JP2002068995A - Diabetes mellitus-treating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a diabetes mellitus-treating agent, exhibiting an excellent insulin-like activity, having a high safety and useful for treating diabetes mellitus and symptoms accompanying with the diabetes mellitus such as hyperglycemia, glycosuria, cataract. SOLUTION: This diabetes mellitus-treating agent is provided by containing a component obtained by making mulberry leaves exhibiting the insulin like activity into single cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for diabetes which contains a component obtained by converting mulberry leaves into a single cell and has an excellent effect in treating diabetes.

[0002]

2. Description of the Related Art Diabetes is a syndrome in which energy metabolism is abnormal due to a wide range of metabolic abnormalities, such as glycoproteins, lipids, water, and electrolytes, due to insufficient action of insulin, which is a glycemic hormone. There are various causes for insufficient action of insulin, and it is said that its treatment is difficult.
Generally, diabetes is classified into insulin-dependent (type 1) and non-insulin-dependent (type 2). The above-mentioned type 1 is caused by destruction of pancreatic β-cells, resulting in insulin secretion deficiency, and type 2 is caused by various factors such as obesity and causes insulin resistance in peripheral parts of the body. Followed by hyperglycemia, showing symptoms of diabetes. An effective treatment for diabetes includes insulin therapy. However, since insulin is broken down in the small intestine and cannot be absorbed into the intestinal tract, insulin therapy requires subcutaneous or intramuscular injection of the patient under the guidance of a physician to control the blood glucose level. The above-mentioned therapy must be performed continuously for a long period of time, and it is a considerable burden on the patient.

[0003] Recently, various diabetic oral drugs have been used as a treatment alternative to insulin therapy. Oral diabetes drugs include sulfonylurea drugs,
Sulfonamide drugs, fast-acting insulin secretagogues, biguanides, insulin sensitizers, and α-glucosidase inhibitors. Of these, appropriate oral medicines are properly used according to various disease states of patients. However, in cases of dry mouth, polydipsia, polyuria, marked hyperglycemia with weight loss, infections, surgery, pregnancy, etc., oral diabetic drugs cannot be used and insulin therapy has to be applied. Absent. Moreover, the above-mentioned known oral drugs for diabetes have side effects such as hypoglycemia and gastrointestinal disorders, and have not been able to obtain a sufficiently satisfactory therapeutic effect as a therapeutic agent for diabetes.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides an excellent insulin-like action and high safety, and is useful for the treatment of diabetes, and other symptoms associated with diabetes such as hyperglycemia, hyperuricemia, and cataract. It is intended to provide a therapeutic agent.

[0005]

Means for Solving the Problems The present invention relates to a therapeutic agent for diabetes, which comprises a component obtained by converting mulberry leaves into a single cell. The above-mentioned agent for treating diabetes is preferably one having an insulin-like action.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Mulberry leaves used in the present invention are Mo
The leaf of the plant belonging to the genus Morus of the raceae family or its cognate plant.
The root bark of lba is referred to as mulberry bark. In Chinese medicine, mulberry bark is used for antitussive and expectorant purposes. The mulberry bark extract is known to have a blood glucose lowering effect, a blood pressure lowering effect, and the like. Mulberry leaves are used privately and are known to have a hypoglycemic effect like roots.

In the present invention, mulberry leaves may be treated with a dilute alkali to remove wax as necessary, and then converted into single cells using an intercellular substance-decomposing enzyme, and the resulting single-cell suspension may be used as it is. it can. Here, the dilute alkali treatment preferably means 0.01 to 3.0% by weight,
More preferably, in an aqueous solution of sodium hydroxide having a concentration of 0.01 to 2.0% by weight, particularly preferably 0.1 to 1.0% by weight, preferably 25 to 50C, more preferably 3 to 50%.
It refers to an operation of immersing and stirring at 0 to 40 ° C, preferably for 15 to 120 seconds, and more preferably for 30 to 100 seconds. Cellulase, e
ndo-polygalacturonase, pec
tin-trans-eliminase, protopectinase and the like. In addition, single cell formation is preferably performed in 1.2 to 3 times, more preferably 1.5 to 2.0 times (weight ratio) water, preferably 0.2 to 5.times. 0% by weight, more preferably 0.3 to
In the presence of 1.0% by weight of an intercellular substance-decomposing enzyme, preferably at 25 to 45 ° C, more preferably 30 to 40 ° C, preferably 70 to 150 minutes, more preferably 90 to 120 minutes.
Stir for a minute. During the single celling step, blanching may be performed as necessary. In addition, at the time of the single cell formation, an antioxidant such as L-ascorbic acid, sodium L-ascorbate, erythorbic acid, and glutathionic acid, and a pH adjuster such as citric acid may be added.

