JPH0780778B2 - Antidiabetic agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a therapeutic agent for diabetes. More specifically, the present invention relates to a dry powder of earthworm and / or a therapeutic agent for diabetes in an earthworm extract portion, which has an excellent activity of lowering blood sugar (hereinafter referred to as blood sugar) and safety. Among them, the present invention relates to an earthworm dry powder prepared by an improved manufacturing method which is novel and innovative, and further relates to a therapeutic / preventive agent for diabetes which contains an extract thereof as an active ingredient.

Prior Art: Earthworms have been the silkworm from the ancient times, mainly in the Eastern countries,
It is called a earth dragon and has been used as a medicine. The pharmacological and pharmacological actions of earthworms reported in conventional literature are shown below.

"Water and life" by Shinryu Obuchi 1947 (Showa 22) 10
Published by Maki Shobo, pages 223-226 and "Reprinted Water"
Shinji Hatai, April 30, 1980, published by Scientist Co., Ltd., pp. 160-163, worms are agents for reducing bladder stones and for discharging them to the outside of the body, therapeutic agents for jaundice and delivery. It has been reported that it has the pharmacological action of drugs, tonics, hair crude drugs, strong sperm agents and antipyretics, while as an earthworm poison, one has a nervous system damage, the other has a hemolytic action of red blood cells. .

"People's Republic of China Anthology" The People's Republic of China Ministry of Health Anthology Committee edition, 1977 edition, some pages 197-198, the following is described. By convention, there are two types of Chiryu products. One of them is Lumbricus Kwangtungesis
Is one in which the abdomen is torn to wash off the internal organs and mud and dried in the sun, shade or low temperature. Other Land Dragon (Lumbricu
s Nativus) is killed by putting it in plant ash and then removing the ash and drying it in the sun, in the shade or at low temperature, and earthworms are clogged with mud. These two types of earth dragons are antipyretic, attracting remedy, blood circulation promoter, hemiplegic treatment,
It is reported to be used as a joint pain reliever, a urinary drug, a bronchial asthma drug and a hypertensive drug at 4.5 to 9 g / day.

"Our Chinese medicine series 3, earth dragons / crow bones /
"Scientific research in China" October 30, 1978, published by Matsuura Pharmaceutical Co., Ltd., page 7 reports that Jilong tincture (ethyl alcohol extract of Jilong) has antihypertensive effect.

"Natural Drug Encyclopedia," edited by Takuo Okuda, April 15, 1986,
Published by Hirokawa Shoten, page 215, Jiryu was a hypothermic agent, painkiller,
It has been reported to be used as a diuretic and antidote.

Mamoru Tanaka [Hokkaido Medical Magazine Vol. 24, pp. 18-24 (1949
)], Silkworms (dried pieces removed from mud) were extracted with boiling water, and the precipitated substance (Lumbrofebrin) obtained by adding ethyl alcohol to the concentrated solution of this extract was added to Ringer's solution. It is reported that when this solution was dissolved and this solution was intravenously injected into an anesthetized cat, a rapid decrease in blood pressure was observed, and accelerated blood coagulation was observed in proportion to shock.

Kenjiro Igawa [Yamaguchi Medical Vol. 9, 571-576 (19
(60 years)], the physiological saline extract of Chiryu, the solution of the extract of Chiryu's ethyl alcohol or acetone in physiological saline was intravenously injected into a mature rabbit, and a decrease in blood pressure was observed.

`` Chuan Andai Dictionary '' edited by Shimaki Jiangsu New Medical School, 1980, published by Shanghai Science and Technology Publishing Co., Ltd., page 2112: Hirochi Ryu tincture, dried silkworm powder powder suspension, hot water immersion liquid of silkworm, decoction of silkworm, etc. Was administered to anesthetized dogs, large mice, cats or mice with chronic renal hypertension, a slowing and persistent hypotensive effect was observed. When the dog or cat under anesthesia was intravenously injected with Chiryu extract, the hypotensive effect was rapidly exhibited. However, it was reported that it was not effective in oral administration and clinical application. Furthermore, on page 2114 of the same magazine, 10 ml of a 40% concentration earth dragon tincture (40 gram of earth dragon is dipped in 100 ml of ethyl call of 60 degrees) each time, 3 times a day [that is equivalent to 12 grams of earth dragon / day. (Converted by the inventor)] Take. For those who cannot drink tincture, add water to pure earth dragon powder to make pills (add a small amount of excipients), 3 to 4 g each time, 3 times a day [equivalent to earth dragon 9 to 12 g / day. (Converted by the present inventor)]
Continued use for 60 days is effective for essential hypertension. or,
Chiryu B ₁ liquid (HgCl ₂ is used to remove hypoxanthine, ion-exchange resin is used to separate the blood pressure-lowering components, and the extracted) 2 ml each time (including Chiryu 8 g of herbal medicine) It has been reported that when taken three times [equivalent to 24 g / day of earth dragon (converted by the present inventor)], it was effective for essential hypertension.

The conventional methods for producing a dried product or a dry powder of earthworms are roughly classified as follows.

i A method in which the abdomen of earthworms is torn to remove the contents (internal organs and mud) inside the body and dried in the sun, shade or at low temperature (usually below 50 ° C).

ii) A method in which earthworms are killed by putting them in plant ash, and then the ash is removed and dried in the sun, in the shade or at a low temperature (usually 50 ° C or lower) to obtain earthworms in which mud is clogged.

iii A method of removing mud from the body of an earthworm and then putting it in plant ash or fire ash to dry it.

And so on. These dried products were crushed and used when needed or used. These manufacturing methods are simple and economical methods, and have the advantage that they can be easily executed at home.

However, the dried product or dry powder of earthworms obtained by these production methods is stored in a refrigerator at 0 to 5 ° C, or at room temperature of 5 to 45 ° C in an open state for about 6 months or less, and when stored in a sealed state. It has the drawback that it becomes unusable due to the growth of mold within a short period of less than a year.

Earthworms dried with mud clogged in the body as in the above method ii or earthworms dried in plant ash or fire ash as in the method iii are extracted with hot water in most cases when used as medicines. Or, after decocting with boiling water, the solution is often taken. In particular, the dried product or dried powder of earthworms produced by the method of ii is rarely taken as a ground dragon tincture or powder, or as a pill.
In addition to taking the earthworms produced by the method i as hot water dip or decoction, take the earth dragon tincture or earth dragon powder as it is, or add a small amount of water or a small amount of excipient to the powder and take it as a pill. There are many. According to the manufacturing method of i and ii,
The yield of dry earthworms with water content of 10 ~ 16% is 5 ~ from live earthworms.
9%, and the yield in the case of the production method of ii is 13 to 19% (measured value by the present inventors).

Recently, one of the inventors of the present invention, Yoichi Ishii, is a health food mainly composed of earthworm proteins and lipids or a method for producing the same [Japanese Patent Application Publication No. 59-216572 (published on Dec. 6, 1984). ] Was reported. This manufacturing method is one of the excellent methods for obtaining earthworm dry powder as a health food. However, it has been found that the method for producing an earthworm dry powder for medicinal purposes such as an arteriosclerotic agent (also known as an antihyperlipidemic agent), an antidiabetic agent and an antihypertensive agent is not sufficient in terms of efficacy. That is, when an external action is applied to remove the excrement remaining in the living body of living earthworm, it is not possible to selectively remove only the fecal soil. It was found that no matter how carefully the procedure is carried out, visceral and body fluids, which contain many components that play an important role in drug efficacy, are removed together with feces and soil, resulting in insufficient drug efficacy. The yield for live earthworms is as low as 10 to 19%. Furthermore, a major problem is that the enzymes in the dry earthworm powder, which have important pharmacological and medicinal effects, are used because the vacuum drying in the final finishing step is carried out at 80 ° C and a vacuum degree of 0.3 torr for a long time of 20 hours or more. Were found to be destroyed or deactivated. Therefore, in comparison with the earthworm dry powder obtained by the production method of the present invention, Japanese Patent Application Publication No. 59-
The effectiveness of the earthworm dry powder obtained in No. 216572 as a therapeutic agent for diabetes was about 50%.

