JPH02215724A - Preparation of earthworm dried powder - Google Patents

Abstract

PURPOSE:To effectively provide an earthworm dried powder exhibiting an excellent medical activity, having no side effect and capable of being stored in a stable state over a long period by grinding the living body of a filth- removed earthworm, vacuum-drying the ground product under such a condition as finally giving a specific temperature range and subsequently pulverizing the dried product. CONSTITUTION:A living earthworm is immersed in fresh water or in weak acidic water adjusted to a pH of 3-6.5 and maintained to a temperature of 6-26 deg.C until feces soil is perfectly discharged from the earthworm and the filth is removed from the body surface and digestion tracts of the earth, followed by grinding the treated earthworm to give a liquid ground product. The ground product is vacuum-dried under a vacuum degree of <=30mmHg, especially <=1mmHg, to give a vacuum-dried product, while heating the ground product stepwisely up to a final temperature of 70-80 deg.C. The method for the drying process includes a method in which the liquid ground product is degased and subsequently dried under the above-mentioned condition and a method in which the liquid ground product is frozen, vacuum-dried under the above-mentioned vacuum degree and subsequently dried under the above-mentioned condition.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application: The present invention provides a method for producing earthworm dry powder having safe and excellent thrombus (the main component of thrombus is fibrin) lytic activity or fibrinolytic activity; The present invention relates to a thrombolytic agent, a therapeutic agent for thrombosis, or an agent for preventing thrombus formation, which contains dry earthworm powder as an active ingredient.

Prior Art: Thrombi (the main component of thrombus is fibrin) are generated every day in the human body and are dissolved by fibrinolytic active enzymes (plasmin) in the body. However, when this balance is disrupted, unnecessary fibrin increases one after another.

In recent years, various diseases caused by fibrin have been attracting attention because they frequently occur in the elderly and elderly.

For example, peripheral arteriovenous thrombosis, pulmonary embolism, coronary artery occlusion,
Various diseases caused by blood coagulation, such as myocardial infarction, cerebrovascular occlusion, and cerebral infarction, are occurring frequently.

Various therapeutic drugs have been developed, among which enzyme preparations such as human urokinase and streptokinase are known to be effective. In particular, human urokinase is widely used because it has relatively few side effects and excellent pharmacological action. Human urokinase is a plasminogen activator that is produced in the human kidney and excreted in the urine.It specifically acts on plasminogen and converts it into plasmin, an enzyme that degrades fibrin. , exerts its effect as a thrombolytic agent.

However, human urokinase has the disadvantage that it is difficult to obtain human urine as a raw material, and it must be administered by intravenous drip, which causes great pain to patients when administering it. .

On the other hand, streptokinase is a metabolite of β-hemolytic streptococci, and like urokinase, it has a plasminogen activation effect, but it is antigenic and has strong side effects such as causing allergies and fever. Regarding administration, it has the same problem of intravenous infusion as the above-mentioned urokinase.

Dried earthworms have been used as medicine since time immemorial, mainly in Eastern countries, where they were called waxworms and earthworms. The conventional manufacturing methods and methods for manufacturing dried earthworm products or powders published in the literature are as follows.

■, r "Record of the People's Republic of China," edited by the Retelling Committee of the Ministry of Health of the People's Republic of China, 1977 edition, part of pages 197 to 19B states the following.

i Open the abdomen of the earthworm to reveal its internal contents (viscera and mud)
Remove from sunlight, shade or low temperature (usually below 50°C)
How to dry with.

ii) A method in which earthworms are killed by placing them in plant ash, the ash is removed, and the earthworms are dried in the sun or in the shade or at low temperatures (usually below 50°C) to obtain earthworms with mud still stuck inside their bodies.

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

■ Manufacturing methods reported in patent publications and other recent literature.

iv Yo Ishii - [One of the inventors of the present invention. Japanese Patent Application Publication No. 59-216572) discloses that after removing the excrement remaining in the body of the earthworm, it is washed several times with water,
Next, it was made into a paste by a machine, this paste was placed in a tray, and after freezing at -30℃ for 48 hours, the vacuum degree was o.
i Tall, heating time 17 hours 40 minutes, temperature -40 ~
The temperature is raised stepwise at 80°C, and after rising to 80°C, the final temperature is 80°C, and the powder is obtained by freezing and vacuum drying at a vacuum degree of 0.3 Torr for more than 20 hours. There is no description in this publication regarding the method for removing excrement remaining in the body (Notes by the present inventors).

■ Kotoshi Mihara [Journal of the Japanese Society of Hematology Vol. 45, No. 2, No. 3
55 pages (1982)] is an earthworm (Lua + bric
After washing approximately 100 kg of the body with tap water several times to wash away the mud adhering to the body surface and thoroughly vomiting out the mud inside the body, the body was homogenized (homogenized).
oge'ze). This homogenization is done in a stainless steel tray (470+w+X320aX3 Qam) 1
Pour into 20 sheets to a thickness of 25 m and heat at -30°C.
They reported that they obtained a freeze-dried earthworm powder by freezing for 30 hours and freeze-drying it for 30 hours in a large freeze dryer.

■ Kotoshi Mihara (Report of the 9th Blood and Blood Vessel Study Group, page 117~
p. 122 (August 1982)] reported as follows. The nature of earthworms is that if the conditions are bad, they will gather together. Once all the soil is removed, only the earthworms are left, so if you wash them with water and leave them for 24 hours, they will vomit out all the dirt inside their bodies. The earthworms were homogenized and freeze-dried to prepare a powder.

Kiwako Kokan (Japanese Patent Application Publication No. 1986-629)
In No. 65), live earthworms were left in clean water for 2 to 4 hours, washed with water, then dried at 80 to 90°C for 2 to 3 hours, and then crushed to obtain dry earthworm powder.

Report: Hisashi Mihara [Environmental Science Research Report Collection B504-R30; Environmental Improvement Technology Research Report Volume 4, pp. 107-112 19
In 1986], the earthworms were washed to remove mud, left in pure water day and night, and after thoroughly excreting the excrement from the fire extinguishing pipe, they were homogenized using an Ultra Homo Mixer (manufactured by Nippon Seiki), and then frozen. It is dried to obtain dry earthworm powder.

Among the above-mentioned known manufacturing methods, dried earthworm products or dry earthworm powders obtained by some of the manufacturing methods are sterile and stored in a refrigerator at 0 to 5°C or in an open state at room temperature of 5 to 45°C. It had shortcomings in its shelf life, as it developed mold or deteriorated within a short period of about six months when stored, and within one year when stored in an airtight state, making it unusable as a medicine. In addition, when an external action is applied to remove excrement from the digestive tract, as in some manufacturing methods, or due to inappropriate drying temperature settings, the yield is reduced and the medicinal efficacy is insufficient. Further, there were disadvantages such as the production time being too long. In general, the above-mentioned known production methods have a low yield of dry earthworm powder of 5 to 19 (wt)% based on the live earthworm raw material, and are difficult to use as an industrial mass production system.

The pharmacology and medicinal effects of earthworms reported in conventional literature are shown 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 describe the pharmacology of bladder stone reduction agents and excretion agents from the body, yellowing treatment agents, birthing agents, tonics, hair herbal medicines, tonics, and antipyretics. On the other hand, as earthworm toxins, one has been reported to affect the nervous system, and the other has been reported to have 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, there are two types of earth dragon products. One such wide dragon (Lumbricus Kwangtungesis)
The abdomen is torn open, the internal organs and muddy sand washed away, and then dried in the sun, shade, or at low temperatures. Other land dragons (Lusb)
ricus nativus) are killed by placing them in plant ashes, then removing the ashes and drying them in the sun or in the shade at low temperatures, and their bodies are filled with mud.

It has been reported that these two types of earth dragons are used as an antipyretic agent, convulsion treatment agent, blood circulation enhancer, hemiplegia treatment agent, joint analgesic agent, urinary agent, bronchial asthma agent, and hypertension agent at 4.5 to 9 g/day. There is.

■ [Our Chinese medicine series 3, earth dragon/squid bone,
Chinese Scientific Research J, October 30, 1978, published by Matsuura Pharmaceutical Co., Ltd., page 7 reports that Earth Dragon tincture (Ethyl air alcohol extract of Earth Dragon) has a hypotensive effect.

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

■ Mamoru Nichichi [Hokkaido Medical Journal Vol. 24, No. 18-2
4 (1949)] is a precipitated material (Lumbrof) obtained by extracting the mud from dried fine pieces with boiling water and adding ethyl alcohol to the concentrated liquid of this extract.
It has been reported that when ebrin) was dissolved in Ringer's civil law 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. The solution was intravenously injected into adult rabbits and a drop in blood pressure was observed.

