KR101146185B1 - reduce blood sugar having echinoid shell powder the water soluble extract - Google Patents

Abstract

The present invention relates to a water-soluble extract of sea urchin powder having a hypoglycemic activity, and more specifically, to a hypoglycemic composition comprising the water-soluble extract of sea urchin shell powder having a hypoglycemic activity as an active ingredient. After sea urchins are removed, the sea urchin shells are collected, washed, dried, and the sea urchin shells are powdered. The water-soluble extracts of the sea urchin shell powders are extracted by a water-soluble solvent extraction method using a powdered sea urchin powder sample as a solvent. The present invention relates to a composition having hypoglycemic activity as an active ingredient, which is a water-soluble extract of sea urchin powder that exhibits excellent hypoglycemic activity in vitro and in vivo and effectively inhibits blood glucose formation.

Since the water-soluble extract of the sea urchin shell powder of the present invention exhibits excellent blood sugar lowering ability, it can be usefully used as an antihyperglycemic pharmaceutical composition and food composition effective for diseases such as diabetes and hypertension due to high blood sugar, circulatory system, and the like.

Sea urchin, water soluble extract, hypoglycemic,

Description

Reduce blood sugar having echinoid shell powder the water soluble extract}

The present invention relates to a hypoglycemic composition comprising a water-soluble extract of sea urchin shell powder having a hypoglycemic activity as an active ingredient. First, the sea urchin is degreased, the sea urchin shell is collected, washed with water, dried, and then the sea urchin shell is powdered. After that, the water-soluble extract of sea urchin shell powder extracted from the powdered sea urchin powder sample using distilled water as a solvent shows an excellent hypoglycemic activity in vitro and in vivo, effectively inhibiting the formation of blood glucose in the blood. The present invention relates to a composition having hypoglycemic activity, wherein the water-soluble extract of sea urchin shell powder is used as an active ingredient.

    In general, diabetes is one of the most common endocrine diseases in modern people. It is a disease caused by impaired production of insulin secreted by beta cells of the pancreas, an organ that regulates blood sugar, or by insulin in vivo due to reduced biological effects (insulin resistance) on target cells. It is one of the chronic degenerative diseases that is fundamentally difficult to heal (Reaven and Laws, 1994; Landau et al., 1996). The prevalence of diabetes is on the rise worldwide and is classified into Insulin Dependent Diabetes Mellitus (IDDM) and Non-Insulin Dependent Diabetes Mellitus (NIDDM) (Kurt et al., 1997 Beckman). et al., 2002). Insulin-independent diabetes mellitus is caused by insulin resistance of insulin target organs such as liver, fat, muscle, etc., and is caused by low insulin secretion and abnormal signaling system for glucose uptake and metabolism of cells. The accompanying metabolic disorders are chronic endocrine and metabolic disorders of long-term condition and can occur in all age groups (Cavaghan et al., 2000). Persistent diabetes is known to be a serious risk factor for cardiovascular system, a major complication of diabetes such as retinal, kidney and nerve lesions, and lipid metabolism abnormalities. Type 2 diabetes can be caused by large amounts of free radicals and oxidative damage. It is known to be a disease associated with various metabolic reductions (Hayden et al., 2005). Diabetes is a refractory disease, and treatment and improvement methods such as drug treatment, exercise therapy, and dietary therapy are being implemented, but the number of diabetics is increasing rapidly. Therefore, the development of diabetes medications is very important given the current situation in which continuous and proper treatment for diabetes is difficult. Current anti-diabetic agents include glycolysis inhibitors that prevent glucose from being absorbed into the body, insulin resistance enhancers that help insulin work, and insulin secretagogues. However, the basic treatment and improvement of existing insulin or hypoglycemic agents of various mechanisms have limitations and are accompanied by economic burdens and risks of drug side effects. Research on the hypoglycemic effect of food-derived and development of related products are being activated.

    The sea urchin's processing by-products are waste and costly to treat as well as environmental pollution. Recently, processing by-products are also recognized as a resource, and efforts and research and development have been made to use such by-products. However, the processing by-products of many marine products, including sea urchins, are still being discarded without being recognized for their use as wastes, and in small enterprises, the waste of these wastes is a burden on corporate management.