Recently, one of the present inventors, Hisashi Mihara and other collaborators have found that the earthworm's fibrinolytic active substance has an optimum pH of 8-10,
Stable pH of 5 to 10, trasylol (trade name), transsamine (trade name), soybean trypsin inhibitor and serum-inhibited, plasminogen activating action and fibrinolytic action, and various substances without fibrinogen lytic action It was confirmed to be an enzyme protein having properties.

Patent application of a method for producing a fibrinolytic active substance consisting of a crude enzyme protein fraction by an extraction method of an aqueous solvent from earthworm and a sugar enzyme protein fraction by the purification treatment, that is, Japanese Patent Application Publication No. 58-148824 ( Filing date: February 27, 1982) and US patent application No. 4 of a foreign application due to the priority claim
70394 (filing date February 28, 1983), British patent application No.
8305359 (filing date February 25, 1983), Italian patent application N
o.47795A (filing date February 25, 1983), French patent application
No.03165 (filing date February 25, 1983), West German patent application
No. P3306944.1 (filing date February 28, 1983) and Canadian patent application No. 422034 (filing date February 21, 1983) are reported.

Furthermore, the application for substance patents of 6 kinds of novel proteases from earthworm by Tsuneyoshi Mihara, that is, Japanese Patent Application Publication No. 59-63184 (filing date, October 2, 1982)
And a patent application for a thrombolytic agent containing these proteases as active ingredients, that is, Japanese Patent Application Publication No. Sho 59-184131 (filing date: March 31, 1983), and priority of these merged applications. Foreign patent applications claiming rights, that is, Korean patent application No. 2990 (filing date June 30, 1983) and the following patent applications (AU.P.App.16293 (filing date June 27, 1983)) ,
CA.P.App.431387 (filing date June 28, 1983), DK.P.App.
3008 (filing date June 29, 1983), EP.P.App.83106288.0
(Filing date June 28, 1983), EP.P.App.523754 (filing date 1
June 30, 983), FI.P.App.832383 (application date June 1983)
29th), No.P.App.2399 (filing date June 30, 1983), PH.
P.App.29151 (filing date June 30, 1983), TW.P.App.7211
983 (filing date June 18, 1983), US.P.App.508163 (filing date June 27, 1983)].

As a result of the investigation by the present inventors, it was not possible to find a document that reported the action activity of a diabetes treatment / prevention agent or a hypoglycemic agent containing an earthworm dry powder or an earthworm extract as an active ingredient.

That is, it was not possible to find a literature that reported that the blood glucose level was significantly lowered when the dry powder of earthworm and the extract of earthworm were administered to experimental diabetic mice caused by alloxan.

In addition, earthworm dry powder was administered to human diabetic patients along with diet for 4 to 9 months. Especially in the case of mild / moderate diabetic patients, the blood glucose level improved from 2 to 3 months after administration, and it was possible to lower the blood glucose to the standard value of healthy people from 4 months after administration. We could not find any literature that reported excellent effects.

Furthermore, it was not possible to find a document reporting a method for producing a dry powder of earthworm and an extract of earthworm having such an effect.

Problems to be Solved by the Invention: Organic synthetic compounds such as sulfonyl urea and biguanide compounds have been widely used as oral therapeutic agents for diabetes.

The inventors of the present invention have long earnestly studied the use of earthworm dry powder for the purpose of creating a highly safe drug without side effects, not from such synthetic organic compounds, but from natural products. Surprisingly, they have found that dry earthworm powder and earthworm extract are effective for the treatment and prevention of diabetes, and completed the present invention.

Means for Solving the Problems The present invention relates to a dry powder of earthworm which contains a dried product of earthworm as a part of the form; an aqueous solvent of the earthworm, a water-miscible organic solvent (meaning an organic solvent which is miscible with water). And a water-immiscible organic solvent (meaning an organic solvent that is immiscible with water) containing at least one extractant selected from the group consisting of earthworms as an active ingredient. Urine fluid treatment / prevention agent. Furthermore, as a result of detailed studies to obtain a high yield of a dry powder of sterile earthworm which has a safe and excellent therapeutic effect on diabetes and can be stored or stored in a sealed state for at least 4 years, the following novel And an improved manufacturing method with an inventive step was established.

Production method 1: Live earthworms in fresh water or organic acids such as acetic acid, citric acid, succinic acid, malic acid, tartaric acid, lactic acid, or phosphoric acid, sulfuric acid,
0.3 (wt)% of at least one compound of inorganic acid such as hydrochloric acid or sodium or potassium salt of these acids
0.5% at a temperature of 1 to 25 ° C in a low-concentration or slightly acidic aqueous solution of pH 3 to 6.5 containing 0.1% by weight or less.
~ 72 hours, preferably at a temperature of 2-15 ° C for 1-40 hours, to excrete the fecal soil in the digestive tract of live earthworms by the excretion power of live earthworms themselves, and then attach it to the body surface of live earthworms with water. The remaining filth was washed off and wet pulverization was performed. Wet-ground earthworms at a low temperature of -5 ° C or lower,
After freezing, preferably at a low temperature of -10 to -60 ° C, freeze / vacuum drying was performed. Freezing and vacuum drying conditions are temperature -60 to + 90 ° C, vacuum degree 100mmHg or less, preferably -40 to
Freezing and vacuum drying were carried out for 5 to 100 hours, preferably 10 to 60 hours, while raising the temperature stepwise at + 80 ° C. and a vacuum degree of 30 mmHg or less to obtain a sterile powder of earthworm.

Process 2: After washing away the dirt attached to the body surface of live earthworms with water, fresh water or organic acids such as acetic acid, citric acid, succinic acid, malic acid, tartaric acid, lactic acid or phosphoric acid, sulfuric acid, hydrochloric acid, etc. Inorganic acid or at least one compound of sodium or potassium salt of these acids is contained in an amount of 0.3 (wt)% or less, preferably 0.1 (wt)% or less in low concentration or pH 3
0.5 to 6.5 in slightly acidic aqueous solution at 1 to 25 ° C
After leaving for 72 hours, preferably at a temperature of 2 to 15 ° C. for 1 to 40 hours, the excrement power of the live earthworm itself excreted the fecal soil in the digestive tract of the live earthworm, and then wet grinding was performed. Wet-ground earthworms are frozen at a low temperature of -5 ° C or lower, preferably -10 to -60 ° C, and then frozen.
Vacuum drying was performed. The freezing and vacuum drying conditions are a temperature of -60 to + 90 ° C and a vacuum degree of 100 mmHg or less, preferably -40 to
Freezing and vacuum drying were carried out for 5 to 100 hours, preferably 10 to 60 hours while raising the temperature stepwise at + 80 ° C. and a vacuum degree of 30 mmHg or less to obtain a sterile dry powder of earthworms.

As a method for wet grinding of earthworms, that is, a method for destroying the tissue (cells) of earthworms, use a homogenizer, a blender, a homomixer, a pickling machine, or a pressure-type cell disruption device to prepare a suspension or homogeneous solution. Is preferred. The temperature during this wet pulverization is preferably 1 to 25 ° C, preferably 2 to 15 ° C.

By the above-mentioned procedure, yellow-brown or brown earthworm dry powder could be obtained from living earthworms in a yield of 20 to 35%. Usually, the water content of this earthworm dry powder is 5 to 16%, preferably 7 to 14%, ash content is 3 to 8%, preferably 4 to 7%, and nitrogen content is 1 to 11%, preferably 6 to 11%. Was prepared. Further, in the earthworm dry powder, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, lysine, histidine, arginine 18 kinds or It contains the amino acids before and after it.

Test Example 1 Table 1 shows the results of rough analysis of the dried earthworm powder obtained by the above-mentioned production methods 1 and 2.

