KR20000071882A - Composition for preventing and treating atherosclerosis comprising extract of alpiniae katsumadaii semen - Google Patents

Abstract

PURPOSE: A composition containing Alpinia Katsumadal semen for prevention, treatment, and cure of atherosclerosis is provided, which inhibits activity of pancreatic cholesterol esterase(pancreatic cholesteryl ester hydrolase; pCEH) specifically, so decreases absorption in body of cholesterol taken from food. CONSTITUTION: A process for the preparation of composition comprises of: pouring pulverized Cinnamomi cortex or pulverized Alpinia Katsumadal and 95% ethanol 1L to a round flask, circulation-cooling while heating at 78°C; filtering, and extracting the residue with ethanol while heating for 2times; adding filtered solution together, and concentrating under decompression to get the ethanol extract; suspending the ethanol extract in 10% methanol, mixing with chloroform; pouring the water layer with chloroform, extracting for 5times again using separate funnel; drying under decompression to get Cinnamomi cortex chloroform extract or Alpinia Katsumadal chloroform extract; removing chloroform in water layer by concentrating under decompression, and mixing with ethylacetate; extracting for 5times again using separate funnel; drying under decompression to get Cinnamomi cortex ethylacetate extract or Alpinia Katsumadal ethylacetate extract; removing ethylacetate in water layer by concentrating under decompression, and mixing with butanol; extracting for 5times again using separate funnel; and drying under decompression to get Cinnamomi cortex butanol extract or Alpinia Katsumadal butanol extract.

Description

Composition for preventing and treating atherosclerosis comprising the extract of cranial bulbs {COMPOSITION FOR PREVENTING AND TREATING ATHEROSCLEROSIS COMPRISING EXTRACT OF ALPINIAE KATSUMADAII SEMEN}

The present invention relates to a composition for the prevention and treatment of atherosclerosis comprising a head cranium extract.

Atherosclerosis is a lesion that becomes hard as the artery wall thickens and loses elasticity. It occurs in all parts of the arterial system, from the aorta to the arterioles. Many factors, such as hyperlipidemia, hypertension, obesity, diabetes and smoking, have been pointed out as the causes of atherosclerosis according to the results of animal experiments, epidemiologic investigations, pathological examinations, and clinical results.

Hypercholesterolemia is important, and it is reported that blood cholesterol has a high positive correlation with the incidence of coronary arteriosclerosis and myocardial infarction, and these reports indicate that mortality due to coronary artery disease or ischemic heart disease is high in cholesterol-rich countries. Reported high (Keys, A., Circulation, 41 (Suppl), 1 (1970); The Lipid Research Clinic Program, JAMA, 251, 365 (1984); and The Expert Panel, Arch.Intern. Med., 148, 36 (1988)).

Cholesterol is an essential component of cells and plays an important role in cell growth, differentiation and development. In adults, cholesterol demand is about 1,300 mg per day, of which about 1,000 mg of cholesterol is produced in almost every cell of the body and about 300 mg of cholesterol is taken from food.

Thus, excess cholesterol from food can meet the needs of the cells (Gurr, MI, Proceedings of Nutrition Society, 47, 277 (1981); Goldstein, JL et al., Circulation, 76, 504 (1987)). Floating along the blood and adsorbed into the vascular lining together with monocytes and megakaryocytes, causing them to turn into foam cells, eventually blocking the arteries and causing a heart attack (Johan, F. et al., Proc. Natl. Acad). Sci., 87, 904 (1990)), accumulate in blood vessels and cause severe cardiovascular diseases such as atherosclerosis and angina pectoris. The Lipid Research Clinics-Coronary Primary Prevention Trial (LRH-CPPT) study of the relationship between cholesterol intake from food (hereinafter referred to as "diet cholesterol") and cardiovascular disease (The Lipid Research Clinic Program, J. Am. Med) Dietary cholesterol, according to Assoc., 251, 365 (1984)) and the results of the Helsinki Heart Study (Frick, MH et al., N. Eng. J. Med., 317, 1237 (1987)). If the intake is reduced by 1%, cardiovascular disease is reduced by 2%.

