JPH02193928A - Antihyperlipidemia drug - Google Patents

Abstract

PURPOSE:To provide an antihyperlipidemia drug having an antihyperlipidemia activity and safety by containing as an active ingredient an earthworm extract prepared by extracting an earthworm with an extraction solvent such as an aqueous solvent, a water-miscible organic solvent or a water-non-miscible organic solvent. CONSTITUTION:An antihyperlipidemia drug contains as an active ingredient an earthworm extract prepared by extracting an earthworm with an extraction solvent comprising an aqueous solvent, a water-miscible organic solvent or a water-non-miscible organic solvent. The earthworm is used in a form of e.g. an earthworm suspension prepared by leaving the earthworm in an aqueous solution containing <=0.3wt.% of a compound such as an organic acid, inorganic acid, organic acid sodium salt, inorganic acid sodium salt, organic acid potassium salt or potassium inorganic acid salt or in pure water to allow the earthworm to stew excrement soils in the digestive tracts, washing filth adhered to the surface of the body with water and subsequently wet-grinding the washed body.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an antihyperlipidemic agent. More specifically, it is a therapeutic/preventive agent for hyperlipidemia and lipid metabolism containing as an active ingredient an earthworm extract or a mixture of earthworm extract and earthworm dry powder, which has excellent antihyperlipidemic activity and safety. The present invention relates to an improving agent or an agent for treating/preventing arteriosclerosis.

[Prior art and problems to be solved by the invention] Hyperlipidemia has been recognized to be a major risk factor for arteriosclerosis, along with hypertension, diabetes, and smoking, and various methods have been proposed for its treatment and prevention. Synthetic organic compound antihyperlipidemic agents have been researched and developed. For example, clofibrate and nicomol were developed, but clofibrate causes muscle pain,
In addition to liver dysfunction, it is known that there is a high possibility of developing gallstones. Nicomol is also known to have side effects such as facial flushing and gastrointestinal disorders. In addition, clofibrate and its derivatives may cause muscle pain, liver dysfunction,
It is known that there is a high possibility of developing gallstones. In addition, clofibrate has been reported to cause liver cancer in animal experiments (D, J, 5voboda, etc.; Cancer Res
, 39.3419 (1979)).

In addition to these safety issues, with regard to drug efficacy and pharmacology, recent advances in research on lipid metabolism, particularly research on the function of serum lipoproteins, which are carriers of serum lipids, have resulted in lower serum lipid concentrations. In addition to the effects on riboproteins, attention has been drawn to the importance of their effects on riboproteins.

Serum cholesterol forms riboproteins together with triglycerides (hereinafter referred to as TG), total phospholipids (hereinafter referred to as PL), and apoproteins. (hereinafter referred to as VLDL), low-density lipoprotein (hereinafter referred to as LDL), and high-density lipoprotein (hereinafter referred to as HDL). Among these, VLDL and LDL are considered to be riboproteins that induce arteriosclerosis. On the other hand, HDL has functions such as transporting cholesterol from peripheral vascular tissues to the liver, producing cholesterol esters, and participating in TG catabolism, and is said to have the effect of preventing and regressing arteriosclerosis.

Therefore, in the future creation of antihyperlipidemic agents, emphasis will be placed on which type of riboprotein cholesterol they act on, in addition to their ability to lower serum total cholesterol (hereinafter referred to as TC). In particular, it has the effect of lowering the cholesterol (hereinafter referred to as LDL-C) value in LDL and increasing the cholesterol (hereinafter referred to as HDL-C) value in HDL, as well as the arteriosclerotic index ((TC-HDL-C)). /H
Calculated using the DL-C formula. Hereinafter referred to as AI. ]
There is a desire for a drug that has the effect of reducing the

As a result of continuing various studies on natural products that have antihyperlipidemic effects, the present inventors found that earthworm extract or a mixture of earthworm extract and dried earthworm powder has excellent antihyperlipidemic effects. Moreover, it has been found that it has the effect of lowering LDL-C, increasing HDL-C, and lowering AI. Furthermore, in addition to the above-mentioned effects, the dried earthworm powder was found to be a highly safe drug without side effects such as liver hypertrophy and liver dysfunction, and the present invention was completed.

Dried earthworms have been used as medicine since time immemorial, mainly in Eastern countries, where they are called ＠矧 or 地郎. The pharmacology and medicinal effects of earthworms reported in conventional literature are described below.

■ “Worms and Life” by Mayu Obuchi, 1947 (Showa 22)
Published by Maki Shobo, October 30, 1980, pp. 223-226, and “Fukoku Mizu” by Shinkiji Hatai, April 30, 1980.
Published by Scientist Co., Ltd., pp. 160-163, earthworms are described as an agent for reducing bladder stones and excreting them from the body, a treatment for yellow sickness, a birthing agent, a tonic, a hair herbal medicine, a tonic, and an antipyretic agent. On the other hand, it has been reported that one of the earthworm toxins affects the nervous system, and the other has a hemolytic effect, i.e., destruction of red blood cells.

■ "The Dictionary of the People's Republic of China" edited by the Dictionary Committee of the Ministry of Health of the People's Republic of China, 1977 edition, part of pages 197-198 states the following. Traditionally, earth dragon products have 2
There are different types. One such wide dragon (Lumbr icus)
Kvangtungesis), the abdomen is torn open, the internal organs and quicksand washed away, and dried in the sun, shade, or at low temperatures. Other Land Dragons (Lumbricus NaLiv)
Earthworms are killed by placing them in plant ashes, then removing the ashes and drying them in the sun, shade, or at low temperatures, and their bodies are filled with mud. These two types of earth dragons are used as an antipyretic, convulsion treatment, blood circulation promoter, hemiplegia treatment, joint analgesic, urinary agent, bronchial asthma agent, and hypertension agent.
It is reported that ~9g/mortar is used7.

■ “Our Chinese Herbal Medicine Series 3, Earth Dragon/Squid Bone/
Chinese Scientific Research Journal, IO, 30th, 1978, published by Matsuura Pharmaceutical Co., Ltd., page 7 reports that Earth Dragon tincture (ethyl alcohol extract of Earth Dragon) has a hypotensive effect.

■ “Natural Medicine Encyclopedia” edited by Takuo Okuda, April 15, 1986
Published by Hiroyo Shoten, p. 215, it is reported that earth dragon is used as a hypopyretic, analgesic, diuretic, and antidote.

■ Mamoru Tanaka [Hokkaido Medical Journal No. 24, pp. 18-24 (1949)] extracted kansei (dried fragments with mud removed) with boiled water and made a concentrate of this extract. The precipitated material obtained by adding ethyl alcohol to
It has been reported that when febrin) was dissolved in Ringer's solution and this solution was injected intravenously into an anesthetized cat, a rapid drop in blood pressure occurred, and an acceleration of blood coagulation was observed in proportion to the shock.

■ Kenji Iyou [Yamaguchi Igaku Vol. 9, pp. 571-576 (1960)] dissolved a physiological saline extract of the earth dragon, an ethyl alcohol or acetone extract of the earth dragon, and dissolved it in physiological saline. When the solution was injected intravenously into adult rabbits, a drop in blood pressure was observed.

■ "Middle and Weak Great Dictionary," Volume 2, edited by Jiangsu New Medical Academy, 1980, published by Shanghai Science and Technology Publishing House, page 2112, contains Guangdirong tincture, Canker dry powder suspension, and Canker hot water soaking solution. When a decoction of ., cypress, etc. was administered to anesthetized dogs, large rats, cats, or mice with chronic renal hypertension, a slow and sustained hypotensive effect was observed.

When earth dragon extract was injected intravenously into a dog or cat under anesthesia,
The blood pressure lowering effect appeared rapidly. However, it has been reported that it was not effective when administered orally or when applied clinically.

Furthermore, on page 2114 of the same magazine, it is stated that 40% concentration of Jiryu tincture (40g of Jiryu soaked in 100 mQ of 60 degree ethyl alcohol) is applied for 10 m each time (2, - 3 times a day [i.e., Jiryu 12
It corresponds to g7th day. (converted by the inventor)]. Those who cannot take tinctures can make pills (with a small amount of excipients) by adding water to pure earth dragon powder and taking 3-4 g each time -
If taken three times a day (equivalent to 9 to 12 g/day (calculated by the inventor)) and continued for 30 to 60 days, it is effective against essential hypertension. In addition, 2 m0 of Jiryu B1 liquid (removed by removing hypoxanthine using HgC1 and separating blood pressure lowering components using an ion exchange resin) was added each time. It has been reported that taking Jiryu (containing 8 g of the crude drug Jiryu) three times a day [equivalent to Jiryu 24 g per 7 days (calculated by the inventor)] was effective against essential hypertension.

Conventional and customary methods for producing dried earthworm products or dry powder can be broadly classified as follows.

i. Rip open the abdomen of the earthworm and extract the internal contents (viscera and mud)
A method of removing and drying in the sun, shade, or at low temperatures (usually below 50°C).

i. A method of killing earthworms by placing them in plant ash, removing the ash and drying them in the sun, shade, or at low temperatures (usually below 50°C) to obtain earthworms with mud still stuck inside their bodies.

A method of removing mud from the earthworm's body and then drying it by placing it in plant ash or fire ash.

etc. These dried products were pulverized and used when needed or used. These manufacturing methods are simple and economical, and have the advantage of being easily carried out at home.

However, dried earthworm products or dried powders obtained by these methods can be stored in a refrigerator at 0 to 5 degrees Celsius or in an open state at room temperature of 5 to 45 degrees Celsius for up to about 6 months when stored in a closed condition. There is a drawback that mold may develop within a short period of one year or less, rendering the product unusable.

When earthworms are dried with their bodies filled with mud, as in method H, or dried in plant ash or fire ashes, as in the foot method, in most cases they are extracted with hot water when used as medicine. Alternatively, it is often decocted with boiled water, filtered, and the filtrate taken. In particular, the dried product or powder of earthworms produced by the manufacturing method of i can be left as Jiryu tincture or powder.
Or it is rarely taken as a pill. Earthworms produced by method i can be taken as a hot water immersion solution or decoction, as well as in the form of Jiryu tincture or Jiryu powder, or as a pill after adding a small amount of water or a small amount of excipient to the powder. There are many things to do. When using the production method of i and
%, and the yield when using the same basket manufacturing method is 13-19%.

Recently, one of the inventors of the present invention, Yo Ishii, has developed a health food containing earthworm proteins and lipids as main components and a method for producing the same [Japanese Patent Application Publication No. 1983-216572 (publication date 1).
December 6, 984)].

This production method is an excellent method for obtaining dried earthworm powder as a health food. However, as a method for producing dried earthworm powder for use as drugs such as arteriosclerotic agents (also known as antihyperlipidemic agents), antidiabetic agents, and antihypertensive agents,
It was found that the medicinal efficacy was not sufficient. That is, when an external action is applied to remove excrement such as feces remaining in the body of a living earthworm as described in the same publication, it is not possible to selectively remove only the feces. It has been found that no matter how careful the procedure is, the medicinal efficacy will be insufficient because the internal organs and body fluids, which contain many components that play an important role in medicinal efficacy, are removed along with the fecal soil. Moreover, the yield for live earthworms is as low as 10-19%. Furthermore, a major problem is that the vacuum drying in the final finishing process is operated for a long time of 20 hours or more at a vacuum level of 80° 0.3 Torr.
It has been found that enzymes in dried earthworm powder, which have important pharmacological and medicinal effects, are destroyed or inactivated. Moreover, some of the other active ingredients were found to be thermally decomposed.

