JPH0780777B2 - Antihyperlipidemic agent - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to an antihyperlipidemic agent. More specifically, it relates to an antihyperlipidemic agent of earthworm dry powder and / or earthworm extract having excellent antihyperlipidemic activity and safety. Among them, it relates to an earthworm dry powder prepared by an improved manufacturing method which is novel and innovative, and a therapeutic / preventive agent for hyperlipidemia containing the extract thereof as an active ingredient; a lipid metabolism improving agent; an arteriosclerotic therapeutic / preventive agent. .

Problems to be Solved by Conventional Techniques and Inventions: Hyperlipidemia is recognized to be a major risk factor for arteriosclerosis along with hypertension, diabetes and smoking, and various synthetic compounds have been used for its treatment and prevention. Organic compound antihyperlipidemic agents have been researched and developed. For example, although clofibrate and nicomol were developed, it is known that clofibrate is highly likely to cause gallstones in addition to muscle pain and liver dysfunction. It is known that Nicomol has side effects such as hot flushes and gastrointestinal disorders. It is known that clofibrate and its derivatives are highly likely to cause gallstones in addition to muscle pain and liver dysfunction.
In addition, it was reported that clofibrate also causes liver cancer in animal experiments [DJ Svoboda et al; Cancer Res., 39 , 3419 (1
979)] has been done.

In addition to these safety issues, research on lipid metabolism in recent years has also progressed, especially as to the function of serum riboprotein, which is a carrier of serum lipids, as a result of advances in lipid metabolism, resulting in lower lipid levels in serum. It has come to be noted that not only the ability but also the action on the riboprotein is important.

Serum cholesterol forms a lipoprotein together with triglyceride (hereinafter referred to as TG), total phospholipids (hereinafter referred to as PL), and avoprotein, and this lipoprotein has an extremely low specific gravity lipoprotein ( Hereinafter, it is classified into VLDL), low-density lipoprotein (hereinafter referred to as LDL) and high-density lipoprotein (hereinafter referred to as HDL). Among them, VLDL and LDL are considered to be lipoproteins that induce arteriosclerosis. In contrast, HDL has functions such as transporting cholesterol from peripheral vascular tissues to the liver, production of cholesterol ester, and involvement in TG catabolism, and is said to have an action of preventing and regressing arteriosclerosis. .

Therefore, in the future creation of antihyperlipidemic agents, importance is attached to which type of lipoprotein cholesterol acts, in addition to the effect of lowering the total cholesterol (hereinafter referred to as TC) of serum. Especially cholesterol in LDL (hereinafter referred to as LDL
-C. ) Lowering the value and raising the cholesterol (hereinafter referred to as HDL-C) value in HDL, and the arteriosclerosis index [TC-HDL-C / HDL-C]. Hereinafter referred to as AI. Is desired.

The present inventors have conducted various studies on natural products having an antihyperlipidemic effect, and as a result, dried earthworm powders containing dried earthworms as a part of their forms; earthworms as an aqueous solvent and a water-miscible organic solvent. (Means an organic solvent having a property of being miscible with water) and an immiscible organic solvent of water (meaning an organic solvent having a property of being immiscible with water), and extracting with at least one kind of extractant selected from the group consisting of The obtained earthworm extract has an excellent antihyperlipidemic effect, lowers LDL-C,
The present inventors have completed the present invention by finding that they are highly safe drugs that not only have the effects of raising DL-C and lowering AI, but also have no side effects such as liver hypertrophy and liver dysfunction.

Earthworms have been used since ancient times, mainly in the Eastern countries
It is called a earth dragon and has been used as a medicine. The pharmacological and pharmacological effects of earthworms reported in conventional literatures are described below.

"Water and life" by Shinryu Obuchi 1947 (Showa 22) 10
Published by Makishobo, pages 223-226 and "Belly Engraving"
Shinki Hatai, April 30, 1980, published by Scientist, Inc., pp. 160-163, worms are agents for reducing bladder stones and for excretion to the outside of the body, therapeutic agents for jaundice, and delivery agents. , Has a pharmacological action of a tonic agent, a hair crude drug, a strong sperm agent and an antipyretic agent, while as an earthworm venom, one has an effect on the nervous system and the other has a destruction of red blood cells, that is, a hemolytic action.

"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, Lumbricus Kwangtungesis, is one that has been torn in the abdomen to wash away the internal organs and mud and dried in the sun, shade or cold. Other Land Dragon (Lumbricus Na
tivus) was killed by putting it in plant ash and then removing the ash and drying it in the sun, in the shade or at a low temperature. These two types of earth dragons are used as antipyretic, attracting therapeutic agent, blood circulation promoting agent, hemiplegic agent, joint painkiller, urinating agent, bronchial asthma agent and hypertensive agent.
It is reported to be used at 4.5-9g / 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
Years)], extracted the silkworms (dried pieces from which mud was removed) with boiling water, and added ethyl alcohol to the concentrated solution of this extract to obtain a precipitate (Lumbrofebrin) in 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.

Ijiro Jiro [Yamaguchi medicine 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.