   Sea urchins are distributed on the east coast of Korea, along the coast of Japan and China, and are called as Haedam in oriental medicine and folk medicine. They are also known to improve tuberculosis, neuralgia, and alcohol degradation, expectorant and tonic. (Yu, 1999; Kim et al., 2002)

  The annual production of sea urchins is 2,500 tons, 20% of which are used for food and about 80% of them are discarded, which is highly likely to cause serious environmental problems if left untreated. Sea urchin has high protein, amino acid and fatty acid composition, and calcium isolated from sea urchin shell is developed as a calcium supplement and marketed as a medicine.In some poultry farms, it is high in calcium and high in unsaturated fatty acids when sea urchin is administered to laying hens. There is a research report suggesting that this is possible (Nam, 1986 Kim et al., 2002). In the East Coast of Korea, the destruction of ecosystems due to the oversaturation of sea urchins is intensifying. This was caused by the local governments and fishermen spraying sea urchins in East Sea in 2003-2005 in order to secure sea urchin resources for export to Japan. This surge, however, was slowed by price competition with Chinese sea urchins, and the excessive concentration of sea urchins resulted in more than 15% of fish farming on the east coast, which caused severe destruction of the ecosystem. . Research and reporting on the overall biofunctionality of sea urchins are weak at home and abroad, and the development of various life science-related products using sea urchins will have a large environmental and economic ripple effect based on national health promotion.

    However, these sea urchins are selectively collected only, and the rest of the sea urchin shells and other wastes are discarded as wastes, and these wastes have been raised as a cause of environmental pollution, and their treatment is emerging as an important problem.

     Therefore, the present inventors have made a diligent research to find a new substance having hypoglycemic activity from natural products. As a result, the extract extracts a water-soluble fraction having a high hypoglycemic activity from the shell of sea urchin, which is a marine organism. Aqueous extract of sea urchin shell powder was used for blood glucose, diabetes, hypertension, cataracts, fatty liver, arteriosclerosis, and cardiovascular system, using GK rat, a model animal of non-obese type insulin-independent diabetes, which has been diagnosed with diabetes, hyperglycemia and impaired glucose tolerance. It is to provide a water-soluble extract of sea urchin shell powder having a hypoglycemic activity that can be usefully used for the prevention and treatment of.

To this end, the Goto-Kakizaki (GK) rats were spontaneously developed type 2 diabetic models that exhibited insulin resistance due to decreased insulin secretion due to pancreatic beta cell dysfunction (Kim and Yokoyama, 1997). ) Was used as an experimental animal.

As described above, since the water-soluble extract of the sea urchin shell powder of the present invention exhibits excellent hypoglycemic activity, effectively inhibiting blood glucose in the composition, the composition containing the same has diabetes, hypertension, cataract, fatty liver, arteriosclerosis, and cardiovascular disease caused by blood glucose. It can be usefully used as a hypoglycemic composition and a food composition effective for diseases such as a machine. Therefore, the present invention not only has the effect of producing high value-added natural materials from sea urchins, which is one of the unused waste resources, but also contributes to the improvement of national health by eliminating pirates and developing new hypoglycemic substances.

In order to achieve the above object, the present invention provides a composition having a hypoglycemic action comprising a water-soluble extract of sea urchin shell powder as an active ingredient, and also has a hypoglycemic action comprising a water-soluble extract of sea urchin powder as an active ingredient To provide a composition.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples, and various modifications can be made without departing from the scope and spirit of the present invention. Is possible.

[Example 1] Production of a sample powder and sea urchin

One. Sea urchin powder  Manufacturing method

 10 kg of sea urchin (Hemicentrotus pulcherrimus) collected from the vicinity of Pohang, Gyeongsangbuk-do, and shelled, washed with sea urchin shell (hereafter referred to as 'sea urchin'), and the washed sea urchin samples are divided into 2 parts at 24 ° C and 60 ° C. Drying step (1) to dry by hot air drying for a time until the moisture content is less than 10%;

 Dry sea urchin is a grinding step (2) of grinding into a powder of 20 ~ 30 mesh using a grinder (A 11 basic Analytical mill, IKA, Germany);

 The sea urchin powder sample powdered in the grinding step (2) is an extraction step (3) of performing reflux cooling extraction at 100 ° C. for 3 hours by adding distilled water 10 times based on dry weight;

The extracted extract is filtered with a filter paper (Watman No. 2) (4);

The filtered extract is concentrated step (5) for producing a concentrated solution at a concentration of 50 ~ 60 ° Brix at 40 ℃ using a rotary vacuum condenser (N-1000S-W, Eyela, Japan);

The concentrated sea urchin concentrate is freeze-dried using a freeze-drier (Free Zone 6, LABCONCO, USA) to freeze-drying step (6) through a sequential step to prepare a sample of concentrated aqueous extract of sea urchin shell powder ( See FIG. 1).

