JP2002272267A - Method for artificially culturing plant worm and extracted composition of isaria japonica and functional food using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for artificially culturing a plant worm by which a fruit body can be formed from spores of a fungal body of the plant worm in a host in a short period with a high possibility and stably fed in a large amount irrespective of the seasons throughout the year, an extracted composition of an Isaria japonica worm plant forming the fruit body obtained by the method for artificial culturing and a functional food using a dried powder of the Isaria japonica worm plant or the extracted composition. SOLUTION: This method for artificial culturing the plant worm is characterized by directly injecting and inoculating a spore suspension of a strain of the plant worm into a body of an aseptically reared insect or an aseptically reared spider or decompressing insect or spider, then forcibly infiltrating and inoculating the spore suspension into the body of the insect or spider when releasing the body to atmospheric pressure in the method for artificially culturing the plant worm comprising forming the fruit body from the spores of the fungal body of the plant worm.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for artificially cultivating Cordyceps sinensis to form fruiting bodies from spores of Cordyceps sinensis cells with high probability and in a short period of time, an extract composition of Cordyceps sinensis and a functional food using the same. .

[0002]

BACKGROUND OF THE INVENTION Cordyceps is composed of a complex of a fruiting body formed by infesting insects with a microorganism belonging to the ascomycetes Ergot Mycorrhidae family and a dead body of an insect host. About 35 in Japan
Although zero species are known, the dried product is mainly produced in China, and has been used in the private sector since ancient times as a tonic, tonic, longevity and nourishing tonic. Recently, it has been increasingly used as a functional food due to changes in lifestyles and health consciousness.

However, looking at the state of supply of cordyceps, most of them are still obtained by collecting those that are present in nature. Furthermore, Cordyceps is difficult to collect because it does not parasitize plants like other mushrooms but parasitizes insects, and a technology that can be stably supplied in large quantities has been expected. Recently, a method has been studied in which microorganisms infested by insects are purely isolated and then cultured in a large amount using a solid medium or a liquid medium to obtain bacterial cells (JP-A-8-125).
No. 88, Japanese Unexamined Patent Publication No. Hei 8-172901, etc.), which are thought to contain a cell component different from Cordyceps caused by symbiotic relationship with insects. In addition, in this technique, since the host is not an insect, it is considered that the added value is lower than that of cordyceps in nature.

[0004] In addition, a method of inoculating a pupa of a silkworm with a cordyceps so as to form a cordyceps in a host rather than a tank culture has been studied (JP-A-10-42691, JP-A-8-172903, (Kaihei 8-75)
However, it takes as long as 30 days to 50 days to several months to form fruiting bodies, and there is a problem in productivity.

[0005] As described above, establishment of an artificial cultivation method of Cordyceps sinensis is expected to form fruiting bodies from spores of Cordyceps sinensis cells with a high probability and in a short period of time. Never before.

[0006]

The first object of the present invention is to allow the host to form fruiting bodies from the spores of Cordyceps sinensis with high probability and in a short period of time, and to stabilize Cordyceps sinensis throughout the year regardless of the season. It is an object of the present invention to provide a method for artificially cultivating Cordyceps sinensis, which can be supplied in large quantities.

A second object of the present invention is to provide excellent pharmacological effects based on physiological activities such as antioxidant activity, activity of promoting tumor necrosis factor production from macrophages, and activity of promoting production of nitric oxide (NO) from macrophages. It is an object of the present invention to provide an artificial cultivation method capable of multiplying, in a short period of time, a Cordyceps sinensis as an expected cordyceps.

[0008] A third object of the present invention is to use aseptically reared silkworm pupa as a host, so that only the target microorganism can be preferentially selected with a high probability and for a short period of time without worrying about propagation and contamination of other microorganisms. It is an object of the present invention to provide an artificial culture method capable of proliferating into an artificial culture.

[0009] A fourth object of the present invention is to make effective use of, for example, pupae of silkworm bred aseptically reared silkworms and fruiting bodies formed on the pupae, which have formed fruiting bodies obtained by the artificial culture method. An object of the present invention is to provide an extract composition of Anopheles velutipes obtained by an appropriate extraction treatment.

[0010] A fifth object of the present invention is to provide the extract composition of Pseudomonas velutipes, which is based on physiological activities such as an activity of promoting tumor necrosis factor production from macrophages and an activity of promoting production of nitric oxide (NO) from macrophages. Proteoglycans (proteins that are expected to have excellent pharmacological effects)
A glycoconjugate).

