KR102488212B1 - Orostachys japonicas and Momordica charantia Linn complex-extract for improving blood sugar and method of manufacturing the same - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating diabetes comprising a composite extract of Waspong and Bitter gourd as an active ingredient.
Accordingly, the present invention can effectively improve or treat diabetes by activating pancreatic beta cells while lowering the blood sugar level to a normal range without side effects using plants.

Description

Wassong·momju complex extract capable of improving blood sugar and manufacturing method thereof {Orostachys japonicas and Momordica charantia Linn complex-extract for improving blood sugar and method of manufacturing the same}

The present invention relates to a composite extract of Waspong and bitter gourd that can improve blood sugar and a method for manufacturing the same, and more particularly, to improve blood sugar using Wasp and bitter gourd bitter gourd, effectively improving, preventing and treating diabetes. It relates to an extract and a method for preparing the same.

The present invention is a task result, and the task information is as follows.

1. Task number: 2017-B-6

2. Department Name: Jeollabuk-do

3. Organization specializing in research management: Jeonbuk Techno Park

4. Research Project Name: 2017 Jeollabuk-do Leading Enterprise Technology Development Capacity Building Support Project

5. Title of research project: Diabetes improvement bio material and product development using Wasong

6. Supervisor: General Bio Co., Ltd.

7. Research period: 2017.10.01 ~ 2018.09.30

Diabetes mellitus is the most representative endocrine disorder that occurs when the action of insulin, which acts on carbohydrate metabolism, is not normally performed. It mainly occurs and is classified as type 2 diabetes in which each tissue of the body shows symptoms of non-responsiveness to insulin and insulin resistance.

As the main treatment for diabetes, orally ingested hypoglycemic agents such as sulfonylureas drugs and biguanides and insulin obtained by DNA recombination are used. However, these drugs have many side effects such as induction of hypoglycemia, renal toxicity, lactic acidosis, diarrhea and stomach upset, and immune rejection, so studies to use natural products with fewer side effects are becoming more active.

On the other hand, pine (瓦松) is called rock brush (Orostachys japonicus A. Berger) and is a perennial herbaceous plant of the Crassulaceae, also known as pine (岩松), jade pine (屋松), hacho (昨葉何草), etc. It is used in oriental medicine for fever, small boils, hemostasis, and dampness [Kim Jae-soo, 1984, Original Color Celestial Plants Daesajang (上卷), Namsan-dang, Seoul, p.447], and is also widely used for cancer treatment as a folk remedy [ Seongsik Bae, 1990, Herbal Medicine and Health, 1, 26].

This plant is a fleshy perennial herb that grows attached to rocks, but it is characterized by withering away when it blooms and bears fruit. Radical leaves spread in a rosette shape, the ends harden and become like thorns, the leaves hang on the main stem, there is no petiole, and the ends do not harden together with the rhizome leaves that appear in summer, but only become sharp, lanceolate, green, but purple or powdered white. there is. Flowers bloom in September, white, racemes are 6-15cm long, flowers without vases are closely attached, bracts are lanceolate, and the ends are sharp. There are 5 calyxes, lanceolate, green, and 5 petals, lanceolate acute, about 6mm long. It has 10 stamens, longer than petals, and 5 ovaries. The pollen is red, but gradually turns black [Lee Chang-bok, 1979, Daehan Plant Research, Hyangmunsa Seoul, p.402]. Wasong is collected from summer to autumn, and the outposts with the roots removed are dried in the sun for medicinal purposes [Kim Jae-woo, 1984, Original Color Celestial Plants Daesagi (上卷), Namsanjang, Seoul, p.447] .

If you look at wasa pine through herbal literature, it is said that it is used for dryness and pain in the mouth in dangboncho (唐本草). It is said to have an astringent effect, and in herbal remedies, it treats white poison and eliminates tumors, and in classification herbal medicine, it is said that all herbs are used to wash hemorrhoids to control hemorrhoidal pain and bleeding. It is said to be effective for (瘡) and Yu (癒) [上海科學技術出版社 小學館編 ;中藥大事典, 小學館, 1985, p.526].