In recent years, cardiovascular disease patients have more than doubled every year due to the increase in meat consumption and the ingestion of high-fat processed foods such as fast foods such as hamburgers, pizzas, and chickens. Internal medicine clinical data) and cholesterol-related atherosclerosis are becoming serious problems. In order to solve this problem, the development of substances that reduce blood cholesterol is urgently needed.

Studies on the absorption of dietary cholesterol in the body show that the ingested cholesterol esters are converted into free cholesterol and fatty acids by pancreatic cholesterol esterase (pCEH) in the pancreatic secretion enzyme solution in the small intestine. Is degraded and absorbed into the small intestine, where absorption of free cholesterol is promoted by pCEH in the small intestine mucosa (Gallo, LL et al., J. Lipid Res., 25, 604 (1984); Bhat, SG et al. , Biochem.Biophys.Res.Commun., 109, 486 (1982); Riley, DJS et al., Biochemistry, 29, 3848 (1990); Vuoristo, M. et al., Gastroenterology, 102, 647 (1992)) . The free cholesterol absorbed by the small intestine mucosa again forms esters with fatty acids, which in turn are CEH and acylcoa: Cholesterol acyltransferase (ACAT) (Gallo, LL et al). , J. Lipid Res., 25, 604 (1984)).

Early studies on the absorption of dietary cholesterol showed decreased CEH activity and cholesterol uptake of the small intestine mucosa in pancreas excised white rats, supporting the hypothesis that CEH from the small intestine mucosa is derived from the pancreas. Experimental results also confirmed that pCEH is absorbed by the small intestinal villi. Removing pCEH from the pancreatic digestive fluid and maintaining ACAT normal inhibits cholesterol absorption into the bloodstream by about 80%. Taken together, these results indicate that absorption of exogenous cholesterol cannot be maintained by ACAT alone, and pCEH is most essential. PCEH is also known to affect intestinal absorption efficiency (Lopez-Candales, A. et al., Biochemistry, 32, 12085 (1993)).

Thus, pCEH inhibitors can prevent and treat arteriosclerosis and cardiovascular diseases caused by ingested cholesterol by preventing the degradation and absorption of dietary cholesterol. WAY-121,751 and WAY-121,898, recently developed by the Wyeth-Ayerst Research, are effective pCEH inhibitors and have been reported to inhibit the induction of cholesterol-induced hypercholesterolemia when administered with cholesterol. (Mckean, MI et al., FASEB J., 6, A1388 Abs. (1992); Clark, DE et al., FASEB J., 6 , A1388 (1992); And Gallo, L. et al., FASEB J., 6, A1388 Abs. (1992)).

In addition, pCEH has been reported to have a large correlation with hepatic CEH. For example, first, CEH purified from rat hepatic triturates also reacted with antibodies to pCEH, and was confirmed to be identical to pCEH in electrophoresis patterns, N-terminal amino acid sequences, and the like. Second, Northern blot of liver mRNA using the known cDNA of pCEH was confirmed as the mRNA of liver CEH of the same size as pCEH. Third, mRNA of liver CEH was injected into Xenopus oocytes to express the same enzyme activity as that expressed from cCE of pCEH in Cos cell or Chinese hamster ovary cell. (Zolfaghari, R. et al., J. Biol., Chem., 268, 13532 (1993)). From these reports it can be seen that there is a high correlation between pCEH and liver CEH.

Cinnamon bark (Cinnamon bark) is a herb belonging to the family Lauraceae, and is mainly produced in southern China and northern Vietnam, and has been shown to have lower heat, central restraint, persistent peripheral vasodilation, platelet aggregation, and skin darkening. It is used for colds, pain relief and dysmenorrhea due to antipyretic fever, aroma health, and anti-causing effect.