Therefore, compared to the dried earthworm powder obtained by the production method of the present invention, the antihyperlipidemic efficacy of the dried earthworm powder obtained in Japanese Patent Application Publication No. 59-216572 is about 50%.
Met.

Recently, Tsune Mihara, one of the inventors of the present invention, and other co-researchers have discovered that the fibrinolytic active substance of earthworms has an optimal pH of 8 to 1.
0. Stable pH is 5-10. Trasylol (trade name)
It is an enzyme protein that is inhibited by soybean trizcin inhibitor and serum, has plasminogen activating action and fibrinolytic action, but does not have fibrinogen dissolving action. confirmed.

A patent application for a method for producing a fibrinolytic active substance consisting of a crude enzyme protein fraction extracted from earthworms using an aqueous solvent and a purified enzyme protein fraction obtained by purification thereof, ie, Japanese Patent Application Publication No. 148824/1983 (applied) (February 27, 1982) and a foreign application claiming priority to the same, U.S. patent application No. 470394 (filing date February 28, 1983), and British patent application No. 830359 (filing date February 1983). 25th), Italian Patent Application No. 4
7795A (filing date February 25, 1983), French Patent Application No. 03165 (filing date February 25, 1983)
Japan), West German Patent Application No. P3306944.1 (
Application date: February 28, 1983) and Canadian patent application no.
, 422034 (filing date: February 21, 1983). Furthermore, Tsune Mihara et al. applied for a substance patent for six new proteases derived from earthworms, namely, Japanese Patent Application Publication No. 1984-63184 (filing date: 1982).
(1982), 1982) and a patent application for a thrombolytic agent containing these proteases as active ingredients, namely, Japanese Patent Application Publication No. 184131 (1982) (filing date, 1982).
8 (March 31, 1983)) and the foreign patent application claiming priority of these combined applications, namely Korean Patent Application No. 299.
No. 0 (filing date: June 30, 1983), the following patent applications have been reported (AU, P, App, l 6293 (filing date: June 27, 1983), CA, P, App, 43
1387 (filing date June 28, 1983), DK, P.
App, :3008 (filing date June 29, 1983),
E P, P, App, 83106288.0 (filing date 1
June 28, 983), E S, P, App, 5237
54 (filing date June 30, 1983), F I, P
, App.

83.2383 (filing date June 29, 1983), NO.
,P.

App, 2399 (filing date June 30, 1983), P
H,P.

App, 29151 (filing date June 30, 1983),
T.W.

P, App, 7211983 (filing date June 1, 1983)
8th), US, P, App, 508163 (issue 1[E
June 27, 1983)].

According to the research results of the present inventors, we could not find any literature reporting the antihyperlipidemic effect of a substance containing a single earthworm extract or a mixture of earthworm extract and dry earthworm powder as an active ingredient. Furthermore, earthworm extract or a mixture of earthworm extract and earthworm dry powder has the effect of lowering TC in serum, lowering LDL-C, increasing I(DL-C), and further lowering AI. Moreover, we could not find any literature reporting that it is a highly safe drug without side effects such as hepatomegaly and liver dysfunction.

[Means for solving problems]

A detailed study was conducted to prepare a single earthworm extract or a mixture of earthworm extract and earthworm dry powder that is safe and has excellent antihyperlipidemic effects.

The earthworm extract of the antihyperlipidemic agent of the present invention is prepared by extracting the earthworm raw material in an aqueous solvent, a water-miscible organic solvent (meaning an organic solvent that is miscible with water), and a water-immiscible organic solvent (meaning an organic solvent that is miscible with water). It is an extract obtained by extraction treatment with at least one type of extractant selected from the group consisting of: This earthworm extract can be obtained by the following production method. That is, raw earthworms are extracted with at least one type 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 form a precipitate. Alternatively, an organic solvent was added to the concentrated extract to obtain a precipitate, or an extract was obtained by completely removing the extraction solvent under normal pressure, increased pressure, or reduced pressure.

The earthworms used as raw materials are dried earthworms and dried powder thereof obtained by the known manufacturing method of i and i in the previous period and the manufacturing method described in Japanese Patent Application Publication No. 59-216572; live earthworms from which excrement has been removed; excrement from which they have been removed. Shitanama worms (meaning earthworms whose excrement is removed from the earthworms as they are collected, and which are not boiled, baked, dried, or pickled) Earthworms can be used.

As a result of detailed research, the present inventors have found that in order to obtain an earthworm extract that is completely sterile, has excellent antihyperlipidemic activity, and does not have the odor inherent to earthworms, it is necessary to It has been found that a method that combines the purification or cleaning method of raw earthworms (collected or cultured earthworms, etc.) with freezing and vacuum drying conditions is a particularly important operation. According to actual measurements by the present inventors, the amount of filth attached to the body surface of raw earthworms, such as collected or cultured earthworms, is approximately 1% per wet earthworm.
The fecal soil in the digestive tract contains 5 to 15% (by weight) of wet earthworms. We have discovered that using clean earthworms from which these filth and excreta have been promptly removed as raw materials for extraction is an important factor in improving medicinal efficacy and yield. That is, the most preferable raw material earthworms are earthworm suspensions obtained by wet pulverization in the processing steps of production methods 1 and 2 described below, and dry earthworm powders produced by freezing and vacuum drying these suspensions. As shown below, the earthworm extract prepared from the earthworm suspension and earthworm dry powder obtained by the methods 1 and 2 has antihyperlipidemic activity superior to that of the earthworm dry powder.

It was also found that a mixture of earthworm extract and earthworm dry powder had more antihyperlipidemic activity than earthworm extract alone. Below, the method for preparing the starting materials, which is an important operational step in the manufacturing method of the product of the present invention, will be explained.

Manufacturing method of earthworm suspension and earthworm dry powder: Manufacturing method 1: Live earthworms are treated with a non-toxic organic acid such as acetic acid, citric acid, succinic acid, malic acid, tartaric acid, lactic acid, etc. or non-toxic acid such as phosphoric acid, sulfuric acid, hydrochloric acid, etc. at least one compound of inorganic acids or sodium or potassium salts of these acids at a concentration of 0.3 (
(wt)% or less, preferably at a low concentration of 0.1 (wt.)% or less, or in a slightly acidic aqueous solution with a pH of 3 to 6.5, or in fresh water at a temperature of 1 to 25°C for 0.5 to 48 hours, Preferably, leave it at a temperature of 8 to 20°C for 1 to 24 hours to fully excrete the fecal soil in the digestive tract of the live earthworm using its own excretory power, and then add water to the body surface of the live earthworm. The soil was washed away, and then wet pulverization was performed to obtain a suspension of earthworms. This earthworm suspension was heated to -5°C.
After freezing at the following low temperature, preferably -1θ to -60°C, freezing and vacuum drying were performed. Freezing/vacuum drying conditions are temperature -60 to +90℃, vacuum degree 100mm.
Hg or less, preferably 39 mmHg at -40 to +80°C
Freezing and vacuum drying were carried out for 5 to 100 hours, preferably 10 to 60 hours, while raising the temperature stepwise at the following degree of vacuum to obtain a sterile dry powder of earthworms.

Manufacturing method 2: After washing off the dirt adhering to the body surface of live earthworms with water, use non-toxic organic acids such as acetic acid, citric acid, succinic acid, malic acid, tartaric acid, lactic acid, or phosphoric acid, sulfuric acid, hydrochloric acid, etc. 13 (by weight) of a non-toxic inorganic acid or at least one sodium- or potassium-blind compound of these acids.
% or less, preferably at a low concentration of 0.1 (weight) % or less, or in a slightly acidic aqueous solution with a pH of 3 to 6.5, or in fresh water at a temperature of 1 to 25°C, preferably for 0.5 to 48 hours. Leave it at a temperature of 8 to 20°C for 1 to 24 hours to fully excrete the fecal soil in the digestive tract of the live earthworm using its own excretory power, and then use water to remove the soil that adheres to the body surface of the live earthworm. The filth was washed off, and then wet pulverization was performed to obtain an earthworm suspension. This earthworm suspension was frozen at a low temperature of -5°C or lower, preferably -10 to -60°C, and then subjected to freezing and vacuum drying. The conditions for this freezing/vacuum drying are: temperature -60 to +90℃, degree of vacuum 100mmH.
g or less, preferably -40 to +80°Os 30mmH
5 to 100 g while increasing the temperature stepwise at a vacuum level of less than 5 g.
Freezing and vacuum drying were carried out for preferably 10 to 60 hours to obtain a sterile dry powder of earthworms.

Ta.

The wet crushing method of earthworms, that is, the method of destroying earthworm tissues (m-cells), involves making a suspension or homogeneous liquid using equipment such as a homogenizer, blender, homomixer, crusher, or pressurized cell destruction device. is preferred. The temperature during this wet grinding is preferably 1 to 25°C, preferably 2 to 15°C.

By the operation in the first stage, it was possible to obtain yellowish brown or brown earthworm dry powder from living earthworms at a yield of 20 to 35%.

Normally, the moisture content of this dry earthworm powder is 5-16%, preferably 7-14%, and the ash content is 3-8%, preferably 4-7%.
%, nitrogen content was adjusted to 1 to 11%, preferably 6 to 11%. In addition, the dried earthworm powder contains aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine,
Contains 18 or more amino acids: inleucine, leucine, tyrosine, phenylalanine, tryptophan, lysine, histidine, and arginine.

Test Example 1 Table 1 shows the crude analysis results of the dried earthworm powder obtained by the methods of the first half production methods 1 and 2 and the production method of the examples described later.

Table 2 shows the results of component analysis of the dry earthworm powder products M-5, M-6, M-8 and M-9 obtained by the method.

(Leaving space below) Test example 2 M-2 and M-4 obtained by the method of manufacturing method 1, Test example 3 of manufacturing method 2 M-2 and M-4 obtained by the method of manufacturing method 1, and M-4 obtained by the method of manufacturing method 2 Amino acid analysis was performed on the crude protein in M-6's dry earthworm powder product, and Table 3 shows the results of comparison with the analytical values for the protein foods fishmeal and soybean flour.

(Left below) Table 3 *P, Mc Donald, R, A, Edward
s and J, F, D Greenhalgh: A
From data described in normalNutrition (1973).

From Table-2 and Table-3, it was found that the dried earthworm powder obtained by Production Methods 1 and 2 contained abundant crude protein, crude lipid, and various metals. The amino acid composition revealed that it contains a large amount of essential amino acids.

The production method 1 produced a slightly more preferable product than the dried earthworm powder obtained by production method 2. Furthermore, it is better to leave live earthworms in an aqueous solution containing at least one compound of the above-mentioned organic acid, inorganic acid, or sodium or potassium salt of both of these acids at a low concentration as above, rather than leaving them in fresh water. Excretion of fecal soil from the digestive tract of live earthworms is faster and the excretion rate is greater than that of live earthworms. According to the research of the present inventors,
For example, when a live earthworm is left in fresh water at 8 to 18 degrees Celsius, it takes 15 minutes to completely expel the feces from the digestive tract.
It took ~48 hours. In this regard, it has been found that when left in the above-mentioned dilute aqueous solution at 8 to 18°C, the excrement is completely discharged in 0.5 to 5 hours.

In the above-mentioned production methods 1 and 2, the finished state of the dried earthworm product after drying is often in an agglomerated state or a mixed state of clumps. However, when this is applied to a pulverizer, it is easily turned into powder.

When administering dried earthworms and earthworm extracts to mammals including humans, powders are preferable to lumps.