[0011] A sixth object of the present invention is to provide a functional food for providing humans with a dry powder of a Hanasagi mushroom or an extract composition of a Hanasagitake mushroom which has formed fruiting bodies obtained by the artificial culture method. is there. The functional foods referred to here are so-called health foods and health drinks that are edible to be effective in maintaining and promoting human health.

[0012]

Means for Solving the Problems The artificial cultivation method of Cordyceps sinensis of the present invention is an artificial cultivation method of Cordyceps sinensis which forms fruiting bodies from spores of the fungus body of Cordyceps sinensis. Directly inoculating the spore suspension of the Cordyceps fungus strain, or forcing the spore suspension into the body of the insect or the spider when the insect or the spider is depressurized and then released to atmospheric pressure. It is characterized by infiltration and inoculation.

In the present invention, insects include not only adults, but also eggs, larvae, and pupae.

In the present invention, by using aseptically reared insects and the like, preferably aseptically reared insects and the like as a host, only the target microorganisms can be prioritized without worrying about propagation contamination of other microorganisms. It becomes possible to proliferate.

Further, a spore suspension of the target microorganism is directly injected into the body of a sterile insect or the like. According to this method, the time required for the spores to enter the insect body can be shortened, and the fruiting body can be obtained in a short time, which is effective.

Alternatively, instead of direct injection, the spore suspension is forcibly infiltrated and inoculated into the body of the insect or spider when the pressure of the insect or spider is reduced and the atmospheric pressure is released. For example, germ-free spores are forced into the spores through the gaps of pupae such as stomata and esophagus by immersing the germ-free insects and the like in the spore suspension when the germ-free insects and the like are released under atmospheric pressure under atmospheric pressure. And inoculate. According to this method, the time required for the spores to enter the insect body and the time required for the bacterial cells to spread throughout the insect body can be reduced, and the time required for obtaining the fruiting body can be significantly reduced. . Further, the direct injection method is preferable in that the fruiting bodies are obtained reliably in a short time, but the above-described depressurization method can secure mass productivity in addition to them. That is, a large number of samples can be processed at one time, a high infection rate can be maintained, and fruiting bodies can be formed in a shorter time than in the conventional technique.

Further, in the method for artificially cultivating Cordyceps according to the present invention,
The fungus body of the cordyceps is Isaria japonic
a It is preferably IM2001 strain (FERM P-18253).

Further, in the method for artificially cultivating a cordyceps cordyceps of the present invention, the aseptically reared insect is preferably a silkworm pupa. This is because silkworm pupae have a well-established production technology and sterile pupae are also available.

The extract composition of Pleurotus ostreatus of the present invention comprises:
Ethanol, water-alcohol mixture or water, or butanol, ethyl acetate, butyl acetate, isobutyl acetate, n
-Using an organic solvent such as hexane or chloroform, xylene, acetone or the like as a solvent, the flowering pupae of Pleurotus ostreatus (pupae of aseptically reared silkworms and the fruit bodies formed on the pupae) are heated at 20 to 140 ° C or heated. It is characterized by extraction processing under pressure heating conditions. By using these solvents, the water-insoluble component, the water-soluble component, or both components of the cordyceps can be extracted according to the purpose.

[0020] The extract composition of Pseudomonas velutipes of the present invention comprises:
It is characterized by containing proteoglycan (protein-sugar complex) purified and fractionated by column chromatography such as ion exchange resin and gel filtration agent.

[0021] The functional food of the present invention is characterized in that it contains a dried powder of the flowering plant of the genus Hanasagi mushroom or the extract composition of the anatomical mushroom of the flowering body. It is preferable to use Cordyceps sinensis artificially cultured in the present invention.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to embodiments and examples, but the present invention is not construed as being limited thereto.

Examples of fungi of Cordyceps sinensis include Hanasagitake, Sanagitake, Usukisanagitake, Tsukutsukuboushitake and Kumotake.

Hosts of Cordyceps include silkworm pupae, cicada,
Examples include insects such as dragonflies and bees, and spiders.

Next, the method for artificially cultivating Cordyceps according to the present invention will be described. The following cells were used as cordyceps fungi. As a result of selecting a strain that forms fruiting bodies faster with good growth, after inoculating the pupa of the aseptic breeding silkworm with repeated culture and pure separation of Cordyceps sinensis collected from Yamanaka of Tan Peninsula, Kyoto Prefecture, furthermore, Isaria japonica IM2001 strain was obtained. In the present invention, Isaria japonica IM2001
This will be described using strains.