Also, Alduia Katsumadai semen is a herbal medicine belonging to the family Zingiberaceae.

The inventors of the present invention, while studying the absorption of dietary cholesterol and triglycerides for the prevention and treatment of various cardiovascular diseases including atherosclerosis, found that cinnamon extract or cedar extract in herbal medicines had pCEH inhibitory activity and completed the present invention. Was done.

An object of the present invention is to provide a composition for preventing and treating atherosclerosis comprising a cinnamon extract or a cranial extract.

Figure 1 shows the process of obtaining the chloroform, ethyl acetate, butanol and water fractions from the ethanol extract of cinnamon or turduol.

Figure 2 is a graph showing the pCEH inhibitory activity of chloroform, ethyl acetate, butanol or water extract of cinnamon or turmeric.

In accordance with the above object, the present invention provides a composition for preventing and treating atherosclerosis comprising a cinnamon extract or a head nut extract and a pharmaceutically acceptable carrier.

As the cinnamon, the bark of the plant of the genus Namos, such as Cinnamomum cassia, Cinnamomum zeylanicum, Cinnamomum burmanii, Cinnamomum obtusifolium, Cinnamomum sieboldii It can be used or by removing the skin.

In addition, the seed head may be a seed part from which the skin of the fruit of Alpinia Katsumadai Hayata is removed.

The cinnamon or turmeric extract is an extract of finely crushed cinnamon or edible ethanol extract extracted again with chloroform (hereinafter referred to as "cinnamon or turmeric chloroform extract"), an extract of water remaining after extraction of chloroform with ethyl acetate (hereinafter referred to as "cinnamon) Or ethyl acetate extract "), or an extract obtained by extracting the water layer remaining after ethyl acetate extraction with butanol (hereinafter referred to as" cinnamon or bean nut butanol extract "), and a chloroform extract is preferable.

Cinnamon or cedar extract can be prepared by boiling or shaking with an extraction solvent such as chloroform, ethyl acetate or butanol. For example, first, cinnamon or cereal bulbs are added to 95% ethanol, cooled to 78-85 ° C. under reflux, filtered, and concentrated under reduced pressure using a rotary evaporator to obtain cinnamon or cereal bulb ethanol extract. The remaining residue may be repeated two or more times. The obtained ethanol extract was suspended in 10% methanol, mixed with the same amount of chloroform in a separatory funnel, shaken and mixed to obtain a chloroform layer and a water layer. In this case, the extraction process for the water layer may be performed by repeating 4 to 5 times. The same amount of ethyl acetate as the remaining water layer after the extraction of chloroform was added to the separatory funnel, and the same process as in the chloroform extraction step was repeated to obtain cinnamon or head nut ethyl acetate extract. The remaining water layer and the same amount of butanol after ethyl acetate extraction were put in a separatory funnel and the same process as in the chloroform extraction step was repeated to obtain a cinnamon or cinnabar butanol extract and a cinnamon or cinnabar water layer.

The extract of the present invention acts to inhibit pCEH activity, which mediates the degradation and absorption of dietary cholesterol in the small intestine, thereby preventing the absorption of dietary cholesterol in the body, thereby preventing an increase in blood cholesterol levels by dietary cholesterol and finally preventing atherosclerosis. Prevent and treat.

In addition, the extract of the present invention has the effect of treating hyperlipidemia by reducing blood triglyceride concentrations in addition to treating arteriosclerosis.

The composition of the present invention may be administered by oral or injection administration, and may be formulated in various forms as follows.

Formulations for oral administration include, for example, tablets, capsules, etc. These formulations, in addition to the active ingredient, may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants ( Eg silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpicolidine, optionally starch, agar, alginic acid or its Disintegrants or boiling mixtures such as sodium salts and / or absorbents, colorants, flavors, and sweeteners. Injectable formulations are preferably aqueous isotonic solutions or suspensions.

