KR100399529B1 - Composition for inhibiting cholesterol esterase comprising galenical extract - Google Patents

Abstract

The present invention is a cinnamon, tofu, pepper, baekbaek, sessile milk, nutmeg, ephedra, peppermint, safflower, purpose, baekryeop, thawed skin, sulphate, chalcedony, rare in the herbal extracts in vitro experiments showing cholesterol esterase (cholesteol esterase) inhibition The present invention relates to a composition comprising a herbal extract that inhibits absorption of cholesterol by selecting 18 Chinese herbal medicines, pagoji, Yanggang, fruit fruit, and inhibiting the absorption of cholesterol, and the composition of the present invention is pancreatic cholesterol esterase. By specifically inhibiting the activity of (pancreatic cholesterol esterase; pancreatic cholesteryl ester hydrolase (pCEH), the body's absorption of cholesterol from food is significantly reduced.

Description

Composition for inhibiting cholesterol esterase comprising galenical extract

The present invention relates to a cholesterol esterase inhibitor composition comprising an herbal extract, and more particularly, to inhibit the intestinal lipid absorption and lowering blood lipids of a cholesterol esterase inhibitor comprising at least one herbal extract of 18 kinds having effective activity. It relates to a composition for preventing and treating cardiovascular diseases such as atherosclerosis through efficacy.

In general, atherosclerosis is a lesion that becomes hard as the wall of the artery thickens and loses its elasticity. 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)). The cholesteryl esters in the arteries are loaded along the blood to show the foam cells of megakaryocytes from monocytes (Gerrity, RG, J. Pathol. 103, 181, (1981)). Continuous accumulation eventually blocks the arteries, leading to serious cardiovascular diseases such as various heart diseases 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) Assoc. , 251 , 365 (1984)) and 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%.

Dietary cholesterol and endogenous cholesterol are mainly synthesized by the liver and small intestine and contribute to the level of cholesterol circulating in the blood. Therefore, the absorption efficiency of plasma cholesterol level is an important factor and also a risk factor involved in the premature develpment of arteriosclerosis.

Studies on the absorption of dietary cholesterol in the body show that the ingested cholesterol ester is released by the pancreatic cholesterol esterase (pancreatic cholesteryl ester hydrolase: hereinafter called "pCEH") contained in the pancreatic secretory fluid in the small intestine. Hydrolyzed into cholesterol and fatty acids to be 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). It is hypothesized that cholesterol is likely to be dissolved in the small intestinal wall by hydrophobic interactions or sterol binding proteins, which is related to absorption. 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)).

ACAT and pCEH are involved in the delivery of cholesterol through the digestive system, ACAT acts as a rate-determining enzyme in the cycle of continuous degradation and esterification, and pCEH plays a major role in hydrolysis of lipids to facilitate absorption. .

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, developed by the Wyeth-Ayerst Research, are effective pCEH inhibitors and have been reported to inhibit 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 a more recent study, the cholesterol-fed rats were observed to reduce plasma cholesterol upon administration of WAY-121,898, and showed a greater reduction in plasma cholesterol when administered with CI-976, an ACAT inhibitor. (Brain R. Krause et al. , Lipid. , 33. 489 (1998)) Meanwhile, cholesylamin, a cholesterol-absorbing agent currently in use, used as a control in our invention, causes gastrointestinal disorders. In some cases, the drug may be taken after the reduction (Maciejko, JJ, et al. Arch. Fam. Mad. , 2, 955 (1994)).

In addition, various studies have examined the effect of high cholesterol diet on the biosynthesis of cholesterol esterase in rat pancreas, and the dietary cholesterol levels showed an increase in cholesterol esterase levels in the pancreas. In rats fed high lipid / high cholesterol at the same time, there was a significant increase in mRNA and at the same time an increase in lipase mRNA was observed (Julia Brodt et al., J. Lipid research 35, 27). , (1994)) Furthermore, CEH purified from rat liver 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.