`` Zhong An University Dictionary '', 2nd volume, Jiangsu New Medical School, 1980, published by Shanghai Science & Technology Publishing Co., Ltd., page 2112, Hirochi dragon tincture, dried silkworm powder powder suspension, hot water immersion liquid of silkworm silkworm, decoction of silkworm silkworm When administered to anesthetized dogs, large rats, cats or mice with chronic renal hypertension, slowing and persistent hypotensive action 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, a concentration of 40%
Take the ground dragon tincture (40 g of the ground dragon in 100 ml of ethyl call at 60 degrees, 10 ml each time, 3 times a day [that is, equivalent to 12 g of ground dragon / day. (Converted by the present inventor)]. For those who cannot drink, add water to pure earth dragon powder to make pills (add a small amount of excipients), 3-4 g each time, 3 times a day [equivalent to earth dragon 9-12 g / day (this book Inventor's conversion))
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 Contents of the body by tearing the abdomen of the earthworm (internal organs and mud)
The method of removing and drying in the sun, shade or low temperature (usually 50 ℃ or less).

ii After killing earthworms in plant ash, remove the ash and dry in the sun, shade or low temperature (usually below 50 ° C),
A method to obtain an earthworm with mud clogged inside.

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 an 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 for about 6 months or less when stored in a sealed state. It has a drawback that it becomes unusable due to the generation of fine particles 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, it is rare that the dried product or dried powder of earthworms produced by the method of ii is taken as a ground dragon tincture or powder as it is, 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 method is one of the excellent methods for obtaining earthworm dry powder as a health food, but it is a medicinal agent for arteriosclerosis agents (also known as antihyperlipidemic agents), antidiabetic agents and antihypertensive agents. It was found that the method for producing an earthworm dry powder for the purpose was 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 earthworms as described in the publication, only the fecal soil cannot be selectively removed. 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. or,
The yield for live earthworms is as low as 10 to 19%. Furthermore, a major problem is that the enzyme in the earthworm dry powder, which has an important pharmacological and medicinal effect in order to operate vacuum drying in the final finishing process at 80 ° C and a vacuum degree of 0.3 torr for a long time of 20 hours or more. It was found that the species were destroyed or deactivated. Therefore, compared with the earthworm dry powder obtained by the production method of the present invention, the efficacy of the earthworm dry powder obtained in Japanese Patent Application Publication No. 59-216572 as an antihyperlipidemic agent was 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 5-10, trasylol (trade name), transsamine (trade name), soybean trypsin inhibitor and serum inhibited, plasminogen activating action and fibrinolytic action, various fibrinogen-free action It was confirmed to be an enzyme protein having properties.

Patent application for a method for producing a fibrinolytic active substance consisting of a crude enzyme protein fraction obtained by extracting an aqueous solvent from earthworms and a purified enzyme protein fraction obtained by the purification treatment, that is, Japanese Patent Application Publication No. 58-148824 (application (February 27, 1982) and US patent application No. 4 of foreign application due to 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. Further, Tsuneyoshi Mihara et al. Filed patents for six new proteases from earthworms, namely Japanese Patent Application Publication No. 59-63184 (filing date: 1982, 1).
(October 2) and patent application for thrombolytic agents containing these proteases as active ingredients, that is, Japanese Patent Application Publication
59-184131 (filed on March 31, 1983)
Foreign patent applications claiming priority of these merged applications, namely, Korean patent application No. 2990 (filing date June 30, 1983) and the following patent applications are reported [AU.P.App.16293 (filing date 1983
June 27), CA.P.App.431387 (filing date June 28, 1983)
Sun), DK.P.App.3008 (filing date June 29, 1983), EP.P.
App.83106288.0 (application date June 28, 1983), ES.P.App.5
23754 (filing date June 30, 1983), FI.P.App.832383 (filing date June 29, 1983), NO.P.App.2399 (filing date June 30, 1983), PH. P.App.29151 (Application date June 30, 1983)
Sun), TW.P.App.7211983 (filing date June 18, 1983), U
SPApp.508163 (filing date June 27, 1983)].

As a result of the investigation conducted by the present inventors, it was not possible to find a document which reported an antihyperlipidemic action using an earthworm dry powder or an earthworm extract as an active ingredient. Furthermore, the earthworm dry powder and the extract of earthworm have an effect of lowering TC in serum, lowering LDL-C in serum, increasing HDL-C, and further lowering AI, and also hypertrophic liver and liver. No literature has been found that reports that it is a highly safe drug without side effects such as dysfunction.

Means for Solving the Problems: The present invention relates to a dry powder of earthworms, which comprises, as a part thereof, a dried product of earthworms; The present invention relates to an antihyperlipidemic agent containing, as an active ingredient, an extract of earthworm obtained by extraction treatment with at least one kind of extractant selected.
Furthermore, as a result of detailed studies to obtain a dry powder of aseptic earthworm which is safe and has an excellent antihyperlipidemic effect, and which can be stored or stored in a sealed state for at least 4 years, in high yield, We have established a new and innovative improved manufacturing method as shown in.