Test Example 2 Table 2 shows the component analysis results of the M-2 and M-4 obtained by the method of the production method 1 and the M-5 dried earthworm powder product obtained by the method of the production method 2.
2 shows.

Test Example 3 M-2 and M-4 obtained by the method of production method 1 and M-5 obtained by the method of production method 2 were subjected to amino acid analysis of the crude protein in the earthworm dry powder product to obtain protein foods, fish meal and soybean flour. The results of comparison with the analytical values of are shown in Table-3.

From Table-2 and Table-3, it was found that the earthworm dry powders obtained by the methods of Production Methods 1 and 2 were rich in crude protein, crude lipid and various metals, and also in the crude protein. It was found that the amino acid composition contained a large amount of essential amino acids.

The production method 1 yielded a slightly more preferable product than the earthworm dry powder obtained in the production method 2. Further, it is better to leave live earthworms in an aqueous solution containing the above-mentioned organic acid or inorganic acid or at least one compound of sodium or potassium salt of these both acids in a low concentration as described above, to leave it in fresh water. Excretion of fecal soil in the digestive tract of live earthworms is faster and the excretion rate is higher than that of live earthworms.

In the above-mentioned production methods 1 and 2, the finished state of the earthworm dried product after the completion of the drying is often an aggregated state or a state in which lumps are mixed. However, when it is crushed, it easily becomes a powder. When the dried earthworm and the extract of earthworm are administered to mammals including humans, the powder is preferable to the mass.

However, there is a drawback that it becomes unusable.

Mihara Tsune et al. [Japanese Patent Application Publication No. Sho 59-63184 and Sho 5]
9-184131], fractionated six novel proteases of fibrinolytic enzymes from earthworms. In other words, Mihara, et al. Added ammonium sulphate to 10 times the amount of physiological saline to the earthworm powder and incubated it for 2 days. The supernatant was subjected to ammonium sulfate fractionation, and then the precipitate. Gel separation with Sephacryl S-200 was performed.
Cellulose ion-exchange chromatography showed that I and I with casein-degrading and fibrin-degrading activities.
Proteins of I and III fractions were obtained. The fractions I, II and III were further purified with DEAE-cellulose, Sephadex G-75, Toyopearl HW55, ACH-Sepharose, Benzamidine-Sepharose and the like to obtain 6 fractions of purified enzyme. When the molecular weight was measured by SDS-PAGE, the molecular weight of the fraction I-0 was the lowest, and it was calculated to be 23,500.
1, I-2, II, III-1, III-2 and molecular weight increase,
III-2 had a molecular weight of 34,200. Moreover, when the isoelectric point of these 6 fractions was measured by isoelectric focusing, I-0 was the highest,
The pH was 4.12.
It was H3.52. These 6-fractions are new proteases different from serine enzymes, and the optimum pH of these 6-fraction proteolytic enzymes is around 8 or pH 8-10, stable pH.
Is 4-12 or 5-12, the optimum temperature is 50 ℃ or 50-60 ℃,
The quenching condition is reported to have been 70 ° C. for 60 minutes.

Earthworm dry powder product M-4 obtained by the above-mentioned production methods 1 and 2
When 10 times the amount of physiological saline was added to M-5 and M-5, and the supernatant was measured with a standard fibrin plate, fibrinolytic activity was immediately observed as shown in Table 4. When the physiological saline solution of the earthworm dry powder of M-4 was incubated at 37 ° C, the fibrinolytic activity of the supernatant was 10 as shown in Table-4.
It becomes about 4 times on the day 5, 5 times on the 50th day, and 5.5 times on the 75th day. This fact is considered to be because a large amount of this proteolytic enzyme precursor is present in the lyophilized powder of earthworm, and the enzyme activity is expressed by autolysis. When the activity of the supernatant on the 50th day was converted into international units of urokinase and compared, it was calculated to be about 8,000 IU / ml. This enzyme also dissolves both plasminogen-free fibrin plates and standard fibrin plates, and the standard frybrin plates have a larger dissolution window than plasminogen-free plates, and the enzyme activity that directly decomposes fibrin, as well as the plasminogen activator activity. Was also shown. In the above operation, M-4
The same results as in Table 4 were obtained as a result of measurement using M-5 earthworm dry powder instead of.

The details of the reason why the dry earthworm powder of the present invention and the extract of earthworm have diabetes treatment / preventive effect or hypoglycemic effect by oral administration to rat and human are unknown, but the dry earthworm powder and earthworm It is considered that it is due to the proteolytic enzyme (protein) itself or the precursor of this enzyme (protein) itself or the protein or lipid substance or other unknown compound itself contained in the extract of (1) or the combination thereof. The nitrogen content of the earthworm dry powder of the present invention most preferred as a therapeutic agent for diabetes is 7-10.
%, That is, a crude protein content of 43.8 to 62.5%.

Next, a preferable specific example of the freeze / vacuum drying operation of the suspension obtained by the wet pulverization of earthworms obtained by the above-mentioned production methods 1 and 2 is as follows.

Wet ground product of earthworms, that is, earthworm suspension -10 ~
After freezing at −60 ° C., preferably −30 to −50 ° C. for 5 to 60 hours, it is freeze-dried at the same temperature under a vacuum of 0.01 to 0.2 mmHg for 5 to 12 hours. Next, at 20-30 ℃ for 5-15 hours, 0.01-0.2mmH
Dry under vacuum of g. Next, it is dried at 35 to 50 ° C. under a vacuum of 0.1 to 0.5 mmHg for 10 to 20 hours. Vacuum drying in the next final finishing step is performed under vacuum of 0.01 to 0.5 mmHg at 70 to 80 ° C., preferably 75 to 80 ° C. for 5 to 10 hours to obtain a sterile earthworm dry powder containing 5 to 15% of water content. I was able to get it. Especially, vacuum drying for final finishing is an important operation. The present inventors have studied in detail in order to make the dry powder of the earthworm aseptic without deactivating the activities of the protease and the precursor of the enzyme contained in the dry powder of the earthworm, and as a result, freeze-vacuum. It was found that the combination of the three factors of vacuum degree, heating temperature and time of the vacuum drying conditions in the final step of drying is an important condition, and the above operating conditions were established.

As described above, all six proteolytic enzymes purified from earthworm dry powder (Japanese Patent Application Publication Nos. 59-63184 and 59-184131) were reported to be inactivated at 70 ° C for 60 minutes. However, the proteolytic enzyme in the earthworm dry powder obtained by the production method of the present invention is not inactivated as shown in Table 4.

Earthworm dry powder obtained by the methods 1 and 2 is 5 to 45 ° C.
In the case where the sample was stored at room temperature in a sealed state for 5 years, no generation of mold and other alterations were observed.

The form of the earthworm dry powder of the therapeutic agent for diabetes of the present invention is, in addition to the earthworm dry powder obtained by the above-mentioned production methods 1 and 2, a dried product of earthworms obtained by the conventionally known production methods i, ii, and iii, and the same. It may be a dry powder.

The earthworm extract, which is a therapeutic agent for diabetes, can be obtained by the following production method.

The earthworm as a raw material is subjected to extraction treatment with at least one kind of extractant selected from the group consisting of an aqueous solvent, a water-miscible organic solvent and a water-immiscible organic solvent, and the obtained extract is concentrated to precipitate as a precipitate. Or, to obtain a precipitate by adding the organic solvent of the other kind or the same kind to the concentrated extract,
Alternatively, the extraction solvent was completely removed under normal pressure, increased pressure or reduced pressure to obtain an extract.

The earthworms of the raw material include the earthworm dry powder obtained by the above-mentioned production methods 1 and 2; the dried earthworm product obtained by the known production method of i, ii and iii and its dry powder; It is possible to use live earthworms or earthworms of these shapes; earthworms in the form of live earthworms and / or ground suspensions of ground earthworms.

Of these, the most preferable form of the material earthworm is a suspension of earthworms obtained by performing wet pulverization in the operation processing steps of the above-mentioned production methods 1 and 2.

