KR870001416B1 - Manufacturing method of anti-cancerous polypeptide - Google Patents

Abstract

An anti-cancerous polypeptide is prepd. by culturing Bacidiomycetes. Thus, Bacidiomycetes is cultured in a medium contg. 50g glucose, 20g peptone, 0.87g KH2PO4, 0.5g MgSO4.7H2O, 0.3g CaCl2, and 10ml B.S.M. in 1 l distilled water (pH=5.0) for 14days. The supernatant obtained from the above culture broth is treated with ethanol to give anti- cancerous polypeptide.

Description

Method for preparing anticancer protein polysaccharide

1 is a chromatogram of H.P.L.C. of monosaccharides constituting the anticancer protein polysaccharide prepared according to the present invention.

2 is a chromatogram of standard amino acids.

3 is a chromatogram by an automatic amino acid analyzer for the amino acids constituting the protein portion of the protein polysaccharide prepared according to the present invention.

The present invention relates to a method for producing a protein polysaccharide having anticancer activity obtained by culturing Korean basidiomycete in a liquid medium in an industrial medium.

Until now, research on the antimicrobial, hallucinogenic, toxic, and cholesterol-reducing components of several species, which are important ingredients, has been conducted from some of the fungi belonging to the higher fungi, but the anti-cancer component has been actively studied recently. Neutrality for these products is increasing.

Studies on the active ingredients contained in basidiomycetes include the report of anti-cancer components of several species from basidiomycetes in 1955 by Bos and artificial culture of psllocybe bacteria in 1964 basidiomycetes by Catalfomo et al. Things. Recently, active studies on anticancer components of basidiomycetes have been carried out, and retinan (lentlnan) extracted from shiitake mushrooms [Lentlnus edodes (Berk) slnger] has strong anticancer activity against Sarcoma-180. I have said.

In 1974, Tugagoshi et al. Announced that protein-bound polysaccharides, one of the ingredients of the Japanese cloud mushroom [Corlolus Verslcolor (Fr.) Quel.], Have anticancer activity against Sarcoma-180. In fiscal year, polysaccharides extracted from Ganoderma lucldum were reported to have anticancer activity against Sarcoma-180.

The cultivation of Japanese basidiomycetes and the extraction of anticancer components include Korean Patent Publication Nos. 81-944 and 98-1708.

Accordingly, the present inventors have completed the present invention as a result of culturing and anticancer component of Ganoderma lucldum (Fr.) Karsten, a native mushroom grown in Korea. .

That is, an object of the present invention is to provide a method of cultivating a mushroom and a method for extracting anticancer components.

The mycelia of the mushrooms used in the present invention are freely available standard strains of the present inventors, which are preserved at the College of Pharmacy, Seoul National University for student education and research purposes. The present invention is carried out by reviewing and establishing various culture conditions for increasing the amount of cell mass produced in the liquid culture, and measuring and confirming the anticancer effect of the components extracted from the cultured mycelium as a proliferation inhibitory effect against Sarcoma-180. It was completed.

Badge composition

1) spawn medium

Sense infusion 10g, glucose 50g, peptone 5g, yeast extract 5g, K ₂ HPO ₄ 1.5g, MnSO ₄ 0.5g added distilled water to 1l, pH adjusted to 5.5 with 0.1N HCl and sterilized by adding 20g agar Afterwards, the slope medium is prepared.

* Potato Infusion is made by slicing 10g of potatoes and putting it in 500g of water and boiling.

2) Preparation of Aqueous Shake Culture Medium

A total of 81 kinds of medium numbers 1-9, which will be described later, are prepared according to each component, and sterilized, and used as a medium for shaking in liquid.

Culture method

1) spawn culture

The mycelia of the stock strains are aseptically isolated, transplanted into a spawn medium for spawn and incubated for one week at 24 ± 1 ° C to prepare spawn.

2) Primary incubation

Aseptic mycelia of the slope medium were added as a small amount of incubation medium, homogenized with a blender, and then transplanted into a 500 ml Erlenmeyer flask to which 125 ml of incubation medium was added, followed by orbital shaking at a speed of 180 rpm at 24 ± 1 ° C. Incubate for one week in the incubator.

3) Secondary liquid culture

After homogenizing the whole culture solution containing the mycelium for 10 seconds, take 10ml each with a sterile pipette and transplant into a 1l Erlenmeyer flask to which 250ml of culture medium was added and incubate for 2 weeks in an orbital shaking culture apparatus at a speed of 180rpm at 24 ± 1 ℃.