Process 1: Living earthworms in fresh water or acetic acid, citric acid, succinic acid, malic acid, tartaric acid, organic acids such as 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
The following content is preferably 0.1 (wt)% or less in a low concentration or slightly acidic aqueous solution of pH 3 to 6.5 at a temperature of 1 to 25 ° C.
After leaving for 5 to 72 hours, preferably at a temperature of 2 to 15 ° C. for 1 to 40 hours, the excrement power of the live earthworm itself excretes the fecal soil in the digestive tract of the live earthworm, and then the surface of the live earthworm is watered. The filth adhering to was washed off and wet pulverized. The wet-ground earthworms were frozen at a low temperature of -5 ° C or lower, preferably at a low temperature of -10 to -60 ° C, and then freeze-dried in a vacuum. The conditions of freezing and vacuum drying are temperature -60 to + 90 ° C, vacuum degree of 100 mmHg or less, preferably -40 to + 80 ° C and vacuum degree of 30 mmHg or less while increasing temperature stepwise for 5 to 100 hours, preferably 10 to 60 hours. Freezing and vacuum drying were performed to obtain a sterile earthworm dry powder.

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 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, then
Freezing and vacuum drying were performed. The conditions of this freezing / vacuum drying are temperature -60 to + 90 ° C, vacuum degree of 100 mmHg or less, preferably -40 to + 80 ° C, 30 mmHg or less while increasing temperature stepwise for 5 to 100 hours, preferably 10 to 60 hours. Freezing and vacuum drying were carried out 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 crusher, or a pressure-type cell disruption device to prepare a suspension or homogenous 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. Also, in the earthworm dry powder, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, lysine, histidine, 18 kinds of arginine 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 a protein food, fishmeal 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 than that of live earthworms, and the excretion rate is high.

In the above-mentioned production methods 1 and 2, the finished state of the dried earthworm 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.

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. That is, T. Mihara et al. Performed ammonium sulfate fractionation on the supernatant obtained by adding 10 times the amount of physiological saline to earthworm dry powder and incubating for 2 days. The precipitate was subjected to gel filtration with Sephacryl S-200, and the resulting protein fraction was analyzed by DEAE.
-As a result of performing cellulose ion exchange chromatography, I having casein decomposition and fibrin decomposition activity,
The proteins of the II 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, as shown in Table 4, a true fibrinolytic activity was recognized. 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 as shown in Table-4.
It will be about 4 times on the 10th day, 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 plate and standard fibrin plate.The standard fibrin plate has a larger dissolution window than the plasminogen-free plate, and the enzyme activity that directly decomposes fibrin is
Activator activity was also shown. In the above operation, M-4
As a result of measurement using M-5 earthworm dry powder instead of, the same result as in Table 4 was obtained.

The details of the reason for exhibiting the antihyperlipidemic effect by orally administering the earthworm dry powder and earthworm extract of the present invention to rats and humans are unknown, but in the earthworm dry powder and earthworm extract. It is considered that it is due to the contained proteolytic enzyme (protein) itself, the precursor of this enzyme (protein) itself or the protein or lipid substance or other unknown compound itself, or the combination thereof. The most preferable anti-hyperlipidemic agent of the present invention has an earthworm dry powder having a nitrogen content of 7 to 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 carried out under a vacuum of 0.01 to 0.5 mmHg at 70 to 80 ° C, preferably 7 to 80 ° C.
Aseptic and water content 5 when vacuum-dried at 5-80 ℃ for 5-10 hours.
It was possible to obtain an earthworm dry powder containing ~ 15%. Especially, vacuum drying for final finishing is an important operation. 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, the present inventor conducted detailed research, and It was found that the combination of the vacuum degree, the heating temperature, and the time 3 elements of the vacuum drying conditions in the final step of vacuum 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 a case where the sample was stored at room temperature in a sealed state for 5 years, slight occurrence or other alteration was not observed.

The form of the earthworm dry powder of the antihyperlipidemic agent of the present invention is, in addition to the earthworm dry powder obtained by the above-mentioned production methods 1 and 2, dried earthworms obtained by the conventionally known production methods i, ii and iii. The thing and its dry powder may be sufficient.

The extract of earthworm, which is an antihyperlipidemic agent, 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. An organic solvent was obtained, or an organic solvent of the same kind was added to the concentrated extract to obtain a precipitate, or the extract 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; Living earthworms or these earthworms;
Earthworms in the form of crushed suspensions of these live and / or green earthworms can be used. Among these, the most preferable form of the raw material earthworm is the earthworm suspension 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 earthworms, and the active substance as an antihyperlipidemic agent can be extracted within a short time, and further treatment is possible. The extraction efficiency is excellent, such as the large amount of substance extracted per unit of time. The next preferable raw material form is the earthworm dry powder obtained by the above-mentioned production 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 kinds of buffer solutions having various compositions such as phosphoric acid, acetic acid, boric acid, citric acid, Tris / hydrochloric acid and having a pH of 5 to 10, preferably pH 6 to 8, can be used. In addition, the pH 5-10, preferably pH 6-8 preparations are water-soluble inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, succinic acid, malic acid or tartaric acid, organic acids and sodium, potassium. It means a dilute aqueous solution prepared in-house from a hydroxide or carbonate of an alkali metal such as. The most preferred aqueous solvents are saline, buffer and fresh water. When using these aqueous solvents, it is preferable to use 0.01 to 0.3 (weight)% of preservatives such as parahydroxybenzoic acid ethyl ester, sodium benzoate, and sodium azide.

