Abstract:
Asn-linked glycoprotein having antitumor activity, with molecular weight of 32-84 kDa, its producing strain, production and use, adsorbs to Con. A. The process for its production comprises culturing a microorganism belonging to genus Aspergillus and isolating from the culture medium Asn-linked glycoprotein having a molecular weight of 32-84 kDa which adsorbs to Con. A, or a mixture thereof. The substance having antitumor activity is effective for treatment of solid tumors, ascites tumors, multiple cytoma and oval tumors and for the suppression of tumors.

Description:
FIELD OF THE INVENTION 
     This invention relates to Asn-linked glycoproteins having antitumor activity and molecular weights of 32-84 kDa, a mixture thereof, their producing microorganism, and the production thereof. 
     PRIOR ART 
     Fungi Aspergillus have been known for use as food additives to Miso and soysauce (Pathogenic Mycology, p. 78-80, 1987, Nanzando Publ., Tokyo) and are known to have bactericidal activities (White, E. C. et al. J. Bacteriol. 45: p. 433-422, 1942). Studies on application of these activities seemed, however, to be few (Dutcher, J. G. J. Biol. Chem. 171: 321-339, 1947). 
     PROBLEMS TO BE SOLVED BY THE INVENTION 
     We have tried to study the activities of Aspergillus and have examined its actions on cancer cells and gingival cells. 
     We have concentrated on the physiological activities of plant compositions on human and animals, and hypothesized that parasitic microorganisms of plants might have such activities. Accordingly, we have tried to isolate various microorganisms from plants and in the course of doing so have completed the present invention. 
     SUMMARY OF THE INVENTION 
     The present invention relates to a bioactive substance having antitumor activity, producing microorganism, production and use thereof. The present invention relates, therefore, to Asn-linked glycoproteins having molecular weights of 32-84 kDa and adsorbing to Con. A, or mixtures thereof. 
     According to the embodiments of the present invention, the glycoproteins or mixtures thereof are Aspergillus-derived substances, and the microorganism is Aspergillus Flavus strain SRT. 
     According to another embodiment of the present invention, the invention relates to a microorganism belonging to genus Aspergillus which produces Asn-linked glycoproteins having molecular weights of 32-84 kDa. 
     According to another embodiment of the present invention, the invention relates to a process for production of Asn-linked glycoproteins having molecular weights of 32-84 kDa, which adsorb to Con. A, comprising culturing a microorganism belonging to genus Aspergillus which produces Asn-linked glycoproteins having molecular weights of 32-84 kDa, which adsorb to Con. A, and isolating the Asn-linked glycoprotein thus produced. 
     According to still another embodiment of the present invention, the invention relates to the antitumor active agent used for the treatment of solid tumor, ascites tumor, polymorphocellular sarcoma, the suppression of metastasis of tumors, or the treatment of intraoral tumors. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1: CBS stain of the substance. 
     FIG. 2: PAS stain of the substance. 
     FIG. 3: Separation of the substance of Con. A-Sepharose. 
     FIG. 4: PAS stain of the substance by Con. A-Sepharose. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     We have tried to isolate microorganisms from plants by means of the following procedures. 
     1. Various plants, for example, grasses, trees, crops or seeds are washed with tap-water, twice washed with re-distilled water, and dried in the air in the room for overnight. 
     2. Dried plants are collected in a group. 
     3. The plants are physically crushed using homogenizer (glass-made, 20 ml, Iwaki Glass Works, Japan) or a mortar (porcelain, 120 mm φ, Tokyo Glass Works, Japan). Before crushing, the plant stems and roots are cut. 
     4. Crushed plants are suspended in re-distilled water. 
     5. Concentration and volume of the suspension in the test tubes are adjusted to equal levels. 
     6. The contaminated bacteria in the suspensions are sterilized at 121° C. for 15 minutes using autoclave (Type HA-240 MII, Hirayama Seisakusho, Japan). 
     7. The supernatant obtained from the above is spread with 0.8 ml/plate on the commercially available 5 potato dextrose agar plates containing chloramphenicol 25 mg/l (90 mm φ, Kojin Bio Inc., Japan), and incubated at 37° C. (incubater Type 1H42/42M, Yamato Sci. Inc., Tokyo), after rocking the plates. 
     8. Colonies are observed after 10 days. 
     9. Colonies at diameter 8-15 mm are collected using 5 ml screw tube bottle (Laboran, lot. No. 9-852-04, Seieido Inc., Japan). 
     10. Separated microorganisms are stored in the freezer below -20° C. (EV200N x K, Whirlpool, USA), and can also be stored at room temperature (20-25°C.) for 1-2 years. 
     Microorganism having producing activities of the substance of the present invention belongs to genus Aspergillus. Preferable strain is Aspergillus flavus which was isolated from the plants by us. Taxonomical properties are shown in the following. Example of the strain is Aspergillus Flavus STR. 
     I. The strain shows good growth on potato dextrose agar. Czapek agar and malt extract agar. and conidia are abundantly attached. Observation on the colonies grown on potate dextrose agar shows that Deuteromycotina is formed on the whole of the vesicles, with metulae 13-16×4-4.8 μm, phialide 7-9.5 ×2-2.4 μm and diameter of conidia 2.4-3.2 μm with spherical to oval and rough to warty walls. 
     II. Properties on the cultured medium 
     (1) Culture properties of the strain of the present invention are shown in Table 1. Results are shown with macroscopical observation after 10 days culture on various media at 25° C. 
     
