KR20210075705A - Composition comprising extracts of Orostachys japonicus as an active ingredient for preventing, alleviating or treating diabetes - Google Patents

Abstract

The present invention relates to a food composition for preventing or alleviating diabetes containing an Orostachys japonica extract as an active component. Accordingly, the present invention can minimize side effects in the human body using plants, and thus can prevent, alleviate or treat diabetes by activating pancreatic beta cells while lowering blood sugar levels to a normal range.

Description

A composition comprising extracts of Orostachys japonicus as an active ingredient for preventing, alleviating or treating diabetes comprising a Wasong extract as an active ingredient.

The present invention relates to a composition for preventing, improving or treating diabetes by containing a Wasong extract as an active ingredient.

Diabetes mellitus is the most representative disease of endocrine disorders caused by the failure of insulin, which acts on carbohydrate metabolism, to function normally. It occurs mainly and is classified as type 2 diabetes in which each tissue in the body exhibits symptoms of insulin resistance and non-responsiveness to insulin.

As the main therapeutic agents for diabetes, sulfonylureas-based drugs, biguanides-based orally ingested hypoglycemic agents, and insulin obtained by DNA recombination are used. However, these drugs have many side effects, such as hypoglycemia induction, renal toxicity, lactic acidosis, diarrhea and upset stomach, and immune rejection.

On the other hand, Wasong (瓦松) is called Orostachys japonicus A. Berger, and is a perennial herbaceous plant of the Crassulaceae family, also called cypress (岩松), jade pine (屋松), and sedumaceae (昨葉何草). It is used in oriental medicine for antipyretic, epilepsy, hemostasis, and eczema [金在佶, 1984, 原色天然藥物大事典(上卷), Namsan堂, Seoul, p.447] and is widely used in cancer treatment as folk remedies [ Bae Seong-sik, 1990, Oriental Medicine and Health, 1, 26].

Although this plant is a fleshy perennial herb that grows by attaching to rocks, it has a characteristic that it dies when flowers bloom and bear fruit. The rosette-shaped leaves spread in a rosette-like shape and become thorn-like with leaves hanging on the main stem, without petioles. Together with the rosette leaves in summer, the end does not harden but only becomes sharp. It is lanceolate, green, but purple or white. have. The flowers bloom in September and are white, and the raceme is 6-15cm long, and the flowers without a vase are closely attached, and the bracts are lanceolate and have a sharp tip. The sepals are 5, lanceolate, green, and 5 petals, lanceolate acute, about 6mm long. Stamens are 10, longer than petals, have 5 ovaries, and the pollen is red, but gradually turns black [Changbok Lee, 1979, Daehanmunsa Seoul, p.402]. Wasong is harvested from summer to autumn, and the whole plant with its roots removed is dried in the sun for medicinal use [金在佶, 1984, 原色天然藥物大事典(上卷), Namsan堂, Seoul, p.447] .

According to the herbalist review of Wasong in the literature, it is said that Dangboncho (唐本草) is used for dry mouth and pain (乾燥), extracting water from the subarachnoid blood of the large intestine and taking it, and applying it to all wounds. It is said to have an astringent effect, and herbal remedies are said to treat white poison (百毒) and eliminate bells (腫). It is said to be effective against (瘡) and oil (癒) [上海科学技術出版社 小学館編; 中藥大事典, 小学館, 1985, p.526].

It is known that such a wasong has an effect of improving skin wrinkles (Patent Document 1) and suppressing a biofilm (Patent Document 2).

The inventors of the present application found that the Wasong extract as above is effective in improving diabetes by lowering blood sugar, and completed the present invention.

KR 10-1685581 B1 (2016. 12. 06.) KR 10-2019-0082704 A (2019. 07. 10.)

An object to be solved in the present invention is to provide a composition capable of improving, preventing and treating diabetes using a plant-based extract.

The composition according to the present invention for solving the above problems is characterized in that it contains a Wasong extract as an active ingredient to provide a food composition for preventing or improving diabetes.

The composition according to the present invention for solving the above problems is characterized in that it contains a Wasong extract as an active ingredient to provide a pharmaceutical composition for preventing or treating diabetes.

The composition according to the present invention can minimize side effects in the human body using plants, and can effectively prevent, improve or treat diabetes by lowering blood sugar levels to a normal range and activating pancreatic beta cells at the same time.

