JPH01228918A - Antidiabetic drug - Google Patents

Abstract

PURPOSE:To provide an antidiabetic drug having a strong insulin action or insulin-releasing action and causing no adverse reaction even when administered for a long period by containing as a main ingredient a protein extracted from Euphausiacea with an aqueous solvent and having a specific range of mol.wt. CONSTITUTION:Euphausiacea is extracted with an aqueous solvent such as cold water, hot water or hydrous ethanol. The obtained extract solution is desalted and dried to give a crude extract, which is subjected to a conventional protein-separating and purifying process to provide an antidiabetic drug containing a protein fraction having a mol.wt. of 20000-40000 as a main ingredient. The preparation is administered, for example, in a dose of 0.1-5g, particularly 1-3g, per day for an adult as the Euphausiacea protein fraction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an antidiabetic agent containing an aqueous krill extract as an active ingredient.

[Conventional technology and its problems]

Diabetes is broadly classified into insulin-dependent type I diabetes and non-insulin-dependent type II diabetes. ■The number of types has increased rapidly in recent years due to the saturation of eating habits and higher social status, and 11
JIl also usually develops at a young age and requires lifelong insulin use, both of which are serious medical problems in our country. ! 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 may cause insulin shock or insulin resistance due to the production of insulin antibodies. Therefore, it has an insulin-like effect or an insulin release activation effect, and there are no functional problems even during long-term administration.It is effective when used alone, or significantly reduces the fecal dose of insulin when used in combination with insulin. There is a need for the development of drugs that can

Since ancient times, many concentrated medicines and various foods have been handed down that are considered effective for diabetes. The inventors also recognized that insulin-like and insulin release activating effects are relatively widespread in various meats and seafood [Pharmaceutical Journal].

107.869 (1987)), these all have low activity, cannot be identified as substances, and have not yet been provided as pharmaceuticals.

[Means to solve the problem]

In view of the above circumstances, the present inventors sought to obtain a substance that has a strong insulin-like action or insulin release activation action, without any production problems even when ingested in large quantities over a long period of time, using foods that are commonly ingested on a daily basis. As a result of intensive screening, it was discovered that an aqueous product of krill satisfies these requirements, and the present invention was completed.

That is, the present invention provides an anti-diabetic agent whose main component is tanno-9, which is extracted from krill with an aqueous solvent and has a molecular weight of 20,000 to 40,000.

Traditionally, krill has been used as food or feed mainly as a protein source, and is known to have excellent nutritional properties.
No. 62524), antihypertensive effect (Japanese Patent Application Laid-open No. 119/1986)
No. 017), antithrombotic effect (JP-A-57-7-355l2)
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 used even in rainwater, and is not limited to any particular type. These are those that have not been frozen as they were caught or those that have been stored in L-frozen storage.
You can also use either whole pieces or those that have been pulverized.

Examples of the aqueous solvent used in the present invention include cold water, hot water, and aqueous ethanol. In addition, the extraction method is not limited, but for example, krill
This is done by boiling in 0 times the volume of hot water for several minutes, and then removing solid matter by filtration, centrifugation, etc. The obtained extract usually contains salts such as common salt, so it is desalted as much as possible by electrodialysis, reverse osmosis, ultrafiltration, gel filtration, etc., and then dried by freeze drying, blow drying, etc. is desirable.

The aqueous extract obtained in this manner exhibits strong insulin-like action and insulin release activating action tNL even in the unpurified stage, and sufficient efficacy as an antidiabetic agent. Molecular weight 20,000-40,000 obtained by subjecting to separation and purification steps
Tanno 9 Ryobu has even better anti-diabetic effect,
preferable. The method for separating and purifying this tannolyte is not particularly limited, and includes known methods such as salting out, organic solvent precipitation, ion collision chromatography, gel filtration, isoelectric focusing, ultracentrifugation, and ultrafiltration. can be used alone or in combination.

The aqueous krill extract and krill protein fraction used in the present invention are mixed as they are or with appropriate pharmaceutical carriers, excipients, diluents, etc., and administered orally in the form of powder, granules, tablets, capsules, injections, etc. Or it can be administered parenterally. It is also possible to mix it with food and have the patient ingest the required amount in the form of food.

For example, in the case of adults, the suitable dose is 0.1 to 5 t, preferably 1 to 3 t, per day of kakiamitan and phlegm fraction, but it may vary depending on symptoms, route of administration, age, body weight, etc. Should.

[Examples] Examples will be described below, but the present invention is not limited thereto.

