KR20210098059A - Composition for preventing, alleviating or treating hyperlipidemia or arteriosclerosis containing extract of Amomum villosum Loureiro as an active ingredient - Google Patents

Abstract

The present invention relates to a food composition for preventing or alleviating hyperlipidemia and arteriosclerosis containing Amomum villosum Loureiro extract as an active ingredient. In addition, the present invention relates to a pharmaceutical composition for preventing or treating hyperlipidemia and arteriosclerosis containing Amomum villosum Loureiro extract as an active ingredient. Accordingly, using a natural product rather than a chemically synthesized drug is able to reduce the amount of total cholesterol to a level resulting in the use of chemically synthesized drugs, while having little problem with toxicity, thereby effectively preventing, alleviating or treating hyperlipidemia and arteriosclerosis.

Description

Composition for preventing, improving or treating hyperlipidemia or arteriosclerosis containing extract of Yangchunsa as an active ingredient {Composition for preventing, alleviating or treating hyperlipidemia or arteriosclerosis containing extract of Amomum villosum Loureiro as an active ingredient}

The present invention relates to a composition containing Yangchunsa extract as an active ingredient, and more particularly, to a composition for preventing, improving or treating hyperlipidemia or arteriosclerosis containing Yangchunsa extract as an active ingredient.

The prevalence of various lifestyle-related diseases and chronic degenerative diseases due to overnutrition, environmental pollution, lack of exercise, and increased stress caused by the improvement of the economic level is emerging as a big problem in our society. Among them, a disease that has recently been attracting attention is metabolic disease. The metabolic disease is a group of diseases caused by lifestyle habits such as overnutrition and lack of exercise, which are represented by the increase in consumption of high-fat and high-carbohydrate processed foods and animal foods of modern people, and include hypertension, diabetes, obesity, hyperlipidemia, and arteriosclerosis. Yes. In particular, excessive intake of fat and cholesterol induces vascular deposition of excessive intake of cholesterol, which is known to be the main cause of circulatory diseases including cardiovascular diseases, due to blockage of arteries or problems with blood supply. there is.

According to the World Health Organization (WHO) report, about 17 million people died from cardiovascular disease in 2008, and this trend continues to increase, and it is expected that more than 23 million people will die from cardiovascular disease by 2030. there is.

In this regard, it is reported that for every 1 mg/dL increase in blood cholesterol level, the incidence of heart disease increases by 2-3%. According to this relationship, management standards related to blood cholesterol have been proposed in order to suppress the incidence of cardiovascular disease.

Preferred blood cholesterol levels for Koreans are defined as less than 200 mg/dL, borderline-high when blood cholesterol levels are 200 to 239 mg/dL, and hypercholesterolemia ( high) is defined. In addition, in the case of blood cholesterol levels taking into account the incidence of coronary heart disease set by the US National Cholesterol Education Program (NCEP), an appropriate level ( optimal), and 100 to 129 mg/dL is close to or slightly above the optimal level (near optimal/above optimal), 130 to 159 mg/dL is borderline high, and 160 to 189 mg/dL is High risk level (high), and 190 mg/dL or more is defined as very high risk level.

Assuming that the daily intake of cholesterol of a normal person is about 400 to 500 mg, it is generally known that the absorption rate of dietary cholesterol is about 60 to 80%, and depending on the limited absorption rate of cholesterol, unabsorbed dietary cholesterol is in the form of cholesterol to the large intestine. It is excreted as metabolites by bacterial enzymatic reactions in the stomach and rectum.

Therefore, patients with cardiovascular disease sometimes take drugs to control blood cholesterol levels, and the cholesterol-regulating action type of these drugs is to inhibit the enzyme activity related to cholesterol hydrolysis and esterification in the small intestine. There are cholesterol and bile acid excretion control or lipid metabolism modulators by regulating the absorption of cholesterol in the intestine or by acting as a sequestrant to promote cholesterol excretion.

However, long-term administration of these drugs causes side effects such as severe liver damage and decreased function, joint muscle pain, constipation and diarrhea. For example, orlistat is a drug that inhibits the activity of a lipolytic enzyme to prevent hydrolysis of cholesterol and triglyceride and helps excrete unoxidized fat in the feces. , XenicalR) have been reported to induce digestive disorders, fatty stools, defecation incontinence, and interference with absorption of fat-soluble vitamins.

Therefore, research on methods using natural products that are expected to relatively minimize the possibility of safety problems are in progress. Research on dietary supplements for improvement is being actively conducted.