                       TABLE 1______________________________________ Growth (Diameter of             Color of    Color of SolubleMedium colony, mm) colony surface                         colony reverse                                  pigment______________________________________Potate Good,       Yellowish-green                         Pale yellowish                                  Nonedextrose Cottony grown,          whiteagar  Conidia, slightly dense attachingCzapek Good,       Yellowish-green                         Pale yellow                                  Noneagar  Cottony growth, Conidia, slightly dense attachingMalt  Good,       Yellowish-green                         Pale yellow                                  Noneextract Cottony growth,agar  Conidia, slightly dense attaching______________________________________ 
    
     (2) Growth conditions on potate dextrose agar at 37° C. for 15 days were good growth (&gt;15 mm ). No growth was observed at 4° C. for 15 days. 
     III Physiological properties 
     (1) Growth temperature: 25-38° C. 
     (2) Optimum growth temperature: 36.5-37° C. 
     (3) Growth pH: 5.4-7.5 
     (4) Optimum pH: 6.7-7.2 
     (5) Aerobic nature: aerobic 
     Based on the above taxonomical properties, the strain is consulted with Illustrated Fungi, Vol. 2 (1991, Kodansha Publ. Tokyo ), and is referred to belong genus Aspergillus and is designated as Aspergillus Flavus SRT. The strain was deposited in National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology of 1-3, Higashi 1 chome Tsukuba-shi, Ibarakiken on Mar. 3th, 1995 and was given permanent accession No. FERM BP-5030. 
     Aspergillus Flavus , preferably Aspergillus Flavus SRT (FERM BP-5030) is cultrued in the medium for obtaining the substance of the present invention. Conventional medium for fungi is used. Submerged aeration culture is preferable used in an industrial culture. Culturing temperature is applied for suitable temperature used in the culture of fungi, i.e. at 18-37° C., preferably at 20-28° C. Culturing time is a suitable time for growth of fungal mycelia and is usually for 5-15 days, preferably 7-12 days. After cultivation, mycelia are separated such as by centrifugation and filter press. 
     In the separation of cells, filter aid, for example diatomaceous earth (Celite etc.) is added. if necessary. Separated cells are, lysed by a method used for mycelial lysis, for example combination of cell wall lysis by enzyme and detergents. Enzyme such as Novozyme 234 (Wako Pure Chemical Works, Japan) is used at 30° C. for 2 hours. Effective lysis of mycelial cells is performed by treatment of detergents such as sodiumdodecyl sulfate after enzyme treatment. 
     Composition of cell lysates was examined by protein analysis and PAS (periodic acid Schiff) stain (hereinafter designates as PAS stain), and glycoprotein is determined by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). 
     [A] Electrophoresis 
     (1) Gel: 4-20% a SDS-PAGE mini-gel (TEFCO, USA). 
     (2) Staining: CBB (Coomassie BrilliantBlue) stain (hereinafter designates as CBB stain) and PAS stain. 
     (3) Electrophoresed samples are shown in Table 2. 
     
                       TABLE 2______________________________________No.         Sample    Charged volume______________________________________1           cell lysates                 10 μl2           cell lysates                 20 μl3           cell lysates                 50 μl______________________________________ 
    
     In Table 2, electrophoresis could not be performed because of large volume of sample No. 3. Accordingly trichloroacetic acid (hereinafter designates as TCA) precipitated protein was supsended in 0.1 N NaOH 20 μl and electrophoresed. 
     [B] Molecular weight 
     Molecular weight of the protein was estimated using image analysis (AE6900M,ATTO Inc., Japan) comparing with molecular weight markers which were electrophoresed simultaneously. CBB stain of the electrophoresis is shown in FIG. 1. Approximately 20-84 kDa protein bands were observed. PAS stain of the bands resulted major 3 bands of proteins [a: estimated molecular weight; 63.8 kDa (a=65±7 kDa), b: estimated molecular weight; 59.7 kDa (b=58±6 kDa) and c: estimated molecular weight; 49.0 kDa (c=48±5 kDa)]. 
     [C] Isolation of glycoprotein 
     Cell lysis was fractionated using lectin column (5 cm×φ01.5 cm, Seikagaku Kogyo Inc., Japan), which adsorb specifically glycoprotein and analysed by SDS-PAGE. 
     (1) Experimental: Separation of glycoprotein by lectin column 
     1 Column: Concanavalin A (Con. A)-Agarose and caster lectin (RCA)-Agarose 
     2 Isolation process: 
     
