KR102093376B1 - USE OF LONGAN PERICARP EXTRACT IN THE MANUFACTURE OF A COMPOSITION FOR THE PROTEIN OF SREBP-1c, ACC, AND SCD-1 MODULATING - Google Patents

Abstract

The present invention provides the use of a long-term peel extract in the manufacture of compositions that reduce the expression of SREBP-1c and ACC and SCD-1 proteins. The longan peel extract significantly inhibits the production of fat, lowers the GPT index and protects the liver.

Description

USE OF LONGAN PERICARP EXTRACT IN THE MANUFACTURE OF A COMPOSITION FOR THE PROTEIN OF SREBP-1c, ACC, AND SCD-1 MODULATING}

The present invention relates to the use of longan peel extracts, in particular to the use of long extract peel extracts in the preparation of compositions for modulating SREBP-1c, ACC and SCD-1 proteins.

The causes of liver disease in Taiwan, Taiwan are viral infections, drug abuse, and long-term drinking. According to the 2015 statistics from the Ministry of Public Administration and Security of the Republic of China, liver diseases such as chronic liver disease and cirrhosis are ranked 10th among the top 10 causes of death among people in the Republic of China.

In recent years, the consumption of alcoholic beverages in Taiwan has increased significantly, and the incidence of alcoholic liver disease has also increased. The biochemical and pathological damage caused by the human body after long-term consumption of alcohol is very complex, and the most affected tissue is the liver, the main tissue that metabolizes alcohol. Liver damage caused by long-term consumption of alcohol includes fatty liver, alcoholic hepatitis, and cirrhosis of the liver, which can lead to liver cancer if not treated in time.

Fatty liver is the first symptom of alcoholic liver disease. Studies have shown that patients with alcoholic fatty liver have a significantly increased chance of having liver fibrosis and cirrhosis. According to another study, fatty liver correlates with the development of liver cancer. Therefore, if the fatty liver can be improved, it is possible to lower the possibility of suffering from liver fibrosis and cirrhosis. However, there is still no effective way to treat fatty liver, and clinically, it is most often recommended to patients to reduce the intake of high-fat foods, consume a lot of vegetables and fruits, and have proper exercise and normal lifestyle. . Of particular importance is the ban on alcohol consumption. However, it is very difficult to demand a short-term abstinence from alcohol-dependent patients with fatty liver, and by planning, reducing alcohol intake in stages and eating foods that can reduce fatty liver will have the greatest therapeutic effect. The liver is an important organ responsible for detoxification and metabolism in the human body. Since unrestrained drinking and infection of the hepatitis virus are two major causes of liver disease, finding antioxidant foods to prevent or reduce liver damage has become a very important task in nutritional medicine.

Longan is mainly shipped from July to September every year, the main production area is Tainan, Taiwan, and the total annual output of Taiwan Province of Taiwan is 100,000 to 130,000 tons. After peeling the Longan Pericarp by baking, it becomes a fruit and can be used as a food or medicinal material and can be stored for a long time. However, after the longan has been processed, a large amount of by-products of the longan peel are formed. Effective use of these by-products, which are now obliged to be discarded, can be said to be a great welfare to mankind if they act as health protection against liver disease.

The present invention is to solve the above problems, the present invention is sterol regulatory element-binding protein-1c (SREBP-1c: sterol regulatory element-binding protein-1c) and acetyl-CoA carboxylase (ACC: acetyl- CoA carboxylase) and stearoyl-CoA desaturase-1 (SCD-1: Stearoyl-CoA desaturase-1) provides the use of a long-sliced peel extract in the preparation of a composition for modulating proteins, wherein the long-sized peel extract It is the extract of long-term rind with solvent of water, alcohol or hydrous alcohol. The composition may further include a pharmaceutically acceptable carrier.

In one embodiment of the invention, the composition may be a powder, granule, liquid, colloidal or paste-like formulation.