The strain Isaria japonica IM2001 was deposited with the Patent Microorganisms Depositary Center, Institute of Biotechnology, Institute of Technology, Ministry of Economy, Trade and Industry (1-1, Higashi 1-1, Tsukuba, Ibaraki Prefecture) under the deposit number FE.
Deposited as RMP-18253.

Next, this strain is dispersed in water to obtain a spore suspension (10 ³ to 10 ⁶ / ml). This spore suspension 5
100 μl, preferably 10 to 30 μl, is injected into the body of a sterile pupa bred aseptically for the entire age of the silkworm by an injector such as a syringe. In addition, it is more preferable that the aseptic pupae be bred aseptically on artificial feed.

The germ-free pupa into which the spore suspension has been injected is kept aseptically in a thermostatic chamber (0 to 20 ° C.) or at room temperature.
By culturing the cells, hyphae appear on the surface of the pupa in about 7 to 8 days, and fruiting bodies can be formed in about 1 month. Furthermore, a technique for forming a large amount of fruiting bodies in a short period of time, which is not possible in the past, such as forming fruiting bodies within 30 days by optimizing the spore concentration of the spore suspension, the amount of inoculum, and the culture temperature after inoculation, has been developed. Could be established. The fruiting body formation rate after infecting the pupa is almost 100%.

In addition to the method of injecting and inoculating spores with the above-mentioned syringe, the sterile silkworm pupae may be spore-suspended when the sterile silkworm pupa is released under atmospheric pressure, for example, under atmospheric pressure by means of an aspirator or a vacuum pump. Suspended liquid (10 ³ to 10 ⁶ /
spores may be forcibly infiltrated and inoculated into the pupa of a sterile silkworm. in this case,
After depressurizing and opening to the atmosphere, any procedure can be adopted as long as it can be forcibly infiltrated and inoculated into the sterile pupa. Therefore, for example, a large number of sterile pupae, which can be sufficiently immersed in a container containing a spore suspension (10 ³ to 10 ⁶ / ml), are put into a larger container, and 200 mmHg to 7 mm.
It is also possible to adopt a method of forcibly infiltrating and inoculating the germ-free pupa body while maintaining the condition under reduced pressure of 60 mmHg for 10 to 60 minutes. It is presumed that the following phenomenon occurs due to the reduced pressure. In other words, the spore suspension is easily evaporated by the reduced pressure, while the air in the pupa expands and diffuses, and the body fluid is volatilized by the reduced pressure to form a void in the pupa. Next, when the pupa is immersed in the spore suspension at the time of opening to the atmosphere, the air in the pupa that has expanded due to the atmospheric pressure contracts, and the spore suspension penetrates into every corner. Alternatively, the spore suspension penetrates into the voids after the bodily fluid has volatilized. The spores can be inoculated into the pupa more uniformly by the reduced pressure inoculation method than by the direct injection method in the pupa. Furthermore, according to the vacuum infiltration inoculation method, it is possible to inoculate a large number of pupae at a time easily. By continuing aseptic cultivation after inoculation, approximately 100% or less within about one month or more as in the case of injection into the body with a syringe.
% Fruiting bodies can be formed.

The microbiological characteristics of the fruit body grown on the pupa are as follows:
It is as shown in Table 1.

[Table 1] These features are consistent with those described in the literature and are not
ia japonica (= Paecilomyces
tenuipes).

The extraction of the active ingredient from the worms that have formed fruiting bodies is performed using ethanol, a water-ethanol mixture or water as a solvent. Preferably 10-90% ethanol in water
Using the ethanol mixture as a solvent, extraction is performed by slowly stirring at room temperature or under warming conditions. The amount of ethanol added and the extraction time are not particularly limited.

The extract is separated by filtration to obtain an ethanol extract. The fat and oil component and the water-soluble component are extracted in this ethanol extract. When only the fat component is selected and extracted, butanol, ethyl acetate, butyl acetate, isobutyl acetate, n-hexane or chloroform,
Used as an organic solvent such as xylene and acetone. However, it is preferable to completely remove this organic solvent in a subsequent step, since functional foods and the like are used. The extracted fat component can be used as a functional food, like the water-soluble component described later. In the above-mentioned organic solvent, it is preferable that the extraction is performed under a pressure and heating condition of 20 to 80 ° C.

Next, water is added to the filtration residue obtained by the ethanol extraction, and the mixture is heated at 50 to 140 ° C. with gentle stirring, or pressurized and heated as necessary to perform extraction. Preferably, hot water extraction is performed under a pressure and heating condition of 80 to 130 ° C. The extract is separated by filtration to obtain a hot water extract (1).