In recent years, sales of dietary supplements have been booming in the United States, which is a natural remedy for maintaining good health as lack of motivation to improve their quality of life and lack of supplementation with synthetic drugs. There are reports that he is paying much attention to things like herbs. However, despite the popularity of these dietary fortifications, the mechanisms, toxicities, and medical effects of medicinal herbs are not yet well known, so it is necessary to reassess them and find out their practical value. (Joseph chang., Biochem. Phama, 59, 211, (2000)) Therefore, the inventors of the present invention intend to experiment with herbal drugs mainly used in herbal medicine with the purpose of inhibiting absorption of cholesterol and various medicinal effects.

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 the ethanol extracts of the herbal medicines have a pCEH inhibitory activity to complete the present invention.

The present invention has been made in view of the above-mentioned problems, and the object of the present invention is to inhibit the activity of pCEH that mediates the decomposition and absorption of dietary cholesterol in the small intestine to prevent the absorption of dietary cholesterol in the body to prevent cardiovascular disease It is to provide a composition comprising a herbal extract useful for the prevention and treatment of.

1 is a graph showing the pCEH inhibitory activity of the ethanol extracts of herbal medicines. In the graph, the vertical axis represents the percent inhibition of cholesterol esterase, the horizontal axis 1 is cinnamon, 2 is larvae, 3 is thick, 4 is baekbaek, 5 is sewage, 6 is nutmeg, 7 is ephedra, 8 is mint, 9 is safflower, 10 is purpose, 11 is temporal lobe, 12 is thawing blood, 13 is turmeric, 14 is scalp, 15 is rare, 16 is pagoji, 17 is Yanggang and 18 is fruit tree.

In order to achieve the above object, the present invention provides an effective amount of cinnamon, tofu, thick pepper, yellow white, sesame oil, nutmeg, ephedra, mint, peppermint, safflower, purpose, white leaf, thawed skin, turmeric, grass nut, rare, pagoji, Yanggang And it provides a composition comprising a herbal extract showing the inhibitory effect on cholesterol esterase comprising at least one extract selected from the group consisting of fruiting and pharmaceutically acceptable carrier.

The herbal medicines can be extracted by adding an extraction solvent, preferably purified water, ethanol, methanol, butanol, ethyl acetate, and a solvent of one species selected from acetone, more preferably 95% ethanol.

In addition, the extract of the present invention is almost the effect of less than 0.01g per day in the weight of the herbal drug itself before extraction, there is almost no difference in the effect of increasing the concentration at 5g or more, so to be administered at 0.01g ~ 5g / kg 1 It may be administered in several divided doses. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate, severity of the disease, and the like of the patient.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The medicinal herbs are finely crushed, and the ethanol extract is extracted from medicinal medicinal ethanol extracts (cinnamon, tofu, baekbak, baekbaek, seomul, nutmeg, ephedra, peppermint, safflower) and ethanol extract (cinnamon, tofu, pakbak) , Yellow and white, sessile, nutmeg, ephedra, peppermint, safflower, purpose, baekryeop, thawed skin, sulphate, chalcedony, rapeseed, pagoji, fruit fruit) are each referred to as A extract and B extract below.

Table 1 lists six herbal drugs that have no activity in vitro .

Table 2 shows the names and efficacy of each of the herbal medicines used in the present invention.

The extract of the present invention was used after concentration under reduced pressure after mixing, and when the actual administration was made in three concentrations so that the weight of the herbal drug itself is 0.05g, 0.1g and 0.2g.

The extract of the present invention acts to inhibit pCEH activity, which mediates the breakdown 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, in addition to the treatment of atherosclerosis, decreases the concentration of triglycerides in the blood and eventually has an effect of treating cardiovascular diseases including hyperlipidemia (hyperlipidemia).