A technology for atorvastatin, which is known to be effective in lowering such cholesterol, is disclosed in 'Patent Document 1'. Atorvastatin is one of the representative drugs used for cholesterol reduction in hyperlipidemia patients.

However, atorvastatin may cause serious side effects, such as myopathy, lupus myolysis, and liver failure, and there is a problem that concerns about toxicity are always accompanied by long-term use due to the nature of the chemical.

KR 10-2004-0025901 A (2004. 03. 26.)

The present invention has been devised to solve the above problems, and an object to be solved in the present invention is to provide a composition containing a substance capable of reducing the possibility of side effects and low toxicity as an active ingredient.

In addition, the problem to be solved in the present invention is to provide a composition containing a natural material, not a chemical synthetic material, as an active ingredient.

The composition according to the present invention for solving the above problems is characterized in that it provides a food composition for preventing or improving hyperlipidemia and arteriosclerosis by containing the extract of Amomum villosum Lour.

In addition, the composition according to the present invention for solving the above problems is characterized by providing a pharmaceutical composition for preventing or treating hyperlipidemia and arteriosclerosis by containing the extract of Amomum villosum Lour.

In addition, the composition according to the present invention for solving the above problems is technically characterized in that the Yangchunsa extract is orally administered at a concentration of 100 to 500 mg/kg based on a mouse.

The composition containing the extract of Yangchunsa according to the present invention as an active ingredient has almost no toxicity problem by using a natural product unlike a chemically synthesized drug, and reduces hyperlipidemia and arteriosclerosis by reducing the amount of total cholesterol to the level of a chemically synthesized drug It can be effectively prevented, ameliorated or treated.

1 is a graph showing the measurement results of triglyceride concentrations in the liver (left) and blood (right) of C57BL/6J mice.
2 is a graph showing the measurement results of total cholesterol concentration in the liver and blood of C57BL/6J mice.
3 is a graph showing the results of measuring cholesterol-related gene expression levels in the liver of C57BL/6J mice.
4 is a photograph and graph showing the measurement results of HMGCR protein (top) and mRNA expression level (bottom) in the liver of C57BL/6J mice.

The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of a term in order to best describe his invention" It should be interpreted as the meaning and concept that

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical ideas of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

COD (砂仁) means the overgrown antler four (綠殼砂Amomum villosum Loureiro var . Xanthioides TLWu et Senjen) or Amomum villosum chunks of ripe fruit or seeds (陽春砂. Amomum villosum Loureiro) over the ginger (Zingiberaceae) Therefore, in the present invention, the fruit of Yangchunsa was used as a material.

For reference, Yangchunsa is mainly produced in Fujian, Guangdong, Guangxi, and Yunnan, and Nokaksa is mainly produced in the Indochina Peninsula region such as Cambodia, India, Laos, Myanmar, Thailand, Vietnam, and some regions of Guangxi and Yunnan in China. .(Flora of China, v24:347-356.2000). Because of the different production areas, in ancient times, Nokgaksa was referred to as ‘Choksain’.

On the other hand, the fruits of Haenamsa (海南砂Amomum longiligulare TLWu), which are recognized as genuine products in China but are treated as fakes in Korea, are also being distributed in large quantities. However, Shen et al. (SHEN Li WANG Yang JIANG Ku et al. Comparison of HPLC Fingerprints of Amomum villosum Lour. , Amomur villosum Lour. var. xanthioides TLWu et Senjen and Amomum longiligulare TLWu. Chinese Pharmaceutical Journal. 2016: 51(12); 1039 -1043.) showed that the three types of causes showed a high degree of similarity in composition, but Haenamsa and Yangchunsa had similar chemical components, while Nokgaksa had a clear difference from the previous two. The comparison should be elucidated.

In the end, the sign of Nokgaksa and the sign of Yangchunsa are clearly different.

Example. Preparation of Yangchunsa extract

Yangchunsa ( Amomum villosum Lour.) was purchased from Gwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, Korea) and its authenticity was verified in the Department of Herbology, College of Oriental Medicine, Wonkwang University. After filtration, the filtrate was concentrated to dryness under reduced pressure to obtain an extract of 9.25 g of sine.

