KR960008227B1 - Antihyperlipemia agents containing panaxidol - Google Patents

Abstract

The panaxidol, a polyacethylene compound with two or more triple bond, is separated from ginseng and shows repression effect on total cholesterol, triglyceride and LDL-cholesterol, which relate to hyperlipemia induction and progression. The used excipient is magnesium stearate and carboxymethyl cellulose etc., the capsule is polysolvate and polyethylene glycol etc. The panaxidol is prepared for drug such as tablet or capsule form, where the adminstration quantity is 20 Pmole/kg B.W.

Description

Use of Panaxidol as an Inhibitor of Hyperlipidemia

The present invention relates to hypercholesterolemia and hyperlipidemia inhibitors containing panaxidole.

Increased cholesterol and lipids in the blood are likely to cause atherosclerosis. Atherosclerosis is a cardiovascular disease that can cause a heart attack, stroke, etc. by blocking the blood flow by forming adenoroma plaques on the walls of blood vessels due to hypercholesterolemia. Prevention and treatment of this should lower blood lipids and cholesterol. Examples of such methods include inhibiting cholesterol biosynthesis, inhibiting cholesterol absorption, and promoting metabolism of cholesterol into bile acids. As a method of inhibiting cholesterol biosynthesis, it is known that it is very effective to lower plasma cholesterol by inhibiting HMG-CoA reductase, a rate-synthesizing enzyme in the biosynthesis step [N. Eng1. J. Med. 305, 515-517 (1981). It has also been demonstrated that lowering LDL-cholesterol can inhibit coronary heart disease. [J. Am. Med. Assoc. 251, 351-374 (1984).

However, the HMG-CoA reductase inhibitor, a rate-synthesizing enzyme in the biosynthetic stage, inhibits the production of physiologically important substances such as ubiquinone and dollyol derived from mevalonate, which leads to significant side effects such as visual impairment and dysfunction. Piol., Chem., 256, 4679-4686 (1981) and Eur. J. Biochem., 87, 313-321 (1978). Therefore, the current focus is on the development of inhibitors for the latter part after squalene during the biosynthesis step that can alleviate side effects rather than inhibitors of HMG-CoA reductase.

a. a. Cures reported that serum total cholesterol, LDL-cholesterol and triglyceride contents were reduced without degrading HDL-cholesterol in chickens treated with water extract and petroleum ether extract of ginseng. Not specifically mentioned. [Atherosclerosis, 48, 81-94 (1983)].

The present invention relates to the use of as an inhibitor of hypercholesterolemia and hyperlipidemia by verifying the effect on the separation of panaxidol from petroleum ether fraction of ginseng on cholesterol metabolism and lipid metabolism.

Panaxidol, a ginseng component, was first isolated by Pollauski et al. As one of the polyacetylene compounds having two or more triple bonds represented by the following structural formula. Chem. 19, 1539-1541 (1980)]

Studies on the physiological activity of panaxidol have been shown to have strong cytotoxicity against cancer cells, but the effect on lipid and cholesterol metabolism has not been reported so far. To function as an inhibitor of hypercholesterolemia and hyperlipidemia, there must be a lowering effect of cholesterol or lipids in animal studies, no toxic effects at the proper dosage used, and a protective effect of cholesterol and hyperlipidemia. The mechanism for the degrading effect should be supported.

According to the results of animal experiments on panaxidol, it was found that serum total cholesterol, triglycerides, and LDL-cholesterol have a lowering effect. It has been reported not to appear [Korean J. Ginseng Soc., 13, 49-55 (1989)]. Therefore, as a result of animal experiments related to the present invention, it can be seen that intraperitoneal administration of panaxidol at a dose of 20μmole / kg BW does not have a toxic effect. appear.

In vitro cholesterol biosynthesis test results showed that panaxidol has the activity of inhibiting the biosynthesis step from lanosterol to late cholesterol, which is the first step in the synthesis of cholesterol in lanosterone. Demonstrates suppressing the migration step. In addition, panaxidol suppressed intestinal cholesterol absorption when administered orally and was shown to be more than twice as potent at the same dose as compared to clofibrate, a conventional drug for treating hypercholesterolemia and hyperlipidemia.

Therefore, in vitro and in vivo experiments revealed that panaxidol has an inhibitory effect on total cholesterol, triglycerides, and LDL-cholesterol related to the induction and progression of hyperlipidemia.

According to the present invention, panaxidol is prepared in the form of a conventional formulation such as tablets or capsules for oral administration.