[0008] The suspension obtained by converting into a single cell is 2 to 10
Observation with a microscope at a magnification of 40 to 600 times that of the cells diluted twice and sufficiently stirred can confirm that the cells are completely separated without adhesion. The number of single cells in the suspension obtained by converting into single cells can be measured using a toma hemacytometer using a 2 to 10-fold diluted solution. The number of single cells in the single cell suspension of the present invention is preferably 1.0 × 10 4
^{6 to} 1.0 × 10 ¹⁰ , more preferably 1.0 × 10 ⁶
1.0 × 10 ⁹ , particularly preferably 5.0 × 10 ⁶
There are 5.0 × 10 ⁸ . The viscosity of the single cell suspension (measurement conditions; 26 ° C., 162 rpm / min) is preferably 1
00 to 7,000 cp, more preferably 200 to 1,
000 cp, particularly preferably 300 to 700 cp. Agar, carrageenan, etc. may be added to adjust the viscosity of the single cell suspension for the purpose of preventing sedimentation of cell particles and the like. The unicellular suspension has a uniform particle size as compared with that obtained by mechanical crushing, and cells having a particle size of 0.02 to 0.04 mm usually have 35% or more, preferably 40% or more. Yes, it has good dispersibility in water and oil, so it is easy to take as it is in the form of a liquid preparation.

The suspension is freeze-dried, spray-dried,
A treatment such as distillation under reduced pressure is performed to obtain a dry powder, which can be used as a powder. Such a single cell component of mulberry leaves can be administered as a therapeutic agent for diabetes in a suspension, powder, or other various administration forms. The administration form of the therapeutic agent for diabetes of the present invention is not particularly limited,
In addition to solid preparations such as tablets, capsules, granules and powders, liquid preparations such as troches and chewable preparations can be mentioned.

[0010] Among the above-mentioned administration forms, additives mainly used as solid preparations are shown below. As inorganic compounds, magnesium aluminate metasilicate, magnesium aluminate silicate, magnesium silicate, aluminum silicate, calcium silicate, silicic acid, magnesium oxide, titanium oxide,
Alumina magnesium hydroxide, magnesium hydroxide, sodium hydrogen carbonate, magnesium carbonate, aluminum hydroxide gel, aluminum hydroxide / sodium hydrogen carbonate coprecipitated product, aluminum hydroxide / magnesium carbonate coprecipitated product, aluminum hydroxide / magnesium carbonate -Calcium carbonate coprecipitation product, calcium phosphate, calcium sulfate and the like. As saccharides, erythritol, mannitol, sorbitol, xylitol, maltitol, maltose, lactitol, reduced maltose,
Examples include starch syrup, sucrose (sucrose), fructose (fructose), arabitol, erythrose, glucose, fructose, xylose, and arabinose. Preferably, erythritol, mannitol, sorbitol, and xylitol, which are non-caloric sugars, are used.

Examples of the carrier for preparations include known inorganic or organic carriers such as lactose, spray-dried lactose, granulated lactose, anhydrous lactose, starch, sucrose, crystalline cellulose, anhydrous calcium phosphate, calcium hydrogen phosphate, mannitol, hydroxypropyl. Excipients such as carboxymethylcellulose (CMC), carboxymethylcellulose calcium, dextran, pullulan, hydrogenated castor oil, talc, gum arabic, and binders such as hydroxypropylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, and partially pregelatinized starch; Lubricants such as, stearic acid, magnesium stearate, calcium stearate, talc, hydrogenated vegetable oil, sodium stearyl fumarate, glycerin monostearate, carnauba wax Modified starch, hydroxypropylmethylcellulose, sodium carboxymethyl starch, disintegrating agents such as croscarmellose sodium, and the like can be used non-ionic surfactant. When used as tablets and the like, a sugar coating may be applied.

Syrups, elixirs, emulsions,
As additives for liquids such as suspending agents, sucrose, xylitol, mannitol, glucose, aspartame, sweeteners such as saccharin, vanilla essence, apple flavor, flavoring agents such as banana flavor, other known excipients, pH adjusters, fresheners, suspending agents, defoamers,
A viscous agent, a solubilizing agent, a coloring agent, a surfactant, a fragrance, and the like can be mixed to form a liquid preparation by a conventional method.

[0013] When the mulberry leaf of the present invention is used for the treatment of diabetes, the ingestion method is 1 to 5 times a day.
It does not matter whether it is a solid preparation or a liquid preparation. The dose of the unicellular component is preferably 100 doses per dose in the case of an adult taking 3 times a day.
3,000 mg, more preferably 500-1,000 mg
0 mg. The method of taking the drug may be any of before, between and after meals, but preferably before meals.

