TW575425B - Extract of shark cartilage and method for producing the same - Google Patents

Abstract

A method for producing an extract of shark cartilage comprises: (a) using an aqueous solution causing no harm to the shark cartilage to extract fragments of shark cartilage for a few hours to a few days and separating from the solid residues in order to obtain a crude extract; (b) separating out effective ingredients with a molecular weight of 8 KDa to 25 KDa from the crude extract; which is characterized in that the extraction temperature in Step (a) is room temperature.

Description

575425 A7 B7 M series λ ^ Άηίι ^ ρι 5. Description of the invention (1) Summary of the invention The present invention relates to a kind, in particular to a shark cartilage extract composition containing 8KDa to 25KDa, its preparation method and use. It is known that shark cartilage extract composition has a curative effect on various diseases, for example, see U.S. Patent Nos. 3895103, 50751 12, 5562535, 5618925, 5843920, and 58835. Among them, those used for anti-neovascular effects and / or tumor suppression are: Patent Nos. 50751 12, 5618925, 5843920, and Patent No. 50751 12 is used after shark cartilage is shredded, and its effective ingredients are not extracted: Patent No. 5618925 is a solid active ingredient after extraction, the molecular weight of which is 2.5KDa, 5.3 ~ 6.25KDa, 7.29 ~ 7.94KDa, 29KDa, 35KDa, 48KDa, 60 KDa, 60 ~ 70KDa, 70 ~ 120KDa and other active ingredients or Its combination; and Patent No. 5843920 uses the complex formed by the protein of shark cartilage and the added oligosaccharide as an active ingredient; both are in accordance with the present invention in the shark cartilage extract with a molecular weight of about 1OKDa and About 14KDa differs as an active ingredient. In addition, the patents related to the treatment or extraction of shark cartilage: Patent Nos. 5562535, 5618925 and 5843920, of which patent No. 5562535 is simply taking shark cartilage, but instead of heating with sound waves when drying and dehydrating, that is, The effective components of shark cartilage are not extracted or isolated; Patent No. 5618925 extracts shark cartilage at about low temperature to obtain solids and crude extract after extraction, and then separates the molecular weight of 1 ~ 2.5KDa from the crude extract. , 5.3 ~ 6.25KDa, 7.29 ~ 7.94KDa, 29 KDa, 35KDa, 48KDa, 60KDa, 60 ~ 70KDa and 70 ~ 120KDa active ingredients, the method must be at about 4 ° C (please read the precautions on the back before filling (This page) * νϋ_— φ.ιϋ ϋϋ ·· I it —ϋϋ · Order -------- II ί I -......- I 1 = 1 1— * I-: ri-— 11 II .....- This paper size applies Chinese National Standard (CNS) A4 (210X 297mm) 575425 A7 B7 _ 5. Description of the invention (2) (Please read the precautions on the back before filling this page) Extraction is more suitable for laboratories and not suitable for mass production. Patent No. 5843920 is based on oligosaccharides and Cartilage complexes formed, if necessary, and then separated from the inorganic salt of the complex effect of anti-angiogenesis protein. The invention extracts shark cartilage at room temperature, removes solids to obtain a crude extract, and then separates an effective ingredient containing 8KDa ~ 25KDa from the crude extract. The effective ingredient is different from the effective ingredient of the previous technology. The extraction method is also different from the above-mentioned prior art. An object