■ Volume 2 of "Middle and Weak Great Dictionary", edited by Jiangsu New Medical Academy, 1980, published by Kamiyu Science and Technology Publishing Co., Ltd., page 2112, contains information on Guangjilong tincture, Waxia dry powder suspension, and Waxyx soaked in hot water. When the liquid, short axis decoction, etc. were 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 anesthetized dogs or cats, a 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, 40% concentration of earth dragon tincture (40 g of earth dragon soaked in 100 m of 60 degree ethyl alcohol) is applied for 101 d each time, -3 times a day [i.e., equivalent to 12 g of earth dragon/day] Converted by the inventors)]. Those who cannot take tinctures can make pills by adding water to pure Earth Dragon powder (adding a small amount of excipients) and administering 3-4 g each time, three times a day [equivalent to 9-12 grams of Earth Dragon/day]. (calculated by the inventors)] If taken and continued for 30 to 60 days, it is effective against essential hypertension. Also, earth dragon B1
Liquid (Hyboxanthin was removed using 1g of HgC, and blood pressure lowering components were separated using ion exchange resin)
2nd every time! It has been reported that taking 3 times a day [equivalent to 24 g of Jiryu/day (calculated by the present inventors)] (containing 8 g of the herbal medicine Jiryu) was effective against essential hypertension.

■Recently, Tsune Mihara et al.
No. 3184] fractionated six new types of fibrinolytic proteases from earthworms. Namely, Tsune Mihara et al. added 10 times the volume of physiological saline to dry earthworm powder and incubated it for 2 days (IncubaLion). After performing ammonium sulfate fractionation on the supernatant, the precipitate was separated into 5 epha
Gel filtration was performed using Cryl S-200, and the obtained protein fraction was subjected to DEAE-cellulose ion exchange chromatography, resulting in proteins in fractions I, ①, and ① having casein-degrading and fibrin-degrading activities. Regarding the I, ■, ■ fractions, DEAE-cellulose, 5ephadexG-75, Toyovar WH5
5, A CH-5epharose 5Benzan+
As a result of purification using 1dine-3epharose and the like, 6 fractions of purified enzyme were obtained. 5DA-P
When the molecular weight was measured using AGE, the molecular weight of fraction 1-0 was the lowest (calculated as 23°500, and then sequentially I-1
, I-2, ■, II [-1, m-2 and the molecular weight increases,
■-2 had a molecular weight of 34,200. Further, when the isoelectric points of these six fractions were measured by isoelectric focusing, 1-0 was the most common, and the pH was 4.12, and then the pH gradually decreased, and at m-2, the pH was 3.52. These six fractions are new proteolytic enzymes that are different from serine enzymes, and
The optimum pH of the proteases in these six fractions is around 8 or pH 8-10, and the stable pH is 4-12 or 5-12.
It is reported that the optimum temperature was 50°C or 50-60°C, and the inactivation conditions were 70°C for 60 minutes. These six types of proteases are considered to be responsible for the clot-dissolving action of dried earthworm powder, and many patent applications have been filed for thrombolytic agents containing proteases as active ingredients, such as Japanese Patent Application Publication No. 1983. The following patent applications have been reported (AU, P, App,
16293 (filing date June 27, 1983), C
A, P, App431387 (filing date June 1983)
(Mon. 28), DK, P.

App, 300B (filing date June 29, 1983)
, EP, P, App, 83106288.0 (filing date 1
June 28, 983), ES, P, App, 5
No. 23754 (filed June 30, 1983), Fl.

P, App, 832383 (filing date June 2, 1983)
9th) No. P. App, 2399 (filing date 1983)
June 30, 2015), PH1P, App, 29151 (
(filing date June 30, 1983), TW, P.

App, 7211983 (filing date June 1, 1983)
8th), US, P. App, 508163 (filing date 1
June 27, 983)].

■ Hisashi Mihara (reference vi above) found that 200 μL of freeze-dried earthworm powder obtained by method i above was put into capsules and orally administered to normal healthy adult volunteers.
After some time, fibrin degradation products (FDP), which were apparently caused by the dissolution of intravascular fibrin clots, increased in the blood. It is reported that the earthworm fibrinolytic enzyme, ie, the aforementioned protease, was absorbed into the intestine and exhibited fibrinolytic activity in the blood.

Problems to be Solved by the Invention: The present inventors had doubts about the protease theory of Mihara (see reference vi above) mentioned above. That is, regardless of the production origin of protease of any proteolytic enzyme originating from animals, plants, or microorganisms, for example, proteases with a molecular weight of 2 to 4 as an anti-inflammatory agent may be used.
Considering the fact that it is generally accepted by academic societies around the world that 10,000 proteases are not absorbed from the human intestinal tract, proteases with a molecular weight of 20,000 to 30,000 in the dried earthworm powder bundle are absorbed from the intestinal tract and have thrombolytic activity. There were many doubts about the theory that this was the case. That is, the present inventors considered that substances other than protease with a molecular weight of 20,000 to 30,000 are involved in intestinal absorption. Therefore, we considered extracting a substance other than 7° rotiase with a molecular weight of 20,000 to 30,000 that has fibrinolytic activity from dried earthworm powder, confirming its structure, manufacturing the substance, and creating a new drug through drug research. However, I learned that it would take a long time to elucidate this problem.

The present inventors believed that the best way to increase the therapeutic effect of dried earthworm powder as a thrombolytic agent by orally administering it to humans is to increase the amount of thrombolytic active substances in the dried earthworm powder itself. .

In this way, it has excellent medicinal efficacy, improves yield, has no side effects such as destruction of red blood cells or hemolysis, is highly safe, and is sterile and odorless and can be stored for at least 5 years in a sealed state. Various studies were conducted in order to establish an industrial production method that would enable mass production of dry earthworm powder. As a result, we succeeded in alleviating and improving the above-mentioned drawbacks, including eliminating the difficulty in obtaining raw materials for conventional thrombolytic agents and avoiding intravenous drip injection. Furthermore, we have succeeded in establishing a method for producing dry earthworm powder that increases the fibrinolytic activity of human peripheral blood and is safe and has excellent effects as a thrombolytic agent by oral administration or administration, resulting in the present invention.

Means to solve the problem: In order to solve the above problem, as a result of detailed research,
We have established the following new and innovative method for producing dry earthworm powder.

Production method 1: Live earthworms are soaked in fresh water or in non-toxic organic acids such as acetic acid, citric acid, malonic acid, succinic acid, malic acid, tartaric acid, lactic acid, or non-toxic inorganic acids such as phosphoric acid, sulfuric acid, hydrochloric acid, etc. At least one compound of sodium or potassium salts of acids is added to a slightly acidic aqueous solution with a pH of 3 to 6.5 at a low concentration of 2 (containing 1% or less, preferably 1% (by weight) or less) at a temperature of 6 to 26%. Leave for 0.5 to 72 hours at a temperature of 8 to 23 degrees Celsius, preferably 1 to 50 hours at a temperature of 8 to 23 degrees Celsius. The dirt adhering to the body surface of the live earthworms was washed off, and then wet pulverization was performed to obtain an earthworm suspension.

Manufacturing method 2: After removing the dirt adhering to the body surface of the live earthworm or washing it off with water, add it to fresh water or a non-toxic acid such as acetic acid, citric acid, malonic acid, succinic acid, sulphate, malic acid, tartaric acid, lactic acid, etc. Contains up to 2% by weight, preferably up to 1% (by weight) of at least one type of chemical organic acid or non-toxic organic acid such as phosphoric acid, sulfuric acid, or hydrochloric acid, or the sodium or potassium salt of these acids. pH 3-6.5 at low concentrations of
in a slightly acidic aqueous solution at a temperature of 6 to 26°C.
After leaving it for 2 hours, preferably at a temperature of 8 to 23°C for 1 to 50 hours, the excretory power of the live earthworms allows the earthworms to fully excrete the feces in their digestive tracts, and then performs wet crushing to obtain an earthworm suspension. Ta.

Production method 3: The earthworm suspension obtained by the above production method 1 or 2 was heated at 15°C.
After freezing at a temperature of C or lower, preferably at a low temperature of -10 to -60°C, freeze drying and vacuum drying were performed. The conditions for freeze drying/vacuum drying are a temperature of -60 to 80°C, a degree of vacuum of 100 μHg or less, preferably a temperature of -40 to +80°C and a temperature of 10
■5 to 1 while increasing the temperature step by step at a vacuum level below Hg.
Freeze drying and vacuum drying were performed for 00 hours to obtain a sterile dry powder of earthworms.