Tsune Mihara et al. [Japanese Patent Application Publications Nos. 59-63184 and 59-184131] fractionated six types of new proteases, fibrinolytic enzymes, from earthworms. Namely, Tsune Mihara et al. added 10 times the amount of physiological saline to dry earthworm powder and
Incubation for days
After carrying out ammonium sulfate fractionation on the supernatant liquid, the precipitate was subjected to gel filtration using 5ephacryl S-200, and the resulting protein fraction was subjected to DEAE-cellulose ion exchange chromatography.
■、■、■ Has casein-degrading and fibrin-degrading activities
Fractionated proteins were obtained. For this I, n, III fraction, DEAE-cellulose, 5ephadex G-7
5, Toyobar HW55, A CH-Sepharo
As a result of purification using selBenzamidine-3 epharose and the like, 6 fractions of purified enzyme were obtained. When molecular weight was measured by 5DS-PAGE, fraction I-
The molecular weight of 0 is the lowest, calculated as 23,500, and then sequentially I-1, I-2, I[, ■-1, I[I-2
and the molecular weight increased, and m-2 had a molecular weight of 34.200. Furthermore, when the isoelectric points of these six fractions were measured by isoelectric focusing, I-0 had the highest pH of 4.12;
The pH gradually decreased, and was pH 3.52 at -2.

These 6 fractions are new proteolytic enzymes that are different from serine enzymes, and the optimum pH of the proteases in these 6 fractions is around 8 or pH 8-10, and the stable pH is 4-1.
2 or 5-12, optimal temperature 50℃ or 50-60℃
, the deactivation conditions were reported to be 70°C for 60 minutes.

Dry earthworm powder product M-4 obtained by the above production methods 1 and 2
When 10 times the amount of physiological saline was added to M-6 and the supernatant was measured using a standard fibrin plate, fibrinolytic activity was immediately observed as shown in Table 4. When this physiological saline solution of M-4 earthworm dry powder is incubated at 37°C, the fibrinolytic activity of the supernatant increases by about 4 times on the 10th day, as shown in Table 4, and on the 50th day. 5 times on the 75th day, and 5.5 times on the 75th day.

This fact suggests that a large amount of the precursor of this protease exists in the freeze-dried earthworm powder, and the enzyme activity is expressed through autolysis.

When the activity of the supernatant on day 50 was converted into international units of urokinase and compared, it was calculated to be about 8.0001 U/mQ. In addition, this enzyme dissolves both plasminogen-free fibrin plates and standard fibrin plates, and the standard fibrin plate has a larger dissolution window than the plasminogen-free plate, and has enzyme activity that directly degrades fibrin as well as plasminogen activator activity. was also shown.

In the above operation, the results of measurements using M-6 earthworm dry powder instead of M-4 were the same as those shown in Table 4.

(Margins below) Table 4: Add 10 times the amount of physiological saline to the dry earthworm powder and hold at 37℃.
The details of why dry earthworm powder and earthworm extract exhibit antihyperlipidemic effects when orally administered to rats and humans are unclear, Proteolytic enzyme (protein) itself, precursor of this enzyme (protein) itself, protein, glycoprotein, metal-binding protein, lipid, carbohydrate, or other unknown compound itself contained in the powder and earthworm extract; This is thought to be due to the effect of the annexation of The nitrogen content of earthworm extract and earthworm dry powder most preferred as antihyperlipidemic agents is 7 to 16%;
That is, it has a crude protein content of 43.8 to 100%.

Next, preferred specific examples of freezing and vacuum drying operations of the suspension obtained by wet pulverization of earthworms obtained by the above-mentioned Production Methods 1 and 2 are as follows.

Wet ground earthworms, i.e., earthworm suspension, are mixed with −1O
~-60°C, preferably -30°C to -50°C for 5-60 hours, and then frozen at the same temperature to 0.01-0.2 mmHH.
Lyophilize under vacuum for 5-12 hours. Next 20-3
Dry under vacuum at 0.01" 0.2 mm Hg for 5-15 hours at 0 °C. Then at 35-50 °C, 0.1-0.5 m
Dry under mHg vacuum for 10-20 hours. The next final finishing step, vacuum drying, is 0. Vacuum drying at 70-80° C., preferably 75-80° C. for 5-10 hours under a vacuum of O1-0.5 mmHH yielded a sterile earthworm dry powder containing 5-15% moisture. In particular, the final finishing step, vacuum drying, is also an important operation. The present inventor conducted detailed research in order to make the dried earthworm powder sterile without deactivating the activities of proteolytic enzymes, enzyme precursors, and other active ingredients contained in the dried earthworm powder. As a result, it was found that the combination of the three elements of vacuum drying conditions in the final step of freezing and vacuum drying, vacuum degree, heating temperature, and time, was an important condition, and the above operating conditions were established.

As shown above, six types of purified proteolytic enzymes from dried earthworm powder (Japanese Patent Application Publication No. 59-6318
It has been reported that the proteolytic enzymes in the dried earthworm powder obtained by production methods 1 and 2 are deactivated in 60 minutes at 70°C (No. 4 and No. 184131/1984), as shown in Table 4. Not being utilized. In addition, the dried earthworm powder obtained by Production Methods 1 and 2 was kept at room temperature of 5 to 45°C in a sealed state for 5 years.
In the preserved examples, no mold growth or other deterioration was observed.

The most preferred raw materials in the present invention are a suspension of earthworms obtained by wet pulverization in the processing steps of Production Methods 1 and 2 as described above, and a dried powder of earthworms produced by freezing and vacuum drying the suspension. . The next preferred raw material earthworms are the above-mentioned publicly known earthworms and the Japanese Patent Application Publication No. 5
These are dried earthworms and their dried powder obtained by the manufacturing method of No. 9-216572. However, the latter three types of raw earthworm extracts are considerably inferior in medicinal efficacy to the former two most preferred raw material extracts, about 30 to 50%
Only 0%. The reason is as follows.

That is, when an external action is applied to remove the manure remaining in the digestive tract of the earthworm (containing about 5 to 15% based on wet earthworms), only the manure cannot be selectively removed. No matter how carefully we manipulate it, internal organs and digestive juices (including various effective enzymes such as cellulases, amylases, groteases, and lipases) and body cavity fluids rich in proteins (containing various effective enzymes such as cellulases, amylases, groteases, and lipases) play an important role in medicinal efficacy.
Coelonic fluid and other body fluids are removed from the body along with the fecal soil, resulting in insufficient medicinal efficacy. or,
As shown in the process description and Examples of Production Methods 1 and 2 above, when raw earthworms are left in a dilute aqueous solution, it is important for medicinal purposes that the feces in the earthworm's digestive tract be completely excreted within the shortest possible time. is important. When the dilute aqueous solution is a concentrated aqueous solution, the earthworms that have been left unattended will have to remove the dorsal surface of the earthworm's body segment before excreting the excrement.
rsum) dorsal foramen (D or s a 1por
e) More yellow or milky body cavity fluid (Coelonicf
luid) and rapidly die, the concentration of the dilute aqueous solution should be as low as 0.3% (by weight) or less or at a pH of 3 to 6.
There is a reason to limit it to a slightly acidic level of 5.

When earthworms are left in a dilute aqueous solution or fresh water for a long time, for example, for 3 days (72 hrs), they excrete a large amount of body fluids such as digestive juices and body cavity fluids in addition to feces, resulting in a decrease in body weight. At this time, the weight of the earthworm decreases in inverse proportion to the length of time it is left in the water. Dry powders produced using such earthworms as raw materials and earthworm extracts thereof lack medicinal efficacy. Therefore, as shown in the examples of the present invention, earthworms are preferable because they can sufficiently (completely) expel the excrement from the digestive tract within a short period of time. Further, dirt adhering to the body surface of earthworms can be relatively easily removed by washing with clean water. Among the two preferred raw material earthworm forms described above, a particularly preferred raw material form is one in which live earthworms are left in the dilute aqueous solution to remove excrement from the digestive tract of the live earthworms, and then washed with water and wet-pulverized. It is a paste-like suspension of earthworms obtained by

When this suspension is used as a raw material, the amount of solvent used can be saved compared to other forms of earthworms, the active substances can be extracted within a short time, and the amount of extract per unit of processing time is large. It has excellent extraction efficiency.

The aqueous extraction solvent is fresh water, distilled water, physiological saline, p
It is preferable to use various buffer solutions of H5 to IO or various prepared waters. The buffer solution is phosphoric acid, acetic acid, boric acid, citric acid, Tris/hydrochloric acid, etc., pH 5-10, preferably pH 6-10.
One or more of the 8 buffer solutions having various compositions can be used. Also, p
H5-10 Preferably pH 6-8 preparation solution is a water-soluble inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, succinic acid or tartaric acid, or an organic acid and an alkali metal such as sodium or potassium. Refers to dilute aqueous solutions prepared in-house from hydroxides or carbonates. The most preferred aqueous solvents are saline, buffer, and fresh water. When using these aqueous solvents, it is preferable to use a preservative such as ethyl hydroxybenzoate, sodium benzoate, or sodium azide in an amount of 01 to 0.3% (by weight).

Examples of water-miscible organic solvents include methyl, ethyl,
Allyl, n-propyl, i-propyl, n-butyl, S
-butyl or t-butyl alcohol with 1 or more carbon atoms
Lower alcohols of 4: Ketones having 3 to 4 carbon atoms such as acetone or methyl ethyl ketone; ethyl ether, 1
．． Ethers having 3 to 4 carbon atoms such as 2-propylene oxide, dioxolane, 4-methyldioxolane or dioxane; and esters having 2 to 4 carbon atoms such as methyl formate, ethyl formate and methyl acetate are preferably used.

Among these, the most preferable water-miscible organic solvent has 1 carbon number.
~4 alcohols.

Examples of water-immiscible organic solvents include n-hexane,
Hydrocarbons having 6 to 10 carbon atoms such as i-hexane, n-heptane, i-hebutane, octane, i-octane, cyclohexane, benzene, toluene, xylene or ethylbenzene; n-hexyl, 2-methylpentylhexyl, Alcohols having 6 to 10 carbon atoms such as S-heptyl, n-octyl, 2-ethylhexyl, 2,6-dimethyl-4-heptyl, and n-decyl alcohol: nothyl-n-amyl ketone, m- or p-methylcyclohexanone, or 7-8 carbon atoms such as methylhexylketone
Ketones; n-butyl acetate, S-butyl acetate, i acetate
-butyl, 2-ethylbutyl acetate, S-amyl acetate,
Esters having 6 to 10 carbon atoms, such as methylamyl acetate or 2-ethylhexyl acetate: chloroform,
1 carbon number such as diclopencune or epichlorohydrin
It is preferable to use halogenated carbonized cords of 5 to 5.

The extraction method may be carried out by a general method and is not particularly limited, but examples thereof are as follows.

For raw earthworms in the above form, an aqueous solvent, concentration 5 to 7
from the group consisting of an aqueous solution of an organic solvent at a concentration of 0% (by volume), preferably from 10 to 60% (by volume), a water-miscible organic solvent at a concentration of 80-100% (by volume), a water-immiscible organic solvent at a concentration of about 100%. At least one type of extractant selected from 1 to 100
Preferably 2 to 30 times the amount (volume/weight) is used, the extraction temperature is -50 to +60°C, preferably -40 to 60°C, and the extraction contact time is from 10 minutes to about 100 days, preferably 30
After stirring occasionally or continuously for a period of minutes to about 40 days,
The extract was separated by standing and filtering. For filtration, a pressurized or reduced pressure filter (machine) or a centrifugal separator may be used. After the extraction residue is thoroughly washed, the extract and washing liquid are combined.