         ______________________________________Equilibrium of column by buffer solution*   ↓Adsorption of cell lysis on column   ↓Fractionation of unadsorbed protein (passed fractions)   ↓Collection of adsorbed fractions and elution by an eluate** (elutedfractions)______________________________________ *Con. AAgarose: 50 mM TrisHCl (pH 7.2), 0.1M NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2 RCAAgarose: 0.1M K--PO.sub.4 (pH 7.2), 0.15M NaCl **eluate: Con. AAgarose: buffer containing 0.2M methylD-mannoside RCAAgarose: buffer containing 0.2M galactose 
    
     [D]Electrophoresis 
     (1) Gel: 4-20% SDS-PAGE mini-gel (TEFCO, USA) 
     (2) Staining: CBB stain and PAS stain 
     (3)Electrophresed samples are shown in Table 3. 
     
                       TABLE 3______________________________________No.  Sample         Charged amount                           Note______________________________________1    cell lysates (pre-fraction)                15 μl   --2    Passed fraction                50 μl   TCA precipitation*3    Passed fraction               100 μl   TCA precipitation*4    Passed fraction               150 μl   TCA precipitation*5    Eluted fraction               100 μl   TCA precipitation*6    Eluted fraction               200 μl   TCA precipitation*7    Eluted fraction               500 μl   TCA precipitation*______________________________________ *TCA (Trichloroacetic acid) precipitation method: Sample ↓ ← 1/10 vol. 0.15% sodium deoxycholate room temp., 10 min. ↓ ← 1/10 vol. 72% TCA 4000 × g, 20 min. centrifuge Precipitation, 20 μl 0.1N--NaOH suspension 
    
     Con. A and RCA of the lectins are used in this invention. Con. A binds with polymannose type and complex type side chains of the sugar chains, which have more than two α-mannose residues of unsubstituted C-3, 3, 6-hydroxyls in asparagine (Asn)-linked glycoprotein, RCA has strong affinity for N-acetylglucosamine (Gal β1-4GlcNAc) in asparagine (Asn)-sugar chain and mucin sugar chain Gal β-3GalNAc. No adsorbed protein in RCA-Sepharose was found. Some protein are adsorbed in the Con. A-Sepharose column (FIG. 3). PAS stain is shown in FIG. 4. As a result, Asn-linked glycoprotein Result of which absorbed in Con. A column is found in cell lysates. 
     Most preferable microorganism in this invention is Aspergillus Flavus SRT (FERM BP-5030). However microorganisms are easily mutated, therefore natural and artificial mutants of Aspergillus Flavus SRT (FERM BP-5030) as well as the strains belonging to Aspergillus Flavus which can produce the compound of the present invention are all involved in the present invention. 
     EMBODIMENTS OF THE INVENTION 
     Following examples illustrate the present invention, but are not construed as limiting. 
     EXAMPLE 1 
     One loopful Aspergillus Flavus SRT (FERM BP-5030) was inoculated in liquid medium 100 ml in 500 ml Sakaguchi flask and shake cultured at 27° C. for 10 days. The cultured broth was inoculated into liquid medium 10 lit. in 30 lit. jar-fermenter and cultured at 27° C. for 10 days under aeration 1 lit./min. with stirring at 150-200 rpm. Cells were separated by centrifugation to obtain wet cells 1 g. Novozyme 234 (Wako Pure Chem. Co., Japan) 0.5 mg/10 mg of dry cells were added to the cells and treated at 30° C. for 2 hours. Treated cells were washed with 50 mM phosphate buffer solution pH 7.2 and the cells were lysed with 1% SDS. The mycelial lysate was centrifuged at 10×g for 20 minutes. Supernatant was lyophilized to obtain lyophilyzate 1 mg. 
     The lyophilyzate was dissolved in a small amount of 50 mM phosphate buffer pH 2.2 and electrophoresed by SDS-PAGE to determine protein compositions. Glycoprotein was detected by PAS stain. Conditions of electro-phoresisis are shown below and SDS-PAGE was performed by the conventional method. 
     (1) Gel: 4-20% SDS-PAGE mini-gel (TEFCO, USA) 
     (2) Staining: CBB stain and PAS stain 
     (3) Electrophresed samples are shown in Table 4. 
     
