TW201822780A - 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 a use of longan pericarp extract in the manufacture of a composition for reducing SREBP-1c and the proteins of ACC and SCD-1 expression. The longan pericarp extract significantly inhibits lipo genesis, reduces GTP index, and liver protecting.

Description

   Use of longan shell extract for preparing composition for regulating SREBP-1c, ACC and SCD-1 protein   

The invention relates to the use of a longan shell extract, in particular to the use of a longan shell extract to prepare a composition for regulating SREBP-1c, ACC and SCD-1 proteins.

Chinese people suffer from liver disease due to viral infection, drug abuse, and long-term alcohol abuse. According to statistics from the Department of Health of the Executive Yuan in 104, liver diseases such as chronic liver disease and liver cirrhosis rank 10th among the top ten causes of death in the country.

In recent years, the consumption of alcoholic beverages in Taiwan has increased significantly, which has also increased the incidence of alcoholic liver diseases. After long-term ingestion of alcohol, the biochemical and pathological damage caused in the human body is very complicated, and the most affected tissue is the liver, the main tissue of alcohol metabolism. Liver damage caused by long-term ingestion of alcohol includes fatty liver, alcoholic hepatitis, and cirrhosis. If left untreated, it can cause liver cancer.

Fatty liver is the earliest sign of alcoholic liver disease. Studies have shown that patients with alcoholic fatty liver have a significantly increased chance of developing liver fibrosis and cirrhosis. Other studies have pointed out that fatty liver is related to the occurrence of liver cancer. Therefore, if the condition of fatty liver can be improved, the chance of liver fibrosis and cirrhosis can be reduced. However, there is currently no effective method for treating fatty liver. Most patients are advised to eat less fatty foods, eat more fruits and vegetables, exercise properly and maintain a regular schedule. The most important thing is to prohibit alcohol intake. However, for patients with fatty liver alcohol dependence, it is very difficult to quit alcohol in a short time. If you can plan to reduce alcohol intake and eat foods that can reduce fatty liver, you can achieve the maximum therapeutic effect. Because the liver is the body's important organ responsible for detoxification or metabolism. Alcoholism and hepatitis virus infection are the two main causes of liver disease, so finding antioxidant foods to prevent or slow liver damage is an important topic in nutrition medicine.

Longan is abundant in July-September, and its main place of production is in Tainan. The annual output of the province is between 100,000 and 130,000 tons. After baking and dehulling the longan, it is longan, which can be used as a tonic and can be stored for a long time. However, after processing longan, a large amount of longan shell by-products are produced. If these by-products that can only be discarded are further effectively used and have a health-care effect on liver disease, it will be a great human welfare.

In order to solve the foregoing problem, the present invention provides the use of a longan shell extract for preparing a composition that regulates SREBP-1c, ACC, and SCD-1 proteins, wherein the longan shell extract is water, alcohol, or water-containing alcohol. Extracted from similar solvents. The composition may further comprise a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the composition may be in the form of a powder, granule, liquid, gel or paste.

In one embodiment of the present invention, the extraction time of the solvent is 0.5 to 3 hours, the extraction temperature of the solvent is 50 to 100 ° C., and the concentration of the longan shell extract is 4 mg / ml or more.

The longan shell extract of the present invention can inhibit fat production, reduce GPT index, and protect the liver.

The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are intended to clarify the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art will not depart from the spirit and scope of the present invention. Within the scope, when some changes and retouching can be done, the protection scope of the present invention shall be determined by the scope of the attached patent application scope.

Figure 1 is the result of quantifying the oil red of HepG2 cells treated with 4mg / mL longan shell extract to inhibit fat production.

Fig. 2 is an oil-red staining diagram of HepG2 cells treated with 4mg / mL longan shell extract to evaluate the inhibition of adipogenesis by longan shell extract.

Figure 3 is the GPT content analysis result of HepG2 cells pretreated with 4mg / mL longan shell extract and then H ₂ O ₂ induced damage.