The concentrated sample of the water-soluble extract of sea urchin shell powder was adjusted by using distilled water as a solvent according to the capacity of each experimental group.

2. Experimental Animal

The experimental animals were 12-week-old male GK rats (Goto-Kakizaki rats, body weight: 228.5 ± 15.4 g, central laboratory animals, Seoul) produced by SLC, Japan, and a control group of Wistar male rats of the same age was used. Used. Animals were reared in a controlled environment with temperature (23 ± 2 ° C, relative humidity (50 ± 5%) and 12 hour contrast cycle (07:00 lit, 19:00 off). The animals were fed solid feeds (purina Korea, Osan) for experimental animals during the experiment, and the drinking water was freely fed with filtered sterilized purified water. It was conducted in accordance with the Center's Standard Operation Procedures (SOP) with the approval of the Care and Use Committee (IACUC).

3. Experimental  Classification

Experimental animals in each experimental group were placed evenly based on the weight of 7 heads, the experimental group was normal group consisting of Wistar rat (●), GK rat (Goto-Kakizaki rats, hereinafter referred to as "GK rat") control (○), GK rats consisted of a total of four experimental groups, including two experimental groups orally administered sea urchins at different doses. The normal group did not receive any treatment, and the urchin group was administered once daily for 4 weeks using an oral syringe at doses of 100 mg / kg (▼) and 200 mg / kg (△), based on dry weight per kg body weight. The GK rat control group was orally administered the same dose of distilled water for the same period. In the experimental group, body weight, blood sugar, plasma insulin concentration, and the like were measured every week.

4. Analyze Sampling and Analysis

For blood collection, the animals were lightly anesthetized with ether every week, blood was collected from the orbital vein, and the blood glucose level was measured. After centrifugation at 1,500 rpm for 20 minutes, only serum was taken at -80 ° C to measure insulin concentration. Stored.

Blood glucose was measured using a blood glucose meter (Super Glucocard II, Japan), and serum insulin content was measured using an ELISA kit (Mercodia rat insulin ELISA kit, Sweden).

5. Oral sugar load  inspection

After 4 hours of dosing, the animals were fasted 12 hours 24 hours before sacrifice, and then 2 g / kg body weight was administered orally using 50% glucose solution. Blood glucose was measured prior to glucose administration (12-hour fasting blood glucose), and at 30, 60, 90, and 120 minutes after glucose administration.

6. Histological Examination

After the final blood collection, the animal was sacrificed and opened, and the extracted pancreas was fixed in 10% neutral formalin solution for at least 7 days, then treated with tissue, embedded in a paraffin embedding machine (Leica EG 116), and embedded in a rotary microtome (Leica 820). 4㎛ sections were made and stained with hematoxylin & eosin and observed with an optical microscope.

7. Immunohistochemical Testing of the Pancreas

The isolated pancreas was fixed in 10% neutral formalin for at least 48 hours, and then embedded in paraffin by performing general histopathological procedures. Paraffin block were attached to the slide to create a fragment with 4 ㎛ interval coated with poly-lysine and subjected to dehydration, vitrification and penetration process using the xylene and ethanol, the tissue is at room temperature in 3% H ₂ O ₂ solution 10 minutes treatment. The tissues were reacted with guinea pig-anti-swine insulin antibody at a dilution of 1: 100 for 30 minutes and treated with horseradish peroxidase-bound dextran polymer for 30 minutes. Washing was carried out every process using Tris buffer (pH 7.6) composed of 0.5% tween 20. Finally, diaminobenzidine was developed and background stained with hematoxylin and 0.3% ammonia water.