In one embodiment of the present invention, the time for extraction with the solvent is 0.5 to 3 hours, the extraction temperature of the solvent is 50 to 100 ° C, and the concentration of the long-term peel extract is 4 mg / ml or more.

Longan peel extract according to the present invention can inhibit the production of fat, lower the GPT index and also protect the liver.

Hereinafter, embodiments of the present invention will be described in detail by combining the accompanying drawings. The examples presented below are intended to illustrate the invention and are not intended to limit the scope of the invention. Those skilled in the art can modify and change the present invention without departing from the spirit and scope of the present invention, and the claims of the present invention are based on what is defined by the claims.

1 is HepG2 cells treated with a 4mg / mL longan peel extract, and the result of quantifying an oil red that the long cut extract extract inhibits fat production.
FIG. 2 is a HepG2 cell treated with 4mg / mL longan peel extract, which is a view stained with oil red that the long extract peel extract inhibits fat production.
3 is a result of analyzing the GPT content after HepG2 cells were pre-treated with 4mg / mL long-term peel extract and then induced to be damaged by H ₂ O ₂ .
FIG. 4 is a fluorescence staining diagram of cells after HepG2 cells were pre-treated with 4 mg / mL long-term peel extract and then induced to be damaged by H ₂ O ₂ .
5 is a result of analyzing the expression of the gene expression related to fat production after HepG2 cells are pre-treated with a 4mg / mL long-term peel extract and then induced to be damaged by H ₂ O ₂ .

The present invention provides the use of the long-sized rind extract in the preparation of a composition for regulating SREBP-1c and ACC and SCD-1 proteins, and the long-run rind extract according to the present invention extracts at different temperatures, and also uses a solvent. It was obtained by extraction and centrifugation of long-term rinds by using, which inhibits the formation of fatty liver, prevents liver damage, lowers the glutamate pyruvate transaminase (GPT) value, and relates to liver fat production. It can be used to reduce genes.

The longan rind extract according to the present invention is obtained by an extraction method, and the extraction method includes the following steps: (a) Extracting a longan rind with an extraction solvent, wherein the extraction is 50 to 100 ° C. Proceeding from the step of the volume ratio of the extraction solvent and the long-term rind 5 ~ 20: 1 ~ 5; (b) centrifuging the obtained product; (c) filtering the supernatant after centrifugation to obtain a filtrate; (d) concentrating the filtrate under reduced pressure at 45-70 ° C. to obtain a concentrated product; And (e) spray drying the concentrated product, wherein the extraction solvent is water, alcohol, hydrous alcohol or a combination thereof.

The present invention also provides compositions for modulating SREBP-1c and ACC and SCD-1 proteins. The composition comprises an effective amount of the long-term peel extract and a pharmaceutically acceptable carrier, the composition is present in a powder form, granule form, liquid form, colloidal form or paste form, and the formulation thereof is food, beverage, drug, It is provided in the form of reagents or supplements.

Hereinafter, the detailed extraction method of the long-sized rind extract according to the present invention and the long-sized rind extract are used to inhibit the formation of fatty liver, prevent liver damage, lower the GPT value and reduce the gene related to liver fat production. By explaining the experiment in detail, the effect of the long-term peel extract will be demonstrated.

Longan Rind  Extraction process:

1. Wash the long-skinned rind to remove foreign substances, and then dry it for later extraction.

2. Proceed with the process of crushing into 0.2 ~ 0.5cm fragments.

3. After pulverization, extract the long-term peel from the solvent at a liquid-solid ratio of 5 to 20: 1 to 5 at 50 to 100 ° C for 0.5 to 3 hours.