That is, live earthworms are organic acids consisting of acetic acid, citric acid, succinic acid, malic acid, tartaric acid or lactic acid; inorganic acids consisting of phosphoric acid, sulfuric acid or hydrochloric acid; sodium salts of these organic acids; potassium salts of these organic acids; Inorganic acid sodium salt; at least one kind of compound selected from the group consisting of these inorganic acid potassium salts is left in a low concentration of 0.3 (wt)% or less or in a slightly acidic aqueous solution of pH 3 to 6.5 or in fresh water. After the fecal soil in the digestive tract of live earthworms is spread, the filth adhering to the body surface of the live earthworms is washed away with water and wet-milled, or the filth adhering to the body surface of the live earthworms is washed with water. After removing the live earthworms in the above-mentioned aqueous solution or in fresh water to spread the excrement in the digestive tract of the live earthworms, wet pulverization is performed, and a paste-like suspension of earthworms is obtained. . When this suspension is used as a raw material, the amount of solvent used can be saved more than other forms of earthworm, and the active substance as a therapeutic agent for diabetes can be extracted in a short time. Excellent extraction efficiency such as large amount of extract. The next preferable raw material form is the earthworm dry powder obtained by the above-mentioned manufacturing methods 1 and 2.

Aqueous solvent of extraction solvent is fresh water, distilled water, physiological saline, pH
It is preferable to use 5 to 10 various buffers or various preparation waters. As the buffer solution, one or more buffer solutions having various compositions such as phosphoric acid, acetic acid, boric acid, citric acid, Tris / hydrochloric acid having a pH of 5 to 10, preferably pH 6 to 8, can be used. Further, the pH 5-10, preferably pH 6-8 preparation liquid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid,
It means a dilute aqueous solution prepared in-house from a water-soluble inorganic acid such as lactic acid, citric acid, succinic acid, malic acid or tartaric acid, an organic acid and an alkali metal hydroxide or carbonate such as sodium or potassium. The most preferred aqueous solvents are saline, buffer and fresh water. When using these aqueous solvents, preservatives such as para-hydroxybenzoic acid ethyl ester, benzoic acid sodium salt and sodium azide should be used.
It is preferable to use 0.01 to 0.3 (weight)%.

Examples of the water-miscible organic solvent include methyl, ethyl,
Allyl, n-propyl, i-propyl, n-butyl, s
-Butyl or t-butyl alcohol and the like having 1 to 1 carbon atoms
Lower alcohols of 4; ketones having 3 to 4 carbon atoms such as acetone or methyl ethyl ketone; ethyl ether, 1,
It is preferable to use C3-C4 ethers such as 2-propylene oxide, dioxolane, 4-methyldioxolane or dioxane; C2-C4 esters such as methyl formate, ethyl formate and methyl acetate. Of these, the most preferred water-miscible organic solvent is an alcohol having 1 to 4 carbon atoms.

Examples of water-immiscible organic solvents include n-hexane and
Hydrocarbons having 6 to 10 carbon atoms such as i-hexane, n-heptane, i-heptane, octane, i-octane, cyclohexane, benzene, toluene, xylene or ethylbenzene; n-hexyl, 2-methylpentyl, 2- Alcohols having 6 to 10 carbon atoms such as ethylbutyl, s-hexyl, s-heptyl, n-octyl, 2-ethylhexyl, 2,6-dimethyl-4-heptyl, n-decyl alcohol; methyl-n-amyl ketone, methyl C7-8 ketones such as cyclohexanone or methylhexyl ketone; s-butyl acetate, i-butyl acetate, acetic acid-2
It is preferable to use esters having 6 to 8 carbon atoms such as ethylbutyl, s-amyl acetate or methyl amyl acetate, and halogenated hydrocarbons having 1 to 5 carbon atoms such as chloroform, dichloropentane or epichlorohydrin.

The extraction method may be a general method and is not particularly limited, but one example is as follows.

For the earthworms of the above-mentioned form, an aqueous solvent, a concentration of 5 to 70
(Volume)% preferably selected from the group consisting of an aqueous solution of an organic solvent having a concentration of 10 to 60 (volume)%, a water-miscible organic solvent having a concentration of 80 to 100 (volume)%, and a water-immiscible organic solvent having a concentration of about 100%. 1 to 100 times volume (volume / weight), preferably 2 to 30 times volume, of at least one extractant used, and the extraction temperature is -40 to +60.
℃, extraction contact time is 10 minutes to about 100 days, preferably 30
The mixture was allowed to stand for 4 minutes to about 40 days with occasional or continuous stirring, and the extract was collected by passing. It is preferable to use a pressure or pressure reducer (machine) or a centrifugal separator for the filtration. After thoroughly washing the extraction residue, the extraction liquid and the washing liquid are combined. The extract or the combined solution was concentrated at a temperature of 60 ° C. or lower under normal pressure, increased pressure or reduced pressure, and the extraction solvent was removed by a concentration method such as ultra-overconcentration or freeze-drying to obtain an extract. However, when, for example, ethanol is used as the extractant, ethanol may remain without being completely removed depending on the type of the final intended dosage form. Further, there is also used a crystal precipitation method in which the extract or the concentrated solution of the combined solution is allowed to stand, or a method in which an organic solvent is added to the concentrated solution to precipitate crystals, and then the extract is dried to obtain an extract.

The yield of the extracted liquid with respect to the raw earthworms in the above operation is 3 to 60 (weight)% in terms of dry matter. This difference in yield depends on the morphology of the raw material earthworm, the type of solvent, the extraction temperature, the extraction time and other extraction conditions. If the extraction temperature is high, the active substance may be decomposed, so the extraction temperature is -40 to + 60 ° C.
Is preferred.

The components contained in the extract are, as shown in Table 2 and Table 3,
18 amino acids, similar to the ingredients in earthworm dry powder
Contains 8 kinds of metals. In particular, the protein in this extract contains a protein consisting of the same amino acid as the starting earthworm, a glycoprotein, a metal-binding protein, and the like. Furthermore, the extract contains free amino acids, lipids, various sugars, ATP and ATP-like substances, and other effective chemical substances of unknown composition.

The earthworms used in the present invention are red earthworms (Lumbricu
s rubellus), LT earthworm (aka Tsurumiwami) (Lumbricu
s terrestris), zebra earthworm (Eisenia foetida), brown earthworm (Allolobophora caliginosa), violet earthworm (Dendrobaena octaedra), sakura earthworm (Allolobophora japonica Michaelssen), hatta earthworm (Drawida hattamimizu hataidiver, groves). (Pheretima communissima), Hatakemimizu (Pheretim
a agrestis), Siebold earthworm (Pheretima sieboldi
Horst), Spotted Earthworm (Pheretima hilgendorf)
i), the earthworm (Pontodrilus matsushimensis Iizuk
a), Itomizu (Tubifex hattai Nomura), Goutouitomizu (aka Yurimizu) [Limnodrilus gotoi Ha
tai ＝ L, socialis Stephenson], and any earthworm that normally grows and is not toxic can be used.

When the earthworm dry powder and earthworm extract of the present invention are administered for clinical treatment, they can be used alone or in combination. The form may be either oral or parenteral, but oral administration is particularly preferable. The oral dosage form of the product of the present invention includes capsules, tablets, granules, scattered (powder), coating agents, dragees, emulsions by mixing the product of the present invention itself or an appropriate pharmacologically acceptable pharmaceutical carrier. Formulations such as Examples of the pharmaceutical carrier include lactose, sucrose, mannitol, butter sugar, den powder as an excipient,
Sorbitol, glycine, calcium phosphate, microcrystalline cellulose, etc .; den powder, gelatin, gum arabic, glucose, sucrose, sorbitol, mannitol as a binder
Tragant, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5-vinylpyridine-methacrylic acid-methylethyl acrylate copolymer, polyvinylpyrrolidone, sodium alginate, etc .; stearic acid as a lubricant, hardened oil , Magnesium stearate, calcium stearate, polyoxyethylene monostearate, talc, silicon oxide, polyethylene glycol and the like; disintegrants such as valesioden powder and den powder containing a surfactant; and wetting agents such as sodium lauryl sulfate. When administered parenterally, it can be used as a suppository. Especially as a base for suppositories, cocoa butter, Witepsol, Subana
l), polyethylene glycol, polypropylene glycol, glycerogelatin, gelatin capsules and the like are used. Others, methyl para hydroxy benzoate,
Known safe preservatives such as propylparahydroxybenzoate, butylparahydroxybenzoate, and butylhydroxyanisole, and other safe dyes are used in combination.