Extraction and Separation of Anticancer Ingredients

The culture solution was filtered under reduced pressure according to the method of Park et al. (1979), and the filtrate and mycelium were separated. The filtrate was concentrated and ethanol of 3 times the volume of the concentrate was added thereto, and the precipitate was left at 4 ° C. for 1 day to generate a precipitate. The precipitate was collected by centrifugation at rpm for 30 minutes, and then freeze-dried using a freeze dryer to obtain Sample A. The mycelium was refluxed for 4 hours at 95 ° C., extracted with water, filtered under reduced pressure, and then the residue was discarded. The filtrate was powdered by the same operation as in the previous method.

Samples A and B were combined to obtain total crude protein polysaccharide.

Analysis of Anticancer Ingredients

1) Analysis of polysaccharide content and constituent sugars of anticancer components

The polysaccharide content was subjected to the antron test with glucose as a control. Anthrone reaction was performed on the glucose and the sample to measure absorbance at 620 nm, and then a weight loss curve was prepared. The polysaccharide content in the sample was calculated using a calibration curve prepared with glucose as a standard.

Constituent sugar was dissolved in 2ml of 0.1N HCl 2ml and put in ampoule, filled with nitrogen and sealed and hydrolyzed at 100 ± 5 ℃ for 5 hours. After filtration and freeze drying, H.P.L.C. was carried out under the following conditions.

Column: μBondapak carbohydrate analysls

Solvent: Ac CN / H ₂ O / BuOH (80/20/15)

Flow rate: 1.5ml / mln

Detector: Rl (X8)

Chart Speed: 1cm / mln

H.P.L.C. was carried out in the same manner for each standard product having a known concentration, and chromatographic holding time was compared and identified between the standard product and the sample to identify and quantify the monosaccharides in the sample.

2) Analysis of Protein Content and Constituent Amino Acids of Anticancer Components

The protein content was subjected to the Raleigh-porin test with albumin as a control. The albumin and the sample were each subjected to a lololipoline reaction, followed by U.V. Absorbance was measured at 750 nm using a spectrophotometer. The protein content in the sample was calculated using a calibration curve prepared with albumin as a standard.

The constituent amino acids were hydrolyzed and analyzed with an amino acid autoanalyzer. 20 mg of sample was dissolved in 5 ml of 6N-HCl, placed in ampoules, filled with nitrogen and sealed. This was hydrolyzed at 110 ± 5 ° C. for 24 hours, filtered to remove precipitates, and concentrated under reduced pressure to dryness. After dissolving this in 2 ml of 0.02N-HCl, 40 μl was injected into an amino acid autoanalyzer and analyzed.

The amino acid autoanalyzer used Hitachi model 835 and the analysis conditions were as follows.

Column: 2.6 × 150mm

Ion-exchange resin: 0.225 ml / mln of # 2619 (Hitachi) buffer

Ninhydrin 0.3 ml / mln

Analysis circle time: 70mln

Column pressure: 80-130kg / ㎠

Ninhydrin pressure: 15-35kg / ㎠

Column Temperature: 53 ℃

N ₂ gas pressure: 0.28kg / ㎠

Reactor temperature: 98 ℃

Wavelength: 570nm, 440nm

Under the same conditions, 40 μl of standard amino acid was injected into the analyzer at a concentration of 3 nmol / 5 μl to obtain a chromatogram, and the constituent amino acids were identified by comparison with that of the sample, and the composition ratio of each amino acid was calculated by the peak height method.

Anticancer test

Animals used in this experiment were male A-straln mice, 25 g, purchased from Seoul National University. 50 mice were injected with 0.1 ml / (1 × 10 ⁶ cells) of a Sarcoma 180 cell suspension. Each group was made up of 10 animals, and 3 groups of control group, 20 mg / kg / day lp, and 100 mg / kg / day lp. Three days after the cancer cell transplantation, the drug was injected continuously for 10 weeks, and all rats were killed at 31 days from the day of the cancer transplantation.

As a marker of anticancer activity, the percentage of tumor inhibition (l.R. = lnhlbltlon ratlo) was calculated by the following equation.

Percentage Supported,

Cw = mean tumor weight of control

Tw = mean tumor weight of treatment group

Effect of Anticancer Compounds on Immunity

1) Experimental material

The mouse used the same mouse as the anticancer experiment.

Protein-bound polysaccharide, which was a combination of the culture filtrate and the sample extracted from the mycelium, was used as a sample.