An extract was obtained by removing the extraction solvent from the extract or combined solution at a temperature of 60° C. or below under normal pressure, increased pressure, or reduced pressure, by a concentration method such as ultrafiltration concentration, or freeze-drying. However, when ethanol is used as the extractant, the ethanol may not be completely removed and may remain depending on the type of final target network. Alternatively, the extract can be obtained by precipitation of crystals by leaving the concentrated solution of the extract or mixture as described above, or by adding other or similar organic solvents to the concentrated solution to precipitate crystals, followed by filtration and drying. Methods are also used.

The yield of the extract based on the raw earthworms in the above operation is 3 to 60% (by weight) in terms of dry matter. This difference in yield depends on the form of the raw earthworm, the type of solvent, extraction temperature, extraction time, and other extraction conditions. If the extraction temperature is high, there is a risk that the active substance will be decomposed, so the extraction temperature should be between -40 and +6.
0°C is preferred.

The components contained in the extract are as shown in Table 2 and Table 3.
It contains 18 types of amino acids and 8 types of metals, just like the ingredients in dry earthworm powder. In particular, the proteins in this extract include proteins made of the same amino acids as the raw earthworms, carbohydrate proteins, metal-binding proteins, and the like. Furthermore, the extract contains free amino acids, lipids, various carbohydrates, ATP and A
Contains TP-like substances, various enzymes, and other effective chemicals of unknown composition.

Production method 3 Preparation of raw earthworms and dry earthworm powder: Live earthworms (
10 kg of red earthworm) was left in 20 ff of an acidic aqueous solution with a pH of 5.8 in which a mixed acid of 2:1 of malic acid and lactic acid was dissolved at a temperature of 10° C. for 2 hours to sufficiently excrete the feces in the digestive tract. After thoroughly washing the live earthworms with water to remove mud, feces, and other impurities adhering to their body surfaces, they are wet-pulverized using an Ultra Homo Mixer (manufactured by Nippon Seiki Co., Ltd.) to suspend the earthworms. 9.3 kg of a cloudy liquid was obtained. This earthworm suspension was divided into six equal parts of 1.55 kg each, and each
-1, -2, -3, -4, -5 and -6.

1.55 kg of E-1 and E-2 earthworm suspension obtained above
Place in each tray and freeze at -40°C for 30 hours.

Next, freeze-dry the product at -40℃ for 6 hours under a vacuum of 1 mmHg, then freeze-dry it at 25℃ for 8 hours under a vacuum of 1 mmHg.
After drying for 1 hour, it was then dried at 45°C for 12 hours under a vacuum of 0.1 mmHH, and finally at 80°C for 0.5 hours. By drying under a vacuum of 1 mHg for 6 hours, 318 g of dried earthworms were obtained in each of the two trays. Let this be ni-1 and ni-2.

Preparation of extract:
After extraction twice, the residue was finally washed with 3 times the amount of 99% methanol 0.945Q, and the combined solution of the extract and washing liquid was heated at 30°C.
88. Concentrate to dryness under reduced pressure and then vacuum dry.
2g of powder extract was obtained. This is referred to as extract X-■.

X-■, 315 g of dried earthworms (K-2), 3 times the amount of chloroform 945Q and 2 times the amount of ethanol 0.
After adding the combined solution of 63Q and extracting three times at 20°C, the residue was finally washed with twice the amount of ethanol 0.630, and the combined solution of the extract and washing liquid was concentrated to dryness under reduced pressure at 30°C. Next, 72.5 g of powder extract was obtained by vacuum drying. This is referred to as extract X-■.

X-■, 6.2 g of fresh water was added to 1.55 kg of earthworm suspension (E-3) and stirred at lOoC for 2 hours.
Filter and separate into extract and residue. After washing the residue with water, a combined solution 7.7Q of the extract and washing solution was obtained. This mixture 7.7
After adding 2.3112 mmol of n-hexane to a and stirring, let it stand and separate into the water-soluble layer and the n-hexane layer.The water-soluble layer was collected, concentrated to dryness under reduced pressure at 30°C, and then frozen. It was dried to obtain 86.8 g of powder extract. This is extracted as Extract
shall be.

X-■, earthworm suspension (E-4) 1.55 kg4:
Add 40% ethanol aqueous solution 1012, stir at 40°C for 12 hours, filter, and separate into extract and residue.

After washing the residue with 0.3ff of 40% ethanol, the combined liquid of the extract and washing liquid was concentrated to dryness under reduced pressure at 40°C.
It was dried under vacuum to obtain 69.7 g of powder extract.

This is referred to as extract X-■.

X-■, earthworm suspension (E-5) 1.55 kg nip
Add 2.50 ml of H6,4 phosphate buffer and incubate at 37°C for 8 hours.
After incubating for a period of time to promote the action of protease originally contained in the earthworm body, it is filtered, and the residue is washed with a phosphate buffer solution of pH 6.4, and the combined solution of the extract and washing solution is 3.9a. I got it. After adding sodium azide Ig to this mixture and incubating at 37°C for 8 hours, it was concentrated under reduced pressure at 30°C, and twice the amount of ethanol was added to the obtained concentrate, and the precipitate was filtered. was vacuum dried and then lyophilized to obtain 64.2 g of powdered extract.

This is referred to as extract X-■.

X-■, after drying 1.55 kg of earthworm suspension (E-6) naturally in the sun, powdered earthworm dry powder C
Named K-3. ) to ethanol and ethyl ether 2:l
900 ml of a mixed solvent was added and extracted three times at 20°C.

Finally, the residue was washed with the mixed solvent from the previous period, and the combined solution of the extract and washing solution was concentrated to dryness under reduced pressure at 30° C., and then vacuum-dried to obtain 61.5 g of an extract. This is extracted as Extract
■.

Manufacturing method 4 Preparation of raw earthworms and earthworm dry powder: Wash 13 kg of cultured live earthworms (red earthworms) with mud thoroughly with water to remove the mud adhering to the body surface, and then add malic acid and citric acid. The live earthworms were left in an acidic aqueous solution 40Q with a pH of 6.2 and dissolved in a mixed acid of 2 hours at a temperature of 12°C for 2 hours to fully excrete the feces in the digestive tract, and then the live earthworms were thoroughly washed with water. After washing off dirt such as mud and feces adhering to the body surface, the live earthworms were wet-pulverized in a mixer to obtain 9.0 kg of mixer suspension.

This earthworm suspension was divided into six equal parts of 1.5 kg each, and these were designated as 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'O for 40 hours, and then frozen at a temperature of -40°C under a vacuum of 0 and 1+ mHg. Freeze-dry for 6 hours, then raise the temperature of the shelf on which the tray is placed to 30'0, dry under a vacuum of O, l mmHg for 6 hours, and then
Raise the shelf temperature to 50°C Q, vacuum dry under 2mmHg vacuum for 8 hours, and finally raise the shelf temperature to 78°C X 0.2mlI
By drying under IHg vacuum for 8 hours, 305 g of each dried earthworm was obtained. This is -7 and -8
shall be.

Preparation of extract: X-■, Mimisuno dried product (K-7) 300g+:, 3
Add 0.9% aqueous sodium chloride solution containing 12% barahydro, xybenzoic acid ethyl ester,
After stirring at ℃ for 96 hours, it was filtered and the residue was reduced to 0. 'l
The extract was washed with 0.9% aqueous sodium chloride solution to obtain 3.8Q clear extract by combining the extract and the washing solution.

This was concentrated by ultrafiltration to a liquid volume of 0.1612,
After adding 0.16α of ethanol to the precipitate and fractionating it, the filtrate was added to the filtrate so that the final concentration of ethanol was 80%, and the resulting precipitate was vacuum-dried to obtain a crystal powder of 14.4
I got g. This is designated as extract X-■-1.

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

X-■, 3ff (
7) Add fresh water containing 0.1% sodium benzoate,
After extraction by stirring at 32° C. for 72 hours, the mixture was filtered and the residue was washed with 0.6 g of fresh water to obtain a clear extract 3.5a which was a combination of the extract and the washing liquid. This extract was concentrated by ultrafiltration, and then concentrated to dryness under reduced pressure to yield 69 g.
A crystalline powder was obtained. This is referred to as extract X-■.

X-■, 40 kg of earthworm suspension (E-9)
Dry earthworm powder (K-9
shall be. ) was added with distilled water containing 0.02% rose hydroxybenzoic acid ethyl ester of 3Q, and heated at 32℃ for 12 hours.
After stirring for 0 hours, it is filtered and separated into an extract and a residue. After thoroughly washing the residue with distilled water, the combined liquid of the extract and washing liquid was concentrated under reduced pressure at 30°C to a volume of about 1/10, and then
After adding twice the amount of ethanol and stirring well, the mixture was allowed to stand overnight, and the resulting precipitate was filtered, washed with ethanol, and dried under vacuum to obtain 72.5 g of a powder extract. This is referred to as extract X-■.

X-@, earthworm suspension (E 10) 6 to 1.5 kg
A 0% ethanol aqueous solution 7.5a was added, and the mixture was allowed to stand at 20 to 22°C for 8 days with occasional stirring in a sealed state. Thereafter, it was filtered, the residue was washed with 0.5a of a 50% aqueous ethanol solution, and the combined liquid of the extract and washing liquid was concentrated to dryness under reduced pressure at 30° C. and then freeze-dried to obtain 40.5 g of a powdered extract. This is designated as extract x-■.

X-■, 5 kg of earthworm suspension (E-11)
A mixed solvent of 0% ethanol aqueous solution 4,5a and 50% isopropanol aqueous solution 3a was added, and the mixture was allowed to stand at 8°C for 16 days with occasional stirring in a tightly stoppered state. Thereafter, it was filtered, and the residue was washed with 0.5 g of 50% ethanol aqueous solution, and the combined liquid of the extract and washing liquid was concentrated to dryness under reduced pressure at 35°C, and then freeze-dried to obtain 28.2 g of a powdered extract. . This is referred to as extract X-■.

X-@, earthworm suspension (E-12) 1.5 kg1:
Add 7.5Q of 60% ethanol aqueous solution, extract three times while stirring at 25°C for 2 hours, and finally reduce the residue to 60%.
The extract was washed with an aqueous ethanol solution 1Q, and the combined solution of the extract and washing solution was concentrated to dryness under reduced pressure at 30° C., and then freeze-dried to obtain 90.5 g of a powdered extract. This is extracted as Extract
shall be.

The earthworms used in the present invention are red earthworms (Lumb.
ricus rubellus), LT earthworm (also known as Lumbricus terrestris)
)), Eiseniafoe Lida,
Cassic tree worm (A I Io lobopho)
racaliginosa), Purple Thrushworm (D
endrobaenaoctaedra), cherry earthworm (Allolobophora japo-nica)
Michaelsen), grasshopper earthworm (Dravi
La-mimizu Hatai), Pheretima diver-gens
Michaelsen), hook earthworm (Phara
timacommun iss jma), Pheretima agres-tis, Siebold earthworm (Pheretima 5ieboldi)
Horst), Pheratima
hilgen-dorfi), Iso-earth (Pont)
odrilus matsushimensisliz
uka), Itomi Mizu (Tubifex)
hattai Nomura), Gotouito earthworm (
Also known as Limnodrilus gotoi
Hatoi=L, 5ocialis 5tephe
Any normally growing earthworm can be used.