Figure 4 is a fluorescence staining diagram of HepG2 cells pretreated with 4 mg / mL longan shell extract and then H ₂ O ₂ -induced damage.

Figure 5 shows the analysis results of the expression of genes related to adipogenesis in HepG2 cells pretreated with 4 mg / mL longan shell extract and then H ₂ O ₂ -induced injury.

The invention provides a longan shell extract for preparing a composition for regulating SREBP-1c, ACC and SCD-1 proteins. The longan shell extract of the present invention is extracted at different temperatures, and the longan shell is extracted with a solvent It is obtained by centrifugation. It can be used to inhibit fatty liver formation, prevent liver damage, reduce GTP (glutamate pyruvate transaminase) value, and reduce genes related to hepatic fatogenesis.

The longan shell extract of the present invention is obtained by an extraction method, which comprises the following steps: (a) extracting the longan shell with an extraction solvent, wherein the extraction is performed at 50-100 ° C, and the extraction solvent The volume ratio to the longan shell is 5-20: 1 ~ 5; (b) the obtained product is centrifuged; (c) the supernatant after centrifugation is filtered to obtain a filtrate; (d) the filtrate is at 45-70 ° C Performing concentration under reduced pressure to obtain a concentrated product; and (e) spray-drying the concentrated product, wherein the extraction solvent is water, alcohols, aqueous alcohols, or a combination thereof.

The present invention also provides a composition for regulating SREBP-1c, ACC and SCD-1 proteins, comprising an effective amount of longan shell extract and a pharmaceutically acceptable carrier. The composition is in the form of powder and granule , Liquid, gel or paste, and its dosage form is provided in the form of food, drink, medicine, reagent or nutritional supplement.

The detailed extraction method of the longan shell extract of the present invention and the test experiments of the longan shell extract for inhibiting fatty liver formation, preventing liver damage, reducing GTP value, and reducing liver fat biosynthesis-related genes will be described in detail below to confirm the Effect of Longan Shell Extract.

Longan shell extraction process:

1. After cleaning the longan shell to remove debris, dry it for subsequent extraction.

2. Perform fragmentation of about 0.2 ~ 0.5cm.

3. Take the crushed longan shell and extract it in a solvent at a temperature of 5 ~ 20: 1 ~ 5 for 0.5 ~ 3 hours.

4. Cool to room temperature.

5. Centrifuge at 5000r.p.m. For 10 minutes, and perform deslag filtration.

6. Filter through a 400 mesh filter.

7. Concentrate under reduced pressure at 45 ~ 70 ° C.

Fatty liver cell test: Experimental Materials:

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

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

(3). Free fatty acid (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 steps:

(1). The cells were implanted in a six-well culture plate at a density of 5 × 10 ⁵ cells per well, and cultured at 37 ° C. overnight.

(2). The cells are treated with 4 mg / mL longan shell extract in a culture solution containing 2% fetal bovine serum for 24 hours.

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

(4). Remove the original culture solution and replace it with the culture solution containing OA-BSA conjugate prepared in step (3.). Add longan shell extract of appropriate concentration and treat for 24 hours.

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

(6). Fix the cells with 10% formaldehyde for 30 minutes.

(7). Wash twice with 1X phosphate buffer solution, and rinse with 50% isopropanol for 15 seconds.

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

(9). Observe through a microscope and dissolve the stain with 100% isopropanol to quantify. Statistics is the Student's t-test using Excel software.

Experimental results Figure 1. HepG2 cells treated with 4mg / mL longan shell extract can inhibit 40.5% of fat formation, which can effectively reduce the amount of fat produced by HepG2 cells, confirming that longan shell extract can effectively inhibit the formation of fat by HepG2 cells. In addition, the stained photo of FIG. 2 shows that the longan shell extract can inhibit fat to prevent fatty liver.

GPT (ALT) detection of liver injury cells: Experimental Materials:

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

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

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

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

(5). ELISA reader (BioTek)

Experimental steps:

(1). The cells were seeded into a 24-well culture plate at a density of 2 × 10 ⁴ cells per well, and cultured overnight at 37 ° C.