Further, water is added to the filtration residue, and the mixture is subjected to hot water extraction under the same pressure and heating conditions as above with gentle stirring, followed by filtration to obtain a hot water extract (2).

Each of the hot water extract (1) and the hot water extract (2) is sterilized by autoclaving at 121 ° C. for 15 minutes and then used as a physiological activity measurement sample or an active ingredient of the composition of the present invention. The amount of water used for extraction is not particularly limited. If necessary, the extract-treated composition without removing the extraction residue can be used as it is as the active ingredient-containing composition.

Further, the powder obtained by drying the insect grass and then finely pulverizing it can be used as it is as a physiologically active composition.

As described in detail in Examples, the ethanol extract and the hot water extract (1) and (2) obtained from the insect vegetation of the present invention can be used for free radical scavenging activity, superoxide scavenging activity or α-linolenic. Antioxidant activity such as acid autoxidation inhibitory activity, tumor necrosis factor (TNF) from macrophages
-Α) has an activity of promoting the production and an activity of promoting the production of nitric oxide (NO) from macrophages, and the physiologically active composition according to the present invention is a functional food utilizing these physiological activities. Useful. In addition, since the same effect can be expected also with respect to the fat component, it is useful as a functional food similarly to the ethanol extract and the hot water extract (1) and (2).

The ethanol extract, the hot water extracts (1) and (2), and the physiologically active composition comprising the fat and oil components can be used alone, respectively. It is possible to further increase the efficacy by using it as an active composition.

When the physiologically active composition alone or as a mixture is used as a functional food, it can be used as it is,
It can be used in combination with other foods or food ingredients, or as a mixture.

When the present composition is used, it may be in the form of liquid or suspension, solid (powder, granule, etc.) or paste. Further, if necessary, it can be used by mixing with a sweetener, an acidulant, or a vitamin, or can be used as a functional food in combination with commonly used ingredients such as health foods or health drinks.

The form of the functional food of the present invention is not particularly limited, and solid preparations such as tablets, granules, fine granules, powders, capsules, troches and the like, liquid preparations such as emulsions, syrups, suspensions, etc. And so on. Preparation of these foods such as dextrin, corn starch, lactose and other excipients, erythritol, trehalose, glucose, xylitol,
PH of sweeteners such as mannitol, citric acid, tartaric acid, etc.
Modifiers, various perfumes, other active ingredients such as ascorbic acid, preservatives such as sorbic acid, emulsifiers and the like can also be used.

The composition of the present invention is of natural origin and has been used as a herbal medicine for a long time, so it has no or very low toxicity and is a very safe functional food for prophylactic and health use. Alternatively, it can be used for therapeutic purposes.

If the active ingredient present in the bioactive composition can be obtained as a more purified substance, it can be used in functional foods as a safer and more stable bioactive substance with effective efficacy. Therefore, when the hot water extract composition was purified and fractionated by column chromatography using an ion exchange resin such as DEAE-Sepharose and DEAE-cellulose and a gel filtration agent such as Toyopearl and Sephadex, the proteoglycan was purified. (Protein-sugar complex)
Examples will be described in more detail including the physiological activities of these components.

From the antioxidant activity of the bioactive composition of Hanasagi mushroom, the analgesic, anti-AIDS,
Improvement of alcohol poisoning, anti-allergy, anti-angina, anti-arrhythmia, anti-atherosclerosis, anti-asthma, antibacterial, anti-diabetes, detoxification, anti-inflammatory, anti-hyperlipidemia, DNA mutation suppression, anti-Parkinson disease,
Anti-psoriasis, anti-rheumatic, anti-ulcer, anti-ulcer, brain function protection, cell growth suppression, skin disease improvement, liver function activation, hypotension, immunosuppression, renal function activation, nerve cell protection, nootropic, ophthalmic disease improvement, radiation protection, It is expected to be used as a functional food effective for prevention, health and treatment of vascular vitality, antivirus, wound medicine, improvement of weakness, etc.

Further, from the activity of promoting the production of tumor necrosis factor from macrophages, it is expected to be used as a functional food effective for enhancing immunity, preventing cancer, antivirus, etc., health, and treatment.

Nitric oxide (NO) from macrophages
From the activity of promoting production, it is expected to be used as a functional food that is effective for prevention, health, and treatment, such as enhancement of intense heart muscle, contractility of myocardium, recovery from fatigue, and recovery of physical and mental fatigue.