4. Cool to room temperature.

5. Centrifuge at 5000 r.p.m. for 10 minutes to remove debris and filter.

6. Filter with 400 mesh filter.

7. Concentrate under reduced pressure at 45 ~ 70 ℃.

Fatty liver cell test:

Experimental material:

(One). Cell line: HepG2 (ATCC, HB-8065)

(2). Culture: Dulbecco's Modified Eagle culture (Gibco, Cat. 12100-038) with 10% fetal calf serum (Gibco, Cat. 10438-026) and 1% penicillin / streptomycin (Gibco, Cat. 15140-122)

(3). Free fatty acids (Oleic acid; Sigma, # O1008)

(4). Bovine serum albumin (Bio Basic Inc., Cat. AD0023)

(5). Oil Red O (Sigma, Cat.O0625)

(6). Isopropanol (Echo chemical, PH-3101)

(7). 10% formaldehyde (Echo chemical, Cat.TG1794-4-0000-72NI)

(8). Phosphate buffered saline (Gibco, Cat. 14200-075)

Experimental procedure:

(One). The cells are transplanted into a 6-well culture plate at a density of 5 x 10 ⁵ cells per well and incubated overnight at 37 ° C.

(2). Cells are treated with 4 mg / mL long-term peel extract for 24 hours in a culture medium containing 2% fetal calf serum.

(3). Prepare a culture solution containing OA-BSA conjugate, 2% fetal calf serum and 1% penicillin / streptomycin.

(4). The existing culture medium is removed and replaced with a culture medium containing the OA-BSA conjugate prepared in procedure (3). It is treated for 24 hours by adding an appropriate concentration of the long-term peel extract.

(5). Remove the culture and wash twice with 1X phosphate buffer.

(6). Cells are fixed with 10% formaldehyde for 30 minutes.

(7). Wash twice with 1X Phosphate Buffer and wash with 50% isopropanol for 15 seconds.

(8). Dye in 60% isopropanol with Oil Red O for 1 hour.

(9). The staining was observed under a microscope, and the stain was dissolved and quantified with 100% isopropanol. Statistics are checked with Student's t in Excel program.

The experimental results are shown in FIG. 1. HepG2 cells treated with 4 mg / mL Longan peel extract can inhibit 40.5% fat formation and effectively lower the fat production of HepG2 cells, and Longan peel extract can effectively suppress HepG2 cells from forming fat. Prove that there is. In addition, as can be seen in the dyeing picture of FIG. 2, the longan peel extract exhibits an effect of preventing fatty liver by inhibiting fat.

Liver damage cell GPT (ALT) detection:

Experimental material:

(One). Cell line: HepG2 (ATCC, HB-8065)

(2). Culture: Dulbecco's Modified Eagle culture (Gibco, Cat. 12100-038) with 10% fetal calf serum (Gibco, Cat. 10438-026) and 1% penicillin / streptomycin (Gibco, Cat. 15140-122).

(3). ELISA Kit of Alanine Aminotransferase (ALT) (USCN., Cat. SEA207Hu)

(4). Phosphate buffer solution (Gibco, Cat. 14200-075)

(5). ELISA Leader (BioTek)

Experimental procedure:

(One). The cells are transplanted into a 24-well culture plate at a cell density of 2 × 10 ⁴ per well and incubated at 37 ° C. overnight.

(2). Cells are pre-treated with 24 mg / ml long-term peel extract in culture for 24 hours prior to treatment with H ₂ O ₂ .

(3). After 24 hours of pretreatment, H ₂ O ₂ was added and treated at a concentration of 500 μM for 6 hours.

(4). Cells are stained for 15-30 min with propidium iodide staining solution (1: 250) and Annexin V (1: 250) Annexin V binding buffer.

(5). Cells are stained for 3 minutes with Hoechst 33342 (1: 20000).

(6). Cells are washed twice with phosphate buffer.

(7). Observe under a fluorescence microscope.

The results are as shown in FIG. 3. In the figure, after the HepG2 cells are damaged by H ₂ O ₂ , the GPT index rises rapidly, but in the group pretreated with 4 mg / ml long-term peel extract, the degradation effect on the GPT index reaches 90%.