of the present invention is to provide a shark cartilage extract composition. Another object of the present invention is to provide a method for preparing a shark cartilage extract composition. Another object of the present invention is to provide a shark cartilage extract composition which can be used for anti-angiogenesis. Yet another object of the present invention is to provide a shark cartilage extract composition that can be used to suppress tumors. Brief Description of the Figures Figure 1 is an SDS / PAGE chart of a shark cartilage extract composition of the present invention. Fig. 2 is a diagram of the benefit of inhibiting HUVEC movement of the shark cartilage extract composition of the present invention. Fig. 3 is a diagram showing the benefit of the shark cartilage extract composition for inhibiting the proliferation of HUVEC. Fig. 4 is a diagram showing the effect of the shark cartilage extract composition of the present invention on inhibiting collagen degradation activity. Fig. 5 is an antiangiogenic effect of the shark cartilage extract composition of the present invention. -4 _ ^ Paper size suitable for China National Standard (CNS) A4 specification (210X297 mm) 575425 A7 B7 i. Description of the invention (3) Figure 6 is a benefit chart of the shark cartilage extract composition of the present invention for inhibiting the growth of tumor cells . Fig. 7 is a diagram showing the benefit of inhibiting the migration of tumor cells by the shark cartilage extract composition of the present invention. Fig. 8 is a diagram showing the benefit of inhibiting the migration of tumor cells by the shark cartilage extract composition of the present invention. The method for preparing a shark cartilage extract composition according to the present invention comprises: (a) extracting shark cartilage fragments with an aqueous solution that does not degrade shark cartilage; for several hours to several days, separating solids to obtain a crude extract; (b) extracting the crude extract from the crude extract; The effective component with a molecular weight of 8KDa to 25Kda is separated in the liquid; it is characterized in that the extraction temperature in step (a) is room temperature. Step (a) can use any aqueous solution that does not degenerate shark cartilage, such as an aqueous solution containing guanidine, an aqueous solution containing MES [ie, 2- (N-morpholino) ethanesulfonic acid], an aqueous solution of a mixture or composition thereof, An aqueous solution of an individual derivative thereof, an aqueous solution of an individual salt thereof (for example, guanidine · HC1), or an aqueous solution of a mixture or composition of the derivative and / or salt. In order to enable step (a) to perform extraction at room temperature, it is preferable to extract shark cartilage using an aqueous solution containing guanidine and MES, and an aqueous solution containing more than 0.1M guanidine and 0.005M to 0.1M is more preferable. The so-called room temperature means that the mother needs to deliberately warm up or cool down the ambient temperature 'or a similar temperature. Of course, step (a) can also be performed under low-temperature cooling or heating, but lowering the temperature will slow down the extraction speed, and excessive heating will change the shark cartilage. The paper size is in accordance with China National Standard (Oyang) 6-4. (2 丨 0/297 male thin) (Please read the notes on the back before filling this page) m Order