1 is a subcutaneous and intra-abdominal adipose tissue autopsy result photograph (based on wasong alcohol extract)
Figure 2 is a picture of the autopsy result of adipose tissue of the liver (based on wasong alcohol extract)
3 is a subcutaneous and intra-abdominal adipose tissue autopsy result photograph (based on wasong hot water extract)
4 is a picture of the autopsy result of adipose tissue of the liver (based on wasong hot water extract)
Figure 5 is a graph showing the weight measurement results by number of weeks (based on wasong alcohol extract)
6 is a graph showing the weight measurement results after autopsy (based on wasong alcohol extract)
7 is a graph showing the liver weight, liver weight / weight ratio, and kidney weight results after autopsy (based on wasong alcohol extract)
8 is a graph showing the weight measurement results for each week (based on wasong hot water extract)
9 is a graph showing the weight measurement results after autopsy (based on wasong hot water extract)
10 is a graph showing the liver weight, liver weight / weight ratio, and kidney weight results after autopsy (based on wasong hot water extract)
11 is a graph showing the measurement results of blood sugar and glycated hemoglobin (based on wasong alcohol extract)
12 is a graph showing the measurement results of blood sugar and glycated hemoglobin (based on wasong hot water extract)
13 is a graph showing the results of an oral glucose tolerance test (based on wasong alcohol extract)
14 is a graph showing the results of the oral glucose tolerance test (based on wasong hot water extract)
15 is a graph showing the measurement results of liver damage levels (ALT, AST, BUN) (based on wasong alcohol extract)
16 is a graph showing the measurement results of liver damage levels (ALT, AST, BUN) (based on wasong hot water extract)
17 is a graph showing the measurement results of triglycerides and total cholesterol (based on wasong alcohol extract)
18 is a graph showing the measurement results of triglycerides and total cholesterol (based on wasong hot water extract)
19 is a photograph showing the results of H & E staining of the liver (based on wasong alcohol extract)
20 is a photograph showing the results of H & E staining of the liver (based on wasong hot water extract)
Figure 21 is a photograph showing the results of H & E staining of the pancreas (based on wasong alcohol extract)
22 is a photograph showing the result of H & E staining of the kidney (based on wasong alcohol extract)
23 is a photograph showing the result of H & E staining of the kidney (based on wasong hot water extract)
24 is a graph showing the expression results of genes related to lipid metabolism and lipid biosynthesis (based on wasong alcohol extract)
25 is a graph showing the expression results of lipid metabolism and lipid biosynthesis related genes (based on wasong hot water extract)
26 is a graph showing the expression results of inflammation-related genes (based on wasong alcohol extract)
27 is a graph showing the expression results of glucose receptors and glucose neosynthesis-related genes (based on Wasong alcohol extract)
28 is a graph showing the expression results of glucose receptors and glucose neosynthesis-related genes (based on wasong hot water extract)
29 is a graph showing the results of gene expression analysis of the insulin resistance experimental model (based on wasong alcohol extract)

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.

Example 1. Wasong hot water extract preparation

Wasong was purchased from the Agricultural Cooperative Corporation located in Namwon. 1 kg of wasong dry powder was put in 10 L of distilled water and extracted by boiling at a temperature of 100° C. for 3 hours. The extract was freeze-dried to complete the Wasong hot water extract.

Example 2. Wasong alcohol extract preparation

Wasong was purchased from the Agricultural Cooperative Corporation located in Namwon. 1 kg of wasong dry powder was put into 10 L of alcohol, and the mixture was extracted under reflux twice while stirring overnight using a homogenizer. The extract was freeze-dried to complete the Wasong alcohol extract.

Experimental Example 1. Comparison of necropsy gross findings and liver weight and body weight

1-1. Experiment preparation

We obtained 24 and 32 db/db mice, a model for expressing type 2 diabetes by genetic predisposition, and db /+ 5-week-old mice, respectively, and after 2 weeks of acclimatization, db /+ mice at 7 weeks of age were obtained. 6 mice per group and 8 mice per group for db/db mice.

Dissolve wasong alcohol extract and hot water extract in physiological saline, and administer 50, 100, and 200 mg/mL for alcohol extract and 100, 200, and 400 mg/ml for hot water extract 5 times a week for 8-10 weeks. did.

1-2. experimental process

After 10 weeks of the experiment, an autopsy was performed.

1-3. Experiment result

(1) Result of autopsy visual findings

Compared to the db/+ group in the diabetic db/db mouse group, It was confirmed that the size of adipose tissue in the subcutaneous and abdominal cavity was increased.

On the other hand, as shown in FIGS. 1 and 2, changes in the shape and size of adipose tissue or liver by the administration of Wasong alcohol extract could not be confirmed.

And as shown in FIGS. 3 and 4, it was confirmed that the adipose tissue was slightly decreased by the administration of the Wasong hot water extract, but the change in the shape and size of the liver could not be confirmed.

