KR101335575B1 - Complex Extracts for Treating Hyperlipidemia - Google Patents

Abstract

The present invention relates to a complex herbal extract for treating hyperlipidemia using a phenylamide compound having an HMG-CoR inhibitory effect and an ACAT inhibitory effect related to the treatment and prevention of hyperlipidemia as an active ingredient. It relates to a composition for. In addition, the present invention relates to a method for producing a phenylamide material for the purpose of treating and preventing hyperlipidemia from each extract without fear of side effects or toxicity.

Description

Complex Extracts for Treating Hyperlipidemia with Phenyamide Compounds

The present invention relates to a complex herbal extract for treating hyperlipidemia using a phenylamide compound which exhibits HMG-CoR inhibitory effect and ACAT inhibitory effect related to the treatment and prevention of hyperlipidemia, and in particular, hyperlipidemia consisting of a natural safflower seed ginger phalanx complex extract A therapeutic composition. In addition, the present invention relates to a method for producing a phenylamide material for the purpose of treating and preventing hyperlipidemia from each extract without fear of side effects or toxicity.

The domestic market for hyperlipidemia drugs is supplying original medicines and technologies, led by overseas global technolo- gies, and local pharmaceutical and bio-ventures are being followed as latecomers. In particular, there are no new drugs developed in Korea, and most of them focus on generic development. In particular, according to the 2009 Pharmaceutical Industry Analysis Report (Korea Health Industry Development Institute), the arteriosclerosis (hyperlipidemia treatment group) in Korea's prescription is about 550 billion won, which ranks 6th among all the pharmaceutical market, but the market growth is 46.4% compared to the previous year. As the most rapidly growing group of therapeutic agents.

The characteristics of the disease of the circulatory system is that the cause of the disease is very diverse, and it is impossible to cure. Therefore, current treatment methods focus on alleviating symptoms by recommending drugs, eating habits, and aerobic exercise. In particular, hyperlipidemia is a symptom that has no subjective symptoms, and most patients become chronic and visit the hospital when it is difficult to improve further. Recently, in Korea, the number of patients with cardiovascular diseases (arteriosclerosis, myocardial infarction, etc.) and the potential patient group have increased very much due to the increase in meat intake, frequent changes in eating habits such as frequent drinking, stress and environmental factors. There is no way to treat it.

Accordingly, therapies are commonly used as drugs, and prescribe mainly statin drugs developed as synthetic drugs. Statin-based drugs mainly inhibit the cholesterol biosynthesis enzymes of hepatocytes, increase the number of LDL receptors on the surface of the tissue cells, increase the migration of blood cholesterol to the tissues and show a clinical tendency to reduce LDL-cholesterol in the blood. However, there is high concern about the side effects of long-term use, and recent clinical studies show that atorvastatin, a statin-based drug, reduces blood lipids, but can cause diabetes by increasing blood insulin and glycated hemoglobin and causing insulin resistance. In particular, statin-based drugs are very careful in the treatment of the elderly due to side effects such as rhabdomyopathy, which decreases the muscle density of the patient during long-term use. Therefore, the method of supplementing the side effects of statin-based drugs and maintaining the efficacy of the situation is ongoing.

Drugs composed of natural extracts have recently been in the spotlight as a drug to alleviate these side effects and supplement the drug.

An object of the present invention is to provide an antihyperlipidemic agent consisting of a natural extract that complements the benefits of a synthetic drug as a pharmaceutical material that reduces the side effects and toxicity of the synthetic drug.

The object of the present invention can be achieved by a composition for treating hyperlipidemia consisting of safflower seed, ginger complex extract. The object of the present invention is more preferably achieved by safflower seed, ginger, and phalanx complex extract.

The composition of the present invention has the advantage of excellent effect in the treatment of hyperlipidemia while reducing side effects and toxicity, which is a problem of synthetic medicines.