Production method 4: After degassing the earthworm suspension obtained by the above production method 1 or 2 under reduced pressure, it was dried at a temperature of -6 using a vacuum rotary drying method.
0~+80℃, degree of vacuum 10mHg or less, heating time 5~5
Vacuum rotary drying was performed for 0 hours to obtain a sterile dry powder of earthworms.

The dried earthworm powders obtained in the above-mentioned production methods 3 and 4 are in the form of lumps or powders containing mixed solids, but these can be easily turned into fine powders by using a dry pulverizer.

When used, powders are preferred over lumps or solids.

In order to solve the above problems, the present inventors considered the ecology and morphology of earthworms.

The outside of the earthworm is a cylindrical body wall, which is made up of connected annular segments (body parts) of exudates. The boundary between the body parts is a slightly concave groove (interbody groove).

If you look at this schematic diagram of the earthworm's body cut into rings, you can see that the earthworm has a double tube. The body wall is quite thick and divided into three layers. The outermost layer is the skin, the innermost layer is a ring-shaped muscle layer (i.e., the circular muscle layer), and the innermost layer is a vertically running muscle layer (i.e., the longitudinal muscle layer). What is the body wall tube and the digestive tract? They are separated, and the spaces between them are filled with coelomic fluid, a yellow or milky mucus. Earthworms have anterior pores in fixed locations on their body walls that open into their body cavities. When an earthworm is teased, it easily releases coelomic fluid from its anterior hole.

According to detailed research conducted by the present inventors, it has been found that the longitudinal muscle layer, coelomic fluid, blood, and gastrointestinal tract of earthworms contain more medicinally important physiologically active components than the skin and circular muscle layer. It was also found that inhibitors and hemolytic substances exist in the skin and the circular muscle layer. Although the details of effective physiologically active substance components are unknown, they include proteins, glycoproteins, metal proteins, low molecular weight proteolytic enzymes that can be absorbed from the intestinal tract, nucleic acids and nucleic acid machine substances, various carbohydrates, various lipids, and It was thought that this was caused by one of the substances of unknown composition or the combined effect of these substances.

One of the objectives of the manufacturing method of the present invention is to obtain all the components of the earthworm's skin, circular muscle layer, longitudinal muscle layer, body cavity fluid, digestive tract, blood, and digestive juices as a dry powder without losing as much as possible. . At the same time, in order to suppress or stop the effects of inhibitors and hemolysis present in the skin and circular muscle layer,
It was found that the finishing conditions of the final vacuum drying, that is, the heating conditions of the three elements of vacuum degree, temperature, and time, are important operations.

In other words, in order to obtain dried earthworm powder that is safe, sterile, odorless, and has excellent medicinal properties, it is necessary to use a purification method or cleaning method for the raw material live earthworms (collected or cultured live earthworms), freeze-drying, and drying.
It has been found that a method in combination with vacuum drying conditions or vacuum rotary drying conditions is a particularly important operation.

According to actual measurements by the present inventors, the filth that adheres to the body surface of raw material live earthworms, such as collected or cultivated earthworms, is usually
It is 10 to 40 (wt)% based on wet earthworms, and the fecal soil in the digestive tract contains 5 to 15 (wt)% based on wet earthworms. Drying with these filth on the body surface and excrement in the digestive tract remaining may cause deterioration of the dried earthworm material or dry powder during storage, or there is a risk of decreasing medicinal efficacy or causing chemical damage. Therefore, using clean earthworms from which these filth and excreta have been promptly removed is useful for stabilizing the quality of earthworm dry powder products, preventing deterioration, and preventing chemical damage, as well as improving medicinal efficacy, preservability, and yield. To,
It was discovered that this is an important prerequisite. In addition, when external action (for example, pressure) is applied to remove the excrement remaining in the earthworm agitation tube, as in the well-known I and stone production methods, only the excrement is selectively removed. I can't. When you tease an earthworm a little, it immediately releases coelomic fluid from its anterior pore, which shows that no matter how careful you are, the earthworm's digestive tract, which contains many components that play an important role in medicinal efficacy, loses its digestive tract, digestive juices, blood, and proteins. If a large amount of body fluid such as body cavity fluid is removed from the body together with feces, the yield and medicinal efficacy will be insufficient. Furthermore, when live earthworms are placed in plant ash or fire ash and dried as in step ii, digestive juices, blood, body cavity fluids, etc. are discharged from the excretory opening and the anterior pore, resulting in insufficient medicinal efficacy and reduced yield.

According to the research of Ta, it has been reported that earthworms are animals that are very sensitive to temperature and light. For example, Eiji Yamaguchi (The story of earthworms, p. 82, published by Hokuryukan in July 1982) reported that the optimum temperature for earthworms to live in soil is 6 to 23°C.

The present inventors used three types of earthworms, red earthworms, striped earthworms, and common earthworms, and clean groundwater to measure the optimum temperature for earthworm activity in groundwater. As a result, the appropriate temperature is 7 to 23
°C, and the next most preferred suitable temperature was 8 to 22 °C1, and a particularly preferred suitable temperature was 15 to 20 °C. 30℃～35℃
Then, just by stretching the whole body thinly, the activity weakens rapidly, 37
°C is the limit of resistance, and at 40 degrees it will die soon. 0~
At 5°C, earthworms become less active and almost cease to be active.

At temperatures below 0°C, they will not die temporarily unless frozen, but they will die within 8 to 10 days.

Blue light and ultraviolet light harm the tissues of earthworms, and earthworms are most active in the dark or at night. Unless the habits of these earthworms are fully utilized, it cannot be said that sufficient conditions have been achieved for removing excrement from the earthworm's digestive tract.

For example, earthworms are grown in clean underground water, tap water, pure water with a pH of 3 to 6.5 at a low temperature below 5°C or a high temperature above 30°C.
48-7 in the dark in the presence of a slightly acidic aqueous solution or air.
Even if left for 2 hours, not only is little excretion of the feces in the digestive tract, but the longer the time is left, the more the body weight decreases. That is, earthworms cease their activity in water, soil, or air at low temperatures of 5° C. or lower or 30° C. or higher. Therefore, the description of only the standing time as a condition for excreting the excrement from the earthworm's digestive tract is incomplete and unreliable. In other words, it cannot be accepted as a technical disclosure of a completed invention that can be easily implemented by a person skilled in the art.

The inventors conducted the following experiment. Red earthworm (Lumbricus rubellus) + striped earthworm (II!1senia foetida), common earthworm (Phereti*acommunissi) for each standing time
10 each of 3 types of grown live earthworms with their body surfaces cleaned
0 g and 500 d of clean groundwater (pH 7, 2) were placed in a plastic container, and placed in a dark place in a constant temperature bath at 15°C and 8°C with a lid that allows air to pass through, and earthworms digested the water after each predetermined time. The excretion rate (wt%) of manure in the pipe was measured. The results were as shown in Table 1.

Below is a margin table-1.In other words, as you can see from table-1, the amount of excrement in the digestive tract is 100
When left in fresh water (groundwater) at 15°C, there is no difference between red earthworms, common earthworms, and striped earthworms (for 36 hours).
When it was left in fresh water at ℃, it was pawned with red earthworms for about 44 hours. In other words, it was found that there were differences in the excretion time of manure due to differences in water temperature.

In addition, after removing 100% of the manure, the earthworms are removed from the water.
The yield (wt%) of salt earthworms after draining is 93% of the raw material for both red earthworm groups A and B, 93% for common earthworms and striped earthworms, and 92-92% for red earthworm groups C and D.
．． 5%, and the longer the time spent in water, the smaller the weight loss was observed.

To this, for example, by adding malic acid alone or a 1:1 mixture of malic acid and citric acid, t)l! I! lt,,ta9
Adding 100 g of each red earthworm with a clean body surface to 500 ad of a slightly acidic aqueous solution of 116.0 and conducting an examination at 15°C under the same conditions as above, 100% of the feces in the digestive tract was excreted in 2 hours and 30 minutes. Moreover, the yield (wt%) of salt earthworms after draining was 95% of the raw earthworms. As shown in the process descriptions and examples of Production Methods 1 and 2 above, when leaving live earthworms in a dilute aqueous solution, it is important for medicinal efficacy that the feces in the digestive tract be completely expelled within the shortest possible time. When the dilute aqueous solution is a concentrated aqueous solution, the earthworms that have been left unattended will sew the dorsum of the earthworm's body (Seg+ment) before excreting the excrement.
) They release yellow or milky white coelonic fluid from the dorsal pore and die rapidly. There is a reason to limit it to slightly acidic.