The composition may contain sterile and / or auxiliaries such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts for regulating osmotic pressure and / or buffers and other therapeutically useful substances.

The formulations may be prepared by conventional mixing, granulating or coating methods and may contain, as active ingredients, cinnamon extract or turmeric extract in the range of about 0.1 to 75%, preferably about 1 to 50%. Cinnamon extract or turmeric extract may be administered in one to several times so as to be administered in an amount of 50 to 300 mg, preferably 100 to 150 mg per kg of body weight per day. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, and the like of the patient.

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.

Reference Example 1 Purification of pCEH

(Step 1) Preparation of pCEH coenzyme liquid

120 g of fresh bovine pancreas was added to 250 ml of Buffer A (50 mM Tris / HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, 2 mM PMSF and 0.1 mM DTT), homogenized and centrifuged at 13,000 x g for 1 hour. 1M acetic acid was added to the supernatant to adjust the pH to 5, and then centrifuged at 13,000 × g for 1 hour. Solid ammonium sulfate was added to the supernatant to make it 60% saturated, and then the precipitate was collected by centrifugation at 13,000 × g for 30 minutes.

This precipitate was dissolved in buffer B (20 mM Hepes / NaOH, pH 7.0, 1 mM EDTA, 1 mM EGTA and 0.1 mM DTT) and used as pCEH coenzyme solution in the next chromatography step.

(Step 2) pCEH Purification Using High Performance Liquid Chromatography (HPLC)

20 ml of the crude enzyme solution obtained in step 1 was injected into a TSKgel phenyl-5PW column (21.5x150 mm, TOSOH) previously equilibrated with buffer B containing 3M potassium chloride, and the protein was mixed using a mixture of buffer B and potassium chloride. Elution was carried out by concentration gradient method while changing the concentration of potassium chloride from 3M to 0M, and again eluted with distilled water for 20 minutes. PCEH activity was determined by reacting 5 μl of the eluted fraction with 200 μl of the reaction mixture (10 μM cholesteryl oleate, 10 μM dipalmitoyl-phosphatidyl choline, 100 mM cholate, 50 mM Tris-hydrochloric acid pH 8.0) to determine cholesterol oleate-hydrolysis capacity. Was determined. As a result, the protein with pCEH activity was eluted from the distilled water elution fraction. After the same chromatography was repeated four times, the pCEH active fractions were collected and concentrated by using an ultra-concentrator (Amicon ultrafiltration apparatus).

The concentrated pCEH active fractions were further purified using a TSKgel heparin-5PW column (7.5 × 75 mm, TOSOH) previously equilibrated with Buffer B. The adsorbed protein was eluted by a concentration gradient method using a mixture of buffer B and sodium chloride while changing the concentration of sodium chloride from 0M to 1M. As a result, pCEH activity was eluted with broad peaks in the 400 mM sodium chloride elution fraction. The fractions were analyzed by SDS-PAGE and identified as two pCEHs, 60 kDa and 70 kDa.

This active fraction was used as an enzyme solution for measuring pCEH inhibitor activity.

Reference Example 2: Determination of pCEH Enzyme Activity and Inhibitory Activity

Cholesteryl oleate labeled with radioisotope [ ¹⁴ C-1] 20,000 cpm, 10 μM cholesteryl oleate, 10 μM phosphatidyl choline, 100 mM sodium cholate, 50 mM tris / hydrochloric acid (pH 8.0) and pCEH obtained in Reference Example 1 After 100 μl of the reaction mixture containing the appropriate amount of enzyme solution was reacted at 37 ° C. for 10 minutes, 1.625 ml of a mixture of methanol / chloroform / heptane (mixing ratio 1.41: 1.25: 1.0) and 50 mM sodium carbonate / 50 mM borate solution (pH 10.0) 0.525 mL was added to stop the reaction. The reaction solution was centrifuged at 1500 xg for 10 minutes at room temperature and the amount of radioactivity contained in 0.75 ml of the aqueous layer was measured using a liquid scintillation counter.

