KR970009150B1 - Liquid culture of phellinus linteus and preparation process ofanticancer material - Google Patents

Abstract

The mycelia of Phellinus linteus (KCTC0082BP) is cultured in liquid medium containing glucose 2-10%, corn steep liquor 0.2-5% and waste yeast extracts 0.1-1.0% as essential components at 24-30 C for 2-4 days. Biomass which is precipitated by centrifuging the culture broth is treated with hot water. Then antitumoral immunity substance is extracted in ethanol precipitation and dialysis for 6-12 hrs therefrom.

Description

Method for manufacturing artificial liquid culture and anti-cancer immunoactive substance of Phellinus linteus mycelium

The present invention is a method for producing an anticancer substance useful for cancer treatment and prevention, including artificial liquid culture method of Pelinus linteus, a type of basidiomycete belonging to the polyporaceae. More specifically, the present invention relates to a new and advanced liquid culture method capable of producing inexpensive, fast and large quantities of mycelia of Felinus linteus, and to a simple method for producing a potent anti-cancer immunoactive activity from mycelium obtained by artificial culture.

Basidiomycete has long been introduced as a sensitive traditional Hanyang, and has been used as a folk remedy for various diseases (Kang Hong-Kyung, circa 500 C., and Hong-Ki-Ki, 1552). In particular, anti-cancer polysaccharides produced by basidiomycete have little side effects and are very safe in terms of toxicity, and have been known to have excellent anti-cancer effects by enhancing the function of the immune system in the human body.

As a method of using basidiomycete as described above, it can be divided into a method of collecting or cultivating fruiting bodies and cultivating the isolated mycelia after culturing the mycelia from the fruiting bodies. Among the methods of collecting and using wild fruiting bodies, there is a problem of depletion of resources and destruction of ecosystems, and in the case of rare basidiomycetes, it is absolutely impossible to secure the required amount. It costs a lot of money and can only be produced for a certain period of time, and it is not preferable because there are many constraints such as a culture period of 3 to 6 months.

Meanwhile, the method of separating and incubating the mycelium from the fruiting body can solve the above problems at one time, but this situation is not generalized due to the following technical difficulties. That is, it is not easy to separate or easily purify mycelia, and secondly, it is very difficult to establish facilities and culture conditions that can be cultured in fermenters without contamination of various germs because the culture conditions are difficult and growth is slow compared to bacteria.

In this regard, the conventional mushroom mycelium culture method is considered as follows. In other words, the mycelium cultivation method (Coriolus versicolor) of the fungi mushroom family (Korea Patent Publication No. 83-1909) is cultured using glucose and yeast extract, but this method requires the use of expensive yeast extract. There are problems in the culture method such as 28 days.

Tokoko Wada also invented a cultivation method of Ganoderma lucium (Ganoderma lucium) strain (Korean Patent Publication No. 88-2476), but this method uses a solid medium containing hardwood, sawdust and rice bran. The preparation of the medium is very cumbersome and the incubation time also usually takes 20 to 30 people.

On the other hand, the present inventors have already invented a method for cultivating mycelia of Felicity Lindeus (Korean Patent Publication No. 92-1194), but since the method used a mixed solid medium such as lumber sawdust, bran, and peptone, the preparation of the medium was very It was complex and could be produced only after 20 days of culture.

On the other hand, the most widely used YMG (YMG) medium, established by Naoaki Wadanabe (Kinoko Experiment Manual, 1991, Kangdam Company Scientific), contains 4g / g of glucose. Malt extract and yeast extract is the main medium composition in the foil, but is used as agar solid medium and the culture period also takes 2 to 3 weeks or more, there are many problems for liquid culture and yield is not very good.

The present invention not only solves the above problems at once, but also develops a revolutionary and advanced liquid culture method capable of producing with high yield and producing a substance having strong anticancer immune activity.

The present invention will be described in more detail as follows.

Felinus linteus mycelium used in the present invention used a strain deposited with KCTC 0082BP in the KIST Genetic Engineering Research Institute Genetic Bank, an international microorganism depositing institution.

In the present invention, the culture of the Felinus linteus KCTC 0082BP strain uses a nutrient source that can be used by conventional microorganisms, but its composition ratio is characteristically different from that of the existing Felinus linteus culture medium. In other words, it does not use rice bran or lumber sawdust at all and increases the amount of glucose to more than 10 times the amount of conventional medium such as YMG medium, and by using a liquid medium containing corn steep liquor and trace elements, the conventional method is greatly improved. Yet it is possible to breakthrough liquid culture that can be mass culture quickly. The liquid medium preferably contains 2 to 10% (w / v) glucose, 0.2 to 5% (w / v) cornspiker, and 0.1 to 1% (w / v) spent yeast extract. Not only glucose but also sucrose, starch syrup, dextrin, starch, molasses can be used. As a nitrogen source, peptone, malt, fish cake, ammonium sulfate, sodium nitrate, urea, etc. can be used. If necessary, it is very effective to add salts, potassium, magnesium, cobalt, chlorine, phosphoric acid and other inorganic salts that promote the formation of ions. As a culture method, shaking culture or aeration stirring in aerobic conditions is recommended.