The component obtained by converting mulberry leaves into a single cell according to the present invention has an excellent antidiabetic action, especially an insulin-like action. In addition, the mulberry leaf single cell-forming component of the present invention has high safety and is useful for treating diabetes, and other symptoms associated with diabetes such as hyperglycemia, hyperuricemia, and cataract. In addition, the mulberry leaf single-cellularized component of the present invention has less energy loss because it does not extract alcohol, and has a higher efficiency than the raw mulberry leaf, as compared with a mulberry leaf extract obtained by extracting alcohol from mulberry leaves by a known method. Can show diabetic effects.

[0015]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. In the examples, parts and%
Are by weight unless otherwise indicated.

Reference Example 1 Fresh mulberry leaves cultivated in Shimane Prefecture were collected, washed with water, and then subjected to a dilute alkali treatment to remove waxy leaves. The dilute alkali treatment was performed by immersing and stirring in a 0.1 to 1.0% aqueous sodium hydroxide solution at 40 ° C. for 90 seconds. After washing the mulberry leaf after dilute alkali treatment, 0.5 ~ 5c
The cells were cut into mx 0.5 to 5 cm, and single cells were formed with an intercellular substance-decomposing enzyme. Unicellularization was performed by stirring at 30 ° C. for 120 minutes in water in a 1.75-fold amount (weight ratio) of mulberry leaf in the presence of 0.5% of intercellular substance-decomposing enzyme. The undiluted solution (suspension) obtained by converting into a single cell was filtered with a 20-mesh filter to separate a suspension from solids such as petiole and leaf vein. The obtained suspension was freeze-dried to obtain a single-cellularized mulberry leaf extract (TK). The yield of TK from the raw mulberry leaves was 10.1%.

Reference Example 2 Mulberry leaves similar to those used in Reference Example 1 were washed with water, cut into small pieces in the same manner, and dried in the shade. The yield after drying in the shade from the raw mulberry leaves was 16.6%. Solid-liquid extraction was performed twice with a 20-fold amount of a 50% aqueous ethanol solution at about 80 ° C. for 2 hours by a digestion method. The obtained extract was filtered, the ethanol in the filtrate was distilled off under reduced pressure, and further freeze-dried to obtain a 50% ethanol extract (KK) of mulberry leaves. The yield of KK from the raw mulberry leaves was 4.7%.

Example 1 (1) Preparation of a streptozotocin (hereinafter referred to as "STZ")-induced diabetes model SLC (Japan SLC, Inc.): Wistar male rats (7 weeks old) fasted for one day were subjected to STZ (SIGMA CHEMICAL). Made, p
H4.5 dissolved in citrate buffer) at a dose of 50 mg / kg intravenously. Two days later, blood was collected from the tail vein and regrouped so that the average values of blood glucose in each group were almost the same. did.

(2) Measurement of Blood Sugar Level Using the regrouped diabetes model, a subject (T
Oral administration of K) was started. Dosage should be 20 per dose
0 mg / kg. Once daily for 30 days
Blood was collected from the tail vein every 10 days after administration, and the blood glucose level was measured. The results are shown in FIG. Example 2 Example 2 was repeated except that the dose of TK was 500 mg / kg. The results are shown in FIG. In addition, 1 in FIG.
The significant difference between the data on day 0 and the data on day 30 is p <0.01 based on the normal rat group of Control Example 1 below.

Control Example 1 (Normal Rat) The blood glucose level was measured in the same manner as in Example 1 except that TK was not used in the untreated group to which STZ was not administered. The results are shown in FIG. Control Example 2 (Diabetes Model) In the treatment group to which STZ was administered, except that TK was not used,
The blood glucose level was measured in the same manner as in Example 1. The results are shown in FIG.

Comparative Example 1 The procedure of Example 1 was repeated, except that KK prepared in Reference Example 2 was used instead of TK. The results are shown in FIG. Comparative example 2 It carried out similarly to Example 2 except having used KK prepared in Reference example 2 instead of TK. The results are shown in FIG. In addition, the significant difference of the data on the 10th day in FIG.
P <0.05 based on the diabetes model of

As shown in FIG. 1, the STZ of Comparative Example 1
The STZ-induced diabetes model of Control Example 2 was compared with the blood glucose level of the untreated group (normal rats) to which no steroid was administered (Control Example 1).
By intravenous administration of TZ, the blood sugar level rises about 4 times,
After that, it gradually rose. However, in Example 1, in the group to which the test subject (TK) was administered at 500 mg / kg (Example 2), an effect of significantly suppressing an increase in blood glucose level was observed 10 days after TK administration. This inhibitory effect was observed 30 days after TK administration. On the other hand, in the group to which KK was administered in Comparative Examples 1 and 2, on the 10th day after KK administration, an effect of significantly suppressing an increase in blood sugar level was observed.
Even in the group administered with mg / kg (Comparative Example 2), no inhibitory effect was observed 30 days after KK administration.