1 is a graph showing the inhibition of cholesterol absorption of peony seed, phalanges, ginger extract.
Figure 2 is a graph showing the inhibition of cholesterol absorption of safflower seed, pepper seed, safflower seed and pepper seed complex extract.
Figure 3 is a graph measuring the IC50 of cholesterol absorption inhibitory ability of ginger extract.
Figure 4 is a graph showing the cholesterol absorption inhibiting ability of the ginger extract-safflower seed extract mixture.
5 is a graph showing the cholesterol synthesis inhibitory ability in safflower seed extract CaCo-2 intestinal endothelial cells.
Figure 6 is a graph showing the cholesterol synthesis inhibitory ability of the ginger extract-safflower seed extract mixture.
Figure 7 is a graph showing the results of comparing the total cholesterol of white rats after oral administration of SD1001 in hyperlipidemia induced animal model.
8 is a graph measuring the change in LDL-cholesterol upon oral administration of SD1001 in a hyperlipidemia induced animal model.
Figure 9 is a graph showing the results of measuring changes in liver function biomarkers when administered SD1001 in hyperlipidemia induced animal model.
10 is a graph showing the weight change of each organ confirmed by oral administration of SD 1001 for 4 weeks in the hyperlipidemia induced animal model.

The present invention relates to an antihyperlipidemic agent consisting of safflower seed and ginger complex extract. The efficacy of the hyperlipidemia therapeutic agent of the present invention can be further improved by the addition of the phalanges extract.

The safflower seed and ginger extract of the present invention, the mixture ratio of safflower seed extract and ginger extract is 4: 1 by weight.

When the phalanges are added and used in the form of three complex extracts, the mixing ratio of safflower seed extract: ginger extract: phalanges extract is preferably 5: 1: 1.

 The present invention will be described in more detail with reference to the following Examples.

Example 1 Preparation of Serotonin Derivative-Containing Extract from Safflower Seed

* Preparation of extracts containing serotonin derivatives, namely N- ( p- coumaroyl) serotonin (CS) and N -feruloylserotonin (FS) from safflower seeds, was carried out as follows. Safflower seed (7.25 Kg) was obtained by milking dry safflower seed (8 Kg), and 80% ethanol aqueous solution (75 L) was added thereto, followed by repeated extraction for 2 hours in a boiling water extractor equipped with a reflux condenser, followed by filtration and concentration. The crude ethanol extract (446.74 g) was obtained. The obtained safflower seed paper ethanol crude extract was purified by Diaion HP-20 column chromatography. 40% ethanol aqueous solution was added to Diaion HP-20 gel, followed by swelling and filling in the column. I was. Next, the sample was dissolved in 40% ethanol aqueous solution, filled in a column, and eluted sequentially with 40% EtOH and 60% EtOH as a mobile phase solvent. The 60% EtOH eluate was concentrated under reduced pressure to give an extract containing serotonin derivative (66.11 g, yield: 0.83%). The contents of two serotonin derivatives of safflower seed extract were measured to be 12.32% CS and 24.61% FS.

Example 2 Preparation of Tyramine Derivative-Containing Extract from Phalanx

Preparation of an extract containing tyramine derivatives, ie, dihydro- N -caffeoyltyramine (DCT) and trans- N -caffeoyltyramine ( t- CT), was carried out as follows from lipocyledic cortex. 80% ethanol aqueous solution (125 L) was added to the phalanges (12 Kg), followed by repeated extraction for 2 hours in an extractor equipped with a reflux condenser, followed by filtration and concentration to obtain crude ethanol extract (993 g). Phalanx ethanol crude extract was purified by Diaion HP-20 column chromatography. 40% ethanol aqueous solution was added to Diaion HP-20 gel, followed by swelling, and the column was filled with 40% ethanol. Next, the sample was dissolved in 40% ethanol aqueous solution, filled in a column, and eluted sequentially with 40% EtOH and 60% EtOH as a mobile phase solvent. The 60% EtOH eluate was concentrated under reduced pressure to obtain an extract containing tyramine derivative (8.54 g, yield: 0.07%). The content of the two tyramine derivatives of the phalanges extract was 26.63% for DCT and 32.18% for t- CT.