The dose of the earthworm dry powder and earthworm extract of the therapeutic agent for diabetes of the present invention varies depending on the administration method, age, weight, condition of the patient and type of disease, but usually from 0.01 g per day to human. About 5 g is preferable. Most preferable is to administer 0.02 g to 2 g per day in 1 to 3 divided doses per day.

Action: The toxicity of the earthworm dry powder of the present invention and the pharmacological test method for the therapeutic effect on diabetes or hypoglycemia and the result thereof will be described in detail below.

A. Acute toxicity test: An acute toxicity test was carried out by oral administration using ddy male mice weighing 30 ± 2 g and Wistar male rats weighing 100 ± 2 g each in groups of five. Earthworm dry powder M of the present invention
-1 (water 10.2%, ash 5.1%, nitrogen 9.4%); Same as M
-2 (water content 10.4%, ash content 5.3%, nitrogen 8.6%); Same M
-3 (water content: 10.7%, ash content: 5.2%, nitrogen content: 9.2%);
-4 (water content: 10.6%, ash content: 5.6%, nitrogen content: 9.6%);
-5 (water content: 9.5%, ash content: 4.5%, nitrogen content: 7.8%);
Each dose of -6 (water 9.2%, ash 4.7%, nitrogen 8.4%) was increased from 0.1g / Kg to 8g / Kg, and the mouse (0.1 to 5g / Kg) and rat (2 to 8g) were added. / Kg) was individually dosed by gavage with a throat dragging rod. Animals were maintained at a room temperature of 22-23 ° C during the study period and observed for 14 days after dosing. No mortality was observed at the dosed dose. I observed toxicosis and behavior after administration over time,
No difference from the normal animal group was observed. Moreover, the weight gain was almost the same as that of the normal group. No macroscopic damage was observed in any part of the main organ in the autopsy performed after the test. Therefore, the earthworm dry powder of the present invention could not determine the LD50 value due to its very low toxicity.

B. Effect on Experimental Diabetic Mice 1. Animals: One group of 5 male ddy mice each weighing 30 ± 2 g was used.

2. Feed and breeding conditions: using solid feed manufactured by CLEA Japan,
One mouse was placed in one cage, and food and water were freely available. The animals were kept at a constant temperature and humidity of 23 ± 1 ° C and a humidity of 55 ± 5%.

After fasting male ddy mice for 16 hours, alloxan 75m
g / Kg was administered intravenously and 48 hours later, the earthworm dry powder M-1 of the present invention (water content 10.2%, ash content 5.1%, nitrogen 9.4%);
Same M-2 (water content 10.4%, ash content 5.3%, nitrogen 8.6%);
-3 (water 10.7%, ash 5.2%, nitrogen 9.2%); M-4
(Water content 10.6%, ash content 5.6%, nitrogen 9.6%); M-5 (water content 9.5%, ash content 4.5%, nitrogen 7.8%); M-6 (water content 9.2)
%, Ash 4.7%, nitrogen 8.4%) 6 kinds (300 mg / Kg) suspended in physiological saline orally and orally administered, and after 150 minutes, blood was collected from the heart and blood glucose was measured by glucose oxidase method. did.

The measurement results are shown in Table-5. It has been found that the earthworm dry powder of the present invention significantly lowers blood glucose.

Manufacturing method 3 Preparation of raw earthworms: 10 kg of live earthworms (red earthworms), malic acid and lactic acid 2: 1
10 ℃ in 20 l of pH 5.8 acidic aqueous solution containing mixed acid
It was allowed to stand for 3 hours to allow the fecal soil in the digestive tract to be excreted sufficiently.
This live earthworm is thoroughly washed with water to remove dirt, feces, and other dirt attached to the body surface of the live earthworm, and then wet pulverized with an ultra homo mixer (manufactured by Nippon Seiki Co., Ltd.) to suspend the earthworm. A suspension of 9.3 kg was obtained. This earthworm suspension
Divide into 1.55kg and divide into 6 equal parts, E-1, -2, -3, -4,
-5 and -6.

1.55 kg of the E-1 and E-2 earthworm suspensions obtained above are placed in each tray and frozen at -40 ° C for 30 hours. Then, freeze-dry at -40 ℃ under vacuum of 0.1mmHg for 6 hours, then 25
After drying under vacuum of 0.1mmHg at ℃ for 8 hours, then at 45 ℃
Dried under vacuum of 0.1mmHg for 12 hours and finally 0.1mH at 80 ℃
Drying under g vacuum for 6 hours yielded 318 g of dried earthworm in each of two trays. These are designated as K-1 and K-2.

Preparation of extract: X-. 5% of 99% of dried earthworm (K-1) 315g
After adding 1.575 l of methanol and extracting 3 times at 25 ° C, the residue was washed with 0.945l of 99% methanol 3 times, and the combined extract and washings were concentrated to dryness at 30 ° C under reduced pressure. After vacuum drying, 88.2 g of powder extract was obtained. This is designated as Extract X-.

X-. To 315 g of dried earthworm (K-2), a mixture of 3 volumes of 0.945 l of chloroform and 2 volumes of 0.63 l of ethanol was added, and the mixture was extracted 3 times at 20 ° C. Wash with 0.63 l of ethanol, and combine the extract and wash at 30 ° C.
Concentrate to dryness under reduced pressure at 72.5% by vacuum drying.
g powder extract was obtained. This is designated as Extract X-.

X-. To 1.55 kg of the earthworm suspension (E-3), 6.2 l of fresh water is added, and the mixture is stirred at 10 ° C. for 2 hours, then passed and separated into an extract and a residue. After the residue was washed with water, 7.7 l of a combined liquid of the extract and the wash was obtained. After adding 2.3 liters of n-hexane to 7.7 liters of this combined liquid and stirring, the mixture was allowed to stand and separated into a water-soluble part layer and an n-hexane layer. The water-soluble part layer was separated and concentrated to dryness under reduced pressure at 30 ° C. Then, it was freeze-dried to obtain 86.8 g of a powder extract. This is designated as Extract X-.

X-. To 1.55 kg of the earthworm suspension (E-4), 10 l of 40% aqueous ethanol solution was added, and after stirring at 40 ° C for 12 hours,
And separate the extract and residue. The residue was washed with 0.3 l of 40% ethanol, and the combined extract and washing solution was concentrated to dryness under reduced pressure at 40 ° C and then vacuum dried to give 69.7 g of powdered extract.
Got This is designated as Extract X-.

X-. 2.5 l of pH 6.4 phosphate buffer was added to 1.55 kg of the earthworm suspension (E-5) and incubated at 37 ° C. for 8 hours to accelerate the action of the protease originally contained in the earthworm body. After that, the residue was washed with a phosphate buffer of pH 6.4 to obtain 3.9 l of a combined solution of the extract and the wash.
After adding 1 g of sodium azide to this combined solution and incubating at 37 ° C. for 8 hours, the mixture was concentrated under reduced pressure at 30 ° C., and the obtained concentrated liquid was added with twice the amount of ethanol, and the precipitate was collected by filtration. Was dried in vacuum and then freeze-dried to obtain 64.2 g of powdery extract. This is designated as Extract X-.