When measuring the pCEH inhibitory activity, the pCEH inhibitor was suspended in distilled water, or the pCEH inhibitor was added to the reaction tube in advance and dried with nitrogen gas, followed by adding a buffer solution (50mM Tris-Cl pH 8.0, 100mM sodium cholate) to an ultrasonic crusher. Homogenized using and then used for the pCEH enzyme reaction.

Example 1: pCEH Inhibitory Activity Fractions of Cinnamon and Turmeric Extracts

The ethanol, chloroform, ethyl acetate, butanol, and water extracts were obtained from cinnamon or cedar according to the procedure of FIG. 1.

(1) Preparation of Ethanol Extract

In order to isolate the pCEH activity inhibitor from cinnamon or scalp, ethanol was first selected as an extractant which does not affect the human body in small amounts.

That is, 500 kg of finely crushed Chinese cinnamon or Chinese bean curd was divided by 500 g, and 500 g of the cinnamon or bean curd and 1 liter of 95% ethanol were placed in a 2 L round bottom flask and heated to reflux at 78 ° C. The solution was passed through a filter paper to obtain a filtrate, and the remaining residue was further extracted twice with ethanol to obtain a filtrate. The filtrates were combined and concentrated under reduced pressure using a rotary evaporator to obtain the following chloroform fraction.

(2) chloroform fraction

The ethanol extract was suspended in 10% methanol and the suspension was added to the separatory funnel with the same amount of chloroform, shaken vigorously, mixed and left to stand, thereby obtaining a chloroform layer, an intermediate layer and a water layer in which chloroform and water were mixed. The water layer was added to the separatory funnel with the same amount of chloroform and extracted five times in the same manner. The obtained layers were combined by chloroform layer, intermediate layer or water layer, and then dried under reduced pressure using a vacuum rotary evaporator to obtain 28.5 g (150 ml) of cinnamon chloroform extract or 62.4 g (120 ml) of chloroform extract from the chloroform fraction.

As a result of measuring 10 μl of the fraction and measuring the pCEH inhibitory activity in the same manner as in Reference Example 2, as shown in FIG. 2, the cinnamon chloroform extract showed 78% and the cedar chloroform extract showed 91% pCEH inhibitory activity. The water layer was used to prepare the following ethyl acetate fractions.

(3) ethyl acetate fraction

The water layer was concentrated under reduced pressure using a rotary evaporator to remove chloroform dissolved in the water layer, and the remaining water layer was placed in a separatory funnel with the same amount of ethyl acetate, mixed vigorously, and left to stand, thereby obtaining an ethyl acetate layer and a water layer. The water layer was added to the separatory funnel with the same amount of ethyl acetate and extracted five times in the same manner. The obtained layers were combined by ethyl acetate layer or water layer and dried under reduced pressure using a vacuum rotary evaporator to obtain 0.48 g (24 ml) of cinnamon ethyl acetate extract or 7.8 g (30 ml) of cinnabar ethyl acetate extract from the ethyl acetate fraction. .

10 μl of this fraction was measured for pCEH inhibitory activity in the same manner as in Reference Example 2. As shown in FIG. 2, cinnamon ethyl acetate extract showed 27% phucho ethyl acetate extract and 80% pCEH inhibitory activity. It was. The water layer was used to prepare the next butanol fraction.

(4) Butanol fraction and water fraction

The water layer was concentrated under reduced pressure using a rotary evaporator to remove the ethyl acetate dissolved in the water layer, and the remaining water layer was placed in a separatory funnel with the same amount of butanol, shaken vigorously, mixed and left to obtain a butanol layer and a water layer. The water layer was added to the separatory funnel with the same amount of butanol and extracted five times in the same manner. The obtained layers were combined by butanol layer or water layer, and dried under reduced pressure using a vacuum rotary evaporator. From the butanol fraction, 0.7 g (35 ml) of cinnamon butanol extract or 5.6 g (35 ml) of cinnabar butanol extract was extracted from the water fraction. 1.4 g (20 mL) of water extract or 8.58 g (17.5 mL) of turmeric water extract were obtained.