The earthworm extract or the mixture of earthworm extract and dry earthworm powder of the drug of the present invention may be administered in either oral or parenteral form for clinical treatment, but oral administration is particularly preferred. The product of the present invention can be used in the oral form as a capsule, tablet, granule, powder, coating agent, sugar-coated tablet, or emulsion. Such formulations are used. Examples of pharmaceutical carriers include excipients such as lactose, sucrose, mannitol, glucose, starch, sorbitol, glycine, calcium phosphate, and microcrystalline cellulose; binders such as starch, gelatin, gum arabic, glucose, sucrose, sorbitol, Mannitol, tragacanth, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5
-Vinylpyridine-methacrylic acid-methylethyl acrylate copolymer, polyvinylpyrrolidone, sodium alginate, etc.; As a lubricant, stearic acid, hydrogenated oil, magnesium stearate, calcium stearate, polyoxyethylene monostearate, talc, oxidation Silicon, polyethylene glycol, etc.; disintegrants include potato starch, starch containing surfactants, etc.; wetting agents include sodium lauryl sulfate, etc. Furthermore, when administered parenterally, it can be used as a suppository. In particular, cocoa butter, Huitepsol (W) is used as a base for suppositories.
iLepsol), Subanal, polyethylene glycol, polypropylene glycol, glycerogelatin, gelatin capsules, etc. are used.

In addition, known safe preservatives such as methylvala hydroxybenzoate, propylvala hydroxybenzoate, butylvala hydroxybenzoate, butylhydroxyanisole, and other safe dyes are used in combination.

The dosage of the antihyperlipidemic agent of the present invention, either alone or as a mixture of earthworm extract and dried earthworm powder, varies depending on the administration method, age, weight, condition, and type of disease of the patient. Generally, it is preferable to use about 01 to 5 g per -H for humans. Most preferably, the dose is 0.02g to 2g per day, divided into 1 to 3 doses per day.

Effects: The toxicity and antihyperlipidemia pharmacological test methods and results of the earthworm extract, the mixture of earthworm extract and dried earthworm powder, and the earthworm dry powder of the present invention will be described in detail below.

A. Acute toxicity test of dry earthworm powder DDY male base weighing 30 ± 2 g and body weight 100 ± 2
An acute toxicity test by oral administration was conducted using 5 mice in each group of Wistar base rats. Earthworm dry powder M-1 (moisture 1002%, ash 5.1%, nitrogen 9.4%); same M-2 (moisture 10.4%, ash 5.
3%, nitrogen 8.6%); same M-3 (moisture 1O87%)
, ash content 5.2%, nitrogen content 9.2%); Same M-4 (moisture content 10.6%, ash content 5.6%, nitrogen content 9.6% content): Same M
-5 (contains 9.5% moisture, 4.5% ash, 7.8% nitrogen): Same M-6 (9.2% moisture, 4.7% ash, 8% nitrogen).
4%); same M-8 (moisture 1001%, ash 4.6%
, containing 8.7% nitrogen) and M-9 (containing 9.8% moisture, 4.8% ash, and 8.5% nitrogen), and γ-oryzanol and soysteel used as control drugs for medicinal efficacy. The respective doses were increased from 0.1 g/kg to 8 g/kg and administered by gavage to the aforementioned mice (0,1 to 5 g/kg) and rats (2 to 8 g/kg) using a throat probe. were individually dosed by. Animals were maintained at an animal room temperature of 22-23° C. during the study period and observed for 14 days after dosing. No deaths were observed at all drug doses administered. Toxicity and behavior after administration were observed over time, but no differences were observed from the normal animal group. Furthermore, there was almost no difference in weight gain from the normal animal group. A post-test necropsy did not reveal any macroscopic damage to any major organs. Therefore, it was not possible to determine the LD value of the earthworm dry powder due to its very low toxicity.

A-1. Acute toxicity test of earthworm extract: Measured by the same method as the acute toxicity test method in A above. That is, weight 3
An acute toxicity test was conducted by oral administration using 0±2 g of ddy male base mice in each group of 5 animals.

For example, the above-mentioned earthworm extracts of the present invention X-■, -■,
-■, -■, -■, -■, -■, and -■ were administered to the above-mentioned mice at increasing doses from 0.1 g/kg to 5 g/kg using a throat probe. Individually dosed. During the test, mice were maintained at an animal room temperature of 22-23°C;
Observations were made for 14 days after administration. No deaths were observed at any given dose. Also, there were no signs of toxicity; no abnormalities were observed at autopsy. Therefore, the aqueous and/or organic solvent extracts from the earthworms of the present invention have very low toxicity, so that no L Dso value could be determined.

A-2. Acute toxicity test of mixture of earthworm extract and earthworm dry powder: Measured by the same method as in A above. That is, the weight is 30
An acute toxicity test was conducted by oral administration using ±2 g of ddy male base mice (5 animals in each group).

A 50:50 (it%) mixture of earthworm extract X-■ obtained in the above-mentioned production methods 3 and 4 and earthworm dry powder and -1; -2; X-
■Toni-3; X-■Toni-7, X-■-1 Toni-7
;X-■ and-9;X-(li) and-7;X-■
Nine types of 50:50 (wt%) mixtures of Toni-8 were administered to the mice at doses ranging from 0.1 g/kg to 5 g/kg.
The drug was administered individually by gavage with a throat probe. Mice were maintained at an animal room temperature of 22-23°0 during the study and observed for 14 days after dosing. No deaths were observed at the doses administered. Moreover, no abnormalities were observed in the findings of toxic symptoms or autopsy. Therefore, the mixture of aqueous and/or organic solvent extracts from earthworms and earthworm dry powder of the present invention has LD, due to its very low toxicity. The value could not be determined.

B. Effect of dry earthworm powder on experimental diabetic mice Pharmacological test 1: l) Animals: A group of 8 male Wistar rats weighing 100±Ig were used.

2) Feed: 1 wt% of cholesterol and 0.5 wt% of hicolic acid were added to powdered feed (GE-2) manufactured by Nippon Talea Co., Ltd. and mixed well, which was used as cholesterol feed.

Four types of earthworm dry powder (M
-2, M-4, M-6 and M-8) at 2 wt% each.
It was mixed and used as test drug feed.

3) Rearing conditions = Rats were placed in one cage, and each feed and water were available ad libitum. Temperature 23±1℃ and humidity 55
The animals were reared for 7 days at constant temperature and humidity of ±5%.

Rats were fasted overnight without food except for water for 7 days.
Blood was collected from the abdominal descending aorta under anesthesia using al)),
Serum was obtained by cold centrifugation.

TC in serum, free cholesterol (referred to as FC)
, LDL-C, and HDL-C using a Toshiba TBA-480 automatic analyzer and each T commercially available from Wako Pure Chemical Industries, Ltd.
Quantification was performed using A480Test reagent. AI was calculated as described above.

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

(Left below) In the group to which 2vL% of earthworm dry powder was added, TC in the serum
, FC, LDL-C and AI were significantly reduced, but HD
LC was constant and did not change significantly.

However, dry earthworm powder was found to have excellent antihyperlipidemic effects.

Pharmacological study 2: l) Animals: Sprague-Da weighing 105 ± 1 g.
Wley (hereinafter referred to as SD) basal type utto group of 8 animals were used.

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

(Note) a, b: The ingredients of the salt mixture and vitamin mixture are as follows: Fukushima et al.: Pharmaceutical Journal, Vol. 89, pp. 857-862 (1)
962) was used.

The ingredients in Table 6 were thoroughly mixed to form a cholesterol feed.The feed for the test drug group consisted of this cholesterol feed and earthworm dry powder M-3 (water content 1007%, ash content 5.2%).
%, nitrogen 9.2%) to 0.25vt%, and the same M-1 (moisture 1O02%, ash 5.1%, nitrogen 9.4%) to 0.5vt%.
Wt% and 0.5 and/or lvt% of γ-oryzanol (trade name: Hijet Fine Granules) or soysterol (trade name: Moristerol Fine Granules) as a control drug were mixed well and used as feed.

3) Rearing conditions: Two rats were placed in a cage, and food and water were available ad libitum. Temperature 23±1℃ and humidity 55±
Rats were kept at a constant temperature of 5% I for 4 weeks; at 64 weeks, they were fasted overnight except for water and treated with bentoparbital sodium [
Blood was collected from the abdominal descending aorta under anesthesia using Nembutal (trade name), and serum was obtained by cold centrifugation.

TClFC, TG, PL, free fatty acids (hereinafter N) in serum
It is called EFA. ), HDL-C, AI, glutamate oxaloacetate transaminase (hereinafter referred to as GOT) and glutamate pyruvate transaminase (
Hereinafter referred to as GPT. ) with the Toshiba TEA-480 automatic analyzer and each TA commercially available from Wako Pure Chemical Industries, Ltd.
Quantification was performed using 48QTest reagent.

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

4) Results: Table 7 shows the effects of dried earthworm powder on experimental hyperlipidemic rats using γ-oryzanol and soysterol as control drugs. In the case of feed containing 0.25 and Q, 5 wt% of dry earthworm powder, serum TC, FCSPL, and NEFA were lower than in the cholesterol-negative group.
was statistically significantly reduced. Although TG showed a decreasing tendency, no significant decrease was observed.

When γ-oryzanol is mixed at 0.5 and lit%,
TC except significantly lowering NEFA.

No significant decrease was observed in the values of FC, TG, and PL. In this regard, when the control drug soysterol was mixed with lit%, TC and FCSPL significantly decreased, but TG
and NEFA values were almost the same as in the cholesterol negative group.

As mentioned above, HDL-C is considered to be a factor that improves arteriosclerosis, and many basic and clinical researchers have found that an increase in this value is a useful property as an antihyperlipidemic agent. is being used. As shown in Table 7, it was confirmed that the earthworm dry powder added group had a statistically significant increase in HDL-C compared to the cholesterol negative group.

On the other hand, no increasing effect was observed with γ-oryzanol in the control group. However, a significant increase was observed in soysterol.

As mentioned above, the effect of lowering AI is useful as an effect of an antihyperlipidemic agent, and it was confirmed that it was significantly reduced in the groups to which dried earthworm powder and the control drug soysterol were added. However, the control drug γ-oryzanol showed a tendency to decrease, but the decrease was not significant.

As mentioned above, some commercially available antihyperlipidemic agents cause liver damage as a side effect. Therefore, it becomes a problem when used continuously for a long period of time. GOT and GPT in serum were measured to examine the effect on the liver. Typically,
It is known that increases in these test values suggest liver dysfunction. The group to which earthworm dry powder was added showed a significant decrease in both GOT and GPT compared to the cholesterol-negative group. On the other hand, in the group containing 0.5 wt% of the control drug γ-oryzanol, although it was not significant, there was a clear upward trend, and 1 wt%
In the added group, both GOT and GPT showed a significant increase.

In this regard, the control drug soysterol lvL% addition group showed lower values for both GOT and GPT than the cholesterol-negative group, but the decrease was not significant.

The weight of the liver showed a significant decrease in the group to which dried earthworm powder was added than in the cholesterol-negative group, but there was almost no difference in the group to which the control drugs γ-oryzanol and soysterol were added compared to the cholesterol-negative group.

TC in liver lipids showed a significant decrease in both the earthworm dry powder and both control drug addition groups compared to the cholesterol negative group.

TG in liver lipids showed a slight tendency to decrease in both the earthworm dry powder and both control drug addition groups, but no essential changes were observed.