575425 A7 B7_ 5. Description of the invention (5) KDa, 35KDa, 48KDa, 60KDa, 60 ~ 70KDa and 70 ~ 120KDa active ingredients. The effective ingredient of the shark cartilage extract composition of the present invention has a molecular weight of 8KDa to 25KDa, and an effective ingredient containing a molecular weight of about 10KDa and / or 14KDa is preferred. The shark cartilage extract composition of the present invention containing an active ingredient with a molecular weight of 8KDa to 25KDa has an anti-angiogenic effect and / or a tumor suppressing function. When necessary, an effective amount of the above effective ingredient can be taken to formulate an anti-angiogenic effect and / or inhibit a tumor Of composition. The effective component of the anti-neovascular effect and / or tumor suppressing composition is preferably an effective component containing a molecular weight of about 1OKDa and about 14KDa. To further illustrate the present invention, preferred embodiments are described below, but these embodiments are not intended to limit the scope of patent application of the present invention. SDS / PAGE (Sodium dodecyl sulfate / polyacrylamide gel-electrophoresis) · Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Polyacrylamide gel, The standard protein is used to judge the molecular weight of the product of the present invention. After electrophoresis, the gel was pre-fixed with 10% (weight / volume ratio) glutaraldehyde for 30 minutes, and stained with CBB (Coomassie Brilliant Blue) for 1 hour, and then discolored. And the molecular weight of the product of the present invention was judged by the standard protein. Cell culture: B16-F10 mouse melanoma cells were obtained from the Institute of Preventive Medicine (Taipei Institute of Preventive Medicine), and the cells were contained in 10% fetal bovine serum (FBS) and 1% L-glutamine (Glutamine) DMEM (Dulbecco's modified Eagle's medium, modified by Dulbecco -7-This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 575425 A7 _________B7_ V. Description of the invention (6) Good Eagle medium) Medium growth. Prior to fusion, cells were seeded and removed for experimental use. Sacoma-180 (sarcoma-180), a mouse sarcoma ', is transplanted into ICR mice by peritoneal (ip) transplantation. To measure antitumor activity, sarcoma-180 cells were transplanted subcutaneously on the back of ICR mice. Cell migration assay: It is slightly modified according to the method described by Leavesley DI, Ferguson GD, Wayner EA and Cheresh DA, et al. In J. Cell Biol ·, 1 17, 1 101-1 107 (1992): Transwell inert (purchased from Costar company) of 6.5mm and 8.0mm pore size was used for cell transfer analysis, but with a slight modification as follows: soluble fibronectin was added to 20Mm Hepes (Purchased from Sigma) pH 7.45, 150 mM NaCl, 1.8 mM CaCl2, 1.8 Mm MgCl2, 5 mM KC1, and 5 mM glucose (HBS) were diluted and placed in a 24-well plate with a penetrating source Inlays, and placed at 37t for 30 minutes before placing the cells. HUVECs that passed earlier were collected with trypsin / EDTA, washed, and suspended in 1 * 106 cells / ml of attachment buffer. Cell movement was measured at different incubation times at 37 °. [3H] -Thymine deoxynucleoside uptake: 10 ng / ml tumor necrosis factor-a (tumor necrosis factor-α, TNF α, purchased from Sigma), with or without the product of the present invention, cultured in 24-well plates HUVEC 48 hours and 72 hours, in the last 8 hours, 5 // Ci of [methyl-3H] thymine deoxynucleoside was added to each well, and the cells were separated and washed 3 times with phosphate buffered saline PBS. Base · 3Η] Thymine paper size applies Chinese National Standard (CNS) Α4 specification (210X297 mm) --------- ^ ------- 1T ------ line (read (Please read the notes on the back before filling this page) 575425 A7 B7 V. Description of the invention (7) Intake of pyridine deoxynucleoside. CAM (chicken chorioallentoic membrane, chicken embryo chorion allantoic membrane) analysis: Incubate 10-day-old chicken embryos at 37 ° C and 60% temperature. At the air sac of the egg, make a hole that penetrates the shell. The second hole is applied with negative pressure to the original hole, and the CAM is taken out from the shell membrane, and then a pseudo airbag is made, and there is a 1.0cm * 1.0cm square hole for accessing the CAM. 200 // 1 of TNFa (5ng / ml) was injected into the CAM. After 24 hours, 100 / zl of HBSS (Hank's balanced salt solution) 100 / z1 with or without the product of the present invention was injected. Two days later, observation was performed with an SZ-PT microscope of Olympus. Collagen inhibitor inhibitor assay: According to Van Wart HE and steinbrink DR, Anal. Biochem. 