Experimental Example 1. Breeding and diet of experimental animals

For the experimental animals, 6-week-old C57BL/6J male mice were purchased (Orientbio, Seongnam, Korea) and acclimatized to the breeding room environment for 1 week. A normal diet control group (Normal diet, ND), a high cholesterol diet control group (HCD), a high cholesterol diet, and an experimental group in which various concentrations of Yangchunsa extract were orally administered to mice with an average body weight of about 22 to 25 g, etc. was divided and administered orally for 4 weeks, and simvastatin (40 mg/kg) as a positive control was intraperitoneally administered for 4 weeks. In the high-cholesterol diet used in this experiment, about 41% of total calories were supplied as fat and 43% carbohydrates, and body weight was measured once a week. The temperature of the breeding room was maintained at 18~24℃ and relative humidity of 50~60%, and the light and dark was adjusted in a 12-hour cycle (8:00~20:00). Animal experiments were approved by Wonkwang University Animal Experiment Ethics Committee (WKU20-01), and the use and management of laboratory animals followed institutional guidelines.

Experimental Example 2. Weighing

After the start of the experiment, the body weight of the mice was measured weekly.

As a result, the body weight of the group administered the high cholesterol diet for 4 weeks showed a significant increase effect compared to the body weight of the group administered the normal diet for 4 weeks. However, the body weight of the group administered with 100, 200, and 500 mg/kg Yangchunsa extract in combination with the high cholesterol diet showed a significant reduction effect compared to the body weight of the high cholesterol diet group (Table 2).

Experimental Example 3. Liver and epididymal triglyceride analysis

After the final weight measurement in Experimental Example 2, anesthetized with ether, an autopsy was performed to extract liver and fat (epidermis fat) tissue, and the weight was measured.

The liver triglyceride content was measured as follows. The liver tissue was homogenized in chloroform-methanol solution (2:1, v/v), reacted at room temperature for 1 hour, centrifuged, and the organic solvent layer was dried for 24 hours. After dissolution in ethanol, the triglyceride content of the liver was investigated using a TG kit (Asan Pharmaceutical, Korea) and normalized to protein concentration.

As a result, the weight of liver and epididymal fat in the group treated with the high-cholesterol diet for 4 weeks showed a significant increase compared to the group treated with the normal diet for 4 weeks. However, the weight of liver and epididymal fat in the group administered with Yangchunsa extract at 100, 200, and 500 mg/kg in combination with a high-cholesterol diet was significantly reduced compared to the weight of the high-cholesterol diet group (Table 3).

Experimental Example 4. Blood Analysis

As in Experimental Example 3, blood was collected from the abdominal vena cava of the autopsied mouse. And the amounts of triglycerides (Asan Pharmaceutical, Korea), LDL cholesterol (EZ DG kit), and HDL cholesterol (Asan Pharmaceutical, Korea) in the blood were analyzed.

The amount of triglyceride (TG) in the liver and blood of the group treated with the high cholesterol diet for 4 weeks was significantly increased compared to the group treated with the normal diet for 4 weeks. However, when 100, 200, 500 mg/kg Yangchunsa extract was administered in combination with a high-cholesterol diet, the amount of triglycerides in the liver and blood was significantly reduced compared to that of the high-cholesterol diet group, as shown in FIG. 1 . In particular, the 500mg/kg Yangchunsa extract group showed a significant reduction effect compared to the simvastatin group administered as a positive control group.

The amount of total cholesterol in the liver and blood of the group treated with the high cholesterol diet for 4 weeks was significantly increased compared to the group treated with the normal diet for 4 weeks. However, when 100, 200, 500 mg/kg Yangchunsa extract was administered in combination with a high cholesterol diet, the total cholesterol amount was significantly reduced compared to the high cholesterol diet group as shown in FIG. 2 (left). In addition, the amount of cholesterol in the LDL blood was significantly increased in the group administered with the high-cholesterol diet for 4 weeks compared to the group administered with the regular diet for 4 weeks. On the other hand, the 100, 200, 500 mg/kg Yangchunsa extract administration group showed a significant decrease by 10.0, 18.5, and 30.9% compared to the group administered with a high cholesterol diet (100%) as shown in FIG. 2 (right).

Experimental Example 5. Gene analysis

Isolation of total RNA from the extracted liver was performed using Trizol reagent (Life Technologies, UK) according to the method provided by the manufacturer. That is, after cutting each tissue finely, it was dissolved with 1000 μl RNAzol B, 200 μl chloroform was added, and reacted on ice for 5 minutes. After the reaction was completed, centrifuged at 13,000 rpm at 4°C for 20 minutes, and 500 μl of the supernatant was transferred to a new tube. The same amount of isopropanol was added to the above supernatant, mixed, and reacted on ice for 30 minutes. After completion of the reaction, centrifugation was performed at 13,000 rpm at 4° C. for 20 minutes, and the precipitate was washed twice with 75% EtOH. After drying the washed RNA, it was dissolved in 20 μl of DEPC water, and the absorbance was measured in a spectrophotometer and quantified. Reverse transcription reaction was performed using 2 μg total RNA and Prime Script TM reagent kit (perfect realtime) (TaKaRa BIOINC.) according to the method provided by the manufacturer. Reverse transcription reaction was performed with a reaction solution containing total RNA (2 μg), oligo d(T)primer (25 pmol), PrimeScript RT enzyme Mix I, and 5X primeScript Buffer at 37°C for 15 minutes and at 85°C for 5 seconds to react cDNA. was synthesized. Real-time reverse transcriptase polymerization reaction was performed using Power SYBY Green PCR Master Mix on 10-fold diluted cDNA. The amount of mRNA of each expressed gene was calculated as a relative amount to GAPDH using LightCycler System software (Roche).