X-. 1.55 kg of the earthworm suspension (E-6) was naturally dried in the sun, and then dried earthworm powder (K-3) was mixed with ethanol and ethyl ether 2: 1 mixed solvent 9
00 ml was added, and the mixture was extracted 3 times at 20 ° C. Finally, the residue was washed with the above mixed solvent, and the combined solution of the extract and the wash was concentrated to dryness at 30 ° C. under reduced pressure, and then vacuum dried to obtain 61.5 g of an extract. This is designated as Extract X-.

Production method 4 13 kg of cultured live earthworms (red earthworms) with mud are thoroughly washed with water to wash off the mud adhering to the body surface, and then a 1: 1 mixed acid of malic acid and citric acid is dissolved in pH 6 .2
After leaving it in 40 liters of the acidic aqueous solution for 3 hours at a temperature of 8 ° C to excrete the fecal soil in the digestive tract sufficiently, the live earthworms are thoroughly washed with water and the mud and feces attached to the body surface of the live earthworms. After the dirt was washed off, live earthworms were wet-milled with a mixer to obtain 9.0 kg of earthworm suspension. This earthworm suspension was divided into 6 equal parts of 1.5 kg each, and they were divided into E-7, -8,-
They are 9, -10, -11 and -12.

1.5 kg of the E-7 and E-8 earthworm suspensions obtained above were placed in each tray and frozen at -30 ° C for 40 hours, and then frozen at -40 ° C under a vacuum of 0.1 mmHg for 6 hours. Dry, then raise the temperature of the tray on which it is placed to 30 ° C and dry under vacuum of 0.1mmHg for 6 hours, then raise the temperature of rack to 50 ° C and vacuum dry under vacuum of 0.2mmHg for 8 hours, and finally to the shelf. The temperature was raised to 78 ° C. and dried under a vacuum of 0.2 mmHg for 8 hours to obtain 305 g of each dried product of earthworm. This is designated as K-7 and K-8.

Preparation of extract: X-. To 300 g of dried earthworm (K-7), 0.02% of 3 l
0.9% Sodium chloride aqueous solution containing para-hydroxybenzoic acid ethyl ester was added, and the mixture was stirred at 30 ° C. for 96 hours and then passed through, the residue was washed with 0.9 l of 0.9% sodium chloride aqueous solution, and the extract and the washing solution were combined. 3.8 l of clear extract
Got This was concentrated by the ultrafiltration method to a liquid volume of 0.16 l, and 0.16 l of ethanol was added to this to separate the precipitate, which was then added to the liquid to a final concentration of 80% ethanol. Was vacuum dried to obtain 14.4 g of crystal powder. This is designated as Extract X-1.

The liquid of Extract X-1 was concentrated under reduced pressure to dryness at 30 ° C. to obtain 72.5 g of crystalline powder. This is the extract X--
Set to 2.

X-. To 300 g of dried earthworm (K-8), 0.1% of 3 l
Fresh water containing sodium benzoate was added, and the mixture was stirred and extracted at 32 ° C. for 72 hours, and then extracted, and the residue was washed with 0.6 l of fresh water to obtain 3.5 l of a clear extract containing the extract and the washing solution. The extract was concentrated by an ultrafiltration method and concentrated under reduced pressure to dryness to give 69 g of crystalline powder. This is designated as Extract X-.

X-. 1.5 kg of the earthworm suspension (E-9) was vacuum dried at 40 ° C., and 3 L of 0.02% para-hydroxybenzoic acid ethyl ester was added to powdered earthworm dry powder (K-9). Distilled water is added, and the mixture is stirred at 32 ° C for 120 hours, passed, and separated into an extract and a residue. Thoroughly wash the residue with distilled water, and then combine the extract and washing solution at 30 ° C.
After concentrating under reduced pressure to about 1/10 volume, adding 4 volumes of ethanol, stirring well, allowing to stand for a whole day and night, filtering the formed precipitate, washing with ethanol, vacuum drying, and powder extract 72.5 g Got This is designated as Extract X-.

X-. To 1.5 kg of the earthworm suspension (E-10), add 7.5 l of 60% aqueous ethanol solution, stirring occasionally with a tight stopper.
It was allowed to stand at 20-22 ° C for 8 days.

Then, the residue was washed with 0.5 l of a 50% aqueous ethanol solution, and the combined solution of the extract and the wash was concentrated to dryness under reduced pressure at 30 ° C. and freeze-dried to obtain 40.5 g of a powder extract. This is designated as Extract X-.

X-. A mixed solvent of 4.5 l of a 50% aqueous ethanol solution and 3 l of a 50% isopropanol aqueous solution was added to 1.5 kg of the earthworm suspension (E-11), and the mixture was allowed to stand still at 8 ° C for 16 days while occasionally stirring with a stopper.

Then, the residue was washed with 0.5 l of a 50% aqueous ethanol solution, and the combined solution of the extract and the wash solution was concentrated to dryness under reduced pressure at 35 ° C. and freeze-dried to obtain 28.2 g of a powder extract. This is designated as Extract X-.

X-. To 1.5 kg of the earthworm suspension (E-12), add 7.5 l of a 60% aqueous ethanol solution, and stir at 25 ° C for 2 hours to mix 3
It was extracted twice, and finally the residue was washed with 60% aqueous ethanol solution 1 and the combined extract and washings were concentrated to dryness under reduced pressure at 30 ° C and then freeze-dried to obtain 90.5 g of a powder extract. It was
This is designated as Extract X-.

Earthworm extract of the present invention, i.e., the raw earthworm is subjected to extraction treatment with at least one extractant selected from the group consisting of an aqueous solvent, a water-miscible organic solvent and a water-immiscible organic solvent, to obtain an extract The pharmacological test method for the toxicity and blood glucose lowering activity and the results are as follows.

A-1. Acute toxicity test: Measured by the same method as the acute toxicity test method of A above. That is, an acute toxicity test by oral administration was carried out using 5 groups of male ddy mice each weighing 30 ± 2 g.

For example, the above-described earthworm extract X-,-,
Eight types of −, −, −, −, −, and − were used to increase the dose from 0.1 g / kg to 5 g / kg, and the mice were individually dosed by gavage with a throat drag pin.

Mice were maintained at a room temperature of 22-23 ° C during the study and 14
Observed for a day. No mortality was observed at the doses administered. Moreover, no abnormalities were found in the findings of poisoning or autopsy. Therefore, the extract with the aqueous and / or organic solvent from the earthworms of the present invention could not determine the LD ₅₀ value due to its very low toxicity.

B-1. Effect on experimental diabetic mouse Pharmacological test: The effect on experimental diabetic mouse of B was measured by the same method as in the pharmacological test. That is, 1. Animals: 5 male ddy mice each weighing 30 ± 2 g were used.

2. Feed and breeding conditions: using solid feed manufactured by CLEA Japan,
One mouse was placed in one cage, and food and water were freely available. The animals were kept at a constant temperature and humidity of 23 ± 1 ° C and a humidity of 55 ± 5%. The ddy male mouse was fasted for 16 hours, and 75 mg / kg of alloxan was intravenously administered, and 48 hours later, 4 kinds of the earthworm dry powder of the present invention [K-1, -3, -7 and -9]. To 300m
g / kg, or 9 kinds of earthworm extract of the present invention [X-
,-,-,-,-,-,-,-And-
50 mg / kg of each of the above] was orally suspended in physiological saline, and after 150 minutes, blood was collected from the heart and the blood sugar amount was measured by the glucose oxidase method.

The measurement results are shown in Table-5-1. The earthworm dry powder and earthworm extract of the present invention were found to significantly lower blood glucose. Earthworm extract is 1/6 of earthworm dry powder
The rate of decrease in blood sugar level was higher than that of dry earthworm powder.

