KR100254908B1 - 3-hydroxy-3-methylglutaryl coa reductase inhibitory composition comprising vitamin c and citrus peel extract - Google Patents

Abstract

PURPOSE: An HMG-CoA reductase inhibiting composition containing vitamin C and orange peel extract is provided, which is useful in the prevention and treatment of hyperlipidemia by decreasing blood cholesterol level. CONSTITUTION: The HMG-CoA reductase inhibiting composition is manufactured by following processes of; washing peel of tangerine of which origin is Korea, drying at normal temperature to get Aurantii Nobilis Pericarpium; adding 95% ethanol, keeping for 24hours at normal temperature, and filtering to get extracted solution and solid residue; extracting the solid residue two more times by the same process, gathering all the extracted solution together, and concentrating under decompression by a rotary vacuum evaporator to get a concentrated extract. The composition can be manufactured as pharmaceutical products and health care foods such as a beverage, a snack, a noodle, a vitamin complex, a candy, etc.

Description

3-hydroxy-3-methylglutaryl koei reductase activity inhibitor composition comprising vitamin C and tangerine extract concentrate as active ingredients

Coronary cardiovascular disease currently accounts for more than 30% of all deaths, and in developed countries, in some cases, up to 40% mortality occurs. On the other hand, as the economic level of Korea is increasing, the mortality rate of heart disease due to excessive weight gain and blood cholesterol increase is increasing due to westernization of eating habits, lack of exercise, and overwork, and especially mortality rate due to cerebrovascular disease such as stroke. 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. Especially, 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. It is known that cholesterol ester transfer protein (CETP) is involved in the conversion of blood cholesterol from high density lipoprotein (HDL) to low density lipoprotein, and there is an opinion that lowering the amount of LDL-cholesterol may result from lowering CETP activity (L. Lagrost). , Biochemical et.Biophysica Acta, 1215, 209-236 (1994)).

On the other hand, it is known that the amount of LDL-cholesterol in the blood may be lowered by inhibiting the activity of acyl CoA: cholesterol acyltransferase (ACAT), an enzyme involved in cholesterol ester formation (DT Witiak, et al. (eds)., Antilipidemic Drugs: Medicinal, Chemical and Biochemical Aspects., pp. 159-195, Elsvier, 1991).

Another best known way to lower the amount of cholesterol in the blood is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), an enzyme involved in cholesterol biosynthesis in the liver. As a way to reduce the amount of cholesterol in the blood by inhibiting reductase, lovastatin, simvastatin, pravastatin, etc., developed by Merck and Sankyo, Japan, form a large market. Doing.

In order to lower blood cholesterol, no successful development of CETP inhibitors or ACAT inhibitors has been reported. HMG-CoA reductase inhibitors, on the other hand, have been industrialized but are too expensive and are known to have some side effects when taken long-term.

The inventors of the present invention have been attempting to develop a safe, non-toxic, antihyperlipidemic-active dietary ingredient, ACAT inhibition and HMG-CoA reductase from the skin of citrus fruits (orange, tangerine, grapefruit, lemon, citron, tanza) Successful extraction of viroflavonoid mixtures showing inhibitory activity was found to be related to hesperidin and naringin. On the other hand, some studies have reported that oxidized LDL is involved in the prevention of atherosclerosis. Therefore, antioxidants including vitamin C may be useful. The ingredients are mixed in an appropriate ratio to produce tablets or capsules in the form of a daily intake of a certain amount will be a health food for preventing hyperlipidemia.

It is an object of the present invention to provide a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity inhibitor composition comprising a tangerine extract concentrate as an active ingredient.

In the present invention, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity comprising vitamin C and citrus peel extract or vitamin C, hesperidin and naringin as active ingredients together with a pharmaceutically acceptable carrier. A pharmaceutical composition for inhibition is provided. Also provided is a food composition and a beverage composition for inhibiting HMG-CoA reductase, including vitamin C and citrus peel extract or vitamin C, hesperidin and naringin.

Citrus fruits in the present invention refers to tangerine, orange, lemon, grapefruit, citron or tanza. Flavonoids contained in citrus fruits may vary in active ingredient ratio depending on the maturity of the fruit or the type of fruit, and their extraction methods are disclosed in the literature and patents such as the Merck Index.