(2) Liver weight and weight measurement results

Compared to the db/+ group in the diabetic db/db mouse group, The results of increased body weight and liver weight were confirmed.

On the other hand, as shown in FIGS. 5 to 7 , it was confirmed that the body weight was significantly reduced by administration of the Wasong alcohol extract, but the statistical significance of the liver and kidney weights could not be confirmed. As a result, it was confirmed that the administration of wasong alcohol extract induces weight loss in diabetic mice.

And as shown in FIGS. 8 and 9 , it was confirmed that the body weight decreased by administration of the Wasong hot water extract, but statistical significance could not be confirmed. As shown in FIG. 10, no change in liver and kidney weights due to administration of wasong hot water extract could be confirmed.

Experimental Example 2. Evaluation of the effect on the antidiabetic index

2-1. Experiment preparation

Mice were prepared as in 1-1.

2-2. experimental process

After fasting for at least 8 hours, (1) blood glucose level and (2) glycated hemoglobin level (HbA1c) were measured for 1-1 mice, and then (3) sugar (2 g/kg body weight) was orally administered and blood glucose level was measured. Glucose levels were measured after 0, 30, 60, 90, and 120 minutes, respectively, to evaluate the ability to control blood sugar.

2-3. Experiment result

(1) Results of blood glucose measurement after fasting

As shown in FIG. 11 , it was confirmed that blood sugar significantly decreased in the diabetic mouse group receiving the Wasong alcohol extract. Based on this result, as a result of comparative analysis of the difference with the initial blood sugar level, it was confirmed that the amount of change in blood sugar was also reduced by the administration of wasong alcohol extract.

And as shown in FIG. 12 , it was confirmed that the blood glucose level was slightly decreased in the diabetic mouse group receiving the Wasong hot water extract. As a result of comparative analysis of the difference with the initial blood sugar level based on these results, it was confirmed that the amount of change in blood sugar was also slightly decreased by the administration of wasong hot water extract.

(2) Glycated hemoglobin (HbA1c) measurement result

As shown in FIG. 11 , it was confirmed that the measured value of glycated hemoglobin significantly decreased in the diabetic-induced mouse group receiving the Wasong alcohol extract.

And, as shown in FIG. 12 , it was confirmed that the measured value of glycated hemoglobin significantly decreased in the diabetic-induced mouse group that received the high concentration of Wasong hot water extract.

(3) Oral glucose tolerance test

In the db/db mouse group, it was confirmed that blood sugar was significantly increased compared to the initial blood sugar even after 120 minutes.

As shown in FIG. 13 , in the case of the Wasong alcohol extract administered group, it was confirmed that the blood sugar decreased significantly over time after the administration of the sugar. These results indicate that the blood sugar control ability is improved in diabetic mice by administration of the Wasong alcohol extract.

And, as shown in FIG. 14 , in the case of the wasong hot water extract administered group, it was confirmed that the blood sugar significantly decreased compared to the non-administered group. These results indicate that the glycemic control ability is improved in diabetic mice by administration of the wasong hot water extract.

Experimental Example 3. Diabetes-induced liver damage and fat deposition

3-1. Experiment preparation

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

3-2. experimental process

Serum was obtained by centrifugation of blood samples collected from mice of 3-1, (1) liver damage-related enzymes ALT (alanine aminotransferase), AST (aspartate transaminase) and BUN (blood urea nitrogen) were measured, (2) ) 3-1 Triglyceride (hereinafter 'TG') and total cholesterol (total cholesterol, hereinafter 'TC') were measured in livers collected from mice.

3-3. Experiment result

(1) Results of liver damage measurement

As shown in FIG. 15 , it was confirmed through serological analysis that the ALT and AST levels were significantly decreased by the administration of the Wasong alcohol extract. This means that liver damage in diabetic mice is reduced by the administration of wasong alcohol extract.

Through serological analysis, it was confirmed that there was no difference in the BUN level by the administration of Wasong alcohol extract.

And, as shown in FIG. 16, it was confirmed through serological analysis that the AST level was significantly decreased by the administration of the Wasong hot water extract. However, no statistically significant results were found in the ALT level. This means that liver damage in diabetic mice was reduced by administration of wasong hot water extract.

Through serological analysis, it was confirmed that the BUN level increased in the diabetic db/db mouse group compared to the db/+ group was decreased by the administration of the Wasong hot water extract.

(2) TG and TC measurement results

As shown in FIG. 17, as a result of measuring the amount of triglyceride and cholesterol in the liver, it was confirmed that the fat accumulation was significantly reduced by the wasong alcohol extract administration.