Examples of excipients used in the preparation of the panaxidol-containing formulations according to the present invention include lactose, corn starch, magnesium stearate, carboxymethylcellulose, and the like in the case of tablets, and polysorbates, polyethylene glycol, and the like in the case of capsules. This is used.

The panaxidol-containing tablet or capsule according to the present invention is prepared to be administered at 20 μmole or less per kg of body at a time.

If a single dose of panaxidol exceeds the above amount, there is a risk of side effects. In general, one tablet or capsule should usually contain 75 mg of panaxidol based on an adult.

Example 1

Panaxidol Separation

500 g of red ginseng was refluxed three times for 3 hours with 2 liters of methanol in a constant temperature water bath, and concentrated by filtration to obtain a methanol extract, dissolved in 1 liter of water, and then extracted and concentrated 3 times for each liter of petroleum (2 g). Obtained by silica gel column chromatography to obtain a P-III fraction as follows.

Petroleum ether 3 times extraction (0.2g)

From the P-III fraction, panacydol, the main compound of polyacetylene, was isolated purely using HPLC Alltech-NH ₂ semipreparation column.

Example 2

Male rats (35 g), which were experimental animals, were divided into a control group (n = 10) and a test group (n = 10) and used for each experiment. 50 μl of dimethyl sulfoxide was administered to the control group for 3 days, and the test group was intraperitoneally administered with dimethyl sulfoxide (50 μl) by dissolving panaxidol (20 μmole / kg, body weight). Three days later, 24 hours after the last dose (17 hours starvation), the rats were anesthetized, blood was collected from the heart, and serum was isolated. Total cholesterol HDL-cholesterol and triglycerides in serum were measured by enzyme method.

Experiment result

(Reference) 1) Mean ± standard error

2) The number of test group when the control group is 100

3) LDL-cholesterol = total cholesterol-HDL-cholesterol-glycerides / 5

Results As shown in Table 1, the panaxidol was lowered by 11% in total cholesterol, 18% in triglycerides and 22% in LDL-cholesterol compared to the control group.

Example 3

To induce hypercholesterolemia in rats, 1% cholesterol and 0.5% cholic acid were fed but mixed to produce high cholesterol food.

The rats were divided into the control group (n = 5) and the test group (n = 5) to feed high cholesterol, while the control group was dimethyl sulfoxide (50 µl) and the test group was 50 µl of dimethyl sulfoxide in the panaxidol (20 µmole / kg, body weight) was mixed and administered to the abdominal cavity once a day for 3 days. At this time, the body weight and sample intake of rats were examined, and after 24 hours (17 hours of starvation) slaughter, the blood was collected from the heart to separate serum, and the liver was excised to remove total cholesterol, triglyceride, and HDL from serum. Cholesterol was measured by enzyme method and cholesterol level was measured in liver.

Experiment result

1) Mean value ± standard error

2) the number of test group when the control group is 100

As shown in Table 2, 51%, 53%, and 59% of triglyceride and LDL-cholesterol in serum of panaxidol-treated test group were lowered as compared to the control group, respectively, which decreased cholesterol content related to hyperlipidemia. On the other hand, HDL-cholesterol, a high cholesterol inhibitor, is increased by about 17%.

1) Mean value ± standard error

2) the number of test group when the control group is 100

Feeding cholesterol in rats significantly increases the amount of ester cholesterol in the liver. As a result of measuring the concentration of cholesterol in the liver, the total cholesterol was decreased by 28% compared with the control group, and the concentration of ester cholesterol, which is easy to accumulate in the liver, was reduced by 36%.

In this example, the weight gain and feed intake of the rats were not significantly different from the control group. The results of Table 3 showed that panaxidol has the effect of lowering the liver high cholesterol content.