That is, after washing the citrus peel with water and dried at room temperature in the shade, 0 to 95% alcohol, for example, 95% ethanol, or water is added, and the active substance is extracted in the temperature range of 5 ℃ -140 ℃. Can be. In the case of using an organic solvent in the extraction, toxic substances may be incorporated into the extract, and as a result, the extract is not suitable for use in medicine or food use. Therefore, it is preferable not to use the organic solvent. In general, when extracting the citrus peel using water or alcohol, the composition of the extract may vary depending on the reaction time, reaction temperature, alcohol concentration, type of fruit, fruit maturity, and the like.

Citrus destruction extract obtained as described above inhibits the activity of HMG-CoA reductase, and some of the blood cholesterol reduction in rats may be attributed to the decrease in HMG-CoA reductase activity. In addition, a combination of hesperidin and naringin, the active ingredient in citrus extracts, also strongly inhibits HMG-CoA reductase activity. Toxicity studies to date have shown that citrus fruit extract, hesperidin or naringin are almost non-toxic when administered orally to mice at doses of 1000 mg / kg, respectively. In addition, citrus peel extracts, hesperidin and naringin have no adverse effects on the function of organs, including the liver.

In order to inhibit the activity of HMG-CoA reductase, HMG-CoA reductase activity inhibitor composition may be prepared by mixing vitamin C and citrus peel extract, concentrate or dry powder thereof with a pharmaceutically acceptable carrier as an active ingredient. . In addition, an HMG-CoA reductase inhibitor composition can also be produced using vitamin C, hesperidin and naringin as active ingredients. These pharmaceutical compositions may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, liquids by conventional methods Or in dosage unit forms or as multi-dose pharmaceutical preparations, such as preparations for parenteral administration.

Vitamin C and citrus rind extract of the present invention or HMG-CoA reductase inhibitor composition containing vitamin C, hesperidin and naringin as an active ingredient can be parenterally or orally administered as desired, vitamins per kg of body weight per day C is 0.01 to 1g, preferably 0.1 to 0.3g, and the citrus peel extract can be administered in one to several times so that the amount of 0.01 to 10g, preferably 1 to 5g. In the case of a composition containing vitamin C, hesperidin and naringin as an active ingredient, the vitamin C per kilogram of body weight per day is 0.01 to 1g, preferably 0.1 to 0.3g, and hesperidin and naringin are 0.001 to 10g, preferably 0.1 Administration may be made in one to several portions to an amount of 1 to 1 g. 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, disease severity, and the like.

Citrus peel extracts of the present invention may also be added to foods or beverages for the purpose of inhibiting the activity of HMG-CoA reductase. Foods to which the citrus peel extract of the present invention can be added for the development of dietary supplements include, for example, various foods, meats, beverages, chocolates, snacks, confectionery, pizzas, ramen noodles, other noodles, gums, ice creams. Alcoholic beverages, vitamin complexes and dietary 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, but the scope of the present invention is not limited to these.

Example 1 Preparation of Citrus Peel Extract Concentrate

Korean citrus peel produced in Jeju Island, Korea was washed and dried at room temperature to obtain a dry weight of 6.7kg. 80 L of 95% ethanol was added thereto, stored at room temperature for 24 hours, and filtered to obtain an extract and a solid residue. The solid residue was extracted twice more in the same way. The obtained extracts were combined and concentrated under reduced pressure with an EYELA Rotary vacuum evaporator N-11 to obtain 1.7 kg of concentrated extract. The density (d) of this extract was 1.3 g / ml.

In order to investigate the components of the skin extract obtained as described above, HPLC analysis was performed under the following conditions. The concentrated extract was diluted to a concentration of 10 mg / ml using a DMSO / MeOH (1: 1 (v / v)) mixed solvent. 100 μl of the resulting solution was pre-equilibrated with Phenomenex Prodigy column with 0.01 M (phosphate / methanol (70:30 (v / v)) mixed solvent (5 μODS (3) 100 μs (250 × 4.6 mm)) Was injected into the HPLC, eluting at a flow rate of 0.6 ml / min using a 0.01 M phosphoric acid / methanol (70:30 (v / v)) mixed solvent, gradually increasing the concentration of methanol to 45% for 55 minutes. 100 μl of a solution prepared by dissolving pure hesperidin and naringin (Sigma Co.) in a solvent of DMSO / MeOH (1: 1 (v / v)) at a concentration of 1 mg / ml was used as a standard. The content was calculated by area comparison analysis of the standard profile and the HPLC profile of the extract and contained 5,950 mg of hesperidin and 280 mg of naringin per kg of extract extract.