Balanced saline solution (= B.S.S) used to suspend splenocytes was prepared as follows.

Solution 1 was dissolved in 10 g of glucose, 0.6 g of KH ₂ PO ₄ , 1.85 g of Na ₂ HPO ₄ , and 2 ml of 0.5% phenolrad solution in 1 l of deionized water.

Solution II dissolved 1.86 g of CaCl ₂ , 4.0 g of KCl, 80 g of NaCl, 2.0 g of MgCl _{2, and} 2.0 g of MgSO _{4 in} 1 L of deionized water. In use, 800 ml of distilled water, 100 ml of solution I, and 100 ml of solution II were mixed and used at pH 7.2.

Conjugatlon buffer used to suspend the antigen was used by dissolving 4.38 g of NaCl, 2.44 g of KH ₂ PO ₄ , and 10.00 g of Na ₂ HPO _{4 in} 1 l of deionized water.

Sheep red blood cells (S.R.B.C) were stored in Alsever's solutln and washed with buffer four times before use.

As a complement, a guinea pig complement (Gulnea plg complement; manufactured by M.A. Bloproducts) was used. The slide chamber was 25 mm × 7 mm, and the ratio of petrolatum and paraffin was 1: 1.

2) Experiment Method

Plaque assays were performed according to the methods of Cunnlngham (1973) and Jerone (1974).

Twenty-four mice were divided into four groups of six animals each, and as a sample administration group, the protein-bound polysaccharide (50 mg / kg / day / l.p.) Was administered as a control group, and the other two groups were administered saline with l.p. for 5 days.

Ten days after the final administration, mice were immunized with 1 × 10 7 cells of sheep red blood cells in one of the administration groups and one of the control groups. Physiological saline was administered to the remaining two groups.

After 5 days, the spleens of the mice were separated, homogenized with 10 ml of BSS, centrifuged at 2,000 rpm for 5 minutes to precipitate splenocytes, and red blood cells were hemolyzed by adding 0.83% NH ₄ Cl 5 ml at 4 ° C.

This was again washed three times with 10 ml of BSS and resuspended with the addition of a certain amount of BSS to calculate the total cell number. A portion of the suspension was taken to make a 10 ⁶ cell / ml splenocyte suspension.

30 μl of BSS, 20 μl of complement, 20% S.R.B.C. 10 μl, 100 μl of the splenocyte suspension were added, mixed well, transferred to a slide chamber, sealed with petroleum jelly and paraffin, incubated at 37 ° C. for 2 hours, and the number of hemolytic plaques formed was counted.

Example 1-10

These examples were prepared using glucose having a composition shown in Table 1 as a control to examine the availability of starch as a carbon source, adding starch to each concentration, and incubating for 14 days with the above-described secondary incubation culture. The anticancer component was analyzed by the aforementioned extraction and analysis methods, and the results are shown in Table 1.

TABLE I

Composition of Medium 1-10

* BMS (basic mineral solution): FeSO ₄ · 7H ₂ O 100mg

MnCl ₂ 4H ₂ O 70mg

ZnSO ₄ 7H ₂ O 40 mg

CuSO ₄ 5H ₂ O 10mg

100ml of distilled water

** Final volume: The volume after distilled water is added to the medium component.

Example 11-20

The present examples were obtained by adding molasses as a carbon source for each concentration, followed by incubation for 14 days using the above-described secondary liquid shaking culture method using the medium of Table II. The anticancer components were extracted and analyzed as described above. Same as 1.

TABLE II

Composition of Medium 11-20

Example 21-30

In the present examples, the Gojijak extract was added to each concentration as a carbon source, and then the availability of the medium in Table III was examined. The culture was carried out for 14 days by the above-mentioned secondary liquid shaking culture, and the anticancer components were analyzed by the above extraction and analysis method. The results are shown in Table 1.

TABLE III

Composition of medium 21-30

* Goji berry extract: Extracted goji berry with water 5 times to the residue extracted at 95-100 ℃ for 4 hours

TABLE 1

Comparison of Mycelia and Protein Bound Polysaccharides (PBP) Produced in Culture Media Using Various Carbon Sources

The unit is g / 100ml (culture medium).

Example 31-40

The present examples were added to each concentration of skim soy flour as a nitrogen source, and then cultured for 14 days by the above-described secondary liquid shaking culture method using the medium of Table IV, and analyzed the anticancer components by the above extraction and analysis method. Is shown in Table 2.