[0047]

EXAMPLES (Formation of fruiting body) [Example 1] 150 pupae produced from silkworms bred aseptically for all ages with artificial feed were prepared in advance by aseptic operation.
Spore suspension of Isaria japonica IM2001 strain (about 10
⁽⁵ cells / ml) was injected into the pupa using a syringe. Place the inoculated pupae in a thermostatic chamber (17 ° C ± 2 ° C, 60
By continuously aseptically cultivating the culture within (-70% humidity), the appearance of hyphae and further fruiting bodies were successively observed, and the culture was terminated on the 29th day to obtain worms forming fruiting bodies. . The fruiting body formation rate was 100%.

[Example 2] Fifty pupae produced from silkworms aseptically bred for an entire age period with an artificial feed were used to prepare a spore suspension of Isaria japonica IM2001 strain (about 1) aseptically prepared.
0 ⁵ / ml) was immersed in a vessel containing 200 ml, placed in a vacuum vessel and the spores left under water jet vacuum for 15 minutes forcibly enters the body of a pupa. After gently removing the surface moisture of the inoculated pupa, a constant temperature room (17 ° C. ± 2 ° C., 60-70 ° C.)
(% Humidity), the cultivation was terminated on the 25th day, and worms forming fruiting bodies could be obtained. The fruiting body formation rate was 100%. Example 1
Although the cultivation period of the worms of Example 2 differs from that of the worms of Example 2, the worms themselves that formed fruiting bodies are not different.

[Comparative Example 1] Fifty pupae produced from silkworms aseptically bred for an entire period of time with artificial feed were used to prepare a spore suspension of Isaria japonica IM2001 strain (about 1) aseptically prepared.
0 ⁵ / ml) was immersed in a vessel containing 200ml were inoculated into pupae body surface. After the surface water of the inoculated pupae was lightly dried and removed, the culture was continued aseptically in a constant temperature room (17 ° C. ± 2 ° C., 60-70% humidity). Insecticidal plants that formed themselves could be obtained. The fruiting body formation rate was 70%.

(Ethanol extract, hot water extract (1),
Preparation of hot water extract (2)) To 100 worms forming fruiting bodies obtained in Example 2, 1 L of a 45% (v / v) aqueous ethanol solution was added, and the mixture was gently stirred at room temperature for 2 days. After the extraction, an ethanol extract was obtained by filtration. 1 L of water was added to the filtration residue, and after gentle stirring, hot water extraction was performed at 102 ° C. for 60 minutes, and a hot water extract (1) was obtained by filtration. Add 1L of water to the filtration residue, stir gently,
Hot water extraction was performed at 02 ° C. for 60 minutes, followed by filtration to obtain a hot water extract (2). Hot water extract (1) and (2)
Was sterilized by an autoclave at 121 ° C. for 15 minutes, and then used in the following Examples.

Example 3 Ethanol extract prepared as described above (19.1 mg / m 2 as lyophilized weight)
l), hot water extract (1) (6.8 m in freeze-dried weight)
g / ml) and the hot water extract (2) (2.1 mg / ml as lyophilized weight) were measured for free radical scavenging activity according to the following method.

(Measurement Method) The free radical scavenging activity was measured by measuring 1 ml of the reaction solution and 0.1 M acetate buffer (pH 5.
5) As 0.5 mM α-diphenyl-β-picryylhydrazyl (DPPH), 20 μg, 40 μg of the extract,
The test was performed on 60 μg. At this time, the reaction was allowed to proceed at room temperature for 30 minutes, and the amount of decrease in DPPH was measured by absorbance at 517 nm. As a control (Comparative Example 2), an acetate buffer was used instead of the extract. The results obtained are shown in Table 2 as free radical scavenging activity according to the above measurement method.

[Table 2]

From the results of Examples 3-1 to 3-9,
It was revealed that the ethanol extract, the hot water extract (1) and the hot water extract (2) had an antioxidant activity with strong free radical scavenging activity.

Example 4 Ethanol extract prepared as described above (19.1 mg / m 2 as lyophilized weight)
l), hot water extract (1) (6.8 m in freeze-dried weight)
g / ml) and the hot water extract (2) (2.1 mg / ml as lyophilized weight) were measured for superoxide scavenging activity according to the following method.