And as shown in FIG. 18, as a result of measuring the amounts of triglycerides and cholesterol in the liver, the difference in fat accumulation could not be confirmed by the administration of the Wasong hot water extract.

Experimental Example 4. Analysis of histopathological changes in the liver

4-1. Experiment preparation

The liver tissue of the mouse autopsied in Experimental Example 1 was prepared.

4-2. experimental process

After formalin fixation, hepatic pathological changes caused by diabetes were observed through H&E staining of tissue sections that had undergone a general treatment process.

4-3. Experiment result

In the case of the Wasong alcohol extract administration group, as shown in FIG. 19 , hepatocellular hypertrophy (ballooning degeneration) and stratosis were observed in the db/db mouse group as a result of liver H&E staining, and this aspect is the Wasong alcohol It was confirmed that it was significantly reduced by administration of the extract.

And in the case of the Wasong hot water extract administration group, as shown in FIG. 20 , hepatocyte degeneration and fat deposition were observed in the db/db mouse group as a result of H&E staining of the liver, and this aspect was slightly reduced by the administration of the Wasong hot water extract. Confirmed.

Experimental Example 5. Analysis of histopathological changes in the pancreas

5-1. Experiment preparation

The pancreas tissue of the mouse autopsied in Experimental Example 1 was prepared.

5-2. experimental process

Pathological changes in the pancreas due to diabetes were observed through H&E staining of tissue sections that had undergone a general treatment after formalin fixation.

5-3. Experiment result

In the case of the Wasong alcohol extract administered group, as shown in FIG. 21 , as a result of pancreatic H&E staining, a marked increase in the area of the islet of Langerhans in the pancreas due to insulin resistance was observed in the db/db mouse group. It was confirmed that these changes were reduced by the administration of wasong alcohol extract.

Experimental Example 6. Analysis of histopathological changes in the kidney

6-1. Experiment preparation

The kidney tissue of the mouse autopsied in Experimental Example 1 was prepared.

6-2. experimental process

After formalin fixation, the tissue sections that had undergone the general treatment process were subjected to H&E staining to observe pathological changes in the kidneys due to diabetes induction.

6-3. Experiment result

In the case of the Wasong alcohol extract-administered group, as shown in FIG. 22, as a result of observing changes in kidney histopathology by administration of the Wasong alcohol extract in diabetic mice through staining, there was no significant difference in renal glomerular damage and glycogen deposition. was confirmed.

And in the case of the wasong hot water extract administration group, as shown in FIG. 23, as a result of observing the change of kidney histopathology by administration of the wasong hot water extract in diabetic mice through staining, there was no significant difference in renal glomerular damage and glycogen deposition. The results were confirmed.

Experimental Example 7. Fat-related gene expression analysis

7-1. experimental process

cDNA was synthesized by extracting mRNA from the liver tissue of the mouse autopsied in Experimental Example 1. 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 Wasong-Yeoju complex extract.

7-2. Experiment result

In the case of the Wasong alcohol extract administered group, as shown in FIG. 24, the expression of Apo B, LPL, L-FABP, and FABP4, which are lipid metabolism-related factors, was comparatively analyzed. As a result, it was significant in the liver of diabetic mice by the Wasong alcohol extract administration. A negative decrease was confirmed.

As a result of comparative analysis of the expression of ACCα, ACCβ, FASN, and SCD1, which are factors related to lipid biosynthesis ( de novo lipogenesis), it was confirmed that the administration of Wasong alcohol extract significantly decreased the liver of diabetic mice.

As a result of comparative analysis of the expression of electronic regulators closely related to these changes and their related factors, SREBP-1, SREBP-2, LXRα, and LXRβ, it was significantly reduced in the liver of diabetic mice by administration of wasong alcohol extract. The result was confirmed. On the other hand, it was confirmed that the expression of PPARα, which is known to have hepatoprotective and antidiabetic effects, slightly increased.

And in the case of the wasong hot water extract administration group, as shown in FIG. 25, as a result of comparative analysis of the expression of L-FABP and ApoB, which are factors related to lipid metabolism, ApoB significantly decreased in the liver of diabetic mice by administration of the wasong hot water extract. The result was confirmed.

As a result of comparative analysis of the expression of ACCα, ACCβ, and FASN, which are factors related to de novo lipogenesis, it was confirmed that the expression of ACCα and ACCβ was significantly reduced in the liver of diabetic mice by administration of wasong hot water extract. .