                       TABLE 4______________________________________No.       Sample        Charged amount______________________________________1         solution of lyophilizate                   10 μl2         solution of lyophilizate                   20 μl3         solution of lyophilizate                   50 μl______________________________________ 
    
     In Table 4, electrophoresis could not be performed because of large volume of sample No. 3. Accordingly trichloroacetic acid (hereinafter designates as TCA) precipitated protein was suspended in 0.1 N NaOH 20 μl and electrophoresed. 
     
         ______________________________________Sample 50 μl↓ ← 5 μl 0.15% sodium deoxycholate↓ room temperature 10 min.↓ ← 5 μl 72% TCA↓ 4000 × g, 20 min. centrifugeprecipitation, dissolved with 20 μl 0.1N NaOH suspension______________________________________ 
    
     Molecular weight of the protein was estimated using image analysis comparing with molecular weight markers which were electrophoresed simultaneously. CBB stain of the electrophoresis is shown in FIG. 1. Approximately 20-84 kDa protein bands were observed. PAS stain of the bands (FIG. 2) resulted major 3 bands of proteins [a: estimated molecular weight; 63.8 kDa (a=65±7 kDa), b: estimated molecular weight: 59.7 kDa (b=58±6 kDa) and c: estimated molecular weight; 49.0 kDa (c=48±5 kDa)]. 
     The major bands were shown high C DB staining and were estimated as glycoprotein. 
     EXAMPLE 2 
     Lyophilized powder 1 mg obtained in Example 1 was dissolved in 50 mM phosphate buffer, pH 7.2, 50 ml. The solution was fractionated using lectin column (length 5 cm×diameter 1.5 cm, Seikagaku Kogyo Inc. Japan) and analysed by SDS-PAGE (TEFCO, USA). 
     1. Separation of glycoprotein by lectin column 
     (1) Column: Concanavalin A (Con. A)-Agarose and caster lectin (RCA)-Agarose 
     (2) Isolation process: 
     
         ______________________________________Equilibrium of column by buffer solution*   ↓Adsorption of lyophilized powder solution obtained in Example 1   ↓Fractionation of unadsorbed protein (passed fractions)   ↓Collection of adsorbed fractions and elution by an eluate**(eluted fractions)The obtained fractions were lyophilized to obtain lyophilizate; a: 0.2mg,b: 0.1 mg, and c: 0.1 mg.______________________________________ *buffer solution: Con. AAgarose: 50 mM TrisHCl (pH 7.2), 0.1M NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2 RCAAgarose: 0.1M K--PO.sub.4 (pH 7.2), 0.15M NaCl **eluate: Con. AAgarose: buffer containing 0.2M methylD-mannoside RCAAgarose: buffer containing 0.2M galactose 
    
     2. Electrophoresis 
     (1) Gel: 4-20% SDS-PAGE mini-gel (TEFCO, USA) 
     (2) Staining: CBB stain and PAS stain 
     (3) Electrophresed samples are shown in Table 5. 
     
                       TABLE 5______________________________________No.  Sample        Charged amount                           Note______________________________________1    Solution of lyophilized               15 μlcell lysates(pre-fractionation)2    Passed fractionation               50 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed3    Passed fractionation              100 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed4    Passed fractionation              150 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed5    Eluted fractionation              100 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed6    Eluted fractionation              200 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed7    Eluted fractionation              500 μl    TCA precipited                           protein was                           suspended in 20 μl                           0.1N NaOH and                           electrophoresed______________________________________ 
    
     TCA precipitation was performed as follows. 
     
         ______________________________________Sample 50 μl↓ ← 1/10 vol. 0.15% sodium deoxycholate↓ room temperature 10 min.↓ ← 1/10 vol. 72% TCA↓ 4000 × g, 20 min. centrifugeprecipitation, dissolved with 20 μl 0.1N NaOH suspension______________________________________ 
    