Table IV

Composition of medium 31-40

Example 41-50

The present examples were added to each concentration of CSL (conestipikriker) as a nitrogen source, followed by incubation for 14 days using the above-described secondary liquid shaking culture method using the medium of Table V, and the anticancer components were extracted and analyzed. Table 2 is as follows.

TABLE V

Composition of medium 41-50

TABLE 2

Comparison of Mycelia and Protein-bound Polysaccharides (PBP) Produced in Cultured Skim Using Most Nitrogen Sources or C.S.L.

The unit is g / 100ml (culture medium).

Example 51-60

In the present examples, the results of the culture experiments were performed using the culture medium of Table VI which changed the concentration of the existing medium to examine the availability of ginseng meal extract as a medium. 39% added showed good mycelial growth.

Table VI

The composition of the medium 51-60

* Solution A: 50g of glucose and 20g of peptone are dissolved in distilled water to make 1000ml.

Panax ginseng extract: A solution extracted at 95-100 ℃ for 4 hours by adding 5 times water to alcohol extract residue of ginseng root.

TABLE 3

As a result of examining the applicability of ginseng gourd extract as a medium, it is as follows, which is not available as a complete medium, but it is thought to have an effect of promoting the growth of mycelia at an appropriate concentration, and thus it can be added to other medium components. I think.

The unit is g / 100ml (culture medium).

Example 61-72

These Examples were cultured in the medium of Table VII by changing the concentration of phosphorus and metal ions by combining medium Nos. 4-9 and 6-8 with excellent mycelium growth in a cheap industrial medium. Same as

[Table VII]

Composition of Medium 61-72

* Goji berry extract-see table

TABLE 4

Growth and PBP Yield of Mycelium by Phosphorus and Metal Ion Concentration

The unit is g / 100ml (culture medium).

Example 73-81

In this example, the culture experiment was performed using the culture medium of Table VII in which only pH was changed under the optimum medium conditions obtained as a result of the experiment in Table VII medium, and the results are shown in Table 5.

[Table VII]

The composition of the badge 73-81

* Goji berry extract-see Table III

TABLE 5

Mycelial Production and PBP Yield with pH Change

The unit is g / 100ml (culture medium).

Example 82

In this example, in order to compare the mycelial growth according to the culture time, the mycelial growth over time was measured while shaking culture using 8-5 having the best mycelium growth in the medium 9-1 to 9-9.

The results are shown in Table 6.

TABLE 6

The unit is g / 100ml (culture medium).

Example 83

Analysis of Anticancer Ingredients

1) Analysis of polysaccharide content and constituent sugars of anticancer components

The results of analysis of the polysaccharide content by spectrophotometer and the constituent sugar by H.P.L.C. after experiment according to the antron method are shown in Table 7, Table 8 and FIG.

2) Protein Content and Amino Acid Analysis of Anticancer Components

U.V. after the Raleigh-Porrin test. Protein content calculated by the spectrophotometer and amino acid composition analyzed by the amino acid autoanalyzer after hydrolysis are shown in Table 9, Table 10 2 and 3.

TABLE 7

Protein-bound polysaccharide content

TABLE 8

Monosaccharide Content

TABLE 9

Protein Content of Protein-bound Polysaccharides

TABLE 10

Amino Acid Content of Protein-bound Polysaccharides

Experimental Example 1

Anticancer test

As a result of the anticancer test using Sarcoma-180 implanted in the cow water, the tumor weight was much smaller in the treatment group than the control group, indicating that the sample had anticancer component (Table 11).

TABLE 11

Effect of Protein Binding Polysaccharide on Sarcoma-180 Inoculated Mice

** mean ± standard deviation

** Statistical significance (P <0.01)

Experimental Example 2

Immunostimulating Effect Test of Anticancer Components

Table 12 shows the results of the immune-stimulating effect of the anticancer component by the assay. In Table 12, although the total splenocyte count was not significant, the number of hemolytic plaque cells increased significantly.

TABLE 12

Hemolytic plaque forming cells in the spleen of mice immunized with exempt red blood cells

Claims (1)

In a known method for producing anticancer protein polysaccharides by culturing the mycelium of Ganoderma Lucldum (Fr. Karsten) in a liquid medium containing an inorganic salt, gojijak extract extract using a carbon source and a nitrogen source among the components of the medium. / v%, a method for producing an anticancer protein polysaccharide using a medium having added conspitip liquor 1.5v / v% and pH of 5.5 to 6.0.

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