[Table 1] Six data of inactivated herbals in vitro

group cpm Negative control 235 Positive control 8160 Sansa 7808 Tosa 7625 Doin 8318 Defeat 8394 Macmundong 7600 Gold 7596

TABLE 2 Name and efficacy of each of the herbal medicines used in the present invention

Herbal medicine genus Effect and action Cinnamon cortex (Cinnamon bark ) Lauraceae It is used for cold, analgesic, and pain relief due to hypothermia, central inhibition, persistent peripheral vasodilation, platelet aggregation inhibition, skin darkening prevention, blood circulation improvement, antipyretic fever, aroma health, and cliche effect Eucommia bark Eucommaceae Sedation, analgesic, lowering blood pressure Magnolia bark And Magnolia (Magnoliaceae) Has strong erosion effect Yellow ( Phellodendron bark ) Rutaceae Antibacterial, antifungal, astringent, anti-inflammatory, diuretic, healthy, subcutaneous bleeding absorption Evidia fruit Rutaceae Strong zone, effective for insecticide Nutmeg (Nutmeg) Myristicaceae Warmer plane Ephedra herb Ephedraceae Sweating, antipyretic, antiviral Peppermint ( Menthae herba ) Labiatae Vasodilation, jingle, skin outbreak, local anesthesia, old wind, etc. Safflower (Safflower) Asteraceae ( Compositae ) Uterine contraction, blood pressure lowering, vasodilation The purpose ( Equiseti herba ) Equisetadeae Pneumonia, astringent, diuretic White leaf ( Biotae orientalis Forium ) Cupressaceae Used for hemostasis, hemostasis, urinary bleeding Kalopanacis cortex Araliaceae (Araliaceae) Sedative, analgesic, antibacterial, antifungal Typhae pollen Typhaceae Hemostatic, uterine contraction, diuretic, anti-tuberculosis Alpinia Katsumadai semen Zingiberaceae Health, clay, convergence Comedy (Siegesbeckiae herba) Asteraceae ( Compositae ) Stroke Pagoji ( Psoraleae semen ) Legumeinosae Coronary artery dilatation, antibacterial melanin pigmentation Lamb ( Alphiniae officinari Rhiaoma ) Zingiberaceae Vascular circulation promoting action, stomach action, analgesic action, antibacterial action Trichosanthis semen Cucurbutaceae Anti-inflammatory, hypo-allergenic, antibacterial, anticancer activity

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, and the like. In addition to the active ingredients, these formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), and suspending agents ( 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 formulation may be prepared by a conventional mixing, granulation or coating method, the effect of A extract or B extract as less than 0.1 as the active ingredient is less than 0.1, at a concentration of more than 75%, there is no difference in effect according to the concentration, 0.1 To 75%, preferably about 1 to 50%.

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.

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 3 M to 0 M and then 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 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 with a concentration gradient method using a mixed solution of buffer B and sodium chloride while changing the concentration of sodium chloride from 0 M to 1 M. 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 pCEH Inhibitory Activity Fraction of A Extract and B Extract

(1) Preparation of Ethanol Extract

In order to separate the pCEH activity inhibitors from the herbal medicines, ethanol was selected as an extractant that does not affect the human body in small amounts.

The herbal extract was extracted with 95g ethanol 100g of 1L to make 1L and then mixed with each extract 1ml as above and concentrated under reduced pressure. At the time of actual administration, three concentrations were prepared such that the weight of the herbal drug itself was 0.05g (low concentration), 0.1g (normal concentration) and 0.2g (high concentration).

Experimental Example: Determination of pCEH inhibitory activity of herbal extracts using animal experiments