     Con. A and RCA of the lectins were used in this invention. Con. A binds with polymannose type and complex type side chains of the sugar chains, which have more than two α-mannose residues of unsubstituted C-3, 3. 6-hydroxyls in asparagine (Asn)-linked glycoprotein. RCA has strong affinity for N-acetylglucosamine (Gal β1-4GlcNAc) in asparagine (Asn)-sugar chain and mucin sugar chain Gal β-3GalNAc. No adsorbed protein in RCA-Agarose was found. Some protein were adsorbed in the Con. A -Agarose column and staining by PAS stain was also observed. As a result, Asn-linked glycoprotein which adsorbed in Con. A column is found in cell lysates. 
     EXAMPLE 3 
     Antitumor Activity and Safety Tests 
     Sarcoma-180 was inoculated in subcutaneously or intraperitoneally in mice and the substance of the present invention was administered to determine life prolongation effect and suppression of tumor growth. 60 mice, STD:DDY, male, 4 weeks old, SLC Inc., Japan were acclimatized for 2 weeks and provided experiment at 6 weeks old. Dose and administration were performed by the following methods. 
     a) Lyophilized powder 1 mg obtained in Example 1 was dissolved in physiological saline 10 ml. 0.1 ml of the solution was subjected to 2-fold dilution and administered (0.2 ml/mouse) intraperitoneally every other days. Control was administered physiological saline (0.2 ml/mouse) i.p. every other days (ascites tumor mice). 
     b) 10 μl of the above solution were administered in the center of solid tumor in every other days using micro syringe (MS-N 100, Itoh Seisakusho Inc., Japan). Same volume of physiological saline was administered as a control (solid tumor mice). Substance of the present invention was started to administer in day 2 after inoculation of tumor (5 times administered 5 group) and day 4 (4 times administered group). 
     Sarcoma-180 was used. Tumor cells 5×10 8  /0.2 ml was administered i.p. for ascites tumor type. Weight of ascites tumor was determined by that ascites tumor was adsorbed in dry cotton and reducted from body weight. Solid tumor weight was determined by that right lower limbs combined with tumor were amputated at the inguinal region and the weight thereof was reducted from mean of the weight of left lower limbs. 
     Type of tumor, terms for administration after transplantation of the tumor and administration routes were defined as follows. 
     A) Ascites tumor (administered on and from day 2 after tumor inoculation, administered every other days for 5 times) 
     1) Ascites control (physiological saline, i.p. 0.2 ml/mouse, n=5) 
     2) Ascites experimental (substance, i.p. 0.2 ml/mouse, n=6) 
     B) Solid tumor (administered on and from day 2 after tumor inoculation, administered every other days for 5 times) 
     3) Solid control (physiological saline, 10 gl/mouse, n=5) 
     4) Experimental (substance administered, in tumor; 10 μl/mouse, n=6) 
     5) Experimental (substance administered, i.p., 0.2 ml/mouse, n=6) 
     C) Solid tumor (administered on and from day 4 after tumor inoculation, administered every other days for 5 times) 
     6) Experimental (substance administered, i.p./in tumor, 0.2 ml/mouse and 10 μl/mouse, respectively, n=6) 
     D) Safety test (substance, administered in normal healthy mice, every other days for 10 times) 
     7) Safety test (substance, i.p., 0.2 ml/mouse, n=6) 
     E) Control (physiological saline, administered in normal healthy mice, every other days for 5 times or 10 times) 
     8) Control (physiological saline, administered every other days, 5 times, i.p., 0.2 ml/mouse, n=6) 
     9) Control (physiological saline, administered every other days, 10 times, i.p., 0.2 ml/mouse, n=5) 
     Results 
     Antitumor Effect of the Substance Obtained in Example 1 for Sarcoma-180 
     Body weights of Sarcoma-180 inoculated i. p. mice were all increased after 4 days of inoculation. Significant differences of the increased body weight in ascites control group (day 4, p&lt;0.01) and substance administered ascites group (day 5, p&lt;0.01) compared to the normal control group (non tumor bearing group) were observed. Body weight of the substance i.p. administered group (ascites tumor group) seemed to be suppressed after 2nd administration (after 4 days of tumor inoculation). Results are shown in Table 6. 
     
                       TABLE 6______________________________________Days after tumor implantation0         2       4        6      8      10______________________________________Control (physiological saline, i.p. n = 6)Mean  30.8    31.4    32.0   32.4   34.8   34.6(S.E.) g (0.34)  (0.38)  (0.38) (0.45) (0.45) (0.49)Control, ascites (physiological saline, i.p. n = 5)Mean  30.1    30.5     34.1**                         37.5**                               39.9** 39.5**(S.E.) g (0.41)  (0.48)  (0.95) (1.20) (1.59) (1.91)Substance administered group, ascites (i.p. n = 6)Mean  30.5    31.7    35.5    35.7**                                38.8***                                      39.3(S.E.) g (0.59)  (0.73)  (0.84) (0.93) (0.53) (2.19)______________________________________ Substance of the present invention administered group: Administration of the substance initiated after 2 days implantation of tumor cells intraperitoneally. Significant difference for the control group **: p &lt; 0.01, ***: p &lt; 0.001 S.E.: Standard error. 
    
     Body weight of the substance administered group (solid tumor group) seemed to increase compared to normal healthy control group after 6 days of tumor inoculation. No body weight difference was observed among the tumor inoculated groups. Results are shown in Table 7. 
     