PL in liver lipids showed a significant decrease in the group to which earthworm dry powder was added compared to the cholesterol-negative group, but no change was observed in the groups to which both control drugs were added.

Regarding the weight of the rats, both the dry earthworm powder and both control drug groups increased their weight steadily, and no significant changes were observed.

(Blank below) B-1. Effect of earthworm extract on experimental hyperlipidemic rats Pharmacological test 3: Measured by the same method as in Pharmacological test 1 of the effect of earthworm dry powder on experimental hyperlipidemic rats in B above. did. That is: l) Animals: Same as pharmacological test l above.

2) Feed: Cholesterol lvt% and Hikol Ilo, 5vt% were added to powdered feed (CE-2) manufactured by Nippon Talea Co., Ltd., and mixed well to prepare a cholesterol diet.

In addition to this cholesterol feed, there are four types of dried earthworm powder [
K-1, -3, -7 and -9] were mixed at 2 wt%, and
Seven types of earthworm extracts of the present invention [X-■, -■, -■, -
■, -・■, -[phase] and -■] were mixed with Q, 4vt% and used as test drug feed.

3) Rearing conditions: Same as the above pharmacological test 1.

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

As shown in Table-7-1, 2wt of earthworm dry powder alone
% added group and the single earthworm extract of the present invention.
There was no difference in effect between the two groups added with %. That is, TC in the serum of both groups.

The values of FC, LDL-C, HDL-C, and AI were almost the same, and TClFC in the serum of both groups.

LDL-C and AI decreased significantly, but HDL-C remained constant with no significant change. However, when compared on a unit weight basis, the earthworm extract of the present invention was found to have excellent antihyperlipidemic activity, and was found to have five times as much antihyperlipidemic activity as dry earthworm powder.

(Blank below) B-2. Effect of the mixture of earthworm extract and dried earthworm powder on experimentally hyperlipidemic rats Pharmacological test 4: Pharmacology of the effect of the dried earthworm powder in B above on experimentally hyperlipidemic rats It was measured by the same method as Test 1. That is: l) Animals: Same as pharmacological test l above.

2) Feed: Cholesterol lvt% and Hicol leo, 5wt% were added to powdered feed (CE-2) manufactured by Nippon Talea Co., Ltd., and mixed well to prepare a cholesterol diet.

The feed for the test drug group was adjusted as follows. That is, 20 wt of the earthworm extract of the present invention was added to the cholesterol feed.
% and 8゜it% of dry earthworm powder.
% well mixed. The combination of this test mixed drug is shown in Table 7.
-2.

(Margins below) Table 7-2 On the other hand, the control drug feed consisted of 2 wt% of single earthworm dry powder (K-1) and earthworm extract (X-1) added to the cholesterol diet.
(2) The individual ingredients were mixed well at 0.4vt% and used as feed.

3) Rearing conditions: Same as pharmacological test 1 above.

4) Results: The results are shown in Table-7-3.

As can be seen from Table 7-3, 2vt% of the single earthworm dry powder and 0.4wt% of the single earthworm extract of the present invention.
There was no difference in effect between the groups to which 0.8 wt% of these mixtures were added. That is, 17) TC,F in the serum of group 3
The values of C, LDL-C, HDL-C and AI were almost the same. In addition, the values of the control drugs shown in Table 7-3, earthworm extract (X-■) and earthworm dry powder (K-1), are as follows:
It agrees well with each value in Table-7-1. However,
The mixture of earthworm extract of the present invention and earthworm dry powder is
．． The addition of 8vt% means that the earthworm extract sail 1
This means that a mixture of 6vt% and 0.64wt% of earthworm dry powder was added. Comparing the antihyperlipidemic activity effects of the drug alone and the mixture on a unit weight basis, the synergistic effect of the mixture is obvious.

(Left below) C. Two oral administration experiments of dry earthworm powder to humans. Four volunteers who agreed to participate in the experiment.
r), capsule D manufactured in Reference Example IO described below
(1 agent contains earthworm dry powder (M-2 above, moisture 10.
4%, ash 5.3%, nitrogen 8.6%) 150rng
(containing)] was administered orally, 3 times a day, within 30 minutes after meals. As a general rule, serum lipids are measured every 3 to 4 months after drug use, and blood samples are collected early in the morning on an empty stomach for 6 to 7 months after drug use, and TC and TG in the serum are measured.

HDL-C and AI were measured. The measurement method used the same kit commercially available from Wako Pure Chemical Industries, Ltd. as described above. This result is shown in Table-
8.

(Leaving space below) Table 8 Standard values of serum lipids (mg/d) of healthy people living in rural areas who conducted an experiment of administering dried earthworm powder to these four volunteers.
Q) is TC130~2301TG 50~170
, HDL-C35-60. As shown in Table-8,
The TC values of all four subjects before administration exceeded standard values. TG and HDL-C values were within standard values.

The results in Table 8 (7) were the results of an experiment in which dry earthworm powder was administered orally to only four people.
The value of 80mg/d12 is 194~4 months after administration.
220 wag/do, which decreased to within the standard value range, and after 6 to 7 months, it significantly decreased to 174 to 209 mg/da. AI clearly decreased 3 to 4 months after administration, and significantly decreased 6 to 7 months after administration along with a decrease in TC.

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

The results of this oral administration experiment to humans showed that TC and AI were significantly decreased, and HDL-C was also slightly increased, similar to the results of the administration experiment to experimental hyperlipidemic rats. However, in the administration experiment to experimental hyperlipidemic rats, no significant decrease in TG was observed, but in the oral administration experiment to humans, a clear decrease was observed after 6 to 7 months. This is considered a noteworthy result.

The oral administration experiment of this dried earthworm powder to humans was successfully completed safely and without any side effects. For example, even after long-term administration for 6 to 7 months, the amount of sugar in the blood (hereinafter referred to as blood sugar) remains at the standard value (60 to 70%).
There was no risk of hypoglycemia that fell below the lower limit of llomg/dQ). As can be seen from Table 8, earthworm dry powder does not seem to be an antihyperlipidemic agent that can rapidly achieve its purpose in a relatively short period of time unlike synthetic organic compound drugs. In other words, it is considered to be a mild-acting antihyperlipidemic agent that requires continuous administration over a relatively long period of time. However, from 3 to 4 months after oral administration to humans, TC
%AI clearly decreased, HDL-C showed a slight upward trend after 6 to 7 months, and TG also clearly decreased after 6 to 7 months.

There are no particular age restrictions on the target patients for the earthworm extract or the mixture of earthworm extract and earthworm dry powder of the present invention. Although the product of the present invention is a multipotent type, the preferred target patients are middle and middle-aged patients.
The patient is elderly. The above results showed that dried earthworm powder is safe and an excellent antihyperlipidemic treatment and prevention agent.

These results demonstrate that earthworm extract, which is an extract from earthworms, is safe for humans and is an excellent antihyperlipidemic treatment and prevention agent, just like dried earthworm powder. Recognize. Similarly, it can be easily seen that a mixture of earthworm extract and earthworm dry powder in any ratio is also safe for humans and is an excellent antihyperlipidemic treatment and prevention agent.

That is, although there is a slight difference in the manufacturing method between earthworm dry powder and earthworm extract, since they are manufactured from the same raw material, it is natural that they exhibit excellent degradation. Furthermore, since the earthworm extract is a selective extraction of the active ingredients in the earthworm suspension or its dry powder, it is believed that it is effective in smaller amounts than the earthworm dry powder. The reason for the synergistic effect of the mixture of earthworm extract and dried earthworm powder was unknown, but it was an excellent and completely unexpected effect.

Example 1. Capsule E Earthworm extract (X-■) 30mg Lactose
28 Microcrystalline cellulose 47 Mannitol 100 Cornstarch 40 Polyvinylpyrrolidone
2-hydroxypropylcellulose
3 total 250 mg After mixing the ingredients other than hydroxypropyl cellulose in the above formulation well using a fluidized bed granulator, a 5% aqueous solution of hydroxypropyl cellulose was sprayed as a binder, and the mixture was dried at 40°C and made into granules. . Hard capsules were prepared by filling 250 mg of each of these granules into hard capsules.

Example 2. Capsule F Granule Earthworm extract (X-■) 30mg Lactose 1
03 Corn starch flour 89 Potato starch flour 22 Tal
the law of nature
3 Magnesium stearate
Total of 3 250+ ng According to the above recipe, granules were produced from the well-mixed powder using an extractor. Hard capsules were manufactured by filling 250 mg of each of the granules into hard capsules.

Example 3. Capsule G Earthworm extract (x-@) 30111
g phosphate - calcium hydrogen 60 phosphate -
Sodium hydrogen 20 mannitol
38 Magnesium Stearate
The above-mentioned formulations (total of 150 mg) were thoroughly mixed, and each 150 mg of this mixed powder was filled into gelatin capsules to produce capsules.

Example 4. Capsule H Earthworm extract (X-@) 30mg
Sodium lauryl sulfate Sodium 4-phosphate l Manitol
113 Magnesium stearate
2 total 150mg Mix the above prescribed ingredients well. This mixed powder was filled into gelatin capsules in an amount of 150 mg each to produce capsules.

Example 5. Capsule ■ Earthworm dry powder (M-2) 75 mg Earthworm extract (X-■ above) 15 mg Sodium lauryl sulfate Sodium 4-phosphate l Manitol
153 Magnesium stearate
250mg in total Mix the above prescribed ingredients well. This mixed powder was filled into gelatin capsules in an amount of 250 mg each to produce capsules.

Example 6. Capsule J Earthworm dry powder (M-4) 50mg Earthworm extract (X-■ above) 20mg Sodium lauryl sulfate 2 Manitol
176 Magnesium Stearate 2 total 250mg Mix the above formulation well. This mixed powder was filled into gelatin capsules in an amount of 250 mg each to produce capsules.

Example 7. Dry earthworm powder (M-1) in capsules 75mg earthworm extract (X-■ above) 15mg sodium lauryl sulfate sodium diphosphate-hydrogen 1 mannitol
55 Magnesium Stearate
2 total 150+og Mix the above formulations well. This mixed powder was filled into gelatin capsules in an amount of 150 mg each to produce capsules.

Example 8. Capsule L Earthworm dry powder (above-7) 50mg earthworm extract (above X@') 20mg sodium phosphate-hydrogen 2 mannitol
76 Magnesium stearate 2 total 150mg Mix the above formulation well. This mixed powder was filled into gelatin capsules in an amount of 150 mg each to produce capsules.

Example 9. Capsule M Earthworm dry powder (above-9) 70mg earthworm extract (above x-o) 15mg milk
sugar 70
Corn starch flour 69 Potato starch flour 22 tarri

2 Magnesium stearate 2 total 250 mg According to the above recipe, granules were manufactured from well-mixed powder using an extruder. Hard capsules were manufactured by filling 250 mg of each of the granules into hard capsules.

Example 10. Tablet A Earthworm extract (X-■ above) 30mg mannitol 22 hydroxypropoxymethylcellulose 4t
Ruri
3 Microcrystalline cellulose
20 hydrogenated castor oil
Total: 80mg Tablet B Earthworm extract (X-■ above) 30mg Cornoxidene powder 30 Milk
Sugar 35t
Ruri
3 Magnesium stearate 2 total 100mg Tablet C Earthworm extract (X-[phase]) 15m
g Earthworm dry powder (-7 above) 50 Soluble starch 20 Corn starch 25 Microcrystalline cellulose
silicon 45 oxide
3 Magnesium stearate 2 total 16
0mg According to the above recipe, the powder was uniformly and thoroughly mixed and used in a tablet machine to produce various tablets.