Example 1 Antarctic krill (Eupha) caught in the Antarctic Ocean
usia 5uperba) -1! ! After boiling for 10 minutes at 90°C in a continuous boiling device, solids were separated in a continuous centrifuge decanter and the broth was boiled (solids 7.75%).
I got it.

Freeze this and take it back to Japan! After 4 freezing, that 5k
G was desalted using an electrodialysis device (Asahi Glass Co., Ltd. Selemion Membrane Decontamination Experimental Equipment FF), and the resulting desalted hydrochloric acid (4.1 Lp) was dried to form a red, powder-like krill hot water extract. (
262t (hereinafter abbreviated as ES) was obtained. Fill determination results obtained by ES high performance liquid chromatography (5hodex WS-803 column) are shown in the full results.

Test Example IES Insulin-like action of ES An insulin-like action test was conducted according to the method of Moody et al. Wi
Atar male rats (body weight 130-1509) were harvested from the frontal and epidermal adipose tissues and cut into small pieces. ? Adipose tissue 209 and 2-519 sliced into Rietelen vials
7-Collagenase (Whorthing On, T
After exchanging the air in the vial with a 95% zero-5% CO2 mixed gas for 11 minutes, the vial was tightly capped and heated at 37℃ for 16 hours.
Digestion was performed for 40 minutes at 0 c/min. The digestive fluid was filtered through a nylon mesh (250 μm) and Krebs-Ri
nger-Bicarbo % nate (KRB
) Buffer C 20mM Hepes S containing 0.55mM glucose and 2% bovine serum albumin (BSA)] and incubated for 30 minutes at 37°C. After adjusting free adipocytes to 5 x 10S cells/- with KRB Yukiho, ES
and 0.4 μCi of 1)-(2-3H,)glucose [Amajam Shirt Qn■.

5 specific activity 94 mci/
Q] was added and incubated at 37°C for 2 hours.

After incubation, 8N H,So, 0.2 m
/ and toluene-based scintillator-5- were added, and the radioactivity of the total lipid extracted in toluene was measured. Total radioactivity was determined by adding 0.4μC1D-[2-3H]-curcose and Instaglu (PackardInstrum) in KRB buffer.
ent Co, Inc.) was added and measured.

1) Conversion from -(2-”H)-glucose to total lipid!
I8″4 was obtained from the following formula.

Conversion rate (%) = A/(B-C)xto.

A: Tsuji radioactivity B: Radioactivity of total lipids after incubation C: Radioactivity of total lipids before incubation Control system with insulin (pig-derived insulin: Sigma
(manufactured by company a) was used. The results are shown in FIG.

From FIG. 2, it is recognized that ES promotes the conversion of D-(2-''H]-glucose into total lipids in a concentration-dependent manner and has a strong insulin-like effect.

Test Example 2 Insulin release activation effect of ES Lac
An insulin release activation effect test was conducted according to the method of Y et al. Male golden hamsters fasted for 24 hours (
7 weeks of age) Under oventoparbital anesthesia, cannulate the common bile duct and inject pancreatic t-g with 10-20 m of Hanka fluid.
It was removed after it was cured. After cutting the removed pancreas into small pieces, one removed pancreas was obtained? ) Collagenase (Whorthington type N
) 51g/2m (Ranks solution containing 15% calf serum) was added and digested at 37°C for 10 minutes. This digestive fluid is
Wash with anks solution and wash with Ficoll-Conray.
Adult pancreatic sacs were isolated by gravity separation method. Isolated adult birds were dispersed using the EDTA-Dispaae dispersion method, adjusted to 5 x 105 cells/- with KRB buffer (containing 20mM Hepes, 5.5% glucose, and 2% BSA), added with ES, and incubated at 37°C. 2 hours, 95% air-s
sco, incubated under gas phase. this,
Centrifuge at 2000 rpm for 3 minutes, release into buffer, fc (7x +7y t-EIA method (MfESA
1N5ULINTEST: MBL■]. Glucose was used as a control system. The results are shown in FIG.

As shown in FIG. 3, ES is found to promote the release of insulin from pancreatic islets of Langerhans in a concentration-dependent manner.

Test Example 3 Hepatic glycogenolysis inhibitory effect of ES Wi
Star male rats (body weight: 230-2501 kg) were used, and liver perfusion was carried out according to the method of Wooden et al., 1 n situ, f
This was done in a low-through manner. Irrigation fluid is KR
B buffer (m7.4) was used, 95% 0, -5% CO
The test was conducted while the 8 mixed gas was being blown. E
S was dissolved in KRB buffer and added at the beginning of perfusion.
It was automatically added into the perfusate from the arm. The flow rate is 25 d
/min, and glucose in 4 m of fluid discharged from the vena cava was measured. Insulin was used as a control system.