(2). Prior to the treatment with H ₂ O ₂ , the cells were pretreated with 4 mg / ml longan shell extract in culture for 24 hours.

(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 were stained with Propidium iodide staining solution (1: 250) and Annexin V (1: 250) in Annexin V binding buffer for 15-30 minutes.

(5). Stain the cells with Hoechst 33342 (1: 20000) for 3 minutes.

(6). Wash the cells twice with phosphate buffer.

(7). Observe with a fluorescence microscope.

The results are shown in Figure 3. The figure shows that the GPT index of HepG2 cells increased sharply after H ₂ O ₂ injury. However, the group pretreated with 4mg / ml longan shell extract can effectively reduce the GPT index by 90%.

Study on the Gene Level of Longan Shell Extract Experimental Materials:

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

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

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

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

(5). ELISA reader (BioTek)

Experimental steps:

(1). In a six-well culture plate, 1 × 10 ⁵ cells per well, 1 mL of culture fluid were implanted, and cultured overnight at 37 ° C.

(2). Treat with longan shell extract at 2mg / ml or 4mg / ml for 24 hours.

(3). Collect stimulated cells.

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

(5). Reverse transcription of RNA with reverse transcriptase to obtain cDNA.

(6). ABI Step One Plus was used to perform qPCR using KAPA SYBR FAST to quantify the target gene.

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

(8). Statistical analysis was performed using Student's t-test of Excel software. (*: P <0.05; **: P <0.01)

The results are shown in Figure 4. The figure shows that HepG2 cells undergo a large number of apoptosis after H ₂ O ₂ injury, and the addition of longan shell extract can improve the cell survival rate. This shows that longan shell extract can protect the liver surface from external stimuli and damage. At the gene level of Longan shell extract, when liver cells are externally stimulated, they will produce the SREBP-1c transcription factor, which will cause the cells to produce the lipid-related proteins ACC and SCD-1. As shown in Figure 5, longan shell extract can significantly reduce SREBP-1c by 78%, reduce ACC by 82%, and SCD-1 by 33% with significant differences. All three proteins can cause triglycerides in liver cells. ) Increased, causing fatty liver.

To sum up, both in cell experiments and gene expression, the longan shell extract of this case has the function of regulating SREBP-1c and ACC and SCD-1 proteins, and can be used to regulate SREBP-1c and ACC and SCD-1 proteins. Food, beverages, medicines, reagents or nutritional supplements, etc.

Therefore, the longan shell extract provided by the present invention can effectively inhibit fatty liver formation, prevent liver damage, reduce GTP value, and reduce genes related to liver fat production and synthesis to achieve the effect of protecting the liver.

Claims (10)

    A longan shell extract is used for preparing a composition for reducing the expression of SREBP-1c, ACC and SCD-1 proteins. The longan shell extract is a longan shell extracted with water, alcohol, or a solvent containing water alcohol. Got.         The use as described in item 1 of the patent application scope, wherein the composition further comprises a pharmaceutically acceptable carrier.         The use according to item 1 of the scope of patent application, wherein the composition is in the form of a powder, granule, liquid, gel or paste.         The use according to item 1 of the scope of the patent application, wherein the concentration of the longan shell extract is 4 mg / ml or more.         The use as described in the first item of the patent application range, wherein the volume ratio of the solvent to the longan shell during the extraction is 5-20: 1-5.         The use as described in item 1 of the patent application range, wherein the extraction time of the solvent is 0.5 to 3 hours.         The use as described in item 1 of the patent application range, wherein the extraction temperature of the solvent is 50 to 100 ° C.         The use according to any one of claims 1 to 7, wherein the longan shell extract can inhibit fat production.         A longan shell extract is used for preparing a composition for protecting the liver, wherein the longan shell extract is obtained by extracting longan shell with water, alcohol, or a water-containing alcohol solvent.         The use as described in item 9 of the scope of patent application, wherein the longan shell extract can reduce the GPT index.