Example 3 Preparation of Gingerol Derivative-Containing Extract from Ginger

Preparation of an extract containing two gingerbread derivatives from ginger, ie, 6-gingerol and 8-gingerol, was carried out as follows. Ginger (10 Kg) was added with 100% ethanol aqueous solution (50 L) at the first extraction with high moisture content and 80% ethanol aqueous solution (50 L) at the second extraction and then for 2 hours in an extractor equipped with a reflux condenser. After extraction twice, filtered and concentrated to give an ethanol crude extract (142.4 g). Ginger ethanol crude extract was purified by Diaion HP-20 column chromatography, equilibrated in Diaion HP-20 gel with 40% ethanol solution, and then dissolved in 40% ethanol solution. Eluted sequentially with 40% EtOH, 60% EtOH and 80% EtOH. The 60% EtOH eluate was concentrated under reduced pressure to obtain a gingerol-containing extract (2.36 g, yield: 0.26%). The contents of the two gingerbreads of the ginger extract were measured to be 44.35% of 6-ginger rolls and 31.30% of 8-ginger rolls.

Example 4 Preparation of Complex Extract

Preparation and production of a complex prescription containing safflower seed, phalanges and ginger extract were carried out as follows. First, safflower seed extract was extracted with safflower seed paper (2.19 Kg) obtained by milking dried safflower seed (2.5 kg) in an boiling water extractor equipped with a reflux condenser with 80% ethanol solution for 2 hours, followed by filtration and concentration. An extract (135.23 g) was obtained. Safflower seed skim ethanol crude extract was subjected to Diaion HP-20 column chromatography to obtain 60% ethanol fraction, that is, safflower seed extract (20 g). The yield of the extract was 0.8% per building, and the content of serotonin derivative was N −. ( p -coumaroyl) serotonin (CS) 13.13% and N -feruloylserotonin (FS) 26.99%.

Next, the phalanges of the phalanges were extracted 80kg of ethanol solution (8.5 kg) twice, repeated two times in an extractor equipped with a reflux condenser for 2 hours, filtered and concentrated to obtain crude ethanol extract (700 g) from the phalanges. 10% ethanol aqueous solution (7 L) was added to the phalanx ethanol crude extract, which was then suspended and partitioned sequentially with dichloromethane and ethyl acetate. Was scored jigolpi extract (6.15 g) The ethyl acetate layer was dried and filtered and concentrated under reduced pressure, the yield amount of 0.07%, tyramine derivatives of extracts dihydro - N - cafe five days tyramine (DCT) 3.05% and the trans - N -Cape oil tyramine ( t- CT) was 8.25%.

On the other hand, the ginger extract ginger (4.0 kg) was extracted twice with an 80% ethanol solution for 2 hours in an extractor equipped with a reflux condenser, filtered and concentrated to obtain a crude ethanol extract (58.64 g). Subsequently, the ginger ethanol crude extract was dissolved in 80% ethanol (1 L), degreased with normal-nucleic acid, and the fraction obtained by dividing the ginger defatted ethanol extract with dichloromethane (1.5 L), that is, a ginger extract (10.79 g), was obtained. The yield of the extract was 0.26%, the content of ginger roll was 6. ginger roll 23.76%.

Based on the safflower seed, phalanges and ginger extract obtained from the above was prepared and produced a composite prescription (30 g) mixed in a ratio of 5: 1: 1.

Test Example 1 Cholesterol absorption inhibitory evaluation

Safflower seeds, phalanges and ginger extracts prepared in Examples 1 to 3, and other natural extracts known to have inhibitory ability to absorb cholesterol, peony seed extract, pepper seed extract, safflower seed extract-pepper seed extract mixed solution (1: 1) It was added at a concentration of 10, 100, 1000 μl / ml, and the cholesterol absorption inhibitory ability was analyzed as follows.