If earthworms are left in a dilute aqueous solution or fresh water for a long time, for example, for 3 days (72 hrs), they will excrete a large amount of body fluids such as digestive juices and body cavity fluids in addition to fecal soil, and their weight will decrease. decreases in inverse proportion to the length of time it is left in water. Dry powders made from earthworms and their earthworm extracts lack medicinal efficacy. Therefore, as shown in the examples of the present invention, short-term exposure to It is preferable to use earthworms that have sufficiently (completely) expelled the excrement from their digestive tracts.Furthermore, the dirt adhering to the earthworm's body surface can be relatively easily removed by washing with clean water; It is preferable to use cooling water at a temperature of 5° C. or lower because earthworms cease to be active under such low-temperature water and are extremely easy to wash.

The wet grinding method of earthworms, that is, the method of destroying earthworm tissues (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.

Previously known drying methods each had the following problems:9 For example, after freezing at -30 to -40°C for 30 to 40 hours,
The method of freeze-drying at the same temperature for 30 to 40 hours is suitable for products produced in small quantities and used within a short period of time.

However, the products produced by this method have poor shelf life and often develop mold within six months to one year, making them unsuitable for industrial mass production. The biggest problem with this method is that the inhibitors (composition unknown, but we believe they are proteins or low molecular weight compounds) present in the earthworm's skin and ring muscles are not decomposed and remain as they are. However, there are drawbacks to suppressing the drug's efficacy. In addition, the degree of vacuum is 0 as the vacuum drying condition for earthworms.
It was found that heating the dried earthworm powder at 33 Torr for 20 hours or more at 80°C improves the shelf life of the dried earthworm powder, but the effective physiologically active ingredients are thermally decomposed and the longer the heating time is, the lower the medicinal efficacy and yield are. In addition, the drying method in which living or raw earthworms in a water-containing state are rapidly heated to 80 to 90°C under normal pressure immediately after washing may cause the effective physiologically active ingredients to hydrolyze or volatilize as in steam distillation. It was found that the efficacy and yield decreased with increasing heating time. Sunawa Chi, 7
When heating to 0 to 80°C or 80 to 90°C, the above dangers can be prevented unless the water in the solid, paste, or powder of earthworms is dehydrated as much as possible before heating under vacuum (10 mHg or less). Can not do it. In order to solve these drawbacks and problems, the present inventors established the freeze drying/vacuum drying method of Production Method 3 or the vacuum rotary drying method of Production Method 4.

Next, a preferred specific example of the freeze-drying/vacuum-drying operation of manufacturing method 3 of the suspension obtained by wet grinding of earthworms obtained by the above method is as follows.

Wet-ground earthworms, i.e., earthworm suspensions, at -1
0~-60″C1 Preferably 5~-30~-50°C
After freezing for 60 hours, 0.01-0.2 m at the same temperature
Freeze dry for 5-12 hours under ug vacuum.

Next, at 20-30℃ for 5-15 hours, 0.01-0.2
Dry under vacuum at 35-50 °C, 0.
Dry under vacuum at 1-0.5 μg for 10-20 hours.

Vacuum drying in the next final finishing step is 0.01~5IIIl
Under vacuum of 11Hg, 70-80°C, preferably 75-80°C
By vacuum drying at 80°C for 0.1 to 19 hours, it was possible to obtain a dry earthworm powder that was sterile and had excellent medicinal effects.

Next, a preferred specific example of the vacuum rotary drying operation of Production Method 4 is as follows. That is, the earthworm suspension is left standing or under weak stirring at a temperature of 0 to 30°C and 10 to 30 mH.
The degassed squid earthworm suspension was degassed under reduced pressure of H for a treatment time of 5 hours or less at a temperature of 0 to 30°C using a vacuum rotary drying method.
Degassed under vacuum of 10-30+ mHg for 0.5-5 hours.

The paste of the degassed earthworm suspension was prepared from −10 to −
After vacuum drying at 50 °C under a vacuum of 1-5 mHg for 1-10 hours with stirring, then drying at 40-50 °C for 0.
Dry for 2-15 hours under vacuum at 0.01-5 mHH, then dry at 70-80 °C under vacuum at 0.01-5μ) Ig.
After vacuum drying for 1 to 19 hours, it was possible to obtain a dry earthworm powder that was sterile and had excellent medicinal effects.

The combination of the three elements of the vacuum degree, temperature, and time in Production Methods 3 and 4 is an important condition. In particular, the vacuum drying conditions of the final finishing process, i.e. 0.01 to 5 mm) Ig vacuum degree, 7
The three combinations of temperature from 0 to 80°C and from 001 to 19 hours are important operating conditions.

The effective physiologically active ingredients that are abundantly contained in the longitudinal muscle layer, body cavity fluid, blood, and digestive tract of earthworms described above do not change in quality or lose their activity under these finishing conditions. On the other hand, inhibitors and hemolytic substances that are abundantly contained in the skin and ring layer of earthworms lose their activity due to alteration or decomposition.

As a result, it was possible to obtain dry earthworm powder, which is the object of the present invention, with excellent sterility, safety, preservability, and medicinal efficacy, at a concentration of 20 to 30 wt% based on the raw material, live earthworms.

The major technical difference is that in manufacturing method 3, the dry material, earthworm suspension or earthworm powder, is dried in a stationary state, and in manufacturing method 4, the material, earthworm suspension or earthworm powder, is dried in a mechanically agitated state. be. Another difference is that the drying time for Production Method 3 is usually 2 to 5 days, whereas the drying time for Production Method 4 is 20 to 40 hours. In manufacturing method 4, the earthworm suspension paste is
If you suddenly spin-dry the earthworm suspension under a vacuum of 0.01-5 mmHg without degassing at ~30℃ under a reduced pressure of 10-30 mHg, the earthworm suspension will scatter and there is a great risk of loss, so degassing is an essential requirement. There is one. Preferred types of vacuum rotary dryers used in the present invention are double cone type vacuum dryers, tumbler type vacuum dryers, and rotating drum type vacuum dryers.

When the dried earthworm powder obtained by the production method of the present invention was stored at a room temperature of 5 to 45° C. in a sealed state for 5 years, no mold formation or other physical or chemical deterioration was observed. From this, it was easily observed that the dried earthworm powder obtained by the production method of the present invention contained a bactericidal substance.

By the above procedure, it was possible to obtain tan 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 0 to 15%.
Preferably 7-14%, ash content 3-8%, preferably 4
The nitrogen content was adjusted to 7% and 1 to 11%, preferably 6 to 11%. In addition, the earthworm dry powder contains 18 types of aspartic acid, threonine, saison, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, lysine, histidine, and arginine. Contains the amino acids before and after.

Test Example 1 Table 2 shows the rough analysis results of the earthworms and dry powder obtained by the combination method of the above-mentioned production methods 1, 2.3 and 4 and the production method of the example below.

Table 2 Test Example 2 W-8 and W-9 obtained by methods 1 and 3, W-11 obtained by methods 2 and 3, W-1 and W obtained by methods 1 and 4
Table 3 shows the results of component analysis of the dried earthworm powder products W-4 and W-6 obtained by the methods 2 and 4.

From Tables 3 and 4 below, it can be seen that the dried earthworm powder contains abundant crude protein, crude lipids, and various metals, and the amino acid composition of the crude protein contains a large amount of essential amino acids. It was found that it contained

The earthworms used in the present invention are red earthworms (Lumb.
ricus rubellus), LT earthworm (also known as Lumbricus terrestri)
s) ) Eisenia foetida
), Alobophos annuus (AI-1alobopho
ra caliginosa), purple earthworm (Dendrobaena octaedra), cherry earthworm (AIlolo-bophora japon)
tca Michaelsen), Hunter's earthworm (Dr.
awida hattamimizu tlatai)
, Pheretima diverge
ns Michelsen), common earthworm (Phe
retia+a coau+unissima), Pheretin+a agrestis
, Siebold earthworm (Pheretima 5iebo)
ldi Horst), Phere
tima htlgendorfi), Isoearth earthworm (P
ontodrilus matsushi+l1ens
is l1zuka), Itomizu (Tubifex
hattai No5ura), Gotouito earthworm (
Limnodrilus go
toiHatai = L, 5ocalis 5te
Any normally growing earthworm such as Phenson can be used.