Examples of the water-miscible organic solvent include methyl, ethyl,
Allyl, n-propyl, i-propyl, n-butyl, i
-C1-C4 lower alcohols such as -butyl, s-butyl, or t-butyl alcohol; C3-C4 ketones such as acetone or methyl ethyl ketone; ethyl ether, 1,2-propylene oxide, dioxolane,
3 carbon atoms such as 4-methyldioxolane or dioxane
It is preferable to use ethers having 4 to 4; esters having 2 to 4 carbon atoms 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 -50 to +60.
℃ preferably -40 to +60 ℃, extraction contact time 10 minutes to about 100 days, preferably 30 minutes to about 40 days, occasionally or after continuous stirring, then allowed to stand, filtered to separate the extract . For filtration, a pressure or vacuum filter (machine) or a centrifuge may be used. 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 ultrafiltration concentration 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. In addition, a crystal precipitation method by leaving the above-mentioned extract or a concentrated solution of the combined solution or adding an organic solvent of another kind or the same kind to the concentrated solution to precipitate crystals, and then filtering and drying to obtain an extract Methods are also used.

The yield of the extract with respect to the raw material earthworm 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.

When the earthworm dry powder and earthworm extract of the present invention are administered as a clinical therapeutic agent, 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, powders (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, glucose, den powder, sorbitol, glycine, calcium phosphate, microcrystalline cellulose, etc. as excipients; den powder, gelatin, gum arabic, glucose, sucrose, sorbitol, etc. as binders. Manitol, tragacanth, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5-vinylpyridine-methacrylic acid-methylethyl acrylate copolymer, polyvinylpyrrolidone, sodium alginate, etc .; stearic acid as a lubricant, Hydrogenated oil, magnesium stearate,
Calcium stearate, polyoxyethylene monostearate, talc, silicon oxide, polyethylene glycol and the like; disintegrants such as valesioden powder and den powder including surfactants; and wetting agents such as sodium lauryl sulfate. When administered parenterally, it can be used as a suppository. Especially as the base of suppositories, cacao butter, Witepsol, Savanal (Su
banal), polyethylene glycol, polypropylene glycol, glycerogelatin, gelatin capsules and the like are used. In addition, known safe preservatives such as methylparahydroxybenzoate, propylparahydroxybenzoate, butylparahydroxybenzoate, and butylhydroxyanisole, and other safe dyes are used in combination.

Although the dose of the earthworm dry powder and the earthworm extract of the antihyperlipidemic agent of the present invention varies depending on the administration method, age, weight, condition of the patient and type of disease, it is usually per day for humans. About 0.01 to 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 antihyperlipidemia and the results 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%);
-6 (water content: 9.2%, ash content: 4.7%, nitrogen content: 8.4%) and the doses of γ-oryzanol and soysterol used as control agents for efficacy were increased from 0.1g / Kg to 8g / Kg. The mice (0.1 to 5 g / Kg) and rats (2 to 8 g / Kg) were 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. Toxicity and behavior after administration were observed over time, but 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. Effects on Experimental Hyperlipidemic Rats Pharmacological Test 1) 1) Animals: 8 Wistar male rats, each weighing 100 ± 1 g, were used.

2) Feed: Cholesterol diet was prepared by adding 1 wt% of cholesterol and 0.5 wt% of cholic acid to powder feed (CE-2) manufactured by CLEA Japan, and mixing them well. Two kinds of the earthworm dry powder of the present invention [M-2
(Water 10.4%, ash 5.3%, nitrogen 8.6%) and M-5
(Water content: 9.5%, ash content: 4.5%, nitrogen content: 7.8%)] was mixed with each other as a test drug feed.

3) Rearing condition: One rat was placed in one cage, and each feed and water were freely ingested. The animals were bred for 7 days at a temperature of 23 ± 1 ° C. and a humidity of 55 ± 5%. Rats fasted overnight except water for 7 days were sodium pentobarbital [trade name:
Blood was collected from the abdominal descending aorta under anesthesia with Nembutal], and serum was obtained by cooling centrifugation. TC in serum, free cholesterol (referred to as FC), LDL
-C and HDL-C were quantified using Toshiba TBA-480 automatic analyzer and each TA480 Test reagent commercially available from Wako Pure Chemical Industries, Ltd. AI was calculated as above.

4) Results The results are shown in Table-5.

In the group to which 2% of the earthworm dry powder of the present invention was added, serum TC, FC, LDL-C and AI were significantly decreased, but HDL-C
Was constant and did not change significantly.

That is, it was found that the dry earthworm powder of the present invention has an excellent antihyperlipidemic effect.

Pharmacological test 2: 1) Animal: Sprague-Dawley (hereinafter referred to as SD) male rat having a body weight of 105 ± 1 g was used.