C. Oral Administration of Earthworm Dry Powder to Humans: Volunteer of 5 people who agreed with this experiment
A capsule C prepared in Example 6 to be described later along with diet was added to 1 capsule of earthworm dry powder (M-3,
Water 10.7%, ash 5.2%, nitrogen 9.2%) 150mg]] 1
1 capsule 3 times a day was taken orally within 30 minutes after eating. Blood collection is performed only for a 62-year-old man every month after administration.
The drug was administered for 9 months and blood was collected. A 59-year-old man took blood before and 2, 3, and 4 months after the administration. A 76-year-old woman collected blood before and 4 months after the administration, and a 79-year-old woman collected blood before and 3 and 6 months after the administration. The 61-year-old woman is before and 1
Blood was collected after 3, 4 and 8 months. The blood sugar level was measured by the glucose oxidase method. This result is
6 shows.

The standard value of blood glucose (mg in healthy people of local people who carried out this experiment)
/ dl) 50 to 100 before breakfast, 150 hours or less after 2 hours, 5 before lunch
0 to 100, 2 hours after lunch, 150 or less, 50 to 100 before dinner, 2 dinners
Less than 150 hours later. All of the five volunteers shown in Table 6 had high blood sugar levels on the fasting state before the drug administration and before breakfast, and were diabetic patients.

In the case of a 62-year-old man who is considered to have moderate diabetes, the blood sugar level was improved from 2 months after the administration of the earthworm dry powder of the present invention, and after 3 months from the administration, eating out of 6 test values was performed. Two
It was improved to almost the standard value except that the value of one item after the time was as high as 185 mg / dl. After 4 months from the administration, all 6 test values reached the standard values. After that, the blood sugar level of the 6th item within the range of the standard value was maintained for 5 months.

A 59-year-old man reached the standard value except that the value of one item before lunch was slightly higher than 114 mg / dl out of the 6 items after 3 months of administration, and all 6 items were standard after 4 months of administration. Reached the value range.

A 79-year-old woman had 4 months 'standard values except 3 months after administration and 6 items' test values 2 hours after lunch and before dinner were slightly higher at 171 and 107 mg / dl. It improved within the range of values and completely reached the standard value 6 months after the administration.

A 76-year-old woman was a mild diabetic patient, and the blood sugar levels in the 6 items reached the standard values 4 months after the administration.

The 61-year-old woman is a fairly severely diabetic. This woman showed an improvement in blood sugar level 4 months after the administration of the earthworm dry powder of the present invention. After 8 months, the values 2 hours after breakfast and before lunch were as high as 197 and 210 mg / dl, respectively. The four items other than the values reached the standard values.

The oral administration experiment of this dried earthworm powder to humans could be safely completed without any side effect. For example, even after long-term administration for 6 to 9 months, there is no risk of hypoglycemia in which the blood sugar level falls below the standard lower limit, and the earthworm dry powder of the present invention is a safe drug. Wakatsuta.

The above results are summarized as follows. That is,
Oral administration of earthworm dry powder was difficult to lower the blood sugar levels of all the above 6 items to the standard value in the case of diabetic patients with severe severity. It was found that there was an improving effect such as reducing the blood sugar levels in the four items to the standard value. However, in the case of light / moderate diabetic patients, the blood sugar levels of all 6 items could be reduced to within the standard value from 4 months after administration. It has been found that the earthworm dry powder of the present invention is a safe and excellent hypoglycemic agent and an excellent therapeutic / preventive agent for diabetes.

Based on the above results, earthworm extract, which is a solvent extract from earthworms, is safe for humans as well as earthworm dry powder, and is an excellent hypoglycemic agent, excellent diabetes
It was easily found to be a preventive agent. That is, although there is a slight difference in the production method between the earthworm extract and the earthworm dry powder, it is considered a natural result because they were produced from live earthworms or ground earthworms of the same starting material.

Example 1. Tablet A Earthworm dry powder 150 mg (the components are the same as M-3 above) Manitol 123 Hydroxypropoxymethylcellulose 7 Talc 5 Microcrystalline cellulose 60 Hydrogenated castor oil 5 total 350 mg Tablet B Earthworm dry powder 150 mg ( Ingredients are the same as M-3) Corn corn flour 60 Lactose 80 talc 7 Magnesium stearate 3 Total 300 mg Tablet C Earthworm dry powder 150 mg (Ingredients are the same as M-3) Soluble den flour 20 Corn den flour 125 Microcrystalline Cellulose 45 Silicon Oxide 6 Magnesium Stearate 4 Total 350 mg According to the above formulation, the powders that were uniformly and well mixed were manufactured into tablets of various weights using a tableting machine.

Example 2. Granules A Dry powder of earthworm 150 mg (the components are the same as M-2) Lactose 20 Microcrystalline cellulose 60 Corn den powder 15 Hydroxypropyl cellulose 5 250 mg in total According to the above formulation, a fluidized bed granulator is used. , Earthworm dry powder, lactose, microcrystalline cellulose and corn den powder were mixed well and sprayed with a 5% aqueous solution of hydroxypropyl cellulose as a binder to obtain low temperature dried granules.

Example 3. Granules B Earthworm dry powder 100 mg (the components are the same as M-2 above) Manitol 10 Microcrystalline cellulose 85 Carboxymethylcellulose calcium 2 Magnesium stearate 1.5 Hardening oil 1.5 Total 200.0 mg Granules C Earthworm dry powder 150 mg (the components are the same as the above M-2) Lactose 53 Corn den powder 39 Valeshioden powder 2 Tark 3 Magnesium stearate 3 Total 250 mg According to the above prescription, the well mixed powder was manufactured into granules by an extruder.

Example 4. Capsule A Dry powder of earthworm 150 mg (the components are the same as M-2) Lactose 28 Microcrystalline cellulose 47 Manitol 10 Corn den powder 10 Polyvinylpyrrolidone 2 Hydroxypropyl cellulose 3 250 mg in total In the above formulation, hydroxypropyl Components other than cellulose were well mixed using a fluidized bed granulator, and then a 5% aqueous solution of hydroxypropyl cellulose was sprayed as a binder, and dried at low temperature to give granules. These granules into hard capsules
A hard capsule was prepared by filling 250 mg each.

Example 5. Capsule B Granules C prepared according to Example 3 are hard capsules, 250m
Each g was filled to prepare a hard capsule.

Example 6. Capsule C Earthworm dry powder 150 mg (the components are the same as M-3 above) Phosphoric acid-calcium hydrogen 60 Phosphoric acid-sodium hydrogen 10 Manitol 28 Magnesium stearate 2 total 250 mg Mix well with the above formulation. No. 1 gelatin capsules were filled with 250 mg each of this mixed powder to produce capsules.

Example 7. Enteric coated tablet Earthworm dry powder 100 mg (the components are the same as M-1) Manitol 10 Microcrystalline cellulose 85 Carboxymethylcellulose calcium 2 Magnesium stearate 1.5 Hardened oil 1.5 Total 200 mg According to the above formulation, they were uniformly mixed. An uncoated tablet was produced from the powder with a tableting machine and then coated with the following enteric coating coating agent to produce an enteric coated tablet.

Coating agent Hydroxypropyl methyl cellulose phthalate 14.8 mg Dioctyl phthalate 2.3 Stearic acid 2.3 Light silicon oxide 0.6 Total 20.0 mg Example 8. Powder A Earthworm dry powder 150 mg (the same ingredients as M-4) Manitol 50 Corn den powder 50 Total 250 mg Powder B Earthworm dry powder 150 mg (the components are the same as M-4) Calcium phosphate-hydrogen hydrogen 20 Corn den powder 80 Total 250 mg The above components were uniformly and well mixed in a conical mixer to obtain a powder.

Example 9. Suppository A Dry powder of earthworm 200 mg (the components are the same as M-5 above) Witepsol 540 (Witepsol) E-85 Witepsol 1,454 (〃) W-35 Methylparahydroxybenzoate 3 Butylparahydroxybenzoate 3 Total 2,200 mg Suppository B Earthworm dry powder 200 mg (the components are the same as M-6 above) Butylhydroxyanisole 6 Semi-synthetic glyceride 2,900 3,106 mg The melts in which the above-mentioned prescriptions are well mixed are poured into an aluminum mold, Upon cooling, suppositories were prepared.