113, 356 ~ 365, 1981. The method is slightly modified: 100 mg FALGPA (purchased from Sigma) is dissolved in 2 ml In methanol. The test buffer contains 0.05M Tricine, 0.4M NaCl, 0.01M CaCl2 and 0.02% NaN3, and the pH is 7.5. Analytical matrix was added with 10 // 1 FALGPA in 990 // 1 FALGPA buffer. When testing for alkali, add collagenase (50 // g / ml), vehicle solution (PBS), printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) and wait The test object was incubated at 25 ° C for 3 hours. Shimadzu's UV-1601 spectrophotometer was used to measure the optical density at 345nm (OD345) at 25 ° C. Growth analysis of tumor cells: Sarcoma-180 cells (4X106) were implanted into the backs of mice After 3 days of tumor transplantation, the product of the present invention or PBS was orally or injected once a day (QD), twice a day (BID) and four times a day (QID). The swelling was measured during 20 days. The paper size is applicable to China National Standard (CNS) A4 specification (21 × 297 mm) 575425 A7 B7___ 5. Description of the invention (8) Tumor diameter. Analysis of tumor cell metastasis: 4X 105 melanoma tumor cells (B16-F10 mice) were slowly injected into the lateral veins of C57BL / 6 mice (6-7 weeks old), and then the product of the present invention or PBS (once a day) was orally or injected. Fourteen days later, the rats were killed and the lungs were removed and placed in 10% formaldehyde. The surface melanin tumor colonies were calculated using an Olympus SZ-PT microscope. Example 1 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Cut the shark fins into small pieces and use 1M guanidine and 0.02M MES at 32t. The aqueous solution was extracted for 3 days and stirred slightly. Take 800 g of the extract and centrifuge at 4 ° C for 4 hours, then take the float. A 3-KDa cutoff ultrafiltration was performed with a PSAC membrane from Millipore to remove guanidine-HCl (guanidine-HC1, guanidine hydrochloride). Then add IN hydrochloric acid dropwise to pH = 2.0, centrifuge at 20,000xg for 30 minutes to remove solids, add 1N sodium hydroxide dropwise to pH = 7.6, and centrifuge at 20,000xg for 30 minutes to remove solids. Add ammonium sulfate to the extract to a saturation of 25% by weight, centrifuge at 1800xg for 30 minutes, and remove the solids. Continue adding ammonium sulfate to a 60% by weight saturation. The obtained solid was dissolved in 0.01 ^ 4 but 1: 5-11 (: 1 (trihydroxymethyl-amino-methane-HCl buffer) »and containing 2M guanidine-HC1, 0.001M Calcium chloride and 0.02% sodium azide in water. Chromatographic separation was performed with a Merck Fractogel TSK HW-50 column. The column was equilibrated with a 0.1M sodium chloride aqueous solution in advance, and It is extracted with 0.1M NaCl solution and collected in 4ml per tube. At 5 ° C, it is continuously monitored at 280nm by LKB UVicord instrument. CAM (chicken -10- This paper scale applies Chinese National Standard (CNS) A4 specification ( (210X297 mm) 575425 A7 B7_ V. Description of the invention (9) Chorioallantoic membrane (Chicken embryo chorioallantoic membrane) test method to monitor the anti-angiogenic activity during the initial purification, and found that the 14th to 25th tubes did have anti-angiogenic effects Example 2. The fractions from tubes 14 to 15 in Example 1 were collected and introduced into a column of Sephadex G-75 from Pharmacia, Sweden, and distilled with distilled water to demineralize them. Anti-neovascular effect after lyophilization, Merck's CM-Sephadex C-50 column was subjected to a three-stage gradient elution with acetic acid under the following conditions: (1) 0 · 05M ammonium acetate (ρΗ = 5.0), 250ml (2 ) 0 · 05M ammonium acid (pH = 5.0) to 0.5M ammonium acetate (pH = 6.8), 500ml 〇 (3) 0 · 6M ammonium acetate (pH = 8.0), 700ml. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the precautions on the back before filling this page) Collect the extract at 3ml per tube at 4 ° C. Freeze-dried and desalted the 78th to 85th tubes, then use Sephadex G-75 Chromatographic separation; elution was carried out by dissolving 0.01M ammonium bicarbonate at 4 ° C, and the eluate was collected in 3ml per tube, of which 12 to 18 tubes (final product) were collected in SDS / PAGE (SDS: 10) Sodium dodecyl sulfate, PAGE: Polyacrylamide gel-electrophoresis shows the bands containing lOKDa and 14KDa (see Figure 1). Figure 1, column 1 (Lane 1), columns 2 and 3 indicate the 12 to 18 tubes of extract in 15% polyacrylamide gel, reduction system (reduced system) and nonreduced system (SDS / PAGE), column 1 shows the lOKDa and 14KDa peptide bands. This paper size applies to China National Standard (CNS) A4 (210X297 mm) 575425