2) Feed: The components of the cholesterol diet in pharmacological test 2 are shown in Table-6.

The feed of the test drug group, which was well mixed with the components of Table 6 and used as a cholesterol diet, was prepared by mixing the cholesterol diet with the earthworm dry powder M-3 (water content 10.7%, ash content) of the product of the present invention.
5.2%, 9.2% nitrogen, 0.25wt%, same M-1 (water content 10.2)
%, Ash 5.1%, nitrogen 9.4%) 0.5 wt% and control drug γ
-Oryzanol (trade name: Hijet fine granules) or soysterol (trade name: Moristerol fine granules) 0.5 and / or
Alternatively, 1 wt% was mixed well and used as feed.

3) Rearing condition: Two rats were placed in one cage, and the feed and water were freely ingested. The animals were bred for 4 weeks at a temperature of 23 ± 1 ° C. and a humidity of 55 ± 5%. On the 4th week, rats fasted overnight except water were anesthetized with sodium pentobarbital [trade name: Nembutal], and blood was collected from the abdominal descending aorta to obtain serum by cooling centrifugation.

Serum TC, FC, TG, PL, free fatty acid (hereinafter referred to as NEFA), HDL-C, AI, glutamate oxaloacetate transaminase (hereinafter referred to as GOT) and glutamate pyruvate transaminase (hereinafter referred to as GPT). Was quantified and calculated using a Toshiba TBA-480 automatic analyzer and each TA480 Test reagent commercially available from Wako Pure Chemical Industries, Ltd.

Lipids in the liver were extracted by the Folch method, and TC, TG and PL in the liver lipids were quantified by the same method as described above.

4) Results: Table 7 shows the effects of the dried earthworm powder of the present invention on experimental hyperlipidemic rats using γ-orzanol and soysterol as control agents. In the case of the feed containing 0.25 and 0.5% of the earthworm dry powder of the present invention, TC, FC, PL and NEFA in serum were all compared to the cholesterol diet group.
Was statistically significantly reduced. TG tended to decrease, but no significant decrease was observed.

When γ-oryzanol was mixed at 0.5 and 1%, no significant decrease in TC, FC, TG and PL values was observed except that NEFA was significantly decreased. In this respect, TC, FC, and PL were significantly reduced when 1% of the control drug, soysterol, was mixed, but the values of TG and NEFA were almost the same as those of the cholesterol diet group.

As described above, HDL-C is considered to be a factor that improves arteriosclerosis, and an increase in this value is a property useful as an antihyperlipidemic agent, and many basic and clinical researchers have found it useful. Have been revealed. The present invention addition group is shown in Table-7.
It was confirmed that HDL-C was statistically significantly increased as compared with the cholesterol diet group as shown in. On the other hand, the control drug γ-oryzanol did not have an increasing effect. However, soysterol showed a significant increase.

As described above, the action of lowering AI is useful as the action of an antihyperlipidemic agent, but it was confirmed that it significantly reduces in the group to which the product of the present invention and the control drug soysterol were added. However, the control drug γ-oryzanol showed a decreasing tendency, but not a significant decrease.

As described above, some commercially available antihyperlipidemic agents cause liver damage as a side effect. Therefore, it becomes a problem in long-term continuous use. GOT and GPT in serum were measured to examine the effect on the liver. It is generally known that an increase in these test values suggests liver dysfunction. GOT in the group to which the present invention product was added
And GPT both showed a significant decrease. On the other hand, the control drug γ-
Although not significant in the 0.5% addition group of oryzanol, there was a clear increase tendency, and in the 1% addition group, both GOT and GPT showed a significant increase. In that respect, soysterol 1 as a control drug
The GOT-added group showed lower GOT and GPT values than the cholesterol-fed group, but it was not significantly decreased.

The liver weight of the present invention-added group was significantly lower than that of the cholesterol-fed group, but the control drug γ-oryzanol and soysterol-added group showed almost no difference from the cholesterol-fed group.

TC in hepatic lipids, in the addition group of the product of the present invention and both control agents,
It showed a significant decrease compared to the cholesterol diet group.

TG in hepatic lipid, in the addition group of the present invention product and both control drugs,
Although it showed a slight downward trend, no essential change was observed.

PL in liver lipids was significantly decreased in the group to which the product of the present invention was added as compared to the cholesterol diet group, but no change was observed in the groups to which both control drugs were added.

The rat body weight was steadily increased in both the present invention product and both control drug addition groups, and no significant change was observed.

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 such as mud and feces adhering to the surface of the live earthworm, and then wet-milled 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 0.1mm at 80 ℃
Drying under Hg 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 three times at 25 ° C, the residue was finally washed with 0.945l of 99% methanol of 3 times volume, and the combined extract and washing solution was concentrated to dryness under reduced pressure at 30 ° C. Then, vacuum drying was performed to obtain 88.2 g of a powder extract. 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 was added, and the mixture was stirred at 10 ° C for 2 hours, filtered, 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. 2.31 l of n-hexane was added to 7.7 l of this mixed solution, and the mixture was stirred and allowed to stand. After separating the aqueous layer and the n-hexane layer, the aqueous 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,
Filter and separate into extract and residue. The residue is washed with 0.3 l of 40% ethanol, and the combined extract and washed solution is concentrated to dryness under reduced pressure at 40 ° C and then vacuum-dried.
got g. This is designated as Extract X-.