As a result of measuring pCEH inhibitory activity in the same manner as in Reference Example 2 by taking 10 μl of butanol fraction or water fraction, as shown in FIG. 2, cinnamon butanol extract and turmeric butanol extract obtained from butanol fraction were 22% and 62%, respectively. PCEH inhibitory activity of, but cinnamon water extract obtained from the water fraction showed no pCEH inhibitory activity, vinegar water extract showed 41% pCEH inhibitory activity.

Example 2: Determination of pCEH inhibitory activity of cinnamon extract or turmeric extract using animal experiment

(1) Diet of experimental animals and administration of cinnamon chloroform extract or cedar chloroform extract

Sprague-Dawley rats (weight 210g) weighed from Korea Experimental Animal Center (negative Chungcheongbuk-do) were 22-26 ℃, 55-60% humidity, 12 hours / 12 shade cycle. Used after adapting under conditions of time.

The rats were fed a diet containing 1% cholesterol and 0.5% sodium-choline acid for 5 weeks to induce dietary hyperlipidemia. Cinnamon chloroform extract or turmeric chloroform extract was added 50, 100, Or orally administered through the needle zone (needle zone) in an amount of 150 mg / kg. At this time, rats without dietary hyperlipidemia were used as a normal group, and rats without dietary hyperlipidemia but not treated with cinnamon chloroform extract or cephalophora chloroform extract were used as a control group.

(2) Effect on Serum Phospholipids and Neutral Lipid Content

In order to determine the effect of cinnamon chloroform extract or cedar chloroform extract on serum phospholipid and triglyceride contents in rats with dietary hyperlipidemia, the contents of phospholipids and triglycerides were measured as follows.

Phospholipid content was measured using a kit (RM 142-K, Iatron Chem. Co.) using the enzyme method of Chen et al. (Chen, P. S. et al., Anal. Chem., 28, 1756 (1956)). That is, an enzyme reagent (containing phospholipase 3.9 U, choline oxidase 5.6 U, peroxidase 3.6 U, and 0.3252 mg of 4-aminoantipyrine) was added to the enzyme reagent dissolution solution [Tris (hydroxymethyl) aminomethane 6.057 mg. Containing] and an enzyme solution is prepared by dissolving it in an ice-cold phase, and adding 20 µl of serum of the dietary hyperlipidemic rat to 3.0 ml of the enzyme solution, reacting at 37 ° C. for 20 minutes, and absorbance at 500 nm. Measured. The control solution containing no serum was used as a control, and the content of neutral lipid was expressed in mg / dl according to the calibration curve.

Neutral lipid content was measured using a kit (AN 157S-K, Asan) using McGowan et al. (McGowan, M.W. et al., Clin. Chem., 29, 538 (1983)). That is, enzyme reagents (lipoprotein lipase 10,800 U, glycerol kinase 5.4 U, peroxidase 135,000 U and L-α-glycerophosphooxidase 160 U) were dissolved in enzyme reagent solution [N, N-bis (2-hydroxy). Ethyl) -2-aminomethanesulfonic acid 0.427] was added to dissolve 72 ml in an ice-cold phase to prepare an enzyme solution. To 3.0 ml of this enzyme solution, 20 µl of the dietary hyperlipidemic rat was added and After reacting for minutes, the absorbance was measured at 500 nm. The control solution containing no serum was used as a control, and the content of neutral lipid was expressed in mg / dl according to the calibration curve.

The results are shown in Table 1.