When the earthworm dry powder of the drug of the present invention is administered for clinical treatment, it may be administered in either an oral or parenteral form, but oral administration is particularly preferred. The oral dosage layer of the present invention product may be the present invention itself or mixed with an appropriate pharmacologically acceptable pharmaceutical carrier to form capsules, tablets, granules, powders, coatings, sugar-coated tablets, and emulsions. 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, acacia, glucose, sucrose, sorbitol, and 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 such as 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 and Uitebsol (W) are used as bases for suppositories.
itepsol), Subanal,
polyethylene glycol, polypropylene glycol,
Glycerogelatin, gelatin capsules, etc. are used. In addition, known safe preservatives such as methylvara hydroxybenzoate, propylvara hydroxybenzoate, butylvara hydroxybenzoate, butylhydroxyanisole, and other safe dyes are blended and used.

The dosage of dried worm powder as a thrombolytic agent of the present invention varies depending on the administration method, patient's age, weight, condition, and type of disease, but is usually 0.001 per day for humans.
About 5 g is preferable. Most preferably, 0.002 g to 3 g per day is administered in 1 to 3 divided doses.

Effect: The toxicity and thrombolytic agent pharmacological test methods and results of the earthworm dry powder of the present invention will be described in detail below.

A. Acute toxicity test of earthworm dry powder: ciay male mice weighing 30±28 and mice weighing 100±
2g Wistar male rats each - Group 5
An acute toxicity test using oral administration was conducted using rats. Earthworm dry powder product W-1 (moisture 10.1%, ash 4.7
%, nitrogen content 9.3%) FW-2 (moisture content 10.2% ash content 4.8%, nitrogen content 9.4% content); W-4 (moisture content (io,
2%, natural content 4.8%, nitrogen 9.3%); W-6 (moisture 10.8%, ash 4.7%, nitrogen 8.8%);
W-8 (moisture 10.2%, ash 5°0%, nitrogen 9.2%
Contains); W-9 (moisture 10.6%, ash 4.8%.

Contains 9.1% nitrogen): W-10 (contains 10.7% moisture, 4.7% ash, and 8.6% nitrogen); W-11 (contains 10% moisture).
6%, ash 4.8%, nitrogen 9.0%) were increased from 0.1 g/kg to 8 g/kg to the mice described above (0.1 to 5 g/kg). and rats (
(2 to 8 g/kg) were administered individually by gavage with a throat probe. During the test period, animals were kept at an animal room temperature of 22 to
The temperature was maintained at 23°C and observed for 14 days after administration. 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 revealed no comparable macroscopic damage to any part of the major organs. Therefore, it was not possible to determine the LD50 value of the earthworm dry powder due to its very low toxicity.

B. Measuring method of thrombolytic activity, total cholesterol and total trigcerite amount: Fibrinolytic activity extracted from dry earthworm powder with physiological saline is mainly measured by Astrup and Muller.
tz(T, Astrup and S, 11ertz
:Arch, Biochem+Bi.

phys, Vol. 40', pp. 346-351 (1952
It was measured using the standard fibrin plate method according to the method of 2010).

In addition, plasminogen-free plates are created using fibrinogen from which plasminogen has been removed by lysine-sepharoazine affinity chromatography (
M, Matsuda etal: Thrombosis
Res, vol. 1, pp. 619-630 (1972)
〕did. The fibrinolytic activity in the blood when the dry earthworm powder of the present invention is administered to humans can be determined using the fibrin plate method and the Euroguprine dissolution time method (J, H, Lewis and J, II
.

Ferguson: J, Cl1n, Inves.
t, Vol. 29, p. 486 (1950)).

Fibrin decomposition products in plasma (FibrinDe
Gradation Products (FDP)
) is a separate HDP (D-Di) ELI kit (Stago
(France)]. In addition, tissue plasminogen activator (tPA) in plasma is
It was measured using a y kit [manufactured by Biopoo 1].

Total cholesterol (TC, ■/d2) and total triglyceride (TG, ■/dl) in serum were measured by the following method. Serum was obtained by cooling centrifugation of the collected blood. TC and TG in serum were Toshiba TBA-480 automatic analyzer and each TA commercially available from Wako Pure Chemical Industries, Ltd.
Quantification was performed using 480Test reagent.

f1 recognition of fibrinolytic active substances in C earthworm dry powder: Product W-1゜-2, -4, - of earthworm dry powder obtained by the above manufacturing method
5, -6, -7, -8, -9, -10, -11 were added with 10 times the amount of physiological saline and stirred and extracted at 30°C for 2 hours. As shown, it was observed that fibrinolytic active substances were dissolved in the supernatant.

Margin table below-5 The components of the saline-soluble substances in this supernatant are unknown, but
The present inventor believes that it consists of proteins, glycoproteins, metalloproteins, $1 prizes, and other low-molecular physiologically active molecules. Also, this water-soluble substance is Plasminoken. Free fibrin plates and standard fibrin plates are also dissolved, and standard fibrin plates contain more braminogen. Compared to a free plate, the dissolution window is larger, directly decomposing fibrin and plasminogen. Activator activity was also demonstrated.

D Hemolytic action: Earthworm dry powder products (W-1, W-2, W-4, W-
6, W-7, W-8, W-9, W-10 and W-11)
Add 5 parts (by weight) of physiological saline to 1 part (by weight) of dry earthworm powder of each of the 9 types of specimens, suspend well, and heat at 5°C.
After leaving it in the refrigerator for 24 hours, it is filtered under reduced pressure and the filtrate is used as a water extract.

The ethyl alcohol or acetone extract component was added to 1 part (heavy ff1) of dry earthworm powder for each of the nine specimens mentioned above, and 5 parts (
Weight) of ethyl alcohol or acetone was added and the mixture was well suspended. After being left at 15-20°C for 24 hours, it was filtered under reduced pressure, and the filtrate was dried under reduced pressure at 40-45°C. A 30% solution of this was prepared in physiological saline to which 1% carboxymethyl cellulose was added. The hemolytic effect test was conducted by the following method. Blood was collected from the ear vein of an adult rabbit weighing approximately 3 kg, and defibrillated blood was obtained by defibrillation treatment.

Each portion of the aqueous extract, ethyl alcohol and acetone extract was diluted with physiological saline for 5.5, 1.0.1.0.01.0.
001.0.0001 (weight)%, and these were diluted to 5
One drop of defibrinated blood was added into the small test tube of d using a pipette, and after 1 hour, the hemolysis results were determined. As shown in Table 6, the test results showed that in all the samples of dried earthworm powder, hemolysis occurred in all cases.In the previous literature, dry earthworm powder (Earthworm) was used as earthworm poison to cause destruction of red blood cells. In other words, it has been reported that it has a hemolytic effect [Worms and Life = written by Mayu Obuchi: October 30, 1947, published by Maki Shobo, Vol.
Pages 23-226 and reprinted version: Written by Shinkiji Hatai, 198
Published by Scientist Co., Ltd. on April 30, 2016; pages 160-163], and it was also reported that water extract and ethyl alcohol extract of dried earthworm (Jiryu) have partial hemolytic effect [Iyyo]. Kenjibu; Yamaro Medical Volume 9, No. 571
576 (1960)], but it has been found that the dried earthworm powder produced by the method of the present invention does not have such hemolytic action.

E. Therapeutic effects on thrombolysis, venous thrombosis, hypertension, hypotension, and hyperlipidemia patients when dry earthworm powder was orally administered to humans: This study was conducted by a doctor at a hospital. W obtained by the above manufacturing method
Capsule A containing 150 fiIg each of -2, -3, -5, -8 and -13 earthworm dry powders.

One of drugs C, D, E and F was used for hypertension and hypotension.

9 patients with hyperlipidemia and venous thrombosis (including 4 males and 5 females)
The drug was administered orally three times daily after each meal to a total of 10 people, including one human volunteer and a normal healthy male volunteer (study number 1).

Four patients with study numbers 2, 3, 9, and 10 had monocomplications of hypertension and hyperlipidemia, or patients with these symptoms alone. Test Nos. 4.5 and 6 were for patients with severe venous thrombosis, and the anticoagulant Mar(arin potassium) was used in combination. Test No. Nα7 and 8 were for patients with hypotension and hyperlipidemia.

Before taking Earthworm Dry Explosive Powder Capsules (Day 0), Dosage 1.2
, 3, 4, 11, 13, 15, 18 or 25 days later, FDP and tpA were measured.

Table 7 shows the results of measuring the maximum blood pressure (systolic blood pressure), minimum blood pressure (diastolic blood pressure), and serum TC and TG of each patient and volunteer before taking the drug and 25 days after taking the drug.

The above-mentioned anticoagulant Marfarinpotassiu
Although m inhibits fibrin formation, it does not have the effect of dissolving the fibrin that has already been formed.In order to impart and promote this fibrin dissolving activity, dried earthworm powder was used in combination, and its therapeutic effect was determined.