On the other hand, in the invention published in Republic of Korea Patent Publication No. 10-2005-0006964 (hereinafter referred to as 'Patent Document 1'), an antidiabetic composition using Wasong is disclosed.

However, Patent Document 1 has a problem of limited efficacy in improving or treating diabetes using only Wasong.

Republic of Korea Patent Publication No. 10-2005-0006964 (published on January 17, 2005)

The present invention has been devised to solve the limited efficacy when using only Waspsong, and the problem to be solved in the present invention is to improve, prevent and treat diabetes efficiently by adding other plants, that is, bitter gourd, in addition to Waspsong. It is to provide an extract and a manufacturing method thereof.

The extract according to the present invention for solving the above problems is technically characterized by containing a composite extract of Waspong and Goji bitter gourd as an active ingredient.

Method for producing an extract according to the present invention for solving the above problems is dissolved in distilled water wasong powder and then reflux extraction; Dissolving bitter gourd powder in distilled water and then extracting under reflux; and mixing the waspsong extract and the bitter gourd extract in a weight ratio of 8:2.

The extract according to the present invention has no side effects by using plants, and can efficiently improve or treat diabetes by lowering blood sugar levels to a normal range and activating pancreatic beta cells at the same time.

1 and 2 are graphs showing blood glucose measurement results
3 is a graph showing the measurement results of glycated hemoglobin
4 and 5 are graphs showing oral glucose tolerance test results
6 and 7 are photographs and graphs showing pancreatic H&E staining results
8 is a photograph showing the results of immunostaining of the pancreas
Figure 9 is a picture of the autopsy results of subcutaneous and intra-abdominal adipose tissue
10 is a photograph of the liver adipose tissue autopsy result
11 is a graph showing the results of weight measurement after autopsy
12 is a graph showing liver weight measurement results after autopsy
13 is a graph showing liver weight / body weight ratio results
14 and 15 are graphs showing liver damage measurement results
16 and 17 are graphs showing fat accumulation measurement results
18 is a photograph showing the results of H&E staining of the liver
19 and 20 are photographs and graphs showing results of Oil-red O staining of liver
21 is a graph showing the expression results of lipid metabolism related genes
22 is a graph showing the expression results of lipid biosynthesis-related genes
23 is a graph showing the expression results of transcriptional regulators related to lipid metabolism and lipid biosynthesis
24 is a photograph showing Akt expression results through western blotting
25 is a graph showing the expression results of genes related to sugar receptors and glycogen synthesis
Figure 26 is a graph showing the results of gene expression analysis of the insulin resistance experimental model to which Wassong extract was administered
Figure 27 is a graph showing the results of gene expression analysis of the insulin resistance experimental model administered with Wassong · Bitter gourd complex extract

The terms or words used in this specification and claims conform to the technical spirit of the present invention based on the "principle that the inventor can appropriately define the concept of terms in order to explain his or her invention in the best way" It should be interpreted as the meaning and concept of

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 of the technical spirit 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.

Example. Wasong·Yeoju Complex Extract

Fruits of Wasong and Yeoju were purchased from a farming association located in Namwon. 1 kg of dry powder consisting of the aerial parts of Japanese pine and bitter gourd was added to 10 L of distilled water and extracted twice under reflux while stirring overnight using a homogenizer. The extract was freeze-dried to complete Wasong·Yeoju composite extract. The mixing ratio of waspsong and bitter gourd used in the present invention was mixed at a ratio of 8:2. As a result of HPLC (High-performance liquid chromatography) analysis of the composition of the Wasson·Yeoju composite extract, five main components were identified, and the results are shown in Table 1 below.

