity inhibitory substance of claim 8 as an active ingredient. The method of claim 11, The effective amount of the CETP activity inhibitory substance is 0.1 to 5 g / kg body weight / day.