Margin Table 7 Below: The thrombolytic activity and therapeutic effects of the earthworm dry powder capsules shown in Table 7 can be classified into the following three groups when judged from the behavior of FDP and tpA values in plasma.

The first group was test number Nα3,7. and 8 patients with a combination of hypertension or hypotension and hyperlipidemia. This patient's fibrin-dissolving ability before taking dry earthworm powder (day 0) was FDP 54-60 ng/d (same value as that of a normal healthy person, see Test No. 1).
On the 2.3rd or 4th day after taking the drug, FDP temporarily increases to about 4 times, but if the drug is continued after that, on the 18th or 25th day, it drops to the value before taking the drug. This indicates that the fibrin (thrombus), which had not dissolved and was deposited in the blood vessels before taking the drug, was dissolved by the action of the physiologically active ingredients in the dried earthworm powder, and the inside of the blood vessels returned to a normal state. .

At the same time, the maximum and diastolic blood pressures of the hypertensive patient in Test No. 11h3 decreased by 15-18 ng to normal values 25 days after taking the drug. In the case of patients with hypotension in test numbers 7 and 8, their maximum and diastolic blood pressures were 16 to 8.
The temperature increased by 20 mmHg and became normal.

Furthermore, in the case of this study number 3.7 and 803 patients, serum TC was 34-46 and TG was 30-3911g/
di decreased to normal values.

The second group includes patients with test numbers 4.5°, 2.9, and 10 other than grade 6. Patients with Nα4 have severe venous thrombosis alone. Patients with Na5 had severe venous thrombosis and hyperlipidemia; patients with Na2, 9, and 10 had complications of thrombosis, hypertension, and hyperlipidemia. That is, before taking the earthworm dry powder, FDP was already 140 to 230 ng/-, but on the first day of taking it, FDP increased and fell by about 5 to 20%, and then
On the 3rd or 4th day of taking the drug, it decreases again to about 13%.
After increasing by ~40%, the FDP gradually decreased to show the same value as a normal healthy person on the 18th or 25th day. This means that while the effective physiologically active ingredients of the ingested dry earthworm powder are absorbed and fibrin dissolves, the blood coagulation ability declines, the fibrinolytic system significantly declines, and metabolic disorders of the blood vessel walls occur.
This indicates that the symptoms that caused thrombosis were improved. 3 patients with hypertension and hyperlipidemia in this second group
A person's systolic blood pressure is 16-17m, and diastolic blood pressure is 15-22m.
Hg decreased to normal values. In addition, this second group of patients with hyperlipidemia complications had a TC of 30 to 50 degrees and a TG of 3.
The values decreased from 7 to 40 .mu./df to normal values.

The third group is the case of patients with complications of venous thrombosis and hyperlipidemia in Study No. 6. This patient's FDP before taking the drug
170ng/#! i! (Direct) increased by 50% on the first day, then decreased, then increased and then gradually decreased.
This patient had TC20°TG15■/dl 25 days after taking the drug.
Although both levels decreased, they were still one step away from normal values.

Furthermore, by continuing to take the drug, it is expected that the complications of venous thrombosis and hyperlipidemia will be completely cured.

Along with FDP, tpA in the blood was measured.

The results are shown in Table 7, and tpA could be continuously increased by oral administration of earthworm dry powder. In particular, a marked increase was observed on or after the 3rd to 4th day of administration. Tsune Mihara et al. [Journal of the Japanese Society of Hematology Vol. 48, Section 514 (1)
[985] found that when normal, healthy male adults took the dried earthworm powder prepared by the freeze-drying method that they had produced, blood FDP decreased by 1 after administration, prior to the increase in tpA.
It is reported that there was a marked increase on the second day. That is, Mihara et al. reported that an increase in tpA occurs after an increase in FDP. However, when taking the dried earthworm powder produced by the manufacturing method of the present invention, as shown in Table 7,
A marked increase in FDP coincided with the maximum value of tpA on or after the 3rd to 4th day of dosing. This means that the manufacturing method of the dried earthworm powder of the present invention is different from the manufacturing method of Mihara et al., and as a result, the type and amount of the active ingredient and inhibitor are different, and as a result, the mechanism of action and effect are different. It is something to prove. That is, Mihara et al. [Environmental Science Research Report Collection B504-R30 Environmental Improvement Technology Research Report Volume 4 Part 10]
pp. 7-112 (1986); Japanese Journal of Hematology Vol. 48, 5
14 (1985)) focused on the fibrinolytic enzyme (protease) possessed by earthworms with a molecular weight of 20,000 to 30,000, tried to apply it as a thrombolytic agent, and thought that earthworms could be used as a thrombolytic agent. .

However, as in the present invention, by orally administering dried earthworm powder to humans, thrombosis, venous thrombosis, hypertension,
The complete recovery of patients with hypotension or hyperlipidemia is an excellent result obtained for the first time by the present invention.

The thrombolytic effect of oral administration of the dried earthworm powder of the present invention may be due to the effect of the physiologically active components of the dried earthworm powder absorbed into the intestine directly dissolving fibrin in blood vessels, or to the activation of plasminogen. Either by acting as an activator that converts into plasmin, or by indirectly activating physiological fibrinolytic ability in the body, plasminogen is activated to plasmin and deposited in blood vessels (
The effect of dried earthworm powder is unclear, whether it dissolves and removes fibrin (or deposited fibrin) or whether it is due to the combined action of two or three of these. However, the results shown in Table 7 indicate that oral administration of the dried earthworm powder produced by the production method of the present invention can improve the effects of venous thrombosis, hypertension, hypotension, or hyperlipidemia alone or in patients with these complications. It has been proven that it has an excellent therapeutic effect on patients. In this clinical trial, there were no side effects due to the administration of dry earthworm powder.

Example 1 1.5 g of potassium dihydrogen phosphate was dissolved in 2.52 g of an acidic aqueous solution with a pH of 5.8 in which citric acid was dissolved, and 1 kg of live earthworms (red earthworms) that had been lightly washed with water were added to the solution.
The pellets were left at °C for 2.5 hours to allow excretion of feces in the digestive tract. Next, the live earthworms are washed 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. The obtained earthworm suspension was inserted into a vacuum rotary dryer, and then degassed for 1 hour under a reduced pressure of 5°Cl2OmmHg. The dryer was continuously agitated and dried at -30°C under a vacuum of 0.2 mmHg for 5 hours. Next, it was dried for 10 hours under a vacuum of 0.2 aallH at 40-45°C, and then dried at 78-80°C for 0.2
By drying under vacuum of mmHg for 2 hours, 280 g of earthworm dry powder product (W-1) was obtained.

Example 2 One ton of live earthworms (red earthworms) lightly washed with water was added to a 3M acidic aqueous solution with a pH of 6.0 in which malic acid was dissolved, and left at 18°C for 2 hours to excrete the feces in the digestive tract.
Next, the live earthworms are washed with water at 5°C to remove dirt adhering to the body surface of the live earthworms, and then wet-pulverized using a homogenizer. While stirring the obtained earthworm suspension,
After degassing for 2 hours at a temperature of 20 mmHg, the sample was inserted into a vacuum rotary dryer at the first gutter. After operating this dryer and drying at -35℃ under a vacuum of 0.2 mHg for 4 hours, it was inserted into the vacuum rotary dryer at the second hole and dried at 45~35℃.
0.1 at 50°C. Dry under vacuum of mmHg for 15 hours. Next, insert it into the vacuum rotary dryer at the third gutter and heat it to 80°C.
By drying for 2 hours under a vacuum of 0.2 mmHg, 283 kg of earthworm dry powder product (W-2) was obtained.

Example 3 Dirt, feces, straw, and other impurities adhering to the body surface of 1 kg of live earthworms (Tsurimimizu) were washed off with water.

Next, the live earthworms were placed in an acidic aqueous solution 32 with a pH of 5.7 containing a 1:1 mixture of malic acid and succinic acid (in which 1 g of sodium acetate and 0.5 g of sodium sulfate were dissolved) at 16°C. After being left for 2.5 hours to excrete the fecal soil in the digestive tract, the body surface of the live earthworms is washed with water to remove the filth, and then wet-pulverized using a homogenizer. The obtained earthworm suspension was placed in a vacuum rotary dryer, and degassed for 1 hour under reduced pressure at 15°C for 25 seconds while stirring. Next, while running this dryer continuously, heat the dryer to -30℃.
After drying for 4 hours under a vacuum of 0.2 mHg,
Dry at 45°C under vacuum for 18 hours with 0.2 mm and 1 g;
Then at 78-80 °C for 0.2 ms +) under a vacuum of Ig for 2
Earthworm dry powder product (W-3) by drying for hours
I got 280.