Experimental Example 1. Evaluation of effects on anti-diabetic indicators

1-1. Experiment preparation

For the experiment, 5-week-old male db/db mice and wild-type C57BL/Ksj-db/+ (db/+) mice of the same age were prepared. The environment maintained general environmental conditions (24 ± 2 ℃, humidity of 50 ± 5%), the diet was supplied as a sterilized standard diet, and water was provided to be freely consumed. The experiments performed below were all conducted according to the guidelines of the Animal Care Ethics Committee of Chonbuk National University (CNNU 2018-038). After a 2-week acclimatization process, db/+ (6 mice per group) or db/db (8 mice per group) mice were randomly divided into 4 groups, and the Waspsong·Mochi chamomile complex extract was administered at the following concentrations. ;0, 100, 200 and 400 mg/kg/day. Wasong·Yeoju extract was dissolved in saline and taken orally. The intake cycle was 5 days a week, once a day, and the experiment was conducted for 6 weeks.

1-2. experimental process

After the mice in 1-1 were fasted for at least 8 hours, (1) blood glucose levels and (2) glycated hemoglobin levels were measured, and then (3) sugar (2 g/kg body weight) was orally administered and blood glucose levels were measured. After 0, 30, 60, 90, and 120 minutes, respectively, blood glucose control ability was evaluated.

1-3. Experiment result

(1) Blood glucose measurement result after fasting

As shown in FIG. 1, it was confirmed that the blood glucose level was significantly reduced in the diabetic mouse group administered with the Wassong·Mongolian complex extract.

Based on this result, as a result of comparative analysis of the difference from the initial blood glucose level, it was confirmed that the change in blood sugar was also reduced by the administration of the extract of Wassong · Goji bitter gourd complex, as shown in FIG.

(2) Glycated hemoglobin (HbA1c) measurement result

As shown in FIG. 3, it was confirmed that the measured value of glycated hemoglobin was significantly decreased in the diabetic mouse group administered with the Wassong·moji chamomile complex extract.

(3) Results of oral glucose tolerance test

As shown in FIGS. 4 and 5 , it was confirmed that the blood glucose level in the db/db mouse group significantly increased after 120 minutes compared to the initial blood glucose level. On the other hand, in the group that received the Wasong·Yeoju complex extract, it was confirmed that blood sugar decreased significantly over time after sugar administration. These results indicate that the blood glucose control ability is improved in diabetic mice by the administration of the Wassong·moji chamomile complex extract.

Experimental Example 2. Analysis of histopathological changes in the pancreas

2-1. Experiment preparation

Pancreatic tissue was prepared.

2-2. experimental process

Pathological changes in the pancreas caused by diabetes were observed through H&E staining of the tissue sections that had undergone normal processing after fixation in formalin.

After normal tissue processing of formalin-fixed tissue, tissue sections were examined for pathological changes in the pancreas caused by diabetes through immunohistochemistry (IHC).

2-3. Experiment result

As a result of pancreatic H&E staining, as shown in Figs. 6 and 7, a significant increase in the area of islets of Langerhans in the pancreas due to insulin resistance was observed in the db / db mouse group. was confirmed to decrease.

As a result of comparative analysis of the expression patterns of insulin and glucagon through immunostaining, as shown in FIG. 8, insulin-positive cells were increased by treating the db / db mouse group with the composite extract of Japanese bitter melon and bitter gourd, and the secretion of glucagon was similar to normal. The cancellation result was confirmed.

Through the results of Experimental Example 2, it can be seen that the Wassong·Mongolia complex extract has a positive effect on the pancreas of individuals with type 2 diabetes.

Experimental Example 3. Comparison of autopsy visual findings, liver weight, and body weight

3-1. Experiment preparation

Mice were prepared as in 1-1.

3-2. experimental process

An autopsy was performed after 6 weeks of the experimental period. They were fasted for 8 hours before necropsy.

3-3. Experiment result

(1) Result of autopsy macroscopic findings

As shown in FIG. 9 , it was confirmed that the diabetic db/db mouse group had increased subcutaneous and intra-abdominal adipose tissue sizes compared to the db/+ group. On the other hand, it was not possible to confirm the change of adipose tissue by administration of Wasong·Yeoju complex extract.