A

7 B V. Description of the invention (10) Examples 3 to 5 The final product of Example 2 was dissolved in phosphate-buffered saline (PBS-buffered saline) according to the conditions in Table 1, and its HUVEC movement was measured. As shown in Figure 2, it is shown that the final product can effectively inhibit HUVEC movement in vitro experiments. Table 1 Example product concentration (// g / ml) in the figure of the example 3 (Control group) 0 4 V 15 5 □ 30 Examples 6-9 The final products of Example 2 were dissolved in PBS according to the conditions in Table 2 And the effect on HUVEC uptake of I3H] -thymine deoxynucleus was measured, and the result is shown in FIG. 3, which shows that the end product has antiproliferation effect on HUVEC Table 2

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Examples 10 ~ 13 The final product of Example 2 has a PBS as a solvent. The solution was prepared by using the -12-dB A- / Ns 6 / IX standard at the OD345nm reading.

Shougong 7 29 X 575425 A7 B7 V. Description of the invention (1 1) (Please read the notes on the back before filling this page) Low, monitor the hydrolysis of FALGPA, measure every 30 minutes to 3 hours, the results are shown in Figure 4 , Showing that the end product has collagenase inhibitory activity. Table 3: Concentration unit β g / ml Example figure Concentration of final product concentration Collagen concentration Note 10 0 0 0 Control group 11 V 0 50 12 □ 30 0 13 0 30 50 Examples 14 to 16 were purchased from Sigma Chemical Company at 200 ml (Sigma Chem. Co.) TNFa (tumor necrosis factor-α) was injected into the 10-day-old chicken embryo mechanical hair allantoic membrane, and 24 hours later, HBSS buffer was injected (Example 14, control group), and (b ) And (c) were injected into the final product of Example 2 30 // g / ml (Example 15) and 50 // g / ml (Example 16), the results are shown in Figure 5 (a), 5 (b), respectively As shown in Fig. 5 (c), it is shown that the end product has anti-neovascular effect and is non-toxic. g / ml Example 17 ~ 20 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, the final product of Example 2 was used to perform the sarcoma-180 tumor cell growth efficacy test according to Table 4. The results are shown in Figure 6, which shows that the injection of this The end product can effectively inhibit the growth of tumor cells (see Examples 21-24), but the effect is poor when taken orally. -1 3-This paper is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 575425 A7 B7 V. Description of the invention (12) Table 4: (200 each time) Example Oral mode 17 Not administered (control group) 18 once a day 19 twice a day 20 times a day Example 21 to 24 Same as Examples 17 to 20, but the injection was changed by oral administration. The results are shown in Fig. 6, which shows that the final product is effective in suppressing tumors, which reduces the tumor cells. The volumes were 34% (QD), 68% (Example 23) and 85% (Example 24). Examples 25 to 27 Melanoma cells from B16-F10 mice were injected intravenously into C57BL / 6 mice, and the melanoma tumor cell metastasis was observed according to the following table. The results are shown in Figures 7 and 8 and show the oral effect. Poor, while the injection effect is significantly effective. (Please read the notes on the back before filling out this page) · ϋ · mi m ..— In-— · mi ίϋ I Γϋ ϋ · ι · 1 mu —ϋϋ m Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5 Example medication 25 None (control group) 26 Oral 200 // g per mouse 27 Inject 200 // g per mouse ------ IT1 — ----- -14- This paper size applies to Chinese national standards (CNS) A4 size (210X297 mm)

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Claims (1)