The sequences of the primers used are as shown in Table 4.

The expression levels of low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA in the liver of the group treated with a high-cholesterol diet for 4 weeks were significantly reduced compared to the group treated with a normal diet for 4 weeks. However, LDLR and PCSK9 mRNA expression levels in the group administered with 100, 200, and 500 mg/kg Yangchunsa extract in combination with a high cholesterol diet were significantly increased compared to that of the high cholesterol diet group, as shown in FIG. 3 . In addition, as a result of examining the mRNA expression level of Sterol regulatory element-binding protein 2 (SREBP2), which is known as a transcription factor that controls the expression of LDLR and PCSK9, the group administered with Yangchunsa extract significantly reduced the decrease in SREBP2 mRNA due to a high-cholesterol diet. .

LDLR and PCSK9 are genes that play a role in moving blood cholesterol to the liver by blocking the regulation of SREBP2 transcription factor and increasing its expression when the cholesterol content in the liver is decreased. Therefore, an increase in LDLR and PCSK9 expression means a decrease in blood cholesterol.

Experimental Example 6. Analysis of mRNA and protein of HMG CoA reductase (HMGCR)

Proteins in liver tissue were isolated using Mammalian Protein Extraction Reagent (M-PER) (Pierce Biotechnology, Rockford, IL, USA). Samples (20㎍) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% acrylamide, and transferred using Hybond™-polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Buckinghamshire, UK). did. Each membrane was blocked using 2% bovine serum albumin or 5% skim milk for 2 hours, and the primary antibody against HMGCR (Cell Signaling, MA, USA) was 1㎍/㎖ of a 1:2000 dilution at 4℃ Incubated overnight in a refrigerator. As the secondary antibody, HRP-conjugated IgG (1:2000 dilution) was used. The protein expression level was confirmed using an image analyzer (EZ-Capture ST, Tokyo, Japan).

As a result, HMGCR protein and mRNA expression in the liver of the group treated with the high cholesterol diet for 4 weeks was significantly reduced compared to the group treated with the normal diet for 4 weeks. However, the HMGCR protein and mRNA expression levels of the group administered with 100, 200, and 500 mg/kg Yangchunsa extract in combination with the high cholesterol diet were significantly increased compared to the high cholesterol diet group, as shown in FIG. 4 . In particular, the expression amount of HMGCR mRNA in the 500mg/kg Yangchunsa group increased by 45.8% compared to the high cholesterol diet group, indicating significance.

HMGCR is an enzyme that controls the synthesis of cholesterol in the liver. In the group administered with a high-cholesterol diet, the cholesterol content in the liver, which controls the total cholesterol pool, increases, so there is no need for new cholesterol synthesis, so the expression level of HMGCR decreases will do

As mentioned above, Yangchunsa extract reduces the LDL cholesterol concentration in the blood, which is known to be the main culprit of arteriosclerosis, by up to 30.9%. The effect mechanism can be explained in two ways.

First, it can be seen that Yangchunsa extract plays the same role as statin, a competitive inhibitor of HMGCR, by significantly inhibiting the decrease in HMGCR expression caused by a high-cholesterol diet in the liver.

Second, it can be seen that Yangchunsa extract reduces blood cholesterol by introducing blood cholesterol into the liver by significantly inhibiting the decrease in LDLR and PCSK9 expression caused by a high-cholesterol diet in the liver.

Claims (3)

A food composition for preventing or improving hyperlipidemia or arteriosclerosis containing an extract of Amomum villosum Lour.
A pharmaceutical composition for preventing or treating hyperlipidemia or arteriosclerosis containing an extract of Yangchunsa ( Amomum villosum Lour.) as an active ingredient.
The method according to claim 1 or 2,
The composition, characterized in that the Yangchunsa extract is orally administered at a concentration of 100 to 500 mg / kg based on the mouse.

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