(1) Dispense CaCo-2 intestinal endothelial cells in a 24-well plate at 0.15 X 10 ⁶ cells / ml and incubate for 4-7 days.

(2) Wash with Ham's F-12 culture medium (0.5% fatty acid-free bovine serum albumin, 25mM / L HEPES pH7.4) and incubate for 1 hour in F-12 culture medium (37 ℃, 5% CO ₂ ).

(3) To measure cholesterol absorption, add F-12 culture (0.1 uci / well [1,2- ³ H]-cholesterol) and the sample to be tested and incubate for 8 hours.

(4) F-12 culture medium ^{(non [1,2- 3 H] -cholesterol} ) by washing twice with [1,2- ³ H] in the free state - to remove cholesterol.

(5) Add 0.5 mL of 0.1% sodium dodecyl sulphate (SDS) to get the cells and put them into the scintillation bial.

(6) Add 3 ml of Ultima Gold scintillant and measure the liquid scintillation with β-scintillation counter.

result:

1) Peony seed extract showed cholesterol-lowering ability of 69% at 1 mg / ml, 54% at 100 ㎍ / ml, and 0% at 10 ㎍ / ml, and phalangeal extract had almost no cholesterol-absorbing ability. Silver inhibited cholesterol absorption of 77% at 1 mg / ml, 15% at 100 μg / ml, and 0% at 10 μg / ml (FIG. 1).

2) Safflower seed extract showed almost no cholesterol absorption inhibitory activity, and red pepper seed extract showed 72.5% cholesterol inhibition rate at 1 mg / ml, 31.0% at 100 ㎍ / ml, 0% at 10 ㎍ / ml, and safflower seed extract-pepper seed In the extract mixture (1: 1), cholesterol inhibitory activity of the red pepper seed extract was slowed down (Fig. 2). As a result, peony seed extract, ginger extract and red pepper seed extract showed similarly excellent cholesterol absorption inhibitory effect, and at 1 mg / ml, it was confirmed that cholesterol absorption inhibitory ability was excellent in the order of ginger extract> pepper seed extract> peony seed extract.

3) To determine the IC50 of cholesterol absorption inhibitory activity in CaCo-2 intestinal endothelial cells of ginger extract, various concentrations from 10 ㎍ / ml to 8 mg / ml were analyzed and then cholesterol absorption inhibitory ability was analyzed. IC50 of cholesterol-inhibiting ability in -2 intestinal endothelial cells was about 120 ㎍ / ml (Fig. 3)

4) The mixture of safflower seed extract with excellent cholesterol synthesis inhibitory ability and ginger extract with excellent cholesterol absorption inhibitory ability was mixed in various ratios, and then analyzed by Ginger extract-safflower seed extract (4: 1) was found to be the best (Figure 4)

Test Example 2 Cholesterol synthesis inhibitory evaluation

Safflower seed extract prepared in the above example, and the cholesterol synthesis inhibitory evaluation of the composite extract of safflower seed and ginger was carried out as follows.

(1) CaCo-2 intestinal endothelial cells were cultured in 2 ml of DMEM-10% (0.1uci / ml, human lipoprotein deficient serum) containing 1 mM [2- ¹⁴ C] acetic acid in a 6-well plate for 37 to 4 hours. do.

(2) Transfer the culture solution of the cultured plate to a glass tube, add 1 ml 0.1N NaOH to the cells, and combine the dissolved cell solution with the culture solution.

(3) 3 ml Et-OH, 0.5 ml 50% KOH and 10 ⁵ cpm of [1,2- ³ H] cholesterol (50 Ci / mmol, internal standard) are mixed in each 3 ml (1 ml NaOH solution).

(4) Saponified each sample (50 to 2 hours), add petroleum ether, and then take supernatant.

(5) The taken supernatant was dried by vacuum drying, and then dissolved in hexane and separated and purified by TLC using chloroform as a developing solvent on plastic-backed silica gel G plate.