In addition, the components of the citrus peel extract were analyzed by standard food analysis methods. Analytical methods consist of drying at 105 ° C, drying, crude protein by the Kjeldahl method, crude fat by the Soxhlet method, free sugar by the Bertrand method, and crude ash by 550-600 ° C. Hesperidin and naringin were analyzed by HPLC, respectively, and as a result, it was confirmed that the citrus peel extract had the composition shown in Table 1 below.

Example 2 Preparation and Animal Administration Experiment of HMG-CoA Reductase Inhibitor Composition

The components of Table 2 were mixed to prepare HMG-CoA reductase activity inhibitory composition.

In order to investigate the effect of the combination formulation of vitamin C and tangerine extract on the activity of HMG-CoA reductase, a cholesterol synthetase, in Sprague-Dawley rats A normal diet (AIN-76 experimental animal diet) was fed for 6 weeks with feed containing 1% cholesterol. As an experimental group, the compound 1 prepared above was orally administered to the rats fed the feed and fed 500 mg daily. Six weeks later, the liver tissues of the rats were extracted. Microsomes were isolated from liver tissue and used as an enzyme source of HMG-CoA reductase. The liver microsomal fraction used as an enzyme source was separated by centrifugation by taking 2.5g of livers extracted from rats whose breeding experiments were completed, and stored in liquid nitrogen until analysis.

HMG-CoA reductase activity measurement using [ ¹⁴ C] HMG-CoA followed the method of Shapiro et al. (Biochimica et Biophysica Acta, 370, 369-377 (1974)) and the procedure is briefly summarized as follows. do. Microsome pellets (100-500 μg protein) stored in liquid nitrogen were reacted with NADPH (50 μmol) at 37 ° C. for 15 minutes. At this time, HMG-CoA is converted to mevalonate and denatures the microsomal protein by adding 10N HCl corresponding to 1/6 of the reaction volume and reacting for 15 minutes at 37 ° C., at this time, mevalonate lactone lactone). After completion of the reaction mixture, the reaction mixture was centrifuged at 10,000 × g for 5 minutes, and the supernatant was added to a silica gel 60G TLC plate (Altech, USA), and then developed in a benzene-acetone (1: 1, v / v) solvent. At this time, mevalonate was measured at Rf 0.6-0.8 using an image analyzer. Finally, the enzyme activity was calculated and expressed in units of mevalonate picomolar / min / mg protein. The enzyme activity thus measured is shown in Table 3 below.

As can be seen from the table, the HMG-CoA reductase activity of the control group fed only the high cholesterol diet was high, and HMG-CoA reductase activity was decreased by 15% in the vitamin C-depletion extract combination group.

Example 3 Oral Toxicity Test of Vitamin C + Citrus Peel Extract Complex]

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 in the experiment. Feed for experimental animals (for Cheil Jedang Co., Ltd., mice and rats) and drinking water were supplied after sterilization and free ingestion.

The vitamin C + citrus peel extract complex prepared in Example 2 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. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss phenomenon of the animals.

The purpose of this study was to determine the degree of acute toxicity in the oral route by combining vitamin C and citrus peel extracts, to provide information on available drug dosages in general pharmacology and efficacy tests, and to derive basic data on toxicity. It carried out and obtained the following result.

In the case of acute oral toxicity, no lethal animals were observed for 10 days at the dose of 1,000 mg / kg of the combination. 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 combination of vitamin C and citrus peel extract did not show toxicity upon oral administration.

HMG-CoA activity inhibitor composition comprising vitamin C and citrus peel extract or vitamin C, hesperidin and naringin of the present invention can be useful as a prophylactic and therapeutic agent for hyperlipidemia because it inhibits HMG-CoA activity in the animal body and reduces blood cholesterol Can be.

Claims (4)

A 3-hydroxy-3-methylglutaryl coeigen (HMG-CoA) reductase activity inhibitor composition in a mammal comprising an effective amount of vitamin C and a citrus peel extract as an active ingredient together with a pharmaceutically acceptable carrier. The composition of claim 1, wherein said mammal is a human. The composition of claim 1, wherein the citrus fruits are tangerine, orange, lemon, grapefruit, citron or tangerine. A 3-hydroxy-3-methylglutaryl coeigen (HMG-CoA) reductase activity inhibitor composition, comprising as an active ingredient an effective amount of vitamin C, hesperidin and naringin in combination with a pharmaceutically acceptable carrier.