Longan pericarp  Exploration in terms of extract genes

Experimental material:

(One). Cell line: HepG2 (ATCC, HB-8065)

(2). Culture: Dulbecco's Modified Eagle culture (Gibco, Cat. 12100-038) with 10% fetal calf serum (Gibco, Cat. 10438-026) and 1% penicillin / streptomycin (Gibco, Cat. 15140-122).

(3). ELISA Kit of Alanine Aminotransferase (ALT) (USCN., Cat. SEA207Hu)

(4). Phosphate buffer solution (Gibco, Cat. 14200-075)

(5). ELISA Leader (BioTek)

Experimental procedure:

(One). In a 6-well culture plate, 1 x 10 ⁵ cells per well are transplanted with 1 mL of the culture medium and incubated overnight at 37 ° C.

(2). Treatment with 2 mg / ml or 4 mg / ml long-term peel extract for 24 hours.

(3). Collect the stimulated cells.

(4). Collect RNA from cells with RNA extraction kit.

(5). RNA is reverse transcribed with reverse transcriptase to obtain cDNA.

(6). Target gene is quantified by performing qPCR using KAPA SYBR FAST by ABI Step One Plus.

(7). The relative amount of gene expression is determined by the 2 ^{- ΔΔCt} method.

(8). Statistics and analysis are performed by Student's t test in the Excel program. (*: P <0.05; **: P <0.01)

The results are as shown in FIG. 4. Figure 4 shows that HepG2 cells are killed by H ₂ O ₂ and then killed in large quantities, but adding the long-term peel extract can increase the survival rate of the cells. Thus, it can be seen that the longan peel extract protects the liver from being damaged by external irritation. When viewed from the gene side of the long-term peel extract, liver cells are stimulated by the outside to generate SREBP-1c transcription factor, and the transcription factor causes the cells to produce ACC and SCD-1, proteins involved in lipid synthesis. As shown in FIG. 5, the longan peel extract reached a 78% reduction effect on SREBP-1c, a 82% reduction effect on ACC, and a 33% reduction on SCD-1. All three of these proteins increase fat in the liver cells (triglycerides), causing fatty liver.

In summary, the long-term peel extract according to the present invention in cell experiments and gene expression shows the effect of regulating SREBP-1c and ACC and SCD-1 proteins, and SREBP-1c and ACC and SCD-1 proteins It can be used as food, beverage products, drugs, reagents or nutritional supplements to control the.

Therefore, the longan peel extract proposed in the present invention effectively suppresses the formation of fatty liver, prevents liver damage, lowers the GPT value, and also protects the liver by lowering the expression of genes related to liver fat production.

Claims (10)

A composition for protecting the liver, comprising an extract of a long-term rind obtained by extracting a longan pericarp using a solvent of water, alcohol or hydrous alcohol,
The composition is characterized by reducing the expression of acetyl-CoA carboxylase (ACC: acetyl-CoA carboxylase) and stearoyl-CoA desaturase-1 (SCD-1: Stearoyl-CoA desaturase-1) proteins. The composition. The composition according to claim 1, characterized in that it further comprises a pharmaceutically acceptable carrier. The composition according to claim 1, wherein the composition is a powder, granule, liquid, colloidal or paste-like formulation. The method according to claim 1, characterized in that the concentration of the long-term peel extract is 4 mg / ml or more, the composition. The composition according to claim 1, characterized in that the volume ratio between the solvent at the time of extraction and the long-term rind is 5 to 20: 1 to 5. The method according to claim 1, characterized in that the time for extracting the long-term peel using the solvent is 0.5 to 3 hours, the composition. The method according to claim 1, The extraction is characterized in that proceeds at a temperature between 50 ℃ ~ 100 ℃, the composition. The composition according to any one of claims 1 to 7, wherein the long-term peel extract is capable of inhibiting lipogenesis. delete delete

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