(2) Liver weight and weight measurement results

Changes in liver size are as shown in FIG. 10 . As shown in FIGS. 11 to 13, it was confirmed that the body weight and liver weight increased in the diabetic db/db mouse group compared to the db/+ group. On the other hand, it was confirmed that the body weight and liver weight or the ratio thereof decreased slightly by the administration of the mixed extract of Wasong·Yeoju. However, statistical significance could not be confirmed.

Experimental Example 4. Confirmation of liver damage and fat deposition caused by diabetes

4-1. Experiment preparation

Mice were prepared in the same way as in 1-1.

4-2. experimental process

Blood samples collected from mice in 4-1 were centrifuged to obtain serum, (1) ALT (alanine aminotransferase) and AST (aspartate transaminase), which are enzymes related to liver damage, were measured, (2) collected from 9-1 mice Triglyceride (hereinafter referred to as 'TG') and total cholesterol (hereinafter referred to as 'TC') were measured in one liver.

4-3. Experiment result

(1) Measurement results of liver damage

As shown in FIGS. 14 and 15 through serological analysis, it was confirmed that the levels of ALT and AST were significantly decreased by the administration of the composite extract of Wassong·Mongolia.

(2) TG and TC measurement results

As shown in FIGS. 16 and 17, it was confirmed that the fat accumulation was significantly reduced by the administration of the Wassong·moji chamomile complex extract.

Experimental Example 5. Analysis of histopathological changes in the liver 1

5-1. Experiment preparation

Liver tissue was prepared.

5-2. experimental process

After formalin fixation, pathological changes in the liver due to diabetes induction were observed through H&E staining of tissue sections that had undergone general processing.

5-3. Experiment result

As a result of H&E staining of the liver, as shown in FIG. 18, hepatocellular hypertrophy and ballooning degeneration and fat deposition (stratosis) were observed in the db/db mouse group, and these aspects were observed by A significant decrease was confirmed.

Summarizing the results of Experimental Examples 3 to 5, it can be seen that the bitter gourd · Wassong · bitter gourd complex extract has an antidiabetic effect by reducing liver damage caused by diabetes and controlling steatosis and blood sugar related to liver damage.

Experimental Example 6. Analysis of histopathological changes in the liver 2

6-1. Experiment preparation

Liver tissue was prepared.

6-2. experimental process

Fat deposition in the liver caused by diabetes was observed at the tissue level through Oil-red O staining of frozen tissue sections.

6-3. Experiment result

As a result of Oil-red O staining of the liver, as shown in FIGS. 19 and 20, it was confirmed that fat deposition was significantly reduced by the administration of the combined extract of Waspong and Bitter gourd, similar to the results of observation of H&E stained tissue.

Experimental Example 7. Fat-related gene expression analysis

7-1. experimental process

cDNA was synthesized by extracting mRNA from liver tissue. Relative quantitative analysis was performed on the expression changes of various genes through qRT-PCR to compare and analyze the changes in gene expression patterns caused by the composite extracts of Wassong·Mongolia.

7-2. Experiment result

As a result of comparative analysis of the expression of Apo B, LPL, and L-FABP, which are lipid metabolism-related factors, as shown in FIG. Confirmed.

As a result of comparative analysis of the expression of ACCα, ACCβ, FAS, and SCD1, which are lipid biosynthesis (de novo lipogenesis) related factors, as shown in FIG. Decreasing results were confirmed.

In addition, as a result of comparative analysis of the expression of electronic regulators closely related to the above changes and their related factors, SREBP-1, SREBP-2 and LXRα, as shown in FIG. A significant decrease in the liver of induced mice was confirmed. On the other hand, it was confirmed that the expression of PPARα, which is known to have hepatoprotective and anti-diabetic effects, significantly increased.