Example 4 After thoroughly washing mud, feces, straw, and other dirt adhering to the body surface of 1 kg of a live earthworm (red earthworm) with water, the live earthworm was treated with a mixed acid of 1:1 of citric acid and phosphoric acid. 2 in a slightly acidic aqueous solution of 2.51 dissolved pus,o
The pellets were left at 0°C for 15 hours to excrete the feces in the digestive tract.

Next, the dirt adhering to the surface of the live earthworm's body is washed off with water, and then it is wet-pulverized using a blender. The ground earthworm suspension is degassed at 8° C. under reduced pressure to 20 ml and then inserted into a rotary vacuum dryer. This dryer was connected and dried for 3 hours under a vacuum of 0.3 mnHg at -30°C, and then dried under a vacuum of 0.2 + mnHg at 43-45°C for 15 hours.
270 g of earthworm dry powder product (W-4) was obtained by drying for 1.5 hours at 75-80 degrees under a vacuum of 0.2 mn11 g for 1.5 hours.

Example 5 After washing dirt such as mud, feces, and straw adhering to the body surface of 1 kg of live earthworms (Phutomimizu) with water,
Next, the live earthworms were placed in a slightly acidic aqueous solution with a pH of 5.9 (pH 5.9) containing 1:1 mixed acid of malic acid and tartaric acid (1:1).
After being left at ℃ for 2 hours to excrete the feces in the digestive tract, the live earthworms are washed with water to remove the filth adhering to the body surface, and then wet-pulverized using a homogenizer. The obtained earthworm suspension was put into a vacuum rotary dryer and dried at 10'C, 20mm.
Degassed under reduced pressure of Hg for 2 hours. While this dryer was operated in a connected manner, it was dried at -35"C under a vacuum of 0.2 M Hg for 3 hours, and then at 40-45 °C under a vacuum of 0.2 M Hg.
Dry under Hg vacuum for 13 hours, then at 75-80 °C
By drying for 3 hours under a vacuum of 0.2 ug, 265 g of dried earthworm powder (W-5) was obtained.

Example 6 After thoroughly washing dirt adhering to the body surface of a live earthworm (red earthworm) Ikg with water, the live earthworm was left in groundwater 31 at 13°C for 40 hours to remove the live earthworm from its digestive tract. The excrement inside was excreted. Next, the dirt adhering to the body surface of the live earthworms was washed off with water, and then wet-pulverized using a homogenizer. The obtained earthworm suspension was degassed at 10° C. under a reduced pressure of 122 mt1 g while stirring, and then inserted into a rotary vacuum dryer. This dryer was operated continuously and dried at -28°C1Q under a vacuum of 3 mtlg for 5 hours, then at 40-45°C under a vacuum of 0.2 owe) Ig for 12 hours, then at 75-45°C. Q, 1m at 80°C
238 g of dried earthworm powder (W-6) was obtained by drying under a vacuum of n+l1 g for 5 hours.

Example 7 I kg of live earthworms (red earthworms) were left in groundwater at 15°C for 40 hours to excrete fecal soil in the digestive tract. Next, the live earthworms are washed with water to remove dirt adhering to the body surface of the live earthworms. Next, it is wet-pulverized using a mixer. The paste of the obtained earthworm suspension was put into a vacuum rotary dryer, and then degassed at 10'C under a reduced pressure of 20 g II g for one hour. Continue to operate this dryer until it reaches zero temperature at -35°C.
．． 2 ml was dried under vacuum for 4 hours. Next 40-4
Dry at 5'C under 0.2 mmHg vacuum for 12 hours, then dry at 75-78 °C under 0.2 mmHg vacuum for 3 hours.
Earthworm dry powder product (W-7) by drying for hours
255g was obtained.

Example: 1 kg of live earthworms (red earthworms) lightly washed with water (
Approximately 2,150 fish were placed in an acidic aqueous solution 41 at pH 6.2 containing a 1:1 mixed acid of malic acid and citric acid at a temperature of 18.
After leaving at °C for 2 hours to fully excrete the feces in the digestive tract, the live earthworms were thoroughly washed with water to remove the mud that was attached to the body surface of the live earthworms.

Wash away dirt such as feces.

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 for 6 hours under vacuum at -40°C in a volume of 0.1ml and 11g, and then placed on a tray. The shelf temperature is 30°C.
After vacuum drying for 6 hours under a vacuum of Q, l mm 11 g, the shelf temperature was raised to 50°C and 0.2 m tl
The earthworm dry powder product (W-8) was dried by vacuum drying for 10 hours under a vacuum of 28 g, then raising the shelf temperature to 80 °C and drying for 8 hours under a vacuum of 0.2 lllfflHg.
I got 0g.

Example 9 1 kg of live earthworms (red earthworms) lightly washed with water was
p containing mixed acids of phosphoric acid, tartaric acid and lactic acid 1:1:1
After leaving the worms in an acidic aqueous solution 31 of H15.5 at a temperature of 13°C for 3 hours to fully excrete the feces in the digestive tract, the live earthworms were thoroughly washed with water to remove the soil attached to the body surface of the live earthworms. Wash off dirt such as mud and feces.

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.
Lower the temperature to -35°C, Q, l l1ff! 111g
Lyophilize for 7 hours under a vacuum of
g vacuum for 13 hours, then dried at 78°C for 0.
By drying under a vacuum of 1 mm and 11 g for 1 hour, 277 g of earthworm dry powder product (W-9) was obtained.

Example 10 1 kg of live earthworms (red earthworms) that have been lightly washed with water are left in fresh water 31 at a temperature of 15° C. for 36 hours to excrete fecal soil in the digestive tract. Wash the live earthworms thoroughly with water to remove dirt such as mud and R adhering to the body surface of the live earthworms. Next, wet grinding is performed using an ultra homo mixer.

Place the obtained earthworm suspension in a tray and freeze at -40°C for 15 hours. Next, lower the temperature to -45°C and freeze at 0.1 atn.
Lyophilize under vacuum at +t1g for 6 hours, then at 30°C for 0,8 wll! After drying under a vacuum of Hg for 10 hours, then at 40°C under a vacuum of 0.2 KH for 15 h, then at 78°C for 0.1 KH.

240 g of a dry earthworm powder product (W-10>) was obtained by drying the product under vacuum for 3 hours.

Example 11 One live earthworm (red earthworm) lightly washed with water was left in an acidic aqueous solution 21 with pH 5.8 in which malic acid was dissolved at a temperature of 15°C for 3 hours to fully excrete the feces in the digestive tract. . Wash the live earthworms thoroughly with water and the mud that adheres to the body surface of the live earthworms,! Wash off dirt such as t.

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 product temperature is -30℃ and the temperature is 0.1
Freeze-dry for 8 hours under a vacuum of mmHg, then increase the temperature of the shelf on which the tray is placed to 25°C, vacuum dry for 7 hours under a vacuum of 0.1 mmHH, and then raise the shelf temperature to 45°C. Dry earthworm powder product (W-11) was obtained by vacuum drying for 12 hours under a vacuum of 1 mm FIH, then raising the shelf temperature to 80 °C and drying under a vacuum of 0.1 mmHg for 7 hours.
275g was obtained.

Example 12 Wash dirt such as mud and straw adhering to the body surface of 1 kg of live earthworm (red earthworm) with water.Next, this live earthworm was left in fresh water 2.5℃ at a temperature of 12°C for 42 hours. After removing the fecal soil from the digestive tract, the live earthworms were lightly washed and then wet-pulverized using an ultra homo mixer. Place the obtained earthworm suspension in a tray and heat at 40°C for 24 hours.
Time frozen.

Next, the product was freeze-dried at -40°C under a vacuum of 0.1 ma+Hg for 5 hours, then at 25°C for 8 hours under a vacuum of 0.1 ma+Hg, and then at 45°C and 0.1 mmH.
Dry under vacuum for 12 h at 80 °C for 0.1 a
235 g of earthworm dry powder product (W-12) was obtained by drying under mHg vacuum for 2 hours.

Example 13 Dirt, feces, and other impurities adhering to the body surface of 1 kg of live earthworms (red earthworms) are thoroughly washed with water 4 times to remove them.
Next, the live earthworms were left in an acidic aqueous solution 2.51 with a pH of 5.7 in which a 1:1 mixture of malic acid and lactic acid was dissolved at a temperature of 15°C for 2.5 hours to excrete the feces in the digestive tract. let Next, after lightly washing the live earthworms, they are wet-pulverized using a mixer. The obtained earthworm suspension was placed in a tray and frozen at -35℃ for 24 hours, then the product temperature was lowered to -35℃ for 0.
．． Freeze-dry for 7 hours under a vacuum of 1 gmlg, then raise the temperature of the shelf on which the tray is placed to 22°C, and add 0.1ml (H
After vacuum drying for 10 hours under a vacuum of
°C and dried under a vacuum of 0.2 InllHg for 10 hours, and finally the shelf temperature was raised to 78 °C and dried under a vacuum of 0.1 mmHg for 3 hours to obtain a dried earthworm powder product (
W-13) 270g was obtained.