The results are shown in FIG.

From FIG. 4, it is recognized that ES significantly suppresses glucose release at a concentration of 200 μt/−.

Test Example 4 Alloxan 60197 and 9 were intravenously injected into the tail vein of ICR male 79 mice (body weight 24-269) in ES alloxan-induced diabetic mice.

Two days later, blood was collected from the mouse orbit (from the eye socket) and blood sugar levels were measured. 3
Mice exhibiting a blood sugar level of 0019/dJ or more were subjected to the test as alloxan diabetic mice. Alloxan administration 5
Days later, ES was administered at 20 and 5 (1 g/day, and tolbutamide sos+y/Kp was administered intraperitoneally as a positive control.
Before administration, 2, 4.8, and 12 hours after administration, blood was collected (from the orbit of the eye) and blood sugar levels were measured.

Normal and control groups received intraperitoneal administration of physiological saline. The results are shown in Table 1.

Below is the margin This result is y, gs is 5019/? It can be seen that the blood glucose level of alloxan diabetic mice was significantly lowered after 4-8 hours at a dose of .

Example 2 Defrosting liquid IK of Antarctic krill broth used in Example 1
P't-Ultrafiltration (Ultrafiltration membrane NTU manufactured by Nitto Denko Corporation)
-325C installed), 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 a krill protein fraction (hereinafter abbreviated as EP) L12. The IQ turn of glue filtration is shown in Figure 5. Incidentally, the l fraction is 15-.

EP has a white, powdery appearance with good solubility in water, and its high performance liquid chromatography (Figure 6) and SDS
& lyacrylamide gel electropherogram (FIG. 7), it was confirmed that the molecule f was 20,000 to 40,000. In addition, EP was mainly composed of protein and protein composed of the amino acids shown in Table 2.

Margin Table 2: Amino acid composition of kakiamitan/Q lithium fraction (gp) (Sample test example 5 Insulin release activation effect of EP (in
Table 3 shows the results measured in the same manner as in Test Example 2.

Table 3 Insulin release activating effect of oxamitan and Q phosphorus fraction (EP) * p<0.05, *book* p<0.00
1 From these results, it was confirmed that EP significantly activated the release of insulin from isolated adult Langerhans hamsters in a concentration-dependent manner.

Test Example 5 EP hyperglycemia lowering test in alloxan-induced diabetic mice The results were measured in the same manner as in Test Example 4 and are shown in Table 4.

Margin below These results show that EP significantly lowers blood sugar levels in alloxan diabetic mice.

Test Example 7 Acute toxicity test 1 group of 10 male DD mice was administered a dose of 5? When each EP was dissolved in physiological saline and administered orally, no abnormality was observed.

Example 3 Tablet Tablets were prepared, each tablet containing the following ingredients:

Krill Hot Water Extract 250” Cornstarch 67 Milk Tong 180”
Amount 500 ■ Example 4 Granules Krill protein fraction 250Q, lactose 465119 and corn starch 265 ■ were mixed uniformly, and 10
Granules were prepared in accordance with the Japanese Pharmacopoeia General Preparations Granules section using a % hydroxyzolobyl cellulose aqueous solution as a sizing agent.

〔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. Furthermore, the antidiabetic agent of the present invention is obtained by separating and purifying krill, which is consumed as a daily food,69 and there are no problems such as side effects due to long-term continuous administration. Therefore, when used in combination with insulin, it is effective in reducing the dose of insulin and reducing the adverse effects of long-term, large-dose administration of insulin.

[Brief explanation of the drawing]

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. Figure 3 shows the insulin release of the hot water extract of krill. FIG. 4 is a diagram showing the test results of the activation effect. FIG. 4 is a diagram showing the test results of the inhibitory effect of hepatic glycogenolysis of krill hot water extract and insulin. FIG. 5 is the krill protein fraction obtained by glufiltration of the krill hot water extract. FIG. 6 is a diagram showing high performance liquid chromatography of the krillatan/Q lithium fraction. FIG. 7 is a diagram showing the 5L) S-? It is a lyacrylamide glucose electropherogram. that's all

Claims (1)

[Claims] 1. Molecular weight extracted from krill by aqueous solvent 2
An antidiabetic agent whose main component is 0,000 to 40,000 proteins.