Example 4

Inhibitory Effect of Panaxidol on In Vitro Cholesterol Biosynthesis

Experimental animals (ICR mice, 25g,) were changed day and night for one week (day; 20: 00-08: 00, night; 08: 20). After digestion, a 50% crushed solution was prepared with 0.1 M phosphate buffer (pH 7.4) containing 30 mM nicotinamide and 4 mM MgCl, centrifuged at 20,000 × g, and the supernatant was used as an enzyme source (30 mg protein). The reaction mixture was prepared with a final volume of 1 ml, enzyme source (20 mg protein), 5 mMMATP, 3 mM glucose-6-phosphate, 1 mM MADP, 0.05 mM melanonolactone and 0.2 μCi R- [2- C] Metalonolactone. Panaxidol was dissolved in propylene glycol, respectively, to 0, 100, 500, and 1000 μM, and propylene glycol was brought to a final concentration of 2%. 1mM of the reaction mixture was added to the cap tube and filled with a mixed gas (O: CO = 95: 5) for 10 seconds, and then the lid was reacted by shaking at 37 ° C. for 60 minutes. The reaction was stopped with 1 ml of 30% KOH dissolved in ethanol and reacted with alkali at 75 ° C. for 1 hour. Next, lipids that can no longer be saponified were extracted three times with petroleum ether, and the extract solution was washed three times with distilled water and concentrated with nitrogen gas. After concentration, it was dissolved in a small amount of chloroformum and developed with a developing solvent (silica gel paper plate (20 × 20 cm)) using a shallow membrane chromatography plate (ethyl acetate: nucleic acid = 15: 85) to prepare cholesterol, lanosterone and squalene. Separated.

Each of the above materials was separated and radioactivity was measured with a scintillation counter (LS-1800).

(Reference) 1) Mean value ± standard error (n = 3)

2) the number of test group when the control group is 100

As can be seen from the experimental results, it was found that panaxidol inhibits cholesterol biosynthesis from lanosterone, which is a late stage of cholesterol biosynthesis, in a concentration-dependent manner. At the concentration of 500 μM of panaxidol, the final product, cholesterol, was reduced by about 50%, while the lanosterone content was increased by 289%. This indicates that it inhibits the demethylation step, which is the first of the cholesterol biosynthesis steps from lanosterone.

Example 5

In the same manner as in Example 4, U- [in place of mevalonolactone using the liver microsomes of Sprague Dawley (200 g) instead of experimental mice. C] The result of examining the step of synthesizing sterol from acetylcoa with acetic acid as a substrate is shown in Table 5.

(Reference) 1) Mean value ± standard error (n = 3)

2) the number of test group when the control group is 100

As can be seen from the experimental results, it was found that panaxidol inhibits cholesterol biosynthesis in a concentration-dependent manner, and inhibits cholesterol biosynthesis from lanosterone. In other words, the total non-glycosylated fat was reduced to 58% at the concentration level of panaxidole 500 μm, while the final product cholesterol was reduced to about 23% while lanosterone was increased to 239%. From the results in this table, it can be seen that a significant decrease in total non-glycosylated fats exhibits inhibitory effects involved in any biosynthetic step from acetic acid to panaxole to squalene.

Example 6

Effect of Panaxidol on Inhibition of Cholesterol Absorption

Panaxidol (5ml / kg, body weight) was dissolved in 1% carboxymethylcellulose intraperitoneally and administered to male rats (Sprague Dawley, 200g) once a day for 3 days. C] -cholesterol (0.5 μCi / 200 g, body weight) injected into the tail vein, simultaneously [1,2- H.-cholesterol (1.64 μCi / 200 g, body weight) was orally administered to measure the amount of radioactivity in the blood 48 hours after the administration of the radioactive substance.

(Reference) 1) Mean value ± standard error (n = 5)

2) the number of test group when the control group is 100

3) Oral administration

4) Intravenous administration

* Cholesterol absorption of non-glycosylated fats H / Calculated at the C ratio.

As described above, panaxidol was found to inhibit cholesterol biosynthesis as well as cholesterol intestinal absorption (25% than control).

Example 7

Effects of Panaxidol on Serum Lipids by Oral Dose

Changes in serum lipids according to the dose of panaxidol were observed in the cholesterol diet (control group) and the panaxidol concomitant group (test group). In male rats (200g), which are experimental stones, the test group dissolves panaxidol in 1% carboxymethyl cellulose by dose and orally administers once a day for 3 days, and separates serum after 24 hours (17 hours starvation) after 3 days. Total cholesterol, triglyceride and HDL-cholesterol were measured by enzymatic method, and the comparative test group of clofibrate, a known high cholesterol lowering agent, was further compared. The control group was administered only 1% carboxymethylcellulose.

Experiment result

1) Mean ± standard error

2) the number of test group when the control group is 100

LDL-cholesterol = total cholesterol-HDL-cholesterol-glycerides / 5

Experimental results As shown in Table 7, the serum total cholesterol of the panaxidol administration group (50 mg / kg, body weight) was inhibited by 4% and LDL-cholesterol was inhibited by 52% compared to the control group.

Claims (2)

Use of panaxidol or a derivative thereof as a cholesterol absorption inhibitor and a biosynthesis inhibitor. Use of panaxidol or a derivative thereof as an agent for preventing and treating hypercholesterolemia and hyperlipidemia.