Example 3 (3) Preparation of Adipocyte Suspension from Rat Epididymal Adipose Tissue Performed according to the method of Rodbell. That is, Wi
Adipose tissue of epididymis (epididymis) of male star rats (about 180 to 200 g) was excised and weighed, and 4% b
ovine serum album (Fr.
V), collagenase (Type II, 3m
g / ml) containing Hanks' Balanced Sa
lt Solution (hereinafter “HBSS”)
(PH 7.4) 2.5 ml / g tissue was added,
Incubate at 37 ° C. for 1.5 hours to digest the tissue,
After filtration through a 250 μm nylon mesh, centrifugation (25
(° C, 500 rpm, 30 sec) to obtain fat cells.
Collagenas which kept the fat cells at 37 ° C
e-free HBSS (4% bovine serum al
(including bumin) three times by centrifugal washing (25 ° C, 500
rpm, 30 sec) and 2.5% bovine al
The suspension was suspended in bumin-containing HBSS at a concentration of 0.4 g / ml to obtain an adipocyte suspension.

(4) Fat mobilization test The fat cell suspension (100 mg of adipose tissue) obtained in (3)
0.25 ml in 4% bovine ser
0.25 ml of HBSS containing um albumin,
0.25 ml of test subject (TK) and adrenalli
Ne (adrenaline) was added in an amount of 0.25 ml [FC (final concentration): 1 μg / ml], incubated at 37 ° C. for 2 hours, and the lipolysis reaction solution was measured using NEFA Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Free fatty acids (FF
A) The amount was measured. The concentration of the subject (TK) is 10 μg
/ Ml.

Example 4 An experiment was carried out in the same manner as in Example 3 except that the concentration of TK was changed to 50 μg / ml in the above (3). Table 1 shows the results. Control Example 3 The same operation as in Example 3 was carried out without using TK. Table 1 shows the results
Shown in Comparative Examples 3 and 4 The same operation as in Examples 3 and 4 was performed using KK prepared in Reference Example 2. Table 1 shows the results. Comparative Examples 5 to 6 Insulin was used as a positive target drug, and the same procedures as in Example 3 were performed at the addition concentrations shown in Table 1. Table 1 shows the results.

[0026]

[Table 1]

*; Significant difference is p <0.01 based on Control Example 3.

As shown in Table 1, when adrenaline was allowed to act on adipocytes in Control Example 3, 14.2
8 ± 0.59 (μEq / g tissue) fatty acids were released (fat mobilization). However, in Comparative Examples 5 to 6, when insulin was added, the amount of released fatty acids was 65 to 68.
% Was suppressed. Then, in Example 4, 50 μl of the single-cellularized mulberry leaf extract (TK) of the present invention was used instead of insulin.
g / ml, the amount of free fatty acids is about 22
% Was suppressed. On the other hand, in Comparative Example 4, when the same amount of 50% ethanol extract (KK) of mulberry leaves was added, the suppression rate was about 19%. Here, comparing the inhibition rate per mulberry leaf of the raw material, TK showed a fatty acid release (fat mobilization) inhibitory effect 2.5 times that of KK.

[0029]

EFFECT OF THE INVENTION The component obtained by converting the mulberry leaf into a single cell according to the present invention is
It has excellent anti-diabetic action, especially insulin-like action. In addition, the mulberry leaf single cell-forming component of the present invention has high safety and is useful for treating diabetes, and other symptoms associated with diabetes such as hyperglycemia, hyperuricemia, and cataract. In addition, the mulberry leaf single-cellularized component of the present invention has less energy loss because it does not extract alcohol, and has a higher efficiency than the raw mulberry leaf, as compared with a mulberry leaf extract obtained by extracting alcohol from mulberry leaves by a known method. Shows diabetic action.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing daily changes in blood glucose levels of rats used in Examples, Comparative Examples, and Control Examples.

Claims (2)

[Claims] 1. A therapeutic agent for diabetes comprising a component obtained by converting mulberry leaves into a single cell. 2. The therapeutic agent for diabetes according to claim 1, which exhibits an insulin-like action.