1) Determination of pCEH Inhibitory Activity Using Cholesterol-Fed Control

(1) Administration of dietary and herbal extracts of laboratory animals

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 with 4% feed containing 1% cholesterol and 0.5% sodium-choline acid for 4 weeks to induce dietary hyperlipidemia. After 2 weeks, three concentrations of herbal extracts were passed through the needle zone. Oral administration. At this time, rats without dietary hyperlipidemia were used as a normal group, and rats with dietary hyperlipidemia but no herbal extract were administered as a control group. Rats were lightly anesthetized with CO ₂ after the administration experiment, and allowed to stand at room temperature for 30 minutes, followed by centrifugation at 3000 rpm for 10 minutes to separate serum, and the phospholipid, triglyceride and cholesterol content of serum were measured using a kit. In addition, the liver is perfused with physiological saline to remove blood, which is freed of blood and other adherents. After that, it is weighed and added to the free Teflon homogenizer by adding 4 times 0.1M potassium phosphate buffer (pH 7.5) per 1g of tissue. Crushed. This supernatant was centrifuged at 600 xg for 10 minutes, and the supernatant from which the nucleus and the unchained portion was removed was ultracentrifuged at 10,000 xg for 1 hour, and the cytosol fraction was suspended by adding the same amount of 0.1M potassium phosphate buffer to the precipitate. The solution was taken as a microsome fraction. All the above operations were performed at 4 degrees C or less unless otherwise specified.

(2) Effect on Serum Phospholipids and Neutral Lipid Content

To determine the effect of A extract or B 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, PS 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, MW 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 ice-cold phase in 72 ml to dissolve the enzyme solution. To 3.0 ml of this enzyme solution, 20 µl of the serum of the dietary hyperlipidemic rat was added, 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 3.

Table 3 Effect of A and B Extracts on Serum Phospholipids and Neutral Lipids in Dietary Hyperlipidemic Rats

Test group Dosage Phospholipids (mg / dl) Neutral Lipid (mg / ㎗) Normal - 116.7 ± 11.0 ¹⁾ 85.5 ± 10.0 ^{1), a} Control - 114.4 ± 23.9 137.4 ± 24.8 ^b A ethanol extract administration group 0.05g 116.1 ± 19.3 138.2 ± 29.3 ^b 0.1g 111.7 ± 16.8 97.7 ± 9.2 ^a 0.2 g 110.7 ± 17.2 99.2 ± 13.0 ^a B ethanol extract administration group 0.05g 105.1 ± 11.8 129.6 ± 19.3 ^b 0.1g 105.6 ± 14.8 90.2 ± 13.8 ^a 0.2 g 109.1 ± 15.9 91.3 ± 19.7 ^{a 1)} Mean ± standard deviation (n = 8). Duncan's multiple range test of ^{a, b, c, e, and f} indicates no significance (p <0.05).

** The quantity indicated above is the weight calculated for each herbal medicine itself.

As shown in Table 3, the control group induced by dietary hyperlipidemia significantly increased the amount of triglycerides compared to the normal group, and the amount of triglycerides decreased significantly as the dosage of the extracts A and B extracts increased. .

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

(3) Effect on serum cholesterol content and arteriosclerosis index

Total cholesterol content, high density lipid (HDL) cholesterol, low density lipid (LDL) cholesterol content, ultra low density lipid (VLDL) cholesterol content and artery in the serum after oral administration of A extract or B extract in rats Curing index was measured as follows.

Total cholesterol content was measured using a kit (AM 202-K, Asan) prepared by an enzyme method such as Richmond (Richmond, W. Clin, Chem, 22, 1579 (1976)). 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 rat using a kit (AM 203-K, Asan) using an enzyme method such as Nakayama (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 Equation 1 and 2, respectively, according to the method of Friedwald et al. (Fridewald, WT et al., Clin. Chem. , 18 , 499 (1972)), Curing index was determined according to the following formula (3).

[Equation 1]

LDL cholesterol amount = [total cholesterol amount- (HDL cholesterol amount + triglyceride amount / 5)]

[Formula 2]

VLDL cholesterol amount = [total cholesterol amount-(HDL cholesterol amount + LDL cholesterol amount)] 1

[Equation 3]

The results are shown in Table 4 below.