As a result of comparative analysis of the expression of electronic regulators closely related to these changes and their related factors, LXRα and LXRβ, statistical significance could not be confirmed in the liver of diabetic mice by the administration of hot water extract of Wasong, but it was slightly decreased. was confirmed. On the other hand, it was confirmed that the expression of PPARα, which is known to have hepatoprotective and antidiabetic effects, slightly increased.

Experimental Example 8. Inflammation-related gene expression analysis

8-1. experimental process

cDNA was synthesized by extracting mRNA from the liver tissue of the mouse autopsied in Experimental Example 1. Relative quantitative analysis was performed on the expression changes of various genes through qRT-PCR to compare and analyze changes in gene expression patterns caused by the Wasong-Yeoju complex extract.

8-2. Experiment result

As a result of confirming the expression of TNF-α, IL-6, and IL-1β, which are representative inflammation-related factors, it was confirmed that the result was significantly decreased in the liver of the mice administered the Wasong alcohol extract as shown in FIG. .

Based on these analysis results, it was confirmed that the treatment of Wasong alcohol extract reduces liver damage by suppressing the expression of inflammatory factors.

Experimental Example 9. Analysis of gene expression related to sugar metabolism

9-1. experimental process

cDNA was synthesized by extracting mRNA from the liver tissue of the mouse autopsied in Experimental Example 1. 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 Wasong alcohol extract.

9-2. Experiment result

In the case of the Wasong alcohol extract, as shown in FIG. 27 , the expression of the sugar receptors (GLUT2, GLUT4) was confirmed. As a result, it was confirmed that there was no significant difference in the expression of the sugar receptors by the administration of the Wasong alcohol extract.

As a result of confirming the expression of gluconeogenesis-related genes (G6Pase, PEPCK), it was confirmed that the expression of G6Pase, a gluconeogenesis-related gene, was significantly decreased in the liver of diabetic mice by the wasong alcohol extract treatment.

Based on these analysis results, it was confirmed that the treatment of Wasong alcohol extract activates insulin signaling and plays a good role in glycemic control by inhibiting hepatic gluconeogenesis and increasing glucose inflow.

And in the case of the wasong hot water extract, as shown in FIG. 28, as a result of confirming the expression of the sugar receptors (GLUT2, GLUT4), it was confirmed that the expression of the sugar receptor was significantly increased by administration of the wasong hot water extract.

As a result of confirming the expression of the gluconeogenesis-related gene (G6Pase), it was confirmed that the expression of G6Pase, a gluconeogenesis-related gene, was slightly decreased in the liver of diabetic mice by the wasong alcohol extract treatment.

Based on these analysis results, it was confirmed that the treatment of Wasong alcohol extract activates insulin signaling and plays a good role in glycemic control by inhibiting hepatic gluconeogenesis and increasing glucose inflow.

Experimental Example 10. Antidiabetic Efficacy in vitro evaluation

10-1. experimental process

HepG2 cells, a hepatocarcinoma cell line, were treated with either PA at a concentration of 0.4 mM or High Glucose (4.5 g/mL) + Insulin (2.5 μg/mL) to induce insulin resistance, fat accumulation and cell damage.

After confirming the concentration of wasong alcohol extract without cytotoxicity, three concentrations were determined and cultured for 24 hours after treatment.

Relative quantitative analysis was performed on the expression changes of various genes through qRT-PCR to compare and analyze changes in gene expression patterns caused by the Wasong alcohol extract at the cellular level.

Three concentrations (10 ng/mL, 100 ng/mL, and 1 μg/mL) at which cytotoxicity does not occur statistically were established.

10-2. Experiment result

Referring to FIG. 29 , it was confirmed that the treatment of Wasong alcohol extract significantly increased GLUT4 in hepatocytes treated with PA or high glucose+insulin, and the expression of G6Pase was significantly decreased in the high glucose+insulin model. was confirmed.

It was confirmed that the treatment of Wasong alcohol extract increased the expression of PPARα in hepatocytes treated with PA or high glucose+Insulin.

It was confirmed that the treatment of Wasong alcohol extract reduced the expression of inflammatory factors in hepatocytes treated with PA or high glucose + Insulin, which was similar to the in vivo experimental results.

Based on these results, it was confirmed that the anti-diabetic effect of Wasong alcohol extract was effective at the cellular level.

Claims (4)

A food composition for preventing or improving diabetes containing Wasong extract as an active ingredient.
The method according to claim 1,
The Wasong extract is a food composition for preventing or improving diabetes, characterized in that it is an alcohol extract.
The method according to claim 1,
The diabetes is a food composition for preventing or improving diabetes, characterized in that type 2 diabetes.
A pharmaceutical composition for preventing or treating diabetes containing a Wasong extract as an active ingredient.

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