X-. After adding 2.5 l of pH 6.4 phosphate buffer to 1.55 kg of the earthworm suspension (E-5) and incubating at 37 ° C for 8 hours, the action of the protease originally contained in the earthworm body was promoted. The mixture was filtered, and 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 twice the amount of ethanol was added to the obtained concentrated solution, and the precipitate was filtered and the separated precipitate Vacuum dried and then freeze dried to a powder extract
64.2 g was obtained. 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 liquid of the extract and the wash was concentrated to dryness under reduced pressure at 30 ° C., and then vacuum dried to obtain 61.5 g of extract.

This is designated as Extract X-.

Manufacturing method 13 13 kg of cultured live earthworms with mud (red earthworm) are thoroughly washed with water to wash off the mud adhering to the body surface, and then a 1: 1 mixture 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 it was divided into E-7,-
8, -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 shelf temperature on the tray to 30 ° C, and under a vacuum of 0.1 mmHg, 6
After drying for an hour, raise the shelf temperature to 50 ° C and vacuum dry under a vacuum of 0.2mmHg for 8 hours. Finally, raise the shelf temperature to 78 ° C and reach 0.2mmH.
By drying under a vacuum of g for 8 hours, 305 g of each dried product of earthworm was obtained. 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% aqueous sodium chloride solution containing para-hydroxybenzoic acid ethyl ester was added, and the mixture was stirred at 30 ° C for 96 hours, filtered, and the residue was washed with 0.9 l of 0.9% aqueous sodium chloride 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, 0.16 l of ethanol was added to this to separate the precipitate, and the final concentration of ethanol was added to the filtrate to give an ethanol concentration of 80%. Was vacuum dried to obtain 14.4 g of crystal powder. This is designated as Extract X-1.

The filtrate of the extract X-1 was concentrated to dryness under reduced pressure 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), 3 l of fresh water containing 0.1% sodium benzoate was added, and the mixture was stirred and extracted at 32 ° C. for 72 hours, filtered, and the residue was washed with 0.6 l of fresh water. 3.5 liters of clear extract combined with the wash was obtained.
The extract was concentrated by an ultrafiltration method and then concentrated under reduced pressure to dryness to obtain 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, filtered, 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 concentration under reduced pressure to about 1/10 volume with, add 4 times volume of ethanol, stir well, let stand for one day and night, filter the formed precipitate, wash with ethanol, and then vacuum dry to obtain powder extract 72.5 got g. 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 it is filtered and the residue is
% Aqueous ethanol solution, and the combined extract and washing solution 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 mixture was filtered, the residue was washed with 0.5 l of a 50% aqueous ethanol solution, the combined liquid of the extract and the wash was concentrated to dryness under reduced pressure at 35 ° C., and then 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 powder extract. Obtained. 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 Toxicity and antihyperlipidemic pharmacological test methods and results are as follows.

A-1. Acute toxicity test: Measured by the same method as the above A. acute toxicity test method. 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 earthworm extract X-,-, of the present invention described above.
The mice were individually dosed by gavage with a throat dragging rod, increasing from 0.1 g / kg to 5 g / kg, using 8 species, −, −, −, −, − and −. During the test, the mice were kept at a room temperature of 22 to 23 ° C. and observed for 14 days after the administration. No mortality was observed at the doses administered. Moreover, no signs of poisoning were observed or no abnormality was observed at 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. Pharmacological test 3 of the effect on experimental hyperlipidemic rats: The effect of the above B on experimental hyperlipidemic rats was measured by the same method as in the pharmacological test 1. That is, 1) Animal: The same as the pharmacological test 1 described above.

2) Feed: Cholesterol diet was prepared by adding 1 wt% of cholesterol and 0.5 wt% of cholic acid to powder feed (CE-2) manufactured by CLEA Japan, and mixing them well. In this cholesterol diet, four kinds of the earthworm dry powder of the present invention [K-
1, -3, -7 and -9] in an amount of 2 wt% and 7 kinds of the extract from earthworms of the present invention [X-,-,-,-
, −, −, And −] were mixed at 0.4 wt% to obtain a test drug feed.

3) Breeding conditions: the same as in the above pharmacological test 1.

4) Results: The results are shown in Table-7-1.

As shown in Table-7-1, 2w of the earthworm dry powder of the present invention was used.
There was no difference in the effect between the group added with t% and the group added with 0.4 wt% of the extract of earthworm. That is, the values of TC, FC, LDL-C, HDL-C and AI in the serum of both groups were almost the same. From this, the extract of earthworms per unit weight is
It was found to have 5 times the antihyperlipidemic activity against dry earthworm powder.

In both groups, serum TC, FC, LDL-C and AI were significantly decreased, but HDL-C was constant and did not change significantly. However, it has been found that the earthworm dry powder and earthworm extract of the present invention have excellent antihyperlipidemic activity.