(6) Remove each band from the plate, add the scintilation cock tail, and quantify with a liquid scitilation counter. (cholesterol RF = 0.31)

result:

1) Safflower seed extract was treated to CaCo-2 intestinal endothelial cells at various concentrations from 40 ㎍ / ml to 1 mg / ml and then cholesterol synthesis inhibitory activity was shown from 200 ㎍ / ml. The IC50 of cholesterol synthesis inhibitory activity in CaCo-2 intestinal endothelial cells was about 600 μg / ml (FIG. 5). This concentration was about 60 times that of atorvastatin (600 μg / ml).

2) The mixture of safflower seed extract with excellent cholesterol synthesis inhibitory ability and ginger extract with excellent cholesterol absorption inhibitory ability was mixed in various ratios and then analyzed by the cholesterol synthesis inhibitory ability to find out which ratio is optimal when used as an actual therapeutic agent. It was found that the mixing ratio of the ginger extract-safflower seed extract (4: 1), which was optimally determined by the inhibition ability, was moderate (FIG. 6).

Test Example 3

(1) Breeding of experimental animals

The experimental animals were ovarian with Sprague Dawley (SD) male rats of 150 ± 10 g of apparently healthy mean weight pre-arranged under optimal conditions (temperature 20 ± 1 ℃, humidity 50 ± 10%, contrast cycle 07: 00 ~ 19: 00). By law, 10 animals were divided into 6 groups and reared for 4 weeks.

(2) Sample processing and diet

Basic dietary diet was a diet and 1.25% high-cholesterol (Research Diets) diet, the treated group was negative control, positive control (Atrovastatin 0.3mg / kg / day), the composite extract of Example 4 (Safflower seed extract: ginger Extract: Phalanx extract 5: 1: 1-SD1001) was dissolved in water at 4, 20, 100 mg / kg / day and orally administered daily using oral sonde.

(3) Analysis item

Samples were administered orally at the same time every day for four weeks, and the body weight, drinking water and feed intake of rats were analyzed for each treatment group each week. After 4 weeks, blood pressure was measured, blood was anesthetized with ether, and then collected by cardiac puncture, and centrifuged at 3000 rpm for 10 minutes to separate serum. Triglyceride, total cholesterol, HDL cholesterol and LDL cholesterol in serum were measured using a blood biochemical analyzer of Hallym University RIC, and GOT and GPT, markers of hepatotoxicity, were measured and compared. After 4 weeks, the animals were euthanized, the weights of various organs were measured, and the state of organs was photographed and compared.

(4) Test result

The total cholesterol of the white rats after oral administration of SD1001 in the hyperlipidemia-induced animal model is shown in FIG. 7. Atovastatin 0.3 mg / kg and SD 1001 were orally administered at doses of 4, 20, and 100 mg / kg, respectively. As a positive control, significant results were observed at 100 mg / kg.

The change in LDL-cholesterol upon oral administration of SD1001 in a hyperlipidemia-induced animal model is shown in FIG. 8. Compared to the positive control group, the overall value was found to be slightly reduced, but the dose of the positive control drug was judged to be somewhat small. However, it was confirmed that the change of the SD1001 100mg / kg administration group was significant compared to the negative control group.

9 shows the results of measuring changes in liver function biomarkers upon administration of SD1001 in hyperlipidemia-induced animal models. There was no particular problem in liver function when SD 1001 was administered for 4 weeks.

Figure 10 shows the weight change of each organ confirmed by oral administration of SD 1001 for 4 weeks in the hyperlipidemia induced animal model. As a result of oral administration of SD 1001 for 4 weeks, no specific changes were found in each organ compared to the normal group.

Claims (3)

N- ( p- coumaroyl) serotonin (CS), N -feruloylserotonin (FS), dihydro- N -cafeoyltyramine (DCT) and trans- N -caoyltyramine ( t- CT) as active ingredients Hyperlipidemia treatment. delete delete

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