(Measurement method) Superoxide scavenging activity was measured by measuring 1 ml of the reaction solution, 0.1 M potassium phosphate buffer (pH 7.4), 20 μM phenazine methosulfate, 156 μM diphosphopyridine nucleotide,
The extraction was performed on 125 μg, 250 μg, and 500 μg of the extract as 50 μM nitroblue tetrazolium. At this time, the reaction was carried out at room temperature for 3 minutes, and the reduction reactant (formazyl compound) of nitro blue tetrazolium with superoxide was measured at an absorbance of 560 nm.
As a control (Comparative Example 3), a potassium phosphate buffer was used instead of the extract. The results obtained are shown in Table 3 as superoxide scavenging activity in accordance with the above measurement method.

[Table 3]

As a result of Examples 4-1 to 4-9, the ethanol extract, hot water extract (1) and hot water extract (2) contained:
It was revealed that a strong antioxidant activity with superoxide scavenging activity was present.

Example 5 Ethanol extract prepared as described above (19.1 mg / m 2 as lyophilized weight)
l), hot water extract (1) (6.8 m in freeze-dried weight)
g / ml) and the hot water extract (2) (2.1 mg / ml as lyophilized weight) were measured for α-linolenic acid autoxidation inhibitory activity according to the following method.

(Measurement method) The antioxidant activity against α-linolenic acid autoxidation was measured by measuring 1 ml of the reaction solution, 50 μl of α-linolenic acid, 40% (v / v) ethanol, 2 m
As an M potassium phosphate buffer (pH 7.8), 300 μg of the extract was used as a target. At this time, at 50 ° C in a dark place
And the formation of lipid peroxide was examined over time by the TBA method. Absorbance of sample in reaction solution 535nm
1 is shown in FIG. 1 as the antioxidant activity against α-linolenic acid autoxidation. Each measured value was shown as an average of three measurements. In FIG. 1, the ethanol extract and the hot water extract (1)
And each of the hot water extract (2) has a smaller absorbance over time as compared with the control (Comparative Example 4), and it is clear that the antioxidant activity against α-linolenic acid autoxidation exists over time. It is. Therefore, the results shown in FIG. 1 indicate that the ethanol extract, hot water extract (1) and hot water extract (2) have strong antioxidant activity against α-linolenic acid autoxidation. Was revealed.

From Examples 3, 4, and 5, it is clear that the composition extracted from the insect grass of Anopheles velutipes has such a strong physiological activity, and its use as a functional food having antioxidant activity can be expected. .

Example 6 Ethanol extract prepared as described above (19.1 mg / m 2 as lyophilized weight)
l), hot water extract (1) (6.8 m in freeze-dried weight)
g / ml) and the hot water extract (2) (2.1 mg / ml as lyophilized weight) to measure the activity of promoting tumor necrosis factor (TNF-α) production from macrophages are described below. Shown in

(Measurement Method) The TNF-α produced by allowing the above sample to act on exudative macrophages prepared from mouse abdominal cavity at 37 ° C. for 24 hours at 5% CO ₂ concentration was measured. The amount of TNF-α was measured by a bioassay using L-929 cells. TNF under each condition
Table 4 shows the -α production amount.

[Table 4]

The concentration of each extract in the reaction solution was standardized by measuring the sugar concentration by the anthrone method and converting it to the amount of glucose. The control (Comparative Example 5) used a phosphate buffer. LPS: lipopolysaccharide was used as a positive control (Comparative Example 6).

Examples 6-1 to 6-6 shown in Table 4
From the results, it is clear that the extract composition obtained from the insect grass of Pseudomonas velutipes, in particular, the hot water extract has a much stronger activity of inducing tumor necrosis factor generation than LPS known as an antitumor polysaccharide. Became.

Example 7 Ethanol extract prepared as described above (19.1 mg / m 2 as lyophilized weight)
l), hot water extract (1) (6.8 m in freeze-dried weight)
g / ml) and hot water extract (2) (2.1 mg / ml as lyophilized weight) to measure the activity of promoting macrophage nitric oxide (NO) production. .

(Measurement Method) Nitric oxide in the reaction solution sample used for the measurement of TNF production ability in Example 6 was measured. The sample was dispensed at 100 μl / well, followed by Gries
ss) 100 μl of reagent was added. After standing at room temperature for 10 minutes, the absorbance at 570 nm was measured. Table 5 shows the results of the amount of nitric oxide (NO) produced from macrophages.
The control (Comparative Example 7) used a phosphate buffer.

[Table 5]

Comparing Example 7-1 to Example 7-6 with Comparative Example 7, it was found that the extract composition obtained from the insect grass of Pseudomonas velutipes has an activity to promote the production of nitric oxide (NO) from macrophages. It is clear that. A pharmacological effect based on this physiological activity can be expected.