Summarizing the results of Experimental Example 6 and Experimental Example 7, the Wassong·moji chamomile complex extract is involved in liver fat metabolism of individuals with type 2 diabetes, and is effective in preventing fat accumulation in the liver, that is, hepatic steatosis can know

Experimental Example 8. Gene expression analysis related to sugar metabolism

8-1. experimental process

Protein was extracted from liver tissue, protein expression was analyzed through Western blot analysis, and cDNA was synthesized by extracting mRNA from liver tissue.

Relative quantitative analysis was performed on the expression changes of various genes through qRT-PCR to compare and analyze the changes in gene expression patterns by the composite extracts of Wassong·Moorbita japonica.

8-2. Experiment result

As shown in FIG. 24, it was confirmed that the activation (phosphorylation) of Akt, which plays an important role in insulin signaling and regulation of glucose receptor expression, was increased in the livers of mice administered with the pine-moistennium composite extract.

As a result of confirming the expression of sugar receptors (GLUT2, 4), it was confirmed that the expression of the sugar receptor was significantly increased by the treatment with the composite extract of Japanese pine and bitter melon (see FIG. 25).

As a result of confirming the expression of genes related to glycogen synthesis (G6Pase, PEPCK), it was confirmed that the expression of G6Pase, a gene related to glycogen synthesis, was significantly reduced in the liver of diabetic mice by the treatment with the Wassong·Mojicha complex extract ( see Figure 25).

Based on the experimental results of Experimental Example 8, it can be seen that the Wassong·Mongolia complex extract inhibits the glucose generation process in the liver and increases insulin sensitivity by regulating sugar-generating enzymes such as G6Pase.

Experimental Example 9. Antidiabetic efficacy in vitro evaluation

9-1. experimental process

Hepatic cancer cell line HepG2 cell was treated with PA (palmitic acid) at a concentration of 0.4 mM to induce insulin resistance, fat accumulation and cell damage. After treatment with 13 ng/mL, 100 ng/mL, and 1 ug/mL (three concentrations at which cytotoxicity does not occur statistically), the cells were cultured for 24 hours.

Relative quantitative analysis was performed on the expression changes of various genes through qRT-PCR, and the changes in gene expression patterns caused by the composite extracts of Wassong and Goji bitter gourd were compared at the cellular level.

9-2. Experiment result

(1) Wasp pine extract treatment result (see Fig. 26)

It was confirmed that the expression of adipogenesis and metabolism-related factors was significantly decreased by the treatment with the hot water extract of Waspsong, and the expression of PPARα was increased.

It was confirmed that the treatment with the Waspong extract increased the expression of glucose receptors and decreased the expression of factors related to sugar production in PA-treated hepatocytes.

Based on these results, it was confirmed at the cellular level that Waspsong extract has an anti-diabetic effect.

(2) Results of treatment of wasabi pine and bitter gourd complex extract (see Fig. 27)

It was confirmed that the treatment with Wasong·Yeoju japonica extract reduced the expression of genes related to lipid accumulation and increased the expression of PPARα in PA-treated hepatocytes.

It was confirmed that the treatment of Wassong·Mongolia complex extract significantly increased GLUT4 in PA-treated hepatocytes, and the expression of G6Pase significantly decreased.

Based on these results, it was confirmed at the cellular level that the Wasong·Yeoju complex extract has anti-diabetic efficacy.

Claims (1)

Catechin 0.7 mg/g, epigallocatechin 0.39 mg/g, epigallocatechin-gallate 0.14 mg/g, epicatechin 0.07 mg/g and epicatechin-gal To contain 0.10 mg/g of epicatechin-gallate, the above ground part of bitter gourd and bitter gourd was mixed at a weight ratio of 8:2, and 10 L of distilled water was added to 1 kg of dry powder to prepare a composite extract of bitter gourd and bitter gourd; and
Preparing a food composition containing 400 mg / kg / day of the composite extract based on oral administration of mice;
Method for producing a food composition for preventing or improving diabetes.

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