8. Statistical Analysis

All data were expressed as mean ± standard error and statistical analysis was performed using the Statistical Package for Social Science (SPSS), version 15.0 (SPSS Inc, Chicago, IL) computer program. Mann-Whitney test was used to compare the two groups. One-way analysis of variance (ANOVA) was used to compare the three groups. P <0.05 was considered statistically significant.

[Experiment result]

1. Effect on weight change

Figure 2 shows the effect of the four-week treatment of sea urchin extract weight change of GK rats, the weight of each group was 76% of the weight of the GK rats compared to the Wistar rat (●) at the start of the experiment and after 4 weeks Reached 85%. The experimental group that received sea urchin was lower than the control group which did not receive any body weight depending on the dose-dependent treatment after 2 weeks, but the GK rat control group (○) and the experimental group which administered sea urchin at the dose of 100 mg / kg (▼) , No statistical significance was found between the experimental groups (△) who received sea urchin at 200 mg / kg.

2. Effect on blood sugar

Figure 3 shows the effect of four-week treatment of sea urchin extract on the blood glucose change in GK rats, the change in blood glucose in the experimental group compared to the blood glucose (111.0 ± 11.5 mg / ㎗) of the normal control group Wistar rat (●) The blood glucose of rat (○) was markedly high hyperglycemia of 197.2 ± 10.5mg / dl, and the hyperglycemia was maintained during the experimental period.

After 2 weeks after the urchin was administered to these GK rats, a decrease in blood glucose was observed. Significance was recognized in the experimental group (△) administered the urchin at 200 mg / kg after 3 weeks. Although statistical significance was not recognized in the experimental group (▼) administered at the dose of 100 mg / kg, the same level of hypoglycemia was shown, although there was a difference in degree from the experimental group administered the high dose (200 mg / kg).

3. In plasma  Changes in insulin concentration

4 shows the effect of four-week treatment of sea urchin extract on the plasma insulin concentration of GK rats. In the experimental group, the plasma concentration of insulin in the control group, the concentration of insulin in Wistar rats (●) It was 22.1 ± 0.9 μU / ml, but the insulin concentration was low at 17.2 ± 1.0 μU / ml in the GK rat control group (○). When sea urchin was administered to GK rats slightly increased after 3 weeks, it was not recognized as a significant change. Therefore, it was confirmed that sea urchin had no effect on direct insulin secretion.

4. Oral sugar load  Changes in blood sugar in the test

Figure 5 shows the effect of the treatment of sea urchin extract for 4 weeks on the change of blood glucose according to the oral glucose loading test of GK rat, In the control of blood glucose by oral glucose loading test, almost no change in blood glucose was observed in the control group of Wistar rats (●) until 120 minutes after oral administration of glucose, but GK rats (○) were treated before and 30 minutes after glucose administration. At 198.5 ± 11.3 ㎎ / ㎗ and 342.7 ± 13.8 ㎎ / 각각, respectively, about 73% of blood glucose was elevated and blood glucose was gradually lowered over time, indicating 299.4 ± 14.2 ㎎ / 에서는 at 120 minutes after glucose administration. However, in the experimental group treated with sea urchin, the highest blood glucose level was lowered after 30 minutes after glucose administration, and then showed the same pattern as the control group. In the experimental group (△) administered at a dose of 200 mg / kg showed a significant decrease in blood glucose compared to the control group at 90 and 120 minutes.

5. Histology and Immunohistochemical Examination of the Pancreas

Histological changes of the pancreas and immunohistochemical changes for insulin are shown in Figure 6 (A, B, C, D). In hematoxylin & Eosin staining, the pancreas (B) of GK rats, pancreatic islet cell colonization, pancreatic islet formation and atrophy and irregular arrangement were observed in pancreas (B) of Wistar rats. These changes were also observed in the sea urchin group, but overall pancreatic islet cell size, phobic changes, and arrangement and size of the progenitor cells showed more normal forms (C, D) than the control group. Could not be confirmed.

In addition, the results of immunostaining for insulin in the pancreas are shown in Fig. As shown in FIG. 6 (E, F, G, H), the distribution of beta cells positive to insulin was significantly suppressed in the GK rat control group compared to the pancreas (E) of the normal group Wistar rats (F). In the experimental group treated with sea urchin, there was an increase in the appearance of insulin-positive cells compared to the GK rat control group, but no clear increase in insulin-positive cells was observed to indicate a decrease in blood glucose (G, H).