Example 8 1.5 g of chloroform was added to 60 g of the dried grass of Pseudomonas velutipes obtained in Example 2, and the mixture was gently stirred for 12 hours, after which the fat-soluble fraction was removed. Residue was obtained. A 10-fold amount (600 ml) of water was added to the residue, and the mixture was heated and extracted at 100 ° C. for 6 hours, and a hot water extract was obtained by filtration. The same operation was further repeated twice to obtain a combined hot water extract for three times, and a four-fold amount of ethanol was added thereto to obtain a sedimentation section. The precipitate was dialyzed and freeze-dried to obtain 3.50 g of dry powder (MII). This powder was dissolved in a 1/15 M phosphate buffer (pH 7.2), filled with DEAE-Sepharose CL-6B, and dissolved in a 1/15 M phosphate buffer (pH 7.2).
7.2) (about 150 ml, diameter 2.
5 cm, 30 cm in length) and two peaks MII-1 and MII-2 in the non-adsorbed section by elution with the same buffer.
And 0.2M sodium chloride-1 / 15M
Elution with phosphate buffer (pH 7.2) yields two peaks, MII-3 and MII-4, eluting with 0.6M sodium chloride-1 / 15M phosphate buffer (pH 7.2). Gave peak MII-5. Preparation of these hot water extracts and the active substance DEAE-Sepharose CL-6
FIG. 2 shows the fractionation by B column chromatography. FIG. 3 shows DEAE-Sepharose CL-6B.
The elution pattern of the active substance by column chromatography was shown. The elution pattern was shown by absorbance at 485 nm by the phenol / sulfuric acid method.

When the peaks of MII-1 to MII-5 were further applied to a Toyopearl HW-55S column, almost all peaks were obtained, and the presence of sugar components and proteins was confirmed. The five components identified were considered proteoglycans (protein-sugar conjugates).

For these five components, tumor necrosis factor (TNF-
α) Production promoting activity was measured, and the results are shown in FIG. FIG.
As is clear from the above, MII-1 to MII-5 showed strong activities. Further, the activity of promoting the production of nitric oxide (NO) from macrophages was measured according to the method described in Example 7, and the results are shown in FIG. As apparent from FIG. 5, MII-1 to MII
-5 showed a strong activity, and it was shown that a stronger promoting activity appeared by fractionation purification. In any case, pharmacological effects based on these physiological activities can be sufficiently expected.

[Example 9] A mixture prepared by the above-mentioned method, in which the ethanol extract, the hot water extract (1) and the hot water extract (2) were mixed at a volume ratio of 10: 5: 5, was mixed at room temperature. Or store under refrigeration, if necessary 3-5
ml could be used as a health drink.

Example 10 1.0 g of a freeze-dried product of the mixed solution prepared in Example 9, 78 g of lactose and 22 g of corn starch were mixed well to prepare a paste and granules. The granules are divided into 1 g portions (containing 10 mg as a dry extract composition), and 1 to 3 capsules per day are taken as needed. Also, 5 g of corn starch and 0.8 g of magnesium stearate were added to the above granules and mixed well, and the mixture was compressed using a compression tablet machine to produce 106 tablets containing 10 mg of the dry extract composition per tablet.
1-3 tablets were taken per day as needed.

[Example 11] Granules were prepared by thoroughly mixing 50 g of powder of the grass of Pseudomonas velutipes produced in Example 2, and then pulverizing the powder, 78 g of lactose and 22 g of corn starch. The granules were divided into 1.5 g portions, and 1 to 3 tablets were taken per day as needed.

[0073]

【The invention's effect】

According to the first aspect of the present invention, the fruiting body is formed from the spores of the fungus of Cordyceps sinensis with high probability and in a short period of time, and the Cordyceps sinensis can be stably formed throughout the year regardless of the season. In addition, a method for artificially cultivating cordyceps, which can be supplied in a large amount, can be provided. That is, it is possible to preferentially grow only the target microorganism without worrying about propagation contamination of other microorganisms, and by directly injecting a spore suspension of the target microorganism into the body of a sterile insect or the like, The time required for spores to enter the insect body can be reduced,
This is effective in that fruiting bodies can be obtained in a short time. Furthermore, after decompression of the insect or spider, the spore suspension is forcibly inoculated and injected into the insect or spider when the atmospheric pressure is released. The time required to spread to the whole body can be reduced, the time required to obtain fruiting bodies can be significantly reduced, and mass productivity can be expected.

