JPH0442369B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an antidiabetic agent containing an aqueous krill extract as an active ingredient. [Prior art and its problems] Diabetes is broadly classified into insulin-dependent type diabetes and non-insulin-dependent type diabetes.
The number of cases of this type has increased rapidly in recent years due to the saturation of the diet and the aging of society, and this type also usually develops at a young age and requires lifelong insulin use, both of which have become serious medical problems in Japan. Insulin preparations exhibit effective therapeutic effects regardless of type or type of diabetes, but there is a problem in that long-term or large-dose administration causes insulin shock and insulin resistance due to the production of insulin antibodies. Therefore, it has an insulin-like effect or an insulin release activating effect, has no side effects even during long-term administration, and is effective when used alone or can significantly reduce the amount of insulin used when used in combination with insulin. There is a need for the development of drugs that can do this. Since ancient times, many Chinese medicines and various foods have been handed down that are considered effective for diabetes. The present inventors also recognized that insulin-like and insulin release activating effects are relatively widely present in various meats and seafood [Pharmaceutical Journal, 107 , 869 (1987)],
All of these have low activity, cannot be identified as substances, and have not yet been provided as pharmaceuticals. [Means for solving the problem] In view of the above-mentioned circumstances, the inventors of the present invention aim to obtain a substance that has a strong insulin-like action or an insulin release activating action, even when ingested in large quantities over a long period of time, without causing side effects. As a result of intensive screening using commonly ingested foods as a search source, it was discovered that an aqueous extract of krill satisfies these requirements, and the present invention was completed. That is, the present invention provides an antidiabetic agent whose main component is a protein with a molecular weight of 20,000 to 40,000 extracted from krill with an aqueous solvent. Traditionally, krill has been used as food or feed mainly as a protein source, and is known to have excellent nutritional properties. Preventive effect (Japanese Patent Application Laid-open No. 119017/1983), antithrombotic effect (Japanese Patent Application Laid-Open No. 1983-119017)
35512) and other physiological activities have also been reported. However, it was not known that specific components of krill have antidiabetic effects. The krill used in the present invention can be any of the genus Euphausia, such as Euphausia superba caught in the Southern Ocean, Euphausia pacifica caught in the sea near Japan, or the genus Thysanoessa, and is not limited to any particular type. These can be used either unfrozen or frozen as they are caught, whole or crushed. Examples of the aqueous solvent used in the present invention include cold water, hot water, and aqueous ethanol. Although the extraction method is not particularly limited, it may be carried out, for example, by boiling krill in 2 to 10 times the amount of hot water for several minutes, and then removing solid matter by filtration, centrifugation, or the like. The obtained extract was
Since it usually contains salts such as common salt, it is desirable to desalt it as much as possible by electrodialysis, reverse osmosis, ultrafiltration, gel filtration, etc., and then dry it by freeze drying, spray drying, etc. The aqueous krill extract thus obtained has strong insulin-like and insulin release activating effects even in its unpurified state, and exhibits sufficient efficacy as an anti-diabetic agent. The molecular weight obtained by subjecting to
A protein fraction of 20,000 to 40,000 has an even better antidiabetic effect and is preferred. This protein separation/purification method is not particularly limited, and for example, known methods such as salting out, organic solvent precipitation, ion exchange chromatography, gel filtration, isoelectric focusing, ultracentrifugation, and ultrafiltration may be used alone. , or in combination. The aqueous krill extract and krill protein fraction used in the present invention can be used as is or mixed with appropriate pharmaceutical carriers, excipients, diluents, etc., and can be used as powder,
It can be administered orally or parenterally in the form of granules, tablets, capsules, injections, and the like. It is also possible to mix it with food and have the patient ingest the required amount in food form. For adults, the dosage is, for example, 0.1 to 5 g of krill protein fraction per day, preferably 1 to 5 g per day.
3g is suitable, but depending on symptoms, administration route, age,
The amount should be increased or decreased depending on body weight, etc. [Examples] Examples will be described below, but the present invention is not limited thereto. Example 1 Antarctic krill (Euphausia superba) caught in the Antarctic Ocean was boiled to 90% in a continuous boiling device.