Effect of Cinnamon Chloroform Extract and Cultivated Chloroform Extract on Serum Phospholipids and Neutral Lipids in Dietary Hyperlipidemic Rats Test group Dose (mg / kg) Phospholipids (mg / dl) Neutral Lipid (mg / ㎗) Normal - 103.3 ± 4.79 75.4 ± 5.07 ^{a, 1)} Control - 107.6 ± 5.13 111.5 ± 4.65 ^b Cinnamon Chloroform Extract Administration Group 50 106.5 ± 5.89 109.4 ± 3.97 ^b 100 105.7 ± 4.24 91.7 ± 3.42 ^c 150 105.2 ± 5.28 90.5 ± 2.92 ^c Scutellor chloroform extract administration group 50 108.2 ± 6.66 98.6 ± 4.31 ^e 100 107.8 ± 4.48 82.6 ± 3.98 ^f 150 106.4 ± 5.51 81.9 ± 4.09 ^{f 1)} Mean ± standard deviation (n = 8). Duncan's multiple range test of ^{a, b, c, e, and f} indicates no significance (p <0.05).

As shown in Table 1, the control group in which dietary hyperlipidemia was induced significantly increased triglyceride content compared to the normal group, and the triglyceride content was significantly increased in the cinnamon chloroform extract administration group and the caudal chloroform extract administration group as the dosage of the extract increased. Decreased.

On the other hand, the phospholipid content was found to be no difference between the normal group, the control group, the cinnamon chloroform extract administration group and the caudal chloroform extract administration group, it can be seen that the phospholipid content and dietary hyperlipidemia are irrelevant.

(3) Effect on serum cholesterol content and arteriosclerosis index

Total cholesterol content, high density lipid (HDL) cholesterol content, low density lipid (LDL) cholesterol content, ultra low density lipid (VLDL) cholesterol in serum The content and arteriosclerosis index were measured as follows.

Total cholesterol content was measured using a kit prepared by the enzyme method (AM 202-K, Asan). That is, an enzyme reagent (cholesterol esterase 20.5 U / l, cholesterol oxidase 10.7 U / l, sodium hydroxide 1.81 g / l) was dissolved in enzyme reagent dissolution solution (1 potassium phosphate 13.1 g / l, phenol 1.88 g / l) 120 After dissolving in ml, the absorbance was measured at a wavelength of 500 nm with a control solution of 20 µl serum. The blood content was expressed in mg / dl according to the standard calibration curve.

The HDL cholesterol content in serum was measured in serum of the hyperlipidemic rats using a kit (AM 203-K, Asan) using Nakayama et al. (Nakayama, NA et al., Clin. Chem., 24, 1504 (1978)). To 20 [mu] l, 0.2 ml of settling reagent (0.1% dextran sulfate, 0.1 M magnesium chloride) was added, mixed well, and left at room temperature for 10 minutes, followed by centrifugation at 3,000 rpm for 10 minutes. 0.1 ml of supernatant was added to 3.0 ml of enzyme solution (cholesterol esterase 20.5 kU / l, cholesterol oxidase 10.7 lU / l, sodium hydroxide 1.81 g / l, potassium dihydrogen phosphate 13.5 g / l, and phenol 1.88 g / l). Dissolved in 150 ml of buffer) and reacted at 37 ° C. for 5 minutes, and then absorbance was measured at 500 nm. The control solution containing no serum was used as a control, and the content of neutral lipid was expressed in mg / dl according to the calibration curve.

The content of LDL cholesterol and VLDL cholesterol in serum was determined by the following equations (1) and (2) according to the method of Friedwald et al. (Fridewald, WT et al., Clin. Chem., 18, 499 (1979)), Arteriosclerosis index was determined according to the following equation (3).

The results are shown in Table 2 below.