                       TABLE 7______________________________________Days after tumor implantation0          2       4        6     8      10______________________________________Control (physiological saline, i.p. n = 6)Mean  30.8     31.4    32.0   32.4  34.8   34.6(S.E.) g (0.34)   (0.38)  (0.38) (0.45)                               (0.45) (0.49)Control, Solid tumor (physiological saline,administered in tumor, n = 5)Mean  30.2     30.6    32.2   33.7  36.0   36.2(S.E.) g (0.51)   (0.42)  (0.44) (0.57)                               (0.62) (0.77)Substance administered group, solid tumor(administered in tumor, n = 6)Mean  31.8     31.8    33.3   34.7   36.8* 36.6(S.E.) g (0.36)   (0.42)  (0.67) (0.71)                               (0.77) (0.98)Substance administered group, solid tumor(i.p. administration, n = 6)Mean  30.9     31.1    31.7   33.2  36.4   36.1(S.E.) g (0.56)   (0.55)  (0.61) (0.67)                               (0.82) (0.87)______________________________________ Substance of the present invention administered group: Administration of the substance initiated after 2 days of implantation of tumor cells intraperitoneally or in the solid tumor. Significant difference for the control group **: p &lt; 0.05. S.E.: Standard error 
    
     The same tendency was observed in the substance administered groups after 4 days of tumor inoculation. Results are shown in Table 8. 
     
                       TABLE 8______________________________________Days after tumor implantation0         2       4        6      8      10______________________________________Control (physiological saline, i.p. n = 6)Mean  30.8    31.4    32.0   32.4   34.8   34.6(S.E.) g (0.34)  (0.38)  (0.38) (0.45) (0.45) (0.49)Control, solid tumor (physilogical saline, administered in tumor, n = 5)Mean  30.2    30.6    32.2   33.7   36.0   36.2(S.E.) g (0.51)  (0.42)  (0.44) (0.57) (0.62) (0.77)Substance administered group (administered in the tumor or i.p. n = 6)Mean  30.8    31.6     33.7*  34.7**                               35.6   36.3(S.E.) g (0.33)  (0.51)  (0.59) (0.49) (0.45) (0.60)______________________________________ Substance of the present invention administered group: Administration of the substance initiated after 4 days of implantation of tumor cells intraperitoneally. Significantly difference for the control group *: p &lt; 0.05, **: p &lt; 0.01 S.E.: Standard error 
    
     Life-prolongation Effect in the Substance i.p. Administered Group 
     In the ascites control group, death was observed after 7 days of tumor inoculation (one animal, 1/5), one animal in day 8 (2/5) and one in day 10 (3/5). No death in the substance administered group was observed (on the day 10 after inoculation). Result is shown in Table 9. 
     
                       TABLE 9______________________________________Days after tumor implantation1    2↓       3      4↓                   5    6↓                             7    8↓                                       9    10↓______________________________________Control (n = 5)Survival rates (%)100  100    100    100  100  100   80   60   60   40(5/5)(5/5)  (5/5)  (5/5)                   (5/5)                        (5/5)                             (4/5)                                  (3/5)                                       (3/5)                                            (2/5)Substance administered group (n = 6)Survival rates (%)100  100    100    100  100  100  100  100  100  100(6/6)(6/6)  (6/6)  (6/6)                   (6/6)                        (6/6)                             (6/6)                                  (6/6)                                       (6/6)                                            (6/6)______________________________________ ↓: administration of the substance of the present invention 
    
     Organ and Tumor Weights of Tumor Bearing Mice 
     Organ weights (liver, kidneys, spleen and thymus) of mice of the ascites control group were decreased to 70-80% of the weight of organs compared to those of normal control group. Thymus weight was decreased 27%. In the substance administered group, increase in the weight of kidneys and spleen (121% and 142%), and significant decrease in the thymus (47%, p&lt;0.001) was observed. Weights of ascites were 16.25 g in the ascites control group and 11.35 g in the substance administered group as shown in 
     
                       TABLE 10______________________________________Liver      Kidneys   Spleen   Thymus  Ascites(mg)       (mg)      (mg)     (mg)    (mg)______________________________________Control (n = 6)Mean  1982     529       166    55      --(S.E.) (39)     (14)      (10)   (3)     (--)Control, ascitesMean  1588     370       130    15      16.25(S.E.) (--)     (--)      (--)   (--)    (--)Substance administered group (n = 6)Mean  1747      641**    236      26*** 11.35(S.E.) (166)    (27)      (30)   (3)      2.75______________________________________ Significant difference for the control group **: p &lt; 0.01, ***: p &lt; 0.001 S.E.: Standard error 
    
     Weights of liver, kidneys and spleen of the substance administered group in the solid tumor groups after 2 days of tumor inoculation were increased compared to normal control, and the thymus weight was slightly decreased. Weight of solid tumor in the substance administered in tumor group is 529±149 mg, which is significant difference (p&lt;0.05) from the solid tumor control of 1564±396 mg Weight of the substance i.p. administered group is 1005±384 mg. which shows decrease in weight without significant difference. Results are shown in Table 11. 
     