According to the invention of claim 2, antioxidant activity,
Highly probable and short-term artificial cultivation of Aspergillus oryzae as a cordyceps with excellent pharmacological effects based on physiological activities such as the activity of promoting tumor necrosis factor production from macrophages and the activity of promoting nitric oxide (NO) production from macrophages A method could be provided.

According to the third aspect of the present invention, by using pupae of silkworms reared aseptically as a host, only the target microorganism can be preferentially selected with high probability and in a short period of time without worrying about propagation and contamination of other microorganisms. It was possible to provide an artificial culture method capable of growing.

According to the fourth aspect of the present invention, in order to effectively utilize the flowering bollworms of the pupae of the silkworm, the pupae of the silkworm bred aseptically and the fruiting bodies formed on the pupae, which formed the fruiting bodies obtained by the artificial culture method. It was possible to provide an extract composition of Anopheles velutipes obtained by an appropriate extraction treatment. In particular, depending on the purpose, a water-insoluble component, a water-soluble component, or both components of the cordyceps can be extracted.

According to the fifth aspect of the present invention, the extract composition of Pseudomonas velutipes is based on physiological activities such as the activity of promoting the production of tumor necrosis factor from macrophages and the activity of promoting the production of nitric oxide (NO) from macrophages. A proteoglycan (protein-sugar complex) expected to have an excellent pharmacological effect could be provided.

According to the sixth aspect of the present invention, it is possible to provide a functional food for providing humans with a dry powder of a Hanasagitake mushroom or an extract composition of a Hanasagitake mushroom which formed a fruiting body obtained by the artificial culture method. Was.

[Brief description of the drawings]

FIG. 1 is a diagram showing the antioxidant activity of α-linolenic acid autoxidation with respect to the ethanol extract and the hot water extract (1) and (2) of the insect plants of Pseudomonas velutipes according to the present invention.

FIG. 2 is a view showing preparation of a hot water extract and fractionation of an active substance by DEAE-Sepharose CL-6B column chromatography in the present invention.

FIG. 3 is a view showing an elution pattern of an active substance by DEAE-Sepharose column chromatography.

FIG. 4 is a diagram showing the activity of promoting TNF-α production from macrophages in the eluted fraction.

FIG. 5 is a graph showing the activity of promoting the production of nitric oxide (NO) from macrophages in the eluted fraction.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasuo Ainozo 7-3-3, Sumiyoshi Yamate, Higashinada-ku, Kobe-shi, Hyogo Prefecture No. 26 Maison Mikage Yamate 301 F term (reference) 2B011 AA07 BA13 GA04 JA01 MA11

Claims (6)

[Claims] 1. A method for artificially cultivating a cordyceps sinensis, wherein fruiting bodies are formed from spores of the fungus cells of a cordyceps sinensis. Or a method for artificially cultivating Cordyceps sinensis, wherein the spore suspension is forcibly infiltrated and inoculated into the body of the insect or the spider when the insect or the spider is depressurized and then released to the atmospheric pressure. 2. The fungus of the cordyceps cordyceps is
aria japonica IM2001 strain (FERM P-182
53. The method for artificially cultivating Cordyceps according to claim 1, wherein the method is 53). 3. The method for artificially cultivating Cordyceps sinensis according to claim 1, wherein the aseptically reared insect is a silkworm pupa. 4. A fruiting body is formed by using ethanol, a water-alcohol mixture or water, or an organic solvent such as butanol, ethyl acetate, butyl acetate, isobutyl acetate, n-hexane or chloroform, xylene or acetone as a solvent. It is characterized in that the insect grass (pupa of the silkworm reared aseptically and the fruit body formed on the pupa) of Isaria japonica IM2001 strain (FERM P-18253) was subjected to an extraction treatment under a heating condition of 20 to 140 ° C. or under a heating condition under pressure. Extract composition of Aspergillus oryzae. 5. The method of claim 1, wherein the flower of the flowering plant isaria japonica IM.
The extract composition of the 2001 strain (FERM P-18253) contains proteoglycan (protein-sugar complex) purified and fractionated by column chromatography such as an ion exchange resin and a gel filtration agent. Item 4. An extract composition of Hanasagi mushroom according to Item 4. 6. An Aspergillus oryzae having formed the fruiting body
ia japonica IM2001 strain (FERM P-1825
3) Dry powder of insect grass or the above-mentioned Isaria jap
Functional food comprising an extract composition of onica IM2001 strain (FERM P-18253).