[Table 4] Effect of A and B Extracts on Serum Cholesterol Content and Atherosclerosis Index

Test group Dosage Cholesterol (mg / ml) Arteriosclerosis index Total cholesterol HLD LDL VLDL Normal - 78.6 ± 9.8 ^{a, 1)} 40.3 ± 4.42 ^a 21.0 ± 3.23 ^e 17.1 ± 3.66 ^c 1.11 ± 0.32 ^a Control - 234.8 ± 33.7 ^b 31.4 ± 2.31 ^b 173.1 ± 10.3 ^a 28.5 ± 4.96 ^a 6.48 ± 1.02 ^b A ethanol extract administration group 0.05g 214.5 ± 35.2 ^b 31.4 ± 5.81 ^b 156.4 ± 7.63 ^b 28.1 ± 3.39 ^a 5.97 ± 1.42 ^b 0.1g 151.6 ± 34.7 39.1 ± 2.71 ^a 94.5 ± 8.20 ^d 19.6 ± 2.07 ^{b, c} 2.88 ± 0.92 ^c 0.2 g 152.4 ± 30.2 ^c 39.2 ± 4.63 ^a 95.3 ± 8.89 ^d 20.5 ± 1.88 ^b 2.94 ± 1.05 ^c B ethanol extract administration group 0.05g 216.9 ± 27.9 ^b 33.5 ± 3.59 ^b 156.8 ± 10.1 ^b 26.9 ± 3.88 ^a 5.50 ± 0.77 ^b 0.1g 160.5 ± 38.4 ^c 38.1 ± 2.30 ^a 105.7 ± 10.6 ^c 19.4 ± 2.44 ^{b, c} 3.22 ± 0.99 ^c 0.2 g 150.3 ± 37.5 ^c 40.3 ± 3.25 ^a 90.6 ± 4.95 ^d 19.6 ± 1.90 ^{b, c} 2.79 ± 1.15 ^{c 1)} Mean ± standard deviation (n = 8). ^{a, b, c, e, f} Duncan's multiple range test indicates no significance (p <0.05).

** The quantity indicated above is the weight calculated for each herbal medicine itself.

As shown in Table 4, the cholesterol content and arteriosclerosis index in the hyperlipidemia-induced group showed a tendency to decrease when the extract was administered. Especially, in the case of 0.1g and 0.2g, the dietary hyperlipidemia showed a tendency to decrease.

2) Determination of pCEH inhibitory activity using a control group fed with cholesterol ester

(1) Administration of dietary and herbal extracts of laboratory animals

The rats were fed with a diet containing high cholesterol esters.

They were fed together for 4 weeks at normal concentrations (0.1 g each calculated on their own). At this time, rats without dietary hyperlipidemia were used as a normal group, and rats with dietary hyperlipidemia but no herbal extract were administered as a control group. Meanwhile, we tried to measure the effectiveness of the extract by comparing the fed with CSR (choelstyramine), which is now known to have a cholesterol inhibitory effect.

(2) Effect on Serum Phospholipids and Neutral Lipid Content

The results of experiments to determine the effect of herbal ethanol extracts on phospholipids and triglycerides in hyperlipidemia induced by cholesterol esters were as follows.

Table 5 Effect of herbal extracts on serum phospholipids and triglycerides in rats fed diets containing cholesterol esters

Test group Dosage Phospholipids (mg / dl) Neutral Lipid (mg / ㎗) Normal - 110.6 ± 13.4 83.3 ± 9.84 ^a Control - 121.3 ± 10.9 136.7 ± 8.43 ^b 6 Ethanol Extracts 0.1g 108.9 ± 14.6 123.4 ± 6.27 ^{b, c} 9 Ethanol Extracts 0.1g 115.9 ± 8.32 114.3 ± 5.24 ^c 18 Ethanol Extracts 0.1g 122.7 ± 18.8 116.9 ± 8.36 ^c CSR (cholestyramine) 100mg / kg 130.6 ± 20.9 110.4 ± 7.96 ^{c 1)} Mean ± standard deviation (n = 8). Duncan's multiple range test of ^{a, b, c, e, and f} indicates no significance (p <0.05).