                       TABLE 11______________________________________Liver     Kidney   Spleen    Thymus Weight of(mg)      (mg)     (mg)      (mg)   tumor (mg)______________________________________Control (n = 6)Mean 1982     529      166     55     --(S.E.) (39)    (14)     (10)    (3)    (--)Control, solid tumor (n = 5)Mean 2434*     597*    284*    47      1564*(S.E.)(153)    (22)     (38)    (4)    (397)Substance administered group, in the tumor (n = 6)Mean 2404*    550      237*    54      529*(S.E.)(132)    (27)     (24)    (3)    (149)Substance administered group, i.p. (n = 6)Mean 2332*    540       273***  46*   1005(S.E.)(131)    (13)     (18)    (1)    (384)______________________________________ Substance administered group: Administration of the substance initiated after 2 days of tumor implantation. Significant difference for the control group *: p &lt; 0.05, ***: p &lt; 0.001 
    
     In the substance administered in tumor and i.p. group, for which the substance as administered after 4 days of tumor inoculation, significant increase in liver and spleen weights was observed. Tumor weight was 1167±244 mg. Results are shown in Table 12. 
     
                       TABLE 12______________________________________Liver       Kidneys Spleen   Thymus Weight of(mg)        (mg)    (mg)     (mg)   tumor (mg)______________________________________Control (n = 6)Mean 1982       529     166    55     --(S.E.) (39)      (14)    (10)   (3)    (--)Control, Solid tumor (n = 5)Mean 2434*       597*    284** 47     1564(S.E.)(153)      (22)    (38)   (4)    (397)Substance administered group in the tumor/i.p. (n = 6)Mean  2505***   513      358** 49     1167(S.E.)(107)      (18)    (29)   (1)    (244)______________________________________ Substance administered group: administration of the substance initiated after 4 days of tumor implantation. Significant difference for the control group *: p &lt; 0.05, **: p &lt; 0.01, ***: p &lt; 0.001. S.E.: Standard error 
    
     Safety Test of Consecutive Administration (10 Times for Every Other Days) of the Substance Obtained in Example 1 
     Body weight changes 
     Body weight changes of the normal mice with the substance administered every other days for 10 times were measured. No difference between the substance administered group and control was observed. In the control group, normal increase in body weight was observed. Decrease in mean body weight in the substance administered group compared to the control group was observed after 12 days (6th administration). Significant difference was observed in day 16 (8th administration, p&lt;0.05), and this was continued to day 20 (10th administration). One animal was died after 8th administration (day 19). Result is shown in Table 13. 
     
                       TABLE 13______________________________________Days0          2↓              4↓                       6↓                             8↓                                    10↓______________________________________Control (physiological saline, i.p. n = 5)Mean  30.6     31.3    32.5   33.4  34.7   35.2(S.E.) g (0.38)   (0.40)  (0.31) (0.38)                               (0.41) (0.39)Substance administered group (i.p., n = 6)Mean  30.9     31.2    33.0   34.2  34.8   35.4(S.E.) g (0.66)   (0.52)  (0.63) (0.75)                               (0.38) (0.49)______________________________________ 12↓          14↓                  16↓                         18↓                               20↓                                      22↓______________________________________Control (physiological saline, i.p., n = 5)Mean  37.0     37.9    38.5   38.5  38.2   38.6(S.E.) g (0.36)   (0.32)  (0.37) (0.37)                               (0.34) (0.70)Substance administered group (i.p., n = 6)(one animal dead on day 19)Mean  35.7     36.5     36.5* 37.7  37.8   38.0(S.E.) g (0.49)   (0.53)  (0.53) (0.64)                               (0.85) (077)______________________________________ ↓: Administration of the substance (number of administration: tota 10) Significant difference for the control *: p &lt; 0.05 S.E.: Standard error 
    
     Weight of liver. kidneys and thymus in both of the administered and the control groups were almost same levels. Weight of the spleen was significantly increased to 165% (p&lt;0.01). Result is shown in Table 14. 
     
                       TABLE 14______________________________________Liver (mg) Kidneys (mg)                  Spleen (mg)                            Thymus (mg)______________________________________Control (n = 5)Mean  2143     583         127     45(S.E.) (73)     (16)        (11)    (7)Substance administered group (n = 6)Mean  2308     532          210**  44(S.E.) (64)     (31)        (13)    (4)______________________________________ Substance administered group: Substance was administered on the every other days, total 10 days. Significant difference for the control **: p &lt; 0.01. S.E.: Standard error. 
    
     Variations of leukocytes counts and erythrocyte count and hemoglobin weights were observed in leukocytes counts 10000/μl (control group) and 124000/μl (administered group). Erythrocyte count and hemoglobin weight were decreased, especially significant decrease in hemoglobin weight of 82% in the administered group compared to the control (p&lt;0.05). Results are shown in Table 15. 
     