The culture temperature is slightly different depending on the conditions when incubated in the above conditions, but it is usually appropriate to incubate at 20 ℃ to 37 ℃, preferably incubated at 24 ℃ to 30 ℃. In addition, the culture period of the shaking culture, the tank culture is usually possible for the desired mass culture by culturing for 2 days to 4 people.

Such a culture method can be incubated not only with Felinus linteus, but also with cloud mushrooms (Coriolus versicolor) or Ganoderma lucium.

After incubation, the culture solution is centrifuged to remove the supernatant, and only the cell parts are recovered. After hydrothermal treatment, the extract is subjected to ethanol precipitation and dialysis to extract and purify anticancer substances.

The temperature at the time of hot water extraction is 90-100 degreeC, Preferably it is 80-100 degreeC, The extraction time is 3-12 hours, Preferably it is 6-12 hours.

The material prepared by the present invention can be formulated as a medicament in a conventional manner using one or more pharmaceutically acceptable carriers. Thus, forms suitable for administration via a variety of routes including oral rectal, transdermal, subcutaneous, intravenous and intramuscular, such as tablets, hard and soft gelatin capsules, solutions, emulsions, suspensions, suppositories or injections Can be formulated and administered.

For the preparation of pharmaceutical preparations, the materials may be combined with a therapeutically inert inorganic or organic carrier. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used, for example, as carriers for tablets and hard gelatin capsules. Examples of suitable carriers for soft gelatin capsules include vegetable oils, waxes, fats, semi-solid and liquid polyols and the like, although in some cases a carrier may not be required. Examples of suitable carriers for the preparation of solutions and syrups are water, polyols, saccharose, invert sugar, glucose and the like. Examples of suitable carriers for the preparation of injectable solutions include water, alcohols, polyols, glycerin, vegetable oils and the like. Suitable carriers for the preparation of suppositories are natural and hardened oils, waxes, fats, semi-liquid-polyols and the like.

The pharmaceutical preparations may also contain preservatives, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for controlling osmotic pressure, buffers, coatings or antioxidants.

Dosages for cancer treatment or immunity can vary widely from a daily dosage of about 1 mg to about 5000 mg per kilogram of body weight, and will, of course, be adjusted to the individual requirements in each particular case.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited thereto, and any modifications apparent to those skilled in the art are included in the scope of the present invention.

Example 1 Liquid Culture of Felinus Linteus KCTC 0082BP Strain

For liquid culture of the Perinus linteus KCTC 0082BP strain, a medium containing 50 g of glucose per liter of medium, 10 g of peptone, 10 g of waste yeast extract, and 0.5 g of second caloric acid was used as a seed medium. Production medium includes 10 g of soluble starch per 1 liter of medium, 50 g of glucose, 5 g of waste yeast extract, 5 g of peptone, 5 g of corn steep liquor, 0.5 g of dicalcium phosphate, 0.3 g of magnesium sulfate and trace elements of MnCl ₂ , FeSO ₄ , ZnSO _A medium containing ₄ and the like was used. Both seed and production media were used after adjusting the pH to 5.0 before sterilization.

After sterilization of a 500 ml liter Erlenmeyer flask containing 50 ml of the seed medium at 121 ° C. for 20 minutes, Pelinus Linteus KCTC 0082BP hyphae on potato-glucose-agar agar medium was inoculated with a small amount of medium and inoculated at 3 ° C. at 28 ° C. Shake culture was carried out daily to obtain the primary seed culture solution. Then, a jar fermenter containing 2 liters of seed medium was sterilized at 121 ° C. for 20 minutes, and the primary seed culture solution was homogenized, inoculated with 50 ml and incubated at 28 ° C. for 3 days to obtain secondary seed culture. .

Subsequently, a 500 L fermentation vessel containing 300 L of production medium was sterilized in advance, and then inoculated with 4 L of the second seed culture solution and pre-incubated at 28 ° C. for 3 days. At this time, the culture conditions were aeration 300ℓ / min, the agitation was 100 ~ 200rpm and after sterilizing the fermentation tank of 5000ℓ volume containing 3000ℓ production medium in advance, the entire culture is aseptically transferred to aeration 3000ℓ / min, agitation is 100 Incubated for 3 days at ~ 200rpm. By carrying out the mycelium liquid culture in a continuous manner as described above it is possible to obtain a mycelia in large quantities in three days after the inoculation, whereby mycelium yield of a dry weight of 25g / ℓ could be obtained.