Example 10. Capsule D Earthworm dry powder 150 mg (the components are the same as M-2) Sodium lauryl sulfate 4 Sodium phosphate-hydrogen hydrogen 1 Manitol 93 Magnesium stearate 2 Total 250 mg The above-prepared ingredients are well mixed. This mixed powder is No.1
Each 250 mg of each gelatin capsule was filled to prepare a capsule.

Example 11 Capsule E Earthworm extract 30 mg (X-) Lactose 28 Microcrystalline cellulose 47 Manitol 100 Corn den powder 40 Polyvinylpyrrolidone 2 Hydroxypropylcellulose 3 250 mg in total In the above formulation, components other than hydroxypropylcellulose were used. After mixing well using a fluidized bed granulator, a 5% aqueous solution of hydroxypropyl cellulose was sprayed as a binder and dried at 40 ° C. to give granules. In this granular hard capsule
A hard capsule was prepared by filling 250 mg each.

Example 12 Capsule F Condyle Granules Earthworm extract 30 mg (X-) Lactose 103 Corn den powder 89 Potato powder 22 Talc 3 Magnesium stearate 3 total 250 mg Extract well mixed powder according to the above formulation The granules were produced on the machine. Hard capsules were manufactured by filling 250 mg each of the granules into hard capsules.

Example 13 Capsule G Earthworm extract 30 mg (X-) Phosphoric acid-calcium hydrogen 60 Phosphate-sodium hydrogen 20 Manitol 38 Magnesium stearate 2 total 150 mg The above-prepared mixture was mixed well and the mixed powder was gelatinized. Capsules were manufactured by filling 150 mg each into capsules.

Example 14 Capsule H Earthworm extract 30 mg (X-) Sodium lauryl sulfate 4 Sodium phosphate-hydrogen hydrogen 1 Manitol 113 Magnesium stearate 2 Total 150 mg The above-prepared ingredients are mixed well. A gelatin capsule was filled with each 150 mg of the mixed powder to produce a capsule.

Example 15 Capsule I Earthworm dry powder 75 mg (same as M-2 above) Earthworm extract 15 mg (X- above) Sodium lauryl sulfate 4 Sodium phosphate-hydrogen 1 Manitol 153 Magnesium stearate 2 Total 250 mg Mix well. A gelatin capsule was filled with 250 mg of each of the mixed powders to manufacture capsules.

Example 16 Capsule J Earthworm dry powder 50 mg (same as M-4 above) Earthworm extract 20 mg (X- above) Sodium lauryl sulfate 2 Manitol 176 Magnesium stearate 2 250 mg A mixture of the above formulations is mixed well. A gelatin capsule was filled with 250 mg of each of the mixed powders to manufacture capsules.

EFFECTS OF THE INVENTION As described above, the present invention relates to a therapeutic agent for diabetes which comprises dry earthworm powder and earthworm extract as active ingredients. Administration of dry earthworm powder and earthworm extract to experimentally diabetic mice with alloxan was able to significantly reduce blood sugar levels.

Next, 5 diabetic volunteers were given one meal of earthworm dry powder capsules (150mg content) once with diet.
The drug was orally administered three times a day after meals for 4 to 9 months. From 1 month, 2 months or 3 to 4 months after administration, at least every 1 month, before breakfast, 2 hours after breakfast, before lunch, 2 hours after lunch,
Blood was collected 6 times before and 2 hours after dinner to measure the blood sugar level. As a result, in the case of mild to moderate diabetic patients, it was improved from 2 to 3 months after the administration of the earthworm dry powder, and from 4 months after the administration, the blood sugar levels of all the above 6 items were the standard values (morning
The standard value of blood glucose before three meals during the day and evening was 50 to 100 mg / dl, and the standard value of blood glucose two hours after the same meal was 150 mg / dl or less). In the case of severe diabetic patients, it has been difficult to lower the blood glucose levels of all 6 items to within the standard range, but after 8 months from the administration, the blood glucose levels of 4 items out of 6 items are lowered to the standard value. The improvement effect was shown. In addition, even after long-term administration of the earthworm dry powder for 6 to 9 months, there was no risk of hypoglycemia in which blood glucose dropped below the lower limit of the standard value, that is, by animal experiments and oral administration experiments to humans. It has been found that the dry powder of earthworm and the extract of earthworm of the present invention are safe and excellent agents for treating and / or preventing diabetes.

Claims (12)

[Claims] 1. A therapeutic agent for diabetes containing earthworm dry powder as an active ingredient. 2. Live earthworm is at least one selected from the group consisting of organic acid, inorganic acid, organic acid sodium salt, inorganic acid sodium salt, organic acid potassium salt and inorganic acid potassium salt.
After standing in an aqueous solution containing 0.3 (wt)% or less of various kinds of compounds or in fresh water to cover the excrement in the digestive tract of live earthworms, wash and remove the dirt attached to the body surface of live earthworms with water, After performing wet pulverization or washing and removing the dirt attached to the body surface of live earthworms with water, the live earthworms are left in the aqueous solution or in fresh water to spread the excrement in the digestive tract of the live earthworms, and then wet. Crush,
Freeze the obtained suspension at -10 to -60 ℃, then -60 to 80 ℃
Freeze and vacuum dry for 10 to 100 hours at a vacuum degree of 10 mmHg or less while raising the temperature stepwise within the range of
An antidiabetic agent containing an earthworm dry powder as an active ingredient, which is characterized in that vacuum drying in the final step is dried at 70 to 80 ° C. under a vacuum of 0.01 to 0.5 mmHg for 5 to 10 hours. 3. The live earthworm is left in the aqueous solution or in fresh water to cover the excrement in the digestive tract of the live earthworm, and then the dirt attached to the body surface of the live earthworm is washed and removed with water, and then wet grinding. The antidiabetic agent according to claim 2, wherein the subsequent steps are performed. 4. The therapeutic agent for diabetes according to claim 3, wherein the live earthworm before being left in the aqueous solution or in fresh water is a live earthworm in which a part of the waste is washed with water. 5. After cleaning and removing the dirt attached to the body surface of live earthworms with water, the live earthworms are allowed to stand in the above-mentioned aqueous solution or fresh water to cover the excrement in the digestive tract of the live earthworms, and then wet grinding Claim 2 which performs the said process
The therapeutic agent for diabetes according to the item. 6. After washing away the dirt attached to the body surface of the live earthworm with water, the live earthworm is left in the aqueous solution or fresh water to spread the excrement in the digestive tract of the live earthworm, and then wash with water. The diabetes treatment agent according to claim 5, further comprising the step of wet pulverization and subsequent steps. 7. An organic acid is acetic acid, citric acid, succinic acid, malic acid, tartaric acid or lactic acid.
Or a therapeutic agent for diabetes according to item 6. 8. The therapeutic agent for diabetes according to claim 2, 3, 4, 5 or 6, wherein the inorganic acid is phosphoric acid, sulfuric acid or hydrochloric acid. 9. Diabetes containing, as an active ingredient, an extract obtained by subjecting earthworms to an extraction treatment with at least one extractant selected from the group consisting of aqueous solvents, water-miscible organic solvents and water-immiscible organic solvents. Therapeutic agent. 10. The therapeutic agent for diabetes according to claim 9, wherein the aqueous solvent is fresh water, distilled water, physiological saline, pH 5 to 10 buffer solution or pH 5 to 10 preparation water. 11. The water-miscible organic solvent has 1 to 4 carbon atoms.
10. The therapeutic agent for diabetes according to claim 9, which is the alcohol, ketone, ether or ester of the above. 12. The water-immiscible organic solvent has 6 to 6 carbon atoms.
The antidiabetic agent according to claim 9, which is 10 hydrocarbons, alcohols, ketones, esters or halogenated hydrocarbons having 1 to 5 carbon atoms.