After boiling at ℃ for 10 minutes, the solid content was separated using a continuous centrifuge decanter to obtain a broth (solid content 7.75%). Freeze this, bring it back to Japan, thaw it, and then
Kg was desalted using an electrodialysis device (Asahi Glass Co., Ltd.'s Selemion membrane equipped experimental device), and the resulting desalted solution (4.1 Kg) was freeze-dried to produce a slightly red, powdered krill hot water extract (hereinafter abbreviated as ES). ) 262g was obtained. Figure 1 shows the measurement results of ES by high performance liquid chromatography (ShodexWS-803 column). Test Example 1 Insulin-like action of ES An insulin-like action test was conducted according to the method of Moody et al. Wistar male rat (weight 130−
After decapitation, the epididymal adipose tissue was removed and cut into small pieces. Put 20 g of shredded adipose tissue and 2.5 mg/ml collagenase (Whorthington, Type) into a polyethylene vial, replace the air in the vial with a 95% O ₂ - 5% CO ₂ mixed gas, then seal the vial, and store at 37°C. Digested for 40 minutes at 160c/〓. Filter the digestive fluid through nylon mesh (250 μm),
Washed three times with Krebs-Ringer-Bicarbonate (KRB) buffer [containing 20mM Hepes, 0.55mM glucose and 2% bovine serum albumin (BSA)];
It was left standing at 37°C for 30 minutes. After adjusting free adipocytes to 5 × 10 ⁵ cells/ml with KRB buffer, ES and 0.4 μCi
D-[ ^2-3H ]glucose (Amasyam Japan Co., Ltd., specific activity 94 mCi/mg) was added and incubated at 37°C for 2 hours. After incubation, 0.2 ml of 8N H ₂ SO ₄ and 5 ml of toluene-based scintillator were added, and the radioactivity of the total lipids extracted in toluene was measured. The total radioactivity is
Instagel with 0.4 μCiD-[ ^2-3H ]-glucose in KRB buffer (Packard Instrument Co.Ink.)
was added and measured. The conversion rate from D-[ ^2-3H ]-glucose to total lipids was determined from the following equation. Conversion rate (%) = A/(B-C) x 100 A: Total radioactivity B: Radioactivity of total lipids after incubation C: Radioactivity of total lipids before incubation Insulin was used as a control drug (Pig-derived insulin:
(manufactured by Sigma) was used. The results are shown in FIG. From Figure 2, ES becomes D-[ ^2-3H ]- in a concentration-dependent manner.
It is recognized that it promotes the conversion of glucose into total lipids and has a strong insulin-like effect. Test Example 2 Insulin release activation effect of ES An insulin release activation effect test was conducted according to the method of Lacy et al. Cannulate the common bile duct of a male golden hamster (7 weeks old) that has been fasted for 24 hours under bentobarbital anesthesia and inject 10-20 ml.
The pancreas was distended with Hank's fluid and then removed. After cutting the excised pancreas into small pieces, 5 mg/2 ml of collagenase (Whorthington type) (Hanks' solution containing 15% calf serum) was added per excised pancreas, and digestion was performed at 37°C for 10 minutes. Wash this digestive juice with Hanks solution,
Pancreatic islets were isolated by Ficoll-Conray gravity separation method. Isolated Lajishima was dispersed using EDTA-Dispase dispersion method, and further added to KRB buffer (20mM Hepes,
5x with 5.5mM glucose and 2% BSA)
After adjusting to 10 ⁵ cells/ml, add ES and incubate at 37℃ for 2 hours.
Incubation was carried out under a gas phase of 95% air-5% _CO2 . This was centrifuged at 2000 rpm for 3 minutes,
Insulin released into the buffer solution was measured by EIA method [MESA INSULIN TEST: MBL Co., Ltd.]. Glucose was used as a control drug. The results are shown in FIG. From FIG. 3, it is recognized that ES promotes the release of insulin from pancreatic islets of Langerhans in a concentration-dependent manner. Test Example 3 Inhibitory effect of ES on hepatic glycogenolysis Wistar male rats (weight 230-250 g) were used, and liver perfusion was performed in situ.flow according to the method of Kimura et al.
-Through method was used. KRB buffer solution (PH7.4) was used as the perfusate, and the test was conducted while bubbling a mixed gas of 95% O ₂ -5% CO ₂ . ES is KRB
was dissolved in a buffer solution and automatically added to the perfusate from the side arm at the start of perfusion. The flow rate was 25 ml/min, and glucose in the perfusate discharged from the inferior vena cava was measured. Insulin was used as a control drug. The results are shown in FIG. From FIG. 4, it is recognized that ES significantly suppresses glucose release at a concentration of 200 μg/ml. Test Example 4 Hyperglycemic lowering test of alloxan-induced diabetic mice in ES 60 mg/Kg of alloxan was intravenously injected into the tail vein of ICR male mice (body weight 24-26 g). Two days later, blood was collected from the mouse orbit and the blood sugar level was measured. 300mg/dl
Mice that showed blood sugar levels above were used for testing as alloxan diabetic mice. Five days after alloxan administration, 20 and 50 mg/Kg of ES and 50 mg/Kg of tolbutamide were administered intraperitoneally as a positive control. Blood was collected from the mouse orbit before administration, 2, 4, 8, and 12 hours after administration, and blood sugar levels were measured. . Normal and control groups received intraperitoneal administration of physiological saline. The results are shown in Table 1.