Example 2 Extraction of Anticancer Immunity Enhancing Active Material

The mycelium obtained in Example 1 above was extracted and purified by the following method. That is, 100 g of the obtained mycelium was placed in distilled water of 500 ml each at 90 to 100 ° C., and extracted by boiling water twice for 2 hours. The mycelium cake was removed and only the hot water extract was recovered. The hot water extract is concentrated in vacuo to 100 ml, and then added to three volumes of 95% ethanol and allowed to stand overnight, followed by centrifugation at 3000 rpm for 30 minutes. The extract thus obtained was dialyzed for about 3 days using a dialysis membrane capable of dissolving in 100 ml of water and dialysis of a substance having a molecular weight of 8000 or less, followed by freeze drying at -70 ° C. to obtain about 3 g of a polymer extract containing polysaccharide.

Example 3) Anticancer Activity

The anticancer activity was examined for the sarcoma 180 of rat sarcoma cancer transplanted with the Perinus linteus extract obtained in Example 2 to 4 weeks old IRC mice. That is, the rice coma 180 cells were transplanted intraperitoneally of the IR mice, and cultured for 7 days, and then 1 × 10 ⁶ cells were transplanted to the left groin subcutaneous of the IR mice. The test drug was dissolved in physiological saline and sterilized with 0.45 μm bacterial filtration membrane and administered intraperitoneally once a day at 100 mg / kg once every 24 hours after cancer transplantation. As a result of calculating the tumor inhibition rate by extracting the solid cancer 30 days after the cancer transplantation, the antitumor activity of about 71.5% was obtained by administering the extract of Felinus linteus as shown in Table 1.

Example 4 Anti-cancer Immunity Over Combat

Rat sarcoma after intraperitoneal fighting of the Perinus linteus extract obtained in Example 2 once daily for 8 days in an amount of 20 mg / kg and 100 mg / kg as shown in Table 1 Cancer rice coma 180 cells were transplanted intraperitoneally by 2 × 10 ⁵ cells. Five days later, the animals were killed and the cancer cells proliferated in the abdominal cavity were recovered and counted. As shown in Table 2, the recovered cancer cells were 395.4 × 10 ⁶ in the control group and 77.9 in the Felinus linteus extract 100mg / kg administration group. × 10 ^6, in the 20mg / kg administered group 184.7 × 10 the ferry Taunus rintewooseu extract administered prior to tumor implantation as ⁶ is 100mg / kg when administered represented a significant tumor growth inhibition rate in the 80.3%, 53.3% during 20mg / kg dose Anticancer immunity effect was seen.

Table 1.Scheduling the Anticancer Immunity Effect of Perinus Linteus Extract

Example 5) Immune Enhancing Activity for Antibody Production Responses

Rice coma 180 cells were transplanted intraperitoneally of the ICE mice and cultured for 7 days, and then 1 × 10 ⁶ cells were transplanted to the left groin subcutaneous of these ICE mice. Felinus linteus extract obtained in Example 2 was dissolved in physiological saline and sterilized with 0.45m bacterial filtration membrane and administered intraperitoneally 5 times at 100mg / kg once daily from 24 hours after cancer transplantation, and then 10 days later 1 × 10 ⁷ erythrocytes were administered intraperitoneally of the experimental animals and immunized by injection. 5 days after immunization, spleens of experimental animals were removed and the method of dresser (Dresser, DW, Assay for immunolobulin-secreting cells, In Handbook of experimental immunology, Vol. 2. Cellular Immunology 4th ed.Eds. DM Weir, Blakwell Scientific Pub , Oxford., Pp. 66.1-63.18, 1986). As a result, as shown in Table 3, administration of Felinus linteus extract showed an immune enhancing effect of 129-fold increase in the number of antibody-producing cells.

Example 6) Toxicity Test of Perinus linteus Extract

The extract obtained in Example 2 was intraperitoneally administered to 10 4 week old ICR mice at a concentration of 10000 mg / kg and observed for 14 days. No abnormalities were found in all 10 animals.

Claims (2)

Culturing the mycelium of Pelinus linteus in a liquid medium containing 2% to 10% glucose, 0.2% to 5% corn steep liquor, and 0.1% to 1.0% waste yeast extract, and culturing the cultured mycelium Method for producing an anticancer substance comprising the step of treating. The method of claim 1, wherein the anticancer substance is further subjected to ethanol precipitation and dialysis after the hydrothermal treatment.