Below is the margin Example 14 Capsule A Earthworm dry powder (W-2) 150 ■Milk
W! 28 Microcrystalline cellulose 47 Manitol
10 corn starch flour
10 polyvinylpyrrolidone
2-hydroxypropylcellulose 3 total 250m
g In the above formulation, the components other than hydroxypropylcellulose were thoroughly mixed using a fluidized bed granulator, and then a 5% aqueous solution of hydroxypropylcellulose was sprayed as a binder, and the mixture was dried at low temperature and made into granules. A hard capsule preparation was prepared by filling 250 μg of the granules into hard capsules.

Below is the margin Example 15 Example 1 Granule A Dry earthworm powder (W-6) 150 ■Milk
tJ! 20 Microcrystalline Cellulose 60 Corn Starch 15 Hydroxypropyl Cellulose 5 Total 250 ■ According to the above recipe, using a fluidized bed granulator, dry earthworm powder, lactose, microcrystalline cellulose, and corn starch were thoroughly mixed to form hydroxypropyl cellulose. A 5% aqueous solution was sprayed as a binder, and granules were obtained after drying at low temperature.

Margins below: Granules B: Dry powder of earthworms (W-3 Manitol Microcrystalline cellulose carboxymethylulose calcium magnesium stearate Hardened oil granules C: Dry powder of earthworms (W Lactose Cornstarch Potato Dental Stearate Magnesium 100) Total: 200° 150° 0 mg According to the above recipe, the well-mixed powder was used to produce granules using an extruder.

Example 17 Capsule B Granules C produced according to Example 16 were put into hard capsules.
Capsules were prepared by filling 0.0 cm each.

Example 18 Capsule C Dry earthworm powder (W-5) Calcium hydrogen phosphate Sodium hydrogen phosphate Manitol Magnesium stearate 150 ■ Total 250 ■ The above formulations were thoroughly mixed, and this mixed powder was mixed with N11
Capsules were prepared by filling 250 μl gelatin capsules into each gelatin capsule.

Example 1 Enteric-coated tablet earthworm dry powder (W-4) Manitol microcrystalline cellulose carboxymethyl cellulose calcium magnesium stearate Hardened oil 100 1.5 1.5 Total 200 Img According to the above recipe, uniformly mixed powder was compressed into tablets. After producing uncoated tablets in a machine, they were coated with the following enteric coat coating agent to produce enteric coated tablets.

The following margin coating agent Hydroxypropyl methyl cellulose phthalate dioctyl phthalate Stearic acid Light silicon oxide 14.8 2.3 2.3 0.6 ■ Example 20 Powder A Earthworm dry powder (W-7) Manitol corn starch powder 150 ■ Total 250 g Total 22.0 ■ Margin Powder Earthworm Dry Powder (W-2) Calcium Hydrogen Phosphate Corn Starch Powder 150 ■ Total 250 ■ The above components were mixed well and uniformly in a conical mixer to form a powder.

Example 2 Example 22 Capsules D Earthworm dry powder (W-3) Sodium lauryl sulfate phosphate-sodium hydrogen mannitol Magnesium stearate 150 ■ Capsules E Earthworm dry powder (W-8) Phosphate-calcium hydrogen phosphate - Sodium hydrogen mannitol Magnesium stearate 150 µm in total 25 (lag) The above-mentioned formulations were mixed. This mixed powder was filled into 250 µl gelatin capsules to produce capsules.

A total of 250 ml of the above-mentioned formulation was thoroughly mixed, and 250 ml of this mixed powder was filled into the first gelatin capsules to produce capsules.

Below margins Below margins Example 23 Example 24 Capsule F Earthworm desiccant powder (W-13) Sodium lauryl sulfate Sodium phosphate-hydrogen phosphate Manitol Magnesium stearate 50 mg Total 250 ■ Mix the above formulations well. This mixed powder is N!
Capsules were prepared by filling 11 gelatin capsules with 250 ml each.

Margins below Tablet A Earthworm desiccant powder (W-10) Manitol hydroxypropoxymethylcellulose tal Microcrystalline cellulose Hydrogenated castor 150■ Total 350+g Tablet B Earthworm desiccant powder (W-11) Corn starch milk powder Sugar talc Magnesium stearate 150 ■ Tablet C Dry earthworm powder (W-12) 150 ■ Soluble starch 20 Corn starch
125 Microcrystalline Cellulose 45 Silicon Oxide 6 Magnesium Stearate 4 Total 350 ■ Tablets of various weights were manufactured from the uniformly mixed powder according to the above recipe using a tablet machine.

Effects of the invention: The present invention has established a novel and innovative method for producing earthworm dry powder. This production method involves soaking earthworms in fresh water or in an aqueous solution with a pH of 3 to 6.5 containing at least one compound selected from the group consisting of organic acids, inorganic acids, sodium salts of these acids, and potassium salts of these acids. After the fecal soil in the digestive tract was allowed to be expelled, the living earthworms were washed with water to remove dirt adhering to their body surfaces, and subjected to wet grinding to obtain an earthworm suspension. This earthworm suspension is freeze-dried, vacuum-dried, or vacuum rotary-dried to obtain dried earthworm powder.

The dried earthworm powder obtained by the production method of the present invention is sterile and does not generate mold even when stored for 5 years. In addition, it was found that there were no side effects such as hemolytic effects, and that it had excellent storage stability and safety. On the other hand, basic experiments confirmed that the dried earthworm powder obtained by the above-mentioned method contained a fibrinolytic active substance and did not contain a hemolytic substance.

Next, capsules of dried earthworm powder obtained by the manufacturing method of the present invention (containing 150 μl of dry earthworm powder) were administered to 3 patients with severe venous thrombosis (anticoagulant Marfarin) three times a day.
Used in combination with Potassium. ) 4 people with hypertension, 3 of them
The person had complications with thrombosis. A total of 9 patients had hypotension, 2 of whom had complications of mild hyperlipidemia. ] and one normal healthy volunteer.
The drug was orally administered to 0 subjects after every meal. The thrombolytic activity and therapeutic effects of dried earthworm powder on these nine patients were determined from the behavior of blood FDP and tpAO values. Marfarin potassjum, an anticoagulant, inhibits fibrin formation but does not have the effect of dissolving fibrin once formed.Dry earthworm powder is used in combination to promote and promote this fibrin dissolving activity, and its therapeutic effect is enhanced. was determined.

As a result, a total of 8 cases of blood clots (fibrin) occurred on the 18th or 25th day of oral administration. The inside of the blood vessels returned to a normal state. At the same time, take 2
On the fifth day, the blood pressure of hypertensive patients decreased, and the blood pressure of hypotensive patients increased, and both reached normal values. Furthermore, the values of TC and TG, which showed symptoms of hyperlipidemia alone or with complications, also became normal values. The improvement effect was also evident in the remaining patients with severe venous thrombosis (complications of hyperlipidemia). Furthermore, Marfarin Potassi
If you continue to take um and dried earthworm powder, you can expect a therapeutic effect.

As shown above, the dried earthworm powder produced by the method of the present invention showed excellent therapeutic effects on patients suffering from severe venous thrombosis symptoms, hypertension, hypotension, or hyperlipidemia alone or with complications.

However, in this clinical trial, there were no side effects of the dried earthworm powder.

Patent applicant: Aimei Co., Ltd. Representative: Yo Ishii -1 Procedural amendment normalized on February 2, 1990

Claims (1)

[Claims] 1. Live earthworms are placed in fresh water or at a pH of 3 to 6. An earthworm suspension obtained by leaving the living earthworms in the aqueous solution of step 5 to expel the excrement from their digestive tracts, and then washing away the dirt adhering to the body surface of the live earthworms with water and performing wet crushing; After removing the filth that adheres to the body surface of the live earthworm, the live earthworm is left in fresh water or the above-mentioned aqueous solution to expel the fecal soil in the digestive tract of the live earthworm, and then the filth that adheres to the body surface of the live earthworm is removed. A method for producing dried earthworm powder, which comprises degassing an earthworm suspension obtained by washing and removing water with water and performing wet pulverization under reduced pressure, followed by vacuum rotary drying.