Example 11 1 kg (approximately 20,000) of live earthworms (red earthworms) lightly washed with water were placed in an acidic aqueous solution 4Q having a pH of 6.2 and dissolving a 1=1 mixed acid of malic acid and citric acid at a temperature of 15°C. So 3
After leaving it for a while to fully excrete the feces in the digestive tract,
Wash the live earthworms thoroughly with water to remove dirt, feces, and other dirt adhering to the body surface of the live earthworms.

Next, it is wet-pulverized using a mixer. The obtained earthworm suspension was placed in a tray and frozen at -30°C for 40 hours, then freeze-dried at -40°C for 6 hours under a vacuum of 1 mm HH. Raise to 30℃ O,
After vacuum drying for 6 hours under a vacuum of 1 mmHg, the shelf temperature was raised to 50°C, and vacuum drying was performed under a vacuum of 0 and 2 mmHg for 10 hours, and then the shelf temperature was raised to 80°C and under a vacuum of 12 mmHg for 8 hours. By drying, 280 g of a dry earthworm powder product (M-1) was obtained.

Example 12゜ Live earthworms (red earthworms) lightly washed with water l kg
2 at a temperature of 10°C in an acidic aqueous solution 3Q of pI (5,5) containing a l:l:1 mixed acid of phosphoric acid, tartaric acid and lactic acid.
After leaving for 5 hours to fully excrete the fecal soil in the digestive tract, the live earthworms are thoroughly washed with water to remove dirt, feces, and other impurities adhering to the body surface of the live earthworms.

Next, it is wet-pulverized using a mixer. The obtained earthworm suspension was placed in a tray and frozen at -25°C for 20 hours, then the temperature was lowered to -35°C and the temperature was reduced to 0. Lyophilize under a vacuum of 1 mmHg for 7 hours, then raise the shelf temperature on which the tray is placed to 28 °C, vacuum dry under a vacuum of 0-1 mmHg for 10 hours, then lyophilize at 40 °C with a vacuum of Q, 2 mmHg. 275 g of earthworm dry powder product (M-2) was obtained by drying under vacuum for 13 hours and then drying at 78° C. for 8 hours under a vacuum of Q, lmmHg.

Example 13゜1 kg of live earthworms (red earthworms) lightly washed with water,
Leave it in 2Q acidic aqueous solution with pH 5.8 in which malic acid is dissolved at a temperature of 13"C for 3 hours to fully excrete the feces in the digestive tract. Wash the live earthworms thoroughly with water and attach them to the body surface of the live earthworms. Wash away dirt such as dirt and feces.

Next, it is wet-pulverized using an Ultra Homo Mixer (manufactured by Nippon Seiki Co., Ltd.). Place the obtained earthworm suspension in a tray and mix for 30 minutes.
After freezing at ℃ for 30 hours, the temperature of each item was reduced to 0.5℃ at -30℃. Freeze-dry for 8 hours under a vacuum of 1 (g), then raise the temperature of the shelf on which the tray is placed to 25°C,
Vacuum dry for an hour, then raise the shelf temperature to 45℃, O, lmmH.
Vacuum dry for 12 hours under a vacuum of
0. 275 g of earthworm dry powder product (M-4) was obtained by drying under a vacuum of lmmHg for 7 hours.

Example 14 Dirt, feces, and other impurities adhering to the body surface of 1 kg of live earthworms (red earthworms) were washed thoroughly with water four times to remove them. Next, the live earthworms are left in an acidic aqueous solution 2.5a having a pH of 5.7 in which a mixed acid of malic acid and lactic acid (1:1) is dissolved at a temperature of 15° C. for 2.5 hours to excrete the feces in the digestive tract. Next, after lightly washing the live earthworms, they are wet-pulverized using a mixer. The obtained earthworm suspension is placed in a tray and frozen at -35°C for 24 hours. Next, Q at the product temperature of -35℃,
Freeze-dry under a vacuum of lmmHg for 7 hours, then raise the temperature of the shelf on which the tray is placed to 22°C. After vacuum drying for 10 hours under a vacuum of 1 mmHg, the shelf temperature was raised to 42°C, and dried for 15 hours under a vacuum of 2 mmHg. Dry powder product of earthworm (M-6) by drying for hours
) 265g was obtained.

Example 15 Dissolve 1.5 g of potassium dihydrogen phosphate in 2.5 Q of an acidic aqueous solution with a pH of 6.0 in which citric acid is dissolved, and add 1 kg of live earthworms (phthurminus) lightly washed with water to this solution. Leave at ℃ for 2 hours to excrete the feces in the digestive tract. Next, the live earthworms are washed twice with water to remove dirt, feces, straw, and other impurities that adhere to the body surface of the live earthworms. Next, it is wet-pulverized using a mixer.

After that, put the obtained earthworm suspension into a tray for 40 minutes.
Freeze for 24 hours at °C. Next, adjust the temperature of the product to 0.1 m at -40°C.
Freeze-dry under mHg vacuum for 5 h, then Q at 25 °C.
After drying under a vacuum of lnoaHg for 8 hours, then
280 g of earthworm dry powder product (M-7) was obtained by drying at 5° C. under a vacuum of 1 mm Hg for 12 hours and then at 80° C. under a vacuum of 0.1 mm Hg for 7 hours.

Example 16: Dirt, feces, straw, and other impurities adhering to the body surface of 1 kg of live earthworms (Tsurimimizu) were washed thoroughly with water 5 times to remove them. Next, the live earthworms were washed with pH 5 solution containing succinic acid.
Dissolve sodium acetate Ig and 0.5 g of sodium sulfate in 3Q of the acidic aqueous solution of . ) at 13°C 2.
The animal was left to stand for 5 hours and the fecal soil in the digestive tract was excreted. Next, the live earthworms are lightly washed with water and then wet-pulverized using a homogenizer. The obtained earthworm suspension was placed in a tray and heated at 35℃ for 2 hours.
Freeze for 4 hours. Next, the product temperature is -35℃ and 0. Freeze-dry for 7 hours under a vacuum of bnmHg, then raise the temperature of the shelf on which the tray is placed to 22°C. After vacuum drying for 10 hours under a vacuum of 1 mmHg, the shelf temperature was then raised to 42°C and 0.2 mmHg.
Dry under vacuum for 15 hours, and finally raise the shelf temperature to 78°C.
, lmmHg vacuum for 7 hours to obtain 275 g of dried earthworm powder product (M-8).

Example 17 Dirt, feces, straw, and other impurities adhering to the body surface of 1 kg of live earthworms (Ptera phthalmica) were thoroughly washed with water 5 times to remove them. Next, the live earthworms were mixed with citric acid and tartaric acid l:l.
0.7 g of potassium citrate is dissolved in 2.5 Q of an acidic aqueous solution of pH 5.9 in which mixed acid of ) in 1
The feces in the digestive tract were excreted after being left at 5°C for 2 hours. Next, after lightly washing the live earthworms with water, they are wet-pulverized using a blender. Place the obtained earthworm suspension in a tray at -35°C.
It was frozen for 24 hours.

Next, freeze-dry for 7 hours at a temperature of -35°C under a vacuum of 0.1no++ Hg, and then reduce the temperature of the shelf on which the tray is placed to 22°0.
After vacuum drying for 10 hours under a vacuum of Q, l + mHg, the shelf temperature was then raised to 42°C and dried under a vacuum of 0.2 mmHg for 15 hours, and finally the shelf temperature was raised to 78°C Q, lmm.
283 g of dry earthworm powder product (M-9) was obtained by drying under Hg vacuum for 7 hours.

Example 1 1 kg of live earthworms (red earthworms) lightly washed with 8° water are left in 3Q fresh water at a temperature of 10° C. for 16 hours to excrete the feces in the digestive tract. Wash the live earthworms thoroughly with water to remove dirt, feces, and other dirt adhering to the body surface of the live earthworms. Next, wet grinding is performed using an ultra homo mixer. The obtained earthworm suspension was placed in a tray and frozen at -25°C for 15 hours.Then, the temperature was lowered to -35°C and freeze-dried for 6 hours under a vacuum of 0.1 mmHg, and then at 30°C for 15 hours. Dry under vacuum at O8mmHg for 10 hours? , m, then 0 at 40℃
．． 245 g of earthworm dry powder product (M-3) was obtained by drying for 15 hours under a vacuum of 2 mmHg and then for 8 hours under a vacuum of Q, lmmHg at 78°C.

Example 19 Dirt, straw, and other impurities adhering to the body surface of 1 kg of live earthworms (red earthworms) were washed thoroughly with water 5 times to remove them. Next, place the live earthworms in 2.5Q of fresh water at a temperature of 15%.
The feces in the digestive tract were excreted after being left at ℃ for 18 hours. Next, after lightly washing the live earthworms, they were wet-pulverized using an ultra homo mixer. Place the obtained earthworm suspension in a tray.
It was frozen at 0°C for 24 hours. Next, reduce the temperature of the product to -40°C and 0. l
Freeze-dry for 5 h under vacuum at mmHg and then dry at 25 °C in O
, lmmHg for 8 hours, then 45
240 g of earthworm dry powder product (M-5) was obtained by drying for 12 hours under a vacuum of 1 m+nHg at 80°C and then for 7 hours under a vacuum of 0.1++ mHg at 80°C.

Reference Example 1゜Tablet A Dry earthworm powder 150mg (Ingredients are the same as M-8 above) Manitol Hydroxypropoxymethyl cellulose Microcrystalline cellulose Talc Hydrogenated castor oil Tablet B Dry earthworm powder (Ingredients are the same as M-8 above) ) Corn starch milk powder Sugar talc Magnesium stearate tablet C Dry earthworm powder (components are the same as M-8 above) Soluble starch Corn starch Microcrystalline cellulose Total 350 mg 50 mg Total 300 mg 50 mg Silicon oxide 6 Magnesium stearate 4 total 350 mg Tablets of various weights were manufactured using a tablet machine from the powder that was mixed well and uniformly according to the above recipe.

Reference example 2. Granule A Dry earthworm powder 150mg (Ingredients are the same as M-2 above) Lactose
2゜Microcrystalline Cellulose 6゜Corn Starch 15Hydroxypropyl Cellulose 5 Total 250mg According to the above recipe, using a fluidized bed granulator, dry earthworm powder, lactose, microcrystalline cellulose and cornstarch were thoroughly mixed, and hydroxyglobil A 5% aqueous solution of cellulose was sprayed as a binder and granulated after drying at low temperature.

Reference example 3. Granule B Dry earthworm powder 100mg (Ingredients are the same as M-2 above) Manitol microcrystalline cellulose carboxymethyl cellulose calcium Magnesium stearate Hydrogenated oil 1.5 1.5 Total 200.0mg Granule C Dry earthworm powder 150mg (Ingredients is the same as M-2 above) Lactose
53 corn starch flour 39 corn starch flour 2 tal.
3 Magnesium stearate
Total of 3 250 mg According to the above recipe, granules were manufactured from the well-mixed powder using an extruder.

Reference example 4. Cagcel agent A Dry earthworm powder 150mg (Ingredients are the same as M-2 above) Lactose
28 Microcrystalline cellulose 47 Mannitol 1O cornstarch 10 Polyvinylpyrrolidone
2-hydroxyglobil cellulose
3 total 250 mg After the components other than hydroxypropylcellulose in the above formulation were thoroughly mixed using a fluidized bed granulator, a 5% aqueous solution of hydroxypropylcellulose was sprayed as a binder, and the mixture was dried at low temperature and made into granules. Hard capsules were prepared by filling 250 mg of each of these granules into hard capsules.