                       TABLE 15______________________________________No. of leukocytes          No. of erythrocytes                        hemoglobin(μl/ml)     (10.sup.4 /ml)                        (g./dl)______________________________________Control (n = 5)Mean  10000        931           14.0(S.E.) (1300)       (33.3)        (0.28)Substance administered group (n = 6)Mean  12400        829            11.5*(S.E.) (3130)       (51.6)        (0.79)______________________________________ Significant difference for the control *: p &lt; 0.05. S.E.: Standard error. 
    
     Result of antitumor effect of the substance obtained in Example 1 is summarized as follows. 
     Intraperitoneal administration of the substance in Sarcoma-180 ascites tumor bearing mice was confirmed by increased life prolongation and suppressive effect against ascites tumor. Intraperitoneally inoculated ascites tumor mice (control) dead on day 7 after inoculation. Survival rate on the day of the biopsy (day 11) is 40% with mean ascites weight of 16.25 g. No death was observed in the substance administered group. Lean ascites weight was 11.35±2.57 g. In the intraperitoneal cavity of the substance administered group, white aggregates, which seemed to be tumor cells, were observed. This was estimated to be a result of an effect of the substance. 
     Significant increase in erythrocyte counts and hemoglobin weight of the ascites control compared to the normal control were observed, which might be a deterioration of the symptom. In the hematological findings of the substance administered group, significant decrease compared to the normal control was observed. However, it showed almost normal value with slight good condition contrary to the ascites control. Decreased weight of the liver, kidneys, spleen and thymus (atrophy) in the ascites control compared to the normal control were observed. The substance administered group showed increased weights of kidneys and spleen, which indicated the homeostasis of the animals. 
     In Sarcoma-180 solid tumor inoculated in the right femoral region subcutaneously, no death was observed in the experimental groups. Tumor weight of the substance administered in tumor group was about 33% ; the substance i.p. administered group was 64% ; and the substance administered in tumor/i.p. group was 74%, compared to the solid tumor control group. Tumor regession below 0.4 g) was observed in the half number of the substance administered in tumor group. Significant decrease (p&lt;0.05) in the mean tumor weight compared to solid tumor control was observed. In the solid tumor control, increase in leukocytes, decrease in erythrocyte (p&lt;0.05) and hemoglobin (p&lt;0.05) compared to the normal control were observed. No significant difference between the normal control and the substance administered in tumor group was observed and the value was almost equal level to the normal mice. 
     Safety of the Consecutive Administration of the Substance 
     The substance 1 mg was dissolved in physiological saline 10 ml. 0.2 ml thereof was administered intraperitoneally in mice on the every other days, total 10 times. No death was observed, except one died on day 19 (after 8th administration). Body weight decrease was observed after 12-20 days of administration compared to the normal control and was to be an effect of the substance. Increase in leukocytes counts and decrease in erythrocyte count without significant difference and significant decrease in hemoglobin (p&lt;0.05) were observed in the substance administered group. From these symptoms, anemia was suspected. Significant increase in spleen weight was observed. 
     Above results indicate no problems on safety of the substance. 
     EXAMPLE 5 
     The substance 1 mg obtained in Example 1 was dissolved in 50 mM phosphate buffer, pH 7.2, 10 ml and was set to the original preparation. Keratinocytes (GK cells) and fibroblasts (GF cells) originated from human gingiva isoalted in Dept. of Preventive and Community Dentistry, School of Dentistry at Tokyo, The Dental University of Nippon were used. HeLa S 3  cells of human uterocervical cancer origin and HT-1080 cells of human lung cancer origin were also used. Cultivations of human gingival fibroblasts and cancer cells were performed in 10% bovine fital serum added Eagle&#39;s MEM and cultivation of keratinocytes was performed in serum-free medium for keratinocytes (SFM, Gibco), at 37° C. under 5%-CO 2  -95% air with 100% humidity. 
     Aliquot of the original extract, in its 10-fold stepwise dilution with PBS (-), was added to the cell cultured liquid. Viability of the cells was made by Trypan blue. DNA synthesis of the cells was measured by after treatment of the cells with the extracts of various concentration for 3 hours, pulse-labelling the cells with 37 KBg/ml of  3  H-thymidine, and radioactivities of acid insoluble fraction of the cells were measured. 
     DNA synthesis after treating with the cellular extracts is shown in Table 16. DNA syntesis was suppressed by the extracts dose-dependently. The suppression was observed higher in cancer cells than in adult gingival cells. 
     
                       TABLE 16______________________________________Concentration of      Uptake of .sup.3 H-tymidine (control %)the substance*      GK cell GF cell  HeLa S.sub.3 cell                               HT-1080 cell______________________________________10.sup.-6  99      103      107     10710.sup.-5  102     103      93      9510.sup.-4  99      101      82      8710.sup.-3  93      93       61      6310.sup.-2  84      85       26      2010.sup.-1  61      64        3       2______________________________________ *Dilution rate of the original extraction