Oral administration of C. earthworm dry powder to humans: Four volunteers (Volunteer) who agreed with this experiment
On the other hand, capsule D produced in Example 10 described later [in one formulation, dry powder of earthworms (M-2 above, water content 10.4%, ash content
5.3%, nitrogen 8.6% included) 150 mg included] 1 drug 3 times a day
The dose was taken orally within 30 minutes after eating. As a general rule, serum lipids should be measured every 3 to 4 months after taking the drug, and the period of taking 6 to
TC, TG, HDL in serum collected by fasting for 7 months in the early morning
-C and AI were measured. As the measuring method, a kit commercially available from Wako Pure Chemical Industries, Ltd. as described above was used. The results are shown in Table-8.

The standard value (mg / dl) of serum lipids of healthy local residents who carried out the administration test of the earthworm dry powder of the present invention to these 4 volunteers was TC130-230, TG50-170, HDL-C35-60. is there. As shown in Table-8, the TC values of all four people before administration exceeded the standard values. The TG and HDL-C values were within standard values.

The results of Table-8 were the results of the oral administration experiment of the earthworm dry powder to only 4 people, but the value of TC238-280mg / dl before administration became 194-220mg / dl 3-4 months after administration, It decreased to within the standard value range, and significantly decreased to 174 to 209 mg / dl after 6 to 7 months. AI was clearly decreased 3 to 4 months after administration, and was markedly decreased 6 to 7 months after administration along with decrease in TC.

On the other hand, HDL-C is almost constant and changes slightly, but 6 to 7
After a month, a slight but clear upward trend was observed.
A tendency for a decrease in TG was observed after 3 to 4 months, and a clear decrease was observed after 6 to 7 months.

In the result of the oral administration experiment to humans, it was confirmed that TC and AI were remarkably lowered and HDL-C was slightly increased, similarly to the experiment result of the experimental hyperlipidemia rat. However, no significant decrease in TG was observed in the experimental hyperlipidemic rat administration experiment, but a clear decrease was observed after 6 to 7 months in the human oral administration experiment. This seems to be a remarkable result.

This oral administration experiment of the earthworm dry powder to humans could be safely completed without any side effect. For example, even in the long-term administration for 6 to 7 months, the blood sugar amount (hereinafter referred to as blood glucose) has a standard value (60 to 60).
There was no risk of hypoglycemia falling below the lower limit of 110 mg / dl). As can be seen from Table-8, the earthworm dry powder does not seem to be an antihyperlipidemic agent which can achieve the purpose within a relatively short period of time and rapidly like the synthetic organic compound drug. That is, it is considered to be a mild action type antihyperlipidemic agent which requires continuous administration for a relatively long period of time. However, from 3 to 4 months after oral administration to humans, TC and AI clearly decreased, HDL-C showed a slight upward trend after 6 to 7 months, and TG also showed a clear decrease after 6 to 7 months. Was given.

The subject patients of the present invention are not particularly limited by age. Although the product of the present invention is functional, the preferred target patients are middle-aged and elderly patients. From the above results, it was found that the earthworm dry powder of the present invention is a safe and excellent antihyperlipidemic therapeutic / preventive agent.

From these results, it is easy to find that the earthworm extract, which is an extract from earthworm, is safe for humans as well as an excellent antihyperlipidemic treatment / prevention agent like earthworm dry powder. Recognize.

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 (the components are Same as the above M-3) Corn den flour 60 Lactose 80 Talk 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 micro Crystalline cellulose 45 Silicon oxide 6 Magnesium stearate 4 Total 350 mg Powders that were uniformly and well mixed according to the above formulation were produced into tablets of various weights using a tableting machine.

Example 2. Granules A Earthworm dry powder 150 mg (the components are the same as M-2 above) Lactose 20 Microcrystalline cellulose 60 Corn den powder 15 Hydroxypropylcellulose 5 Total 250 mg Using a fluidized bed granulator according to the above formulation, Earthworm dry powder, lactose, microcrystalline cellulose and corn den powder were mixed well, sprayed with a 5% aqueous solution of hydroxypropyl cellulose as a binder, and dried at low temperature to give 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 earthworm powder 150 mg (the components are the same as M-2) Lactose 28 Microcrystalline cellulose 47 Manitol 10 Corn den flour 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 250 mg of granules C prepared according to Example 3 in hard capsules
Each was filled to produce 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 The above formulation was mixed well. No. 1 gelatin capsules were filled with 250 mg each of this mixed powder to produce capsules.

Example 7. Enteric coated tablets Earthworm dry powder 100 mg (the components are the same as the above 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 methylcellulose phthalate 14.8 mg Dioctyl phthalate 2.3 Stearic acid 2.3 Light silicon oxide 0.6 Total 22.0 mg Example 8. Powder A earthworm dry powder 150 mg (the same ingredients as M-4) Manitol 50 Corn corn flour 50 Total 250 mg Agent 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 prepare a powder.