Reference example 5. Capsule B Granules C manufactured according to Reference Example 3 were put into hard capsules, 25
Hard capsules were prepared by filling 0 mg each.

Reference example 6. Capsule C Dry earthworm powder 150mg (Ingredients are the same as M-6 above) Calcium hydrogen phosphate 60 Sodium hydrogen phosphate 10 Yanitol
28 Magnesium Stearate
250mg in total Mix the above formulations well and add this mixed powder to No.
Capsules were prepared by filling 250 mg of each gelatin capsule into 1 liter gelatin capsules.

Reference example 7. Enteric-coated tablet earthworm dry powder 100mg (components are the same as above M-1) Mannitol 10 Microcrystalline cellulose 85 Carboxymethyl cellulose calcium 2 Magnesium stearate 1.5 Hydrogenated oil 1
．． 5 200 mg in total According to the above recipe, the uniformly mixed powder was manufactured into uncoated tablets using a tablet press, and then coated with the following enteric-coated coating agent to manufacture enteric-coated tablets.

Coating agent Hydroxyglobil methylcellulose 7 tallate 14.
8mg Dioctyl phthalate 2.3 Stearic acid 2.3 Light silicon oxide 0.6 Total 20.0mg Reference example 8. Powder A Earthworm dry powder (components are the same as M-4 above) Manitol corn starch powder 50 mg Total 250 mg Powder B Earthworm dry powder (components are the same as M-4 above) Calcium phosphate-hydrogen corn starch powder 50 mg Total 259 mg Above ingredients They were each mixed thoroughly and uniformly in a conical mixer to form a powder.

Reference example 9. Suppository A Dry earthworm powder 200mg (Ingredients are the same as M-9 above) Whitteds Sole 540 (W 1t
epsol) E-85 Witepsol l, 454 (tt)
W-35 Methylbarahydroxobenje-1-3butylparahydroxybenzoate 3 total 2,200mg Inhibitor B Earthworm dry powder 200mg (components are the same as above M-6) Butylhydroxyanisole 6 semi-synthetic glyceride 2,900 total 3,106 mg The melt obtained by thoroughly mixing each of the above formulations was poured into an aluminum mold and cooled to produce a suppository.

Reference example 10. Capsule D Earthworm dry powder 150mg (Ingredients are the same as M-6 above) Sodium lauryl sulfate Sodium 4-phosphate l Manitol
93 Magnesium Stearate
2 total 250B Mix the above formulations well. This mixed powder is No.
, 250 mg of each gelatin capsule was filled to produce capsules.

〔Effect of the invention〕

As stated above, the present invention relates to an antihyperlipidemic agent containing an earthworm extract or a mixture of an earthworm extract and earthworm dry powder as an active ingredient.

Rats were fed a high-cholesterol diet containing the earthworm extract of the present invention, a mixture of earthworm extract and dry earthworm powder, and the dry earthworm powder for one week, or rats were fed a high-cholesterol diet containing dry earthworm powder for four weeks. The product of the present invention was found to have an excellent antihyperlipidemic effect through rearing experiments. That is, a one-week hot rice experiment using the product of the present invention and dried earthworm powder, which is one of the starting materials for the product of the present invention. So, compared to the cholesterol group, TC in serum.

FC% LDL-C and AI are significantly reduced, but HDL
-C had no significant change.

On that point, in a 4-week hot meal experiment using dried earthworm powder, TC and FC in serum were lower than in the cholesterol group.

PL and NEFA were significantly reduced. Furthermore, it was confirmed that it significantly increased HDL-C, which is considered to be an arteriosclerosis improving factor, and significantly decreased AI. T
Although G showed a decreasing tendency, the decrease was not significant. or,
GOT and GPT were significantly reduced. Although liver weight and liver lipids TC and PL were significantly reduced, TG remained almost unchanged. In addition, the weight of the rats increased steadily, and no significant changes were observed.

One dose of dry earthworm powder capsules (containing 150mg) 3 times a day, 4 volunteers
r) for 6 to 7 months after meals to reduce TC in serum.
.

TG%HDL-C and AI were measured. The results show that TC and AI clearly decreased after 3 to 4 months.

On the other hand, changes in HDL-C were slow, but a slight upward trend was observed after 6 to 7 months. TG is 6 to 7
A clear decrease was observed after several months.

Animal experiments using earthworm extracts, mixtures of earthworm extracts and dry earthworm powder, and dry earthworm powders, as well as oral administration experiments of earthworm dry powders to humans, revealed that the earthworm extracts, earthworm extracts, and dry earthworm powders of the present invention The mixture was found to be safe and extremely useful as an excellent therapeutic and preventive agent for hyperlipidemia; an excellent serum lipid metabolism improver; and an excellent therapeutic and preventive agent for arteriosclerosis.

Director General of the Japan Patent Office Yoshi 1) Moon Takeshi 1, Indication of the case 1999 Patent Application No. 12036 2, Name of the invention Antihyperlipidemic agent 3, Relationship with 4 cases Patent application population 5, increase due to amendment No number of claims6, “Claims” and “Detailed Description of the Invention” of the specification subject to amendment
column.

Attachment (1) The scope of patent claims will be amended as follows.

2. Claim 1: The active ingredient is an earthworm extract obtained by extracting earthworms 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. Antihyperlipidemic agent containing as.

2. When the earthworms have a fishy odor, they can absorb at least one compound selected from the group consisting of organic acids, inorganic acids, organic acid sodium salts, inorganic acid sodium salts, organic acid potassium salts, and inorganic acid potassium salts by 0.3 (by weight). )% or less in an aqueous solution or in fresh water to remove the excrement from the digestive tract of the live earthworm, and then wash and remove the dirt adhering to the body surface of the live earthworm with water and perform wet crushing. After removing the suspension or dirt adhering to the body surface of the live earthworms by washing with water, the live earthworms are left in the aqueous solution or fresh water to remove the fecal soil in the digestive tract of the live earthworms. Claim 1 consisting of an earthworm suspension obtained by washing and removing dirt adhering to the body surface with water and performing wet grinding.
Antihyperlipidemic agents as described.

3. Earthworms consume the earthworm suspension according to claim 2 from -10 to
After freezing at -60℃, the temperature is raised stepwise within the range of -60 to 80℃ and the vacuum level is 10m+Hg or less.
Freeze and vacuum dry for 0 to 100 hours, including the final step of vacuum drying at 70 to 80 degrees Celsius.
The antihyperlipidemic agent according to claim 1, which comprises dried earthworm powder obtained by drying under flIHg vacuum for 5 to 10 hours.

4. An antihyperlipidemic agent containing a mixture of earthworm extract and earthworm dry powder as an active ingredient.

(2) "External" in the fifth line of page 6 of the specification is corrected to "external".

(3) “Early period” in line 5 on page 16 of the specification, line 11 on page 20, line 4 on page 21, and line 4 on page 43 is corrected to “J above.” To do.

(4) Change “manufacturing” in line 5 on page 16 of the specification to “manufacturing method
I will correct it.

(5) The amino acid "tyroline" in line 17 of Table 3 on page 25 of the specification is corrected to "r-tyrosine."

(6) Line 14 of page 33 and line 4 of page 34 of the specification
r Coe ton ic J of row rceloIl
I will correct it as ic J.

(7) “Succinic acid or” on page 35, line 16 of the specification
Correct it to ``r-succinic acid, malic acid, or''.

(8) “S-butyl” in line 7 on page 36 of the specification is “
1-butyl, S-butyl”.

(9) “Vinegar Wi-2” on page 37, line 9 of the specification
Correct -J to racetic acid 2-1.

(10) About r 30'C on page 46 of the specification, line 2.
Correct it to be approximately 1 at r30℃.

(11) On page 46 of the specification, line 4, “-Leave it undisturbed day and night;
” should be corrected to “-Leave it undisturbed day and night.”

(12) On page 54 of the specification, in line 15, ``fasted and ta'' is corrected to ``r fasted''.

(13) Correct "value" in line 20 of page 59 of the specification to read "r value."

(14) The serum TC value r188.7±19.2 when the drug dose was 0.5 wt% for the earthworm dry powder addition group in Table 7 on page 63 of the specification. 19.
I will correct it as 2”Iゞ】.

(15) The AI value of the normal diet group in Table 7 on page 63 of the specification is ro, ai ± 0°12'', ro, 81 ± 0.12 *
＊ ＊” I corrected it.

(16) Liver weight r of the earthworm dry powder added group in Table 7 on page 63 of the specification when the drug dose was 0.25 wt%
Correct "11.3±0.3°1" to rll, 3±0.3°j.

(17) Liver TC value r34.2±1.3 when the drug dose o of the γ-oryzanol addition group in Table 7 on page 63 of the specification was 5 wt%. 3* *
I will correct it as j.

(18) Liver TC-M when the drug dose was 1.0 wt% in the γ-oryzanol addition group in Table 7 on page 63 of the specification
(1) r32.8+o, 9 J o r32.8+ 0
．． 9”J I will correct it.

(19) In Table 7 on page 63 of the specification, the hepatic MTC value of the soysterol-added group, r34.3±1.311mm, has been corrected to r34.3±1.3°.

(20) r23 of the liver PL value of the soysterol addition group in Table 7 on page 63 of the specification. Of 0.6”
J to r23. [lf: Corrected to 0.6J.

(21) "Adjustment" in line 11 on page 67 of the specification is corrected to "preparation."

(22) Mixture Oni 5 of rK-7J of earthworm dry powder 80W1% in Table 7-2 on page 68 of the specification to rK-9
” I am corrected.

(23) “I took it” on page 71 of the specification, line 7.
is corrected to "taken as a drug."

(24) "Corn" in line 20 on page 81 of the specification is corrected to "maize."

(25) rM-6J in the first line from the bottom of page 93 of the specification
Correct it to rM-3J.

(26) rM-6J on page 97 of the specification, line 4
I will correct it to -2J.

(27) "No. 24" in line 12 on page 7 of the specification is corrected to "Volume 24."

Claims (1)

[Claims] 1. An active ingredient is an earthworm extract obtained by extracting earthworms 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. Antihyperlipidemic agent containing as. 2. When earthworms feed raw earthworms with at least one compound selected from the group consisting of organic acids, inorganic acids, sodium salts of organic acids, sodium salts of inorganic acids, potassium salts of organic acids, and potassium salts of inorganic acids, 0.3 (by weight) The earthworm suspension is obtained by leaving it in an aqueous solution or fresh water containing less than After removing the turbid liquid or the dirt adhering to the body surface of the live earthworm with water, the live earthworm is left in the aqueous solution or fresh water to remove the fecal soil in the digestive tract of the live earthworm, and then the body of the live earthworm is removed. 2. The antihyperlipidemic agent according to claim 1, which comprises an earthworm suspension obtained by washing and removing dirt adhering to the surface with water and performing wet grinding. 3. Earthworms consume the earthworm suspension according to claim 2 from -10 to -
After freezing at 60°C, the temperature was raised stepwise within the range of -60 to 80°C and the vacuum level was 10 mmHg or less.
Freeze and vacuum dry for ~100 hours, including the final step of vacuum drying at 70 to 80°C to a thickness of 0.01 to 0.5 mm.
The antihyperlipidemic agent according to claim 1, comprising dried earthworm powder obtained by drying under Hg vacuum for 5 to 10 hours. 4. An antihyperlipidemic agent containing a mixture of earthworm extract and earthworm dry powder as an active ingredient.