Finally, diabetes can be divided into insulin-independent and insulin-independent, which are slowly progressed over a long period of time depending on pathophysiology, and other malnutrition, pregnancy-related or other secondary diabetes, etc. (Kim and Min, 2005; Seok et al., 2007). Most patients suffering from domestic diabetes mellitus are insulin-independent diabetes mellitus induced after middle age, and hyperglycemia and urine glucose excretion are caused by decreased insulin secretion and action, and therefore proper treatment and dietary management are required (Cambell & Steil, 1998). The underlying factors of insulin-independent diabetes are the lack of insulin and the desensitization of insulin in tissues. (Taylor, 1994) Insulin resistance is a hallmark of insulin-independent diabetes and is an essential factor in the hyperglycemic state of diabetes. (DeFronzo, 1992). Therefore, it is important to consider the improvement of glucose homeostasis when using a therapeutic drug that inhibits insulin resistance.

Currently, oral hypoglycemic agents such as sulfone urea, biguanide-based drugs, alpha-glucosidase inhibitors, and thiazolidinedione-based drugs, insulin therapy, and pancreas transplantation are used for the treatment of diabetes (Kim and Min, 2005). The drugs that are applied to can cause side effects such as hypoglycemia and hepatotoxicity if used for a long time, and the efficacy is limited. Therefore, a lot of research has been focused on natural ingredients that have no side effects and are effective for diabetes symptoms.

On the other hand, GK rats are spontaneously developed diabetic rats established by repeatedly mating selection as indicators of decreased glucose tolerance from Wistar rats, and are specific insulin-independent diabetic model animals that simultaneously exhibit pancreatic insulin secretion as well as insulin resistance. (Goto et al., 1976).

This study was conducted to investigate the effect of sea urchin on the blood glucose and insulin resistance of diabetes using GK rat, a model animal of insulin independent diabetes. After four weeks of oral administration of sea urchins at 100 and 200 mg / kg in GK rats, body weights were not significantly different among experimental groups, but they tended to decrease in proportion to the dose of sea urchins. It was judged that there is a possibility of formation and substitution.

In the experimental group, the change in blood glucose was observed in the dose-dependent form of sea urchin administration, and in the 200 mg / kg dose group, significant hypoglycemia was observed from 3 weeks after administration. It was confirmed that the hypoglycemia of the sea urchin appears to be dose-time dependent on the dose and time. However, the change in plasma insulin in the same blood was slightly increased in the experimental group of sea urchin, and there was no increase in insulin corresponding to the change in blood glucose. There was no direct connection. This fact suggests that the hypoglycemic effect of sea urchins promotes other mechanisms for hypoglycemia rather than increased insulin secretion. In general, as a mechanism for lowering blood glucose, it may be thought to promote insulin release from beta cells of the pancreas or to convert to metabolism and glycogen in peripheral tissues (Eriksson and Lindgrade, 1991). In this experiment, the effect of promoting the secretion of insulin by the administration of sea urchin was not observed, but the decrease in blood glucose was confirmed. Therefore, the sea urchin does not change the absolute amount of insulin, but it is believed that the insulin sensitivity is increased or blood glucose is lowered through other glycolytic systems, and it may be estimated that there will be subsequent improvement in insulin resistance. The glucose load test was performed on GK rats treated with sea urchins for 4 weeks. As a result, GK rats treated with sea urchins were inhibited after 30 minutes of glucose loading in proportion to the dose of sea urchins compared to the control group of GK rats. Significance was recognized. These results suggest that the use of sea urchin increases the glucose utilization rate and susceptibility to insulin in diabetic GK rats.

Further studies on the effects of improving insulin resistance were necessary, such as through the use of glucose clamp tests.

The morphological changes of the pancreas showed that pancreas of GK rats with urchins were observed in the pancreatic islet cells, colony formation of pancreatic islets, atrophy and irregular arrangement of the islet cells. Improvement effects were observed in which the fear changes were normalized and the arrangement and size of the cells were clearly constant. These results suggest that the treatment of sea urchin causes hypoglycemia, and the pancreatic endocrine system undergoes some morphological repair during the 4-week period. On the other hand, by performing immunostaining on insulin, the appearance of insulin-positive beta cells, which are significantly reduced in the pancreas of GK rats, showed a slight increase by the treatment of sea urchin. These results suggest that the production of insulin in the beta cells of the pancreatic islets that are necrotic or do not function normally by the treatment of sea urchins does not improve or enhance.