Example 9. Suppository A Earthworm dry powder 200 mg (the components are the same as M-5) 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 above) Sodium lauryl sulfate 4 Phosphate-sodium 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 Hydroxypropyl cellulose 3 250 mg in total In the above formulation, components other than hydroxypropyl cellulose 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. A hard capsule was manufactured by filling 250 mg of each of the granules into a hard capsule.

Example 12 Capsule F Granules Earthworm extract 30 mg (X-) Lactose 103 Corn Den powder 89 Potato powder 22 Talc 3 Magnesium stearate 3 total 250 mg According to the above formulation, the well mixed powder was granulated by an extruder. The agent was manufactured. 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 Phosphoric acid-sodium hydrogen 20 Manitol 38 Magnesium stearate 2 total 150 mg The above-prepared mixture was mixed well and the mixed powder was mixed. 150 mg each was filled into a gelatin capsule to produce a capsule.

Example 14. Capsule H Earthworm extract 30 mg (X- described above) Sodium lauryl sulfate 4 Phosphate-sodium 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 things 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 sulphate 2 Manitol 176 Magnesium stearate 2 250 mg A mixture of the above formulation 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 an antihyperlipidemic agent containing, as an active ingredient, an earthworm dry powder and an earthworm extract. From the experiments in which rats were raised for 1 week and 4 weeks on a high-cholesterol diet in which the earthworm dry powder and earthworm extract of the present invention were mixed, it was found that the present invention has an excellent antihyperlipidemic effect. .

In a 1-week feeding experiment of earthworm dry powder and earthworm extract, TC, FC, LD in serum were higher than those in the cholesterol diet group.
LC and AI were significantly decreased, but HDL-C was not significantly changed. In the 4-week feeding experiment of the earthworm dry powder, TC, FC, PL and NEFA in serum were significantly decreased as compared with the cholesterol diet group.

Furthermore, it was confirmed that HDL-C, which is an arteriosclerosis-improving factor, was significantly increased and AI was significantly decreased. TG tended to decrease, but not significantly.
Moreover, GOT and GPT were significantly decreased.

The liver weight and TC and PL in liver lipids were significantly decreased, but TG was almost unchanged.

The rat body weight increased steadily and no significant change was observed.

Earthworm dry powder capsules (containing 150 mg) once a day, 3 times a day, for 4 volunteers (Volunteer) for 6 to 7 months orally after meal, TC, TG, HDL-C in serum
And measured AI. This result shows that after 3 or 4 months
TC and AI are clearly reduced. On the other hand, HDL-C showed a slight change, but a slight upward trend was observed after 6 to 7 months. The TG was clearly reduced after 6 to 7 months.

According to animal experiments and oral administration experiments to humans, dry earthworm powder and earthworm extract are safe and have excellent therapeutic / preventive agents for hyperlipidemia, excellent serum lipid substitution improvers, and excellent arteriosclerosis treatment / It is extremely useful as a preventive agent.

Claims (12)

[Claims] 1. An antihyperlipidemic agent containing dry earthworm 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 of the live earthworm extinguishing pipe, wash and remove the dirt attached to the body surface of the live earthworm with water, After performing wet pulverization, or after removing the dirt attached to the body surface of the 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, Wet pulverization was performed, the obtained suspension was frozen at -10 to -60 ° C, and then -60 to
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 80 ° C. An antihyperlipidemic agent containing an earthworm dry powder as an active ingredient, characterized by being dried for 5 to 10 hours. 3. The live earthworms are left in the aqueous solution or in fresh water to cover the digestive tract of the live earthworms, and then the dirt attached to the body surface of the live earthworms is washed and removed with water, followed by wet grinding. The antihyperlipidemic agent according to claim 2, wherein the subsequent steps are performed. 4. The anti-hyperlipidemic agent according to claim 3, wherein the live earthworm before being left in the aqueous solution or in fresh water is a live earthworm obtained by washing a part of waste 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 antihyperlipidemic agent according to the item. 6. After washing dirt on the body surface of live earthworms with water, the live earthworms are allowed to stand in the aqueous solution or fresh water to dislodge the excrement in the digestive tract of the live earthworms, and further washed with water, Next, the antihyperlipidemic agent according to claim 5, wherein the steps after wet pulverization are performed. 7. An organic acid is acetic acid, citric acid, succinic acid, malic acid, tartaric acid or lactic acid.
The antihyperlipidemic agent according to 4, 5, or 6. 8. The antihyperlipidemic agent according to claim 2, 3, 4, 5 or 6, wherein the inorganic acid is phosphoric acid, sulfuric acid or hydrochloric acid. 9. An anti-bacterial composition comprising an extract obtained by subjecting earthworms to an 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, as an active ingredient. Hyperlipidemic agent. 10. The antihyperlipidemic agent according to claim 9, wherein the aqueous solvent is fresh water, distilled water, physiological saline, a buffer solution having a pH of 5 to 10 or a preparation water having a pH of 5 to 10. 11. The water-miscible organic solvent has 1 to 4 carbon atoms.
The antihyperlipidemic agent 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.
10. The antihyperlipidemic agent according to claim 9, which is 10 hydrocarbons, alcohols, ketones, esters or halogenated hydrocarbons having 1 to 5 carbon atoms.