** The quantity indicated above is the weight calculated for each herbal medicine itself.

As shown in Table 5, the control group fed dietary hyperlipidemia by feeding cholesterol ester significantly increased the triglyceride content compared to the normal group, and the extract showing the inhibitory effect in vitro was slightly decreased compared to the control group. The decrease was similar to that of CSR. On the other hand, the six extracts that did not show an inhibitory effect was somewhat higher than the other extracts. In the previous experiment, it was found that the reduction effect was somewhat lower than the result of using cholesterol as a diet.

On the other hand, the phospholipid content was not significantly different between the normal group and the control group, so the effects of other extracts could not be compared.

(3) Effect on serum cholesterol content and arteriosclerosis index

Changes in total cholesterol content, high density lipid (HDL) cholesterol, low density lipid (LDL) cholesterol content, ultra low density lipid (VLDL) cholesterol content and atherosclerosis index of crude ethanol extracts in rats fed cholesterol ester Measured.

TABLE 6 Effect of herbal extracts on serum cholesterol in rats fed diets containing cholesterol esters

Test group Dosage Cholesterol (mg / ml) Arteriosclerosis index Total cholesterol HDL LDL VLDL Normal - 73.6 ± 4.87 ^a 38.2 ± 3.46 ^a 19.8 ± 3.03 ^e 17.9 ± 2.75 ^d 0.92 ± 0.08 ^a Control - 239.2 ± 6.49 ^b 29.8 ± 2.11 ^b 180.7 ± 6.97 ^a 31.0 ± 5.08 ^a 4.00 ± 0.77 ^b 6 Ethanol Extracts 0.1g 223.7 ± 5.49 ^c 31.6 ± 1.98 ^{b, c} 167.8 ± 5.73 ^b 25.7 ± 2.96 ^b 2.91 ± 0.19 ^c 9 Ethanol Extracts 0.1g 101.7 ± 6.73 ^d 35.9 ± 2.09 ^{a, c} 48.8 ± 2.76 ^c 23.4 ± 3.48 ^{b, c} 1.83 ± 0.13 ^d 18 Ethanol Extracts 0.1g 98.4 ± 8.28 ^d 33.2 ± 1.87 ^{b, c} 40.9 ± 1.69 ^d 21.0 ± 2.53 ^{c, d} 1.96 ± 0.10 ^d CSR (Cholestylamine) 100mg / kg 80.7 ± 5.23 ^a 36.1 ± 3.20 ^{a, c} 19.7 ± 1.74 ^e 17.6 ± 2.64 ^d 1.23 ± 0.15 ^{a 1)} Mean ± standard deviation (n = 8). ^{a, b, c, e, f} Duncan's multiple range test indicates no significance (p <0.05).

** The quantity indicated above is the weight calculated for each herbal medicine itself.

As shown in Table 6, cholesterol content and arteriosclerosis index were increased in the hyperlipidemia-induced control group with cholesterol ester and decreased when the 9 and 18 extracts were administered. The effect was greatest in CSR, but similarly decreased in 9 and 18 extracts. In the six extracts, which had little inhibitory effect in vitro , the cholesterol content was similar to that of the control group, and there was a slight decrease in the atherosclerotic index.

The extract of the present invention inhibits the activity of pCEH, which mediates the degradation and absorption of dietary cholesterol in the small intestine, thereby preventing the absorption of dietary cholesterol in the body and is useful for the prevention and treatment of cardiovascular diseases including atherosclerosis.

Claims (5)

A composition for the prevention and treatment of atherosclerosis or hyperlipidemia comprising an effective amount of an herbal extract consisting of cinnamon, tofu, thick pepper, yellowish white, sesame oil, nutmeg, ephedra, mint and safflower as an active ingredient and a pharmaceutically acceptable carrier. The method of claim 1, The composition of the composition for preventing and treating atherosclerosis or hyperlipidemia, characterized in that it further comprises a purpose, baekryeop, thawed blood, turmeric, turmeric, rare, pagoji, Yanggang and fruiting. delete delete The method according to claim 1 or 2, A composition for preventing and treating atherosclerosis or hyperlipidemia, wherein the effective amount of the herbal extract is 0.01 g to 5 g / kg body weight / day.