Recently, studies on genetic factors causing diabetes have been actively conducted. As a candidate gene, a series of proteins related to the calcium ion channel of pancreatic beta cells and the extracellular release of insulin have been discussed (Schulla et al., 2003). ). Insulin secretion in pancreatic beta cells is due to intracellular influx of calcium through the L-type voltage-dependent calcium channel (LTCC). In other words, insulin secretion stimulation of blood glucose, amino acids, sulfonylurea antidiabetics, etc. causes cell membrane depolarization of beta cells, and LTCC is opened to cause extracellular secretion of insulin by calcium introduced into cells (Smith et al. , 1989; Ashcroft et al., 1994; Sher et al., 2003). It is well known that calcium ions in the extracellular fluid play an important role in the secretion process in cells containing secretory granules. The concentration of calcium in the extracellular fluid plays an important role in the insulin secretion of glucose-stimulated pancreatic beta cells. It has been shown through several studies that insulin secretion by glucose stimulation is at the highest level under appropriate calcium concentration. (Curry et al., 1968) One of the most important factors in the intracellular metabolism of insulin secretion is the regulation of calcium metabolism by islet beta cells. In addition, the presence of extracellular calcium is known to be absolutely necessary for insulin secretion by glucose and insulin secretion stimulant. (Rajan et al., 1990) The serum calcium level of diabetic patients was significantly lower than that of normal subjects, and urine calcium was higher in diabetic patients. Insulin pump therapy in diabetic patients showed significantly lower urine calcium levels than normal subjects. Glucose control has been reported to reduce calcium and phosphorus excretion for more than a week (Joo et al., 1996). Recent studies have reported that pancreatic beta-cell sensitivity of GK rats to intracellular influx of calcium ions caused by depolarization decreases, resulting in a deficiency in insulin secretion of glucose (Rose et al., 2007). Sea urchins contain a lot of calcium in protein, amino acids, fatty acids, and shells. The isolated calcium is developed and marketed as a calcium-assisted medicine. Production of eggs with high calcium content and rich in unsaturated fatty acids when sea urchins are administered to laying hens (Nam, 1986 Kim et al., 2002), and the authors previously reported that calcium ion water has antidiabetic effects in a model animal of insulin-independent diabetes (Lee et al., 2006). Based on this, the high concentration of calcium in sea urchins may have contributed to lowering blood sugar levels and increasing insulin sensitivity.

Taken together, sea urchins lowered blood glucose and improved glucose tolerance and insulin resistance in GK rat diabetes. As a result, it is highly likely that the sea urchin will be used as a raw material for food and functional biotechnological products for lowering blood sugar and improving insulin sensitivity, and further research on safety and other antidiabetic mechanisms is needed. It is judged that

1 is a flow chart of the method for producing a water-soluble extract of sea urchin powder having a hypoglycemic activity of the present invention

2 shows the effect of sea urchin extract treatment on body weight in GK rats for 4 weeks.

Figure 3 Effect of treatment of sea urchin extracts for 4 weeks on blood glucose changes in GK rats

4 shows the effect of four-week treatment of sea urchin extract on the plasma insulin concentration of GK rats.

5 shows the effect of treatment of sea urchin extract for 4 weeks on the change of blood glucose according to oral glucose tolerance test of GK rats.

Figure 6 is a photograph showing the results of histological changes of the pancreas and immunohistochemical changes (A, B, C, D) and insulin immunostaining (E, F, G, H) in the pancreas

Claims (4)

 The water-soluble extract powder of sea urchin shell powder is 10 ~ 30 mesh, and 10 times distilled water is added to the weight of sea urchin powder, and it is concentrated under reduced pressure with 50 ~ 60˚Brix after extraction by reflux cooling for 3 hours at 100 ℃. , A composition having a hypoglycemic action, obtained by lyophilization and powdering delete The method of claim 1, A composition having a hypoglycemic action comprising an aqueous extract of sea urchin shell powder as an active ingredient. delete

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