[Table] Each value is the mean ± standard error Significant difference test was performed using Student's t test for blood sugar levels of the control group at the same time, a) p<0.05
The results show that ES significantly lowers blood glucose levels in alloxan diabetic mice after 4-8 hours at a dose of 50 mg/Kg. Example 2 1 kg of the thawed liquid of the Antarctic krill broth used in Example 1 was subjected to ultrafiltration (Nitto Denko Corporation ultrafiltration membrane).
(NTU-325C equipped), and the filtrate was concentrated under reduced pressure at 50°C. Next, gel filtration was performed using a Sephadex G-25 column, and the first eluted peak was fractionated and freeze-dried to obtain 1.7 g of a krill protein fraction (hereinafter abbreviated as EP). The pattern of gel filtration is shown in FIG. Note that 1 fraction is 15 ml. EP has a white, powdery appearance with good solubility in water, and its molecular weight is 20,000 to 40,000 based on its high performance liquid chromatography (Figure 6) and SDS polyacrylamide gel electropherogram (Figure 7). was recognized. Furthermore, EP was mainly composed of proteins composed of the amino acids shown in Table 2.

【table】

[Table] Test Example 5 Insulin release activation effect of EP (in vitro) Table 3 shows the results measured using the same method as Test Example 2.

[Table] These results demonstrate that EP significantly activated insulin release from isolated islets of Langerhans in hamsters in a concentration-dependent manner. Test Example 6 Hyperglycemia lowering test in EP alloxan-induced diabetic mice The results were measured in the same manner as in Test Example 4 and are shown in Table 4.

〔Effect of the invention〕

The antidiabetic agent of the present invention has an antidiabetic effect based on insulin-like action and insulin release activation action, and is effective in improving the condition of diabetes. Also,
The antidiabetic agent of the present invention is isolated and purified from krill, which is ingested as a daily food.
There are no problems with side effects due to long-term continuous administration. Therefore, when used in combination with insulin, it is effective to reduce the dose of insulin and reduce the adverse effects of long-term, large-dose administration of insulin.

[Brief explanation of drawings]

Figure 1 shows the high performance liquid chromatography of the hot water extract of krill, Figure 2 shows the test results of the insulin-like action of the hot water extract of krill, and Figure 3 shows the insulin-like effect of the hot water extract of krill. Figure 4 is a diagram showing the test results of the release activation effect. Figure 4 is a diagram showing the test results of the hepatic glycogen decomposition inhibitory effect of hot water extract of krill and insulin. Figure 5 is the result of gel filtration of krill protein by hot water extract of krill. FIG. 6 is a diagram showing the separation of fractions. FIG. 6 is a diagram showing high performance liquid chromatography of the krill protein fraction. FIG. 7 is an SDS-polyacrylamide gel electropherogram of the krill protein fraction.

Claims (1)

[Claims] 1. An antidiabetic agent whose main component is protein with a molecular weight of 20,000 to 40,000 extracted from krill with an aqueous solvent.