Effect of Cinnamon Chloroform Extract and Cultivated Chloroform Extract on Serum Cholesterol Content and Atherosclerosis Index Test group Dose (mg / kg) Cholesterol (mg / ㎗) Arteriosclerosis index Total cholesterol HLD LDL and VLDL Normal - 69.8 ± 2.11 ^{a, 1)} 35.4 ± 2.09 ^a 41.7 ± 3.46 ^a 0.98 ± 0.06 ^a Control - 110.6 ± 3.40 ^b 30.1 ± 2.53 ^b 68.9 ± 13.5 ^b 2.69 ± 0.20 ^b Cinnamon Chloroform Extract Administration Group 50 108.7 ± 2.69 ^b 29.4 ± 2.43 ^b 68.2 ± 2.66 ^b 2.72 ± 0.22 ^b 100 108.6 ± 2.40 ^b 29.9 ± 2.16 ^b 54.6 ± 2.40 ^c 2.32 ± 0.20 ^c 150 93.1 ± 3.58 ^c 33.4 ± 1.82 ^a 48.2 ± 2.54 ^d 1.73 ± 0.07 ^{d, e} Scutellor chloroform extract administration group 50 109.2 ± 3.68 ^b 29.7 ± 1.62 ^b 67.2 ± 3.45 ^b 2.69 ± 0.18 ^b 100 97.5 ± 2.12 ^b 33.9 ± 2.03 ^a 51.1 ± 2.59 ^{c, d} 1.84 ± 0.06 ^d 150 87.6 ± 2.57 ^c 34.8 ± 2.50 ^a 47.6 ± 2.11 ^d 1.53 ± 0.11 ^{e 1)} Mean ± standard deviation (n = 8). ^{a, b, c, e, f} Duncan's multiple range test indicates no significance (p <0.05).

As shown in Table 2, the 150 mg / kg cinnamon chloroform extract administration group, 100 and 150 mg / kg caudal gourd extract administration group showed a tendency to reduce dietary hyperlipidemia.

Test Example: Toxicity Test of Cinnamon Chloroform Extract and Cultivated Extract

30 cinnamon chloroform extract obtained in (2) of Example 1 (250 mg / kg, 500 mg / kg, 750 mg / kg, 1,000 mg / kg, 1,250 mg / kg, 1,500 mg / kg and 1,750 mg / kg) The number of animals that died 24 hours after intraperitoneally injection into ICR-based mice (body weight about 20 g) was measured.

The results are shown in Table 2 below, and the calculated LD ₅₀ value is 1,175 mg / kg.

Dose (mg / kg) 250 500 750 1,000 1,250 1,500 1,750 death rate 0/30 6/30 6/30 12/30 12/30 18/30 30/30

In addition, 24 hours after intraperitoneal injection of mice with 1000 mg / kg, 1,250 mg / kg, 1,500 mg / kg, 1,750 mg / kg, 2,000 mg / kg and 2,250 mg / kg in the same manner as above. The number of animals that died at that time was measured.

The results are shown in Table 4 below, and the calculated LD ₅₀ value is 1625 mg / kg.

Dose (mg / kg) 1,000 1,250 1,500 1,750 2,000 2,250 death rate 0/30 6/30 12/30 18/30 24/30 30/30

The extract of the present invention is useful in the prevention and treatment of atherosclerosis by inhibiting the absorption of dietary cholesterol in the body by inhibiting the activity of pCEH that mediates the degradation and absorption of dietary cholesterol in the small intestine.

Claims (4)

A composition for the prevention and treatment of atherosclerosis, comprising an effective amount of chondula extract and an pharmaceutically acceptable carrier as an active ingredient. The method of claim 1, The chodugu extract is a composition characterized in that it is prepared by extracting the ethanol extract of chodugu with chloroform. The method of claim 1, The chodugu extract is characterized in that the extract is extracted by extracting the ethanol extract of chodugu with chloroform and the remaining water layer with ethyl acetate. The method of claim 1, The composition comprising the extract of ethanol extract of the ethanol extract of the vulgaris extracted with chloroform and the remaining water layer with ethyl acetate and the remaining water layer with butanol.