Marriage 575425 Repaired in November 2003. A8, B8, C8, D8, j. Application for Special Purposes> (] Give Lei Yiyi --- 1. A method for producing shark cartilage extract composition, which includes 1α) is essentially 10 ° C ~ 40 At a temperature of ° C, an aqueous solution containing guanidine, an aqueous solution of guanidine derivatives, an aqueous solution of guanidine acid salt, an aqueous solution of 2- (N-morpholinyl) ethanesulfonic acid, and 2- (N-morpholine Aqueous solution of ethanesulfonic acid derivative or 2- (N-morpholinyl) ethanesulfonic acid alkali metal salt solution, or an aqueous solution of a mixture or composition thereof, extracting shark cartilage for 2 to 15 days, separating solid matter , To obtain a crude extract; (b) separating the effective components of the crude extract with a molecular weight of 8 KDa to 25 KDa by chromatography. 2. The method according to item 1 of the scope of the patent application, wherein the aqueous solution contains guanidine or a derivative or salt thereof, and 2- (N-morpholinyl) ethanesulfonic acid or a derivative or salt thereof. Aqueous solution. 3. The method according to item 2 of the scope of the patent application, wherein the aqueous solution is an aqueous solution containing guanidine and 2- (N-morpholinyl) ethanesulfonic acid. 4. The method as described in item 3 of the scope of the patent application, wherein the aqueous solution is an aqueous solution containing more than 0.1M guanidine and 2- (N-morpholinyl) ethanesulfonic acid from 0.005M to 0.1M. 5. The manufacturing method as described in the scope of patent application No. 丨, 2, 3 or 4, wherein after step (a), some impurities in the crude extract are removed by ultrafiltration. 6. The manufacturing method described in item 5 of the scope of patent application, after ultrafiltration, first acidify with an inorganic acid, remove the solids generated after acidification, and then use inorganic-15-this paper applies the Zhongguanjia standard ( CNS) A4 specification (21G X 297 mm) '------ T --------------- L ------- ^ (Please read the precautions on the back first (Fill in this page again.) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 575425 A8 B8 C8 D8 Amended in November 2003. 6. Scope of patent application: The crude extract after alkali neutralization and acidification, and the solids are removed. Sexual crude extract. (Please read the precautions on the back before filling this page) 7. The manufacturing method as described in item 6 of the scope of patent application, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt MOH, where M is an alkali metal, and the pH after neutralization is 6.0 to 9.0. 8. The manufacturing method as described in item 7 of the scope of patent application, which adds ammonium salt to the substantially neutral crude extract to make it a low-saturation ammonium salt solution, and removes solids. 9. The method according to item 8 of the scope of the patent application, wherein the ammonium salt is ammonium sulfate or ammonium chloride, and the low saturation is 30% by weight or less. 10. The manufacturing method described in item 9 of the scope of patent application, which further adds the ammonium salt to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid substance, separates the solid substance, and makes This solid is dissolved in an aqueous solution that does not degrade shark cartilage. Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 11 · The manufacturing method described in item 10 of the scope of patent application, wherein the aqueous solution that does not degenerate shark cartilage is an aqueous solution containing guanidine or its derivatives or its salts. 12. The manufacturing method described in item 1, 2, 3 or 4 of the scope of patent application, which applies to the Chinese National Standard (CNS) A4 specification (21G X 297 mm) at -16-I paper size. 575425 A8 B8 C8 D8 Amended in November 2003. 6. After applying for ultrafiltration in the scope of patent application, first acidify with an inorganic acid and remove the solids generated after acidification. Then neutralize the acidified crude extract with an inorganic base and remove the solids. It is a neutral crude extract. 13. The method according to item 12 of the scope of the patent application, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt is MOH, where M is an alkali metal, and after neutralization The pH value is 6.0 to 9.0. 14. The manufacturing method as described in item 13 of the scope of patent application, in which the substantially neutral crude extract is added with an ammonium salt to make it into a low-saturation ammonium salt solution, and the solids are removed. 15. The method according to item 14 of the scope of patent application, wherein the ammonium salt is ammonium sulfate or ammonium chloride, and the low saturation is 30% by weight or less. 16. The manufacturing method as described in item 15 of the scope of patent application, which further adds the ammonium salt to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid substance, separates the solid substance, and makes This solid is dissolved in an aqueous solution that does not degrade shark cartilage. 17. The method according to item 16 of the scope of the patent application, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine or a derivative or a salt thereof. -17-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the notes on the back before filling this page) f-L ------- line. Ministry of Economic Affairs Printed by the Intellectual Property Bureau employee consumer cooperatives 575425 ABCD Printed by the Intellectual Property Bureau employee consumer cooperatives of the Ministry of Economic Affairs November 2003 Amended the scope of patent application 18.-A shark cartilage extract containing active ingredients with a molecular weight of 10KDa and / or 14KDa (A) at a temperature of substantially 10 ° C to 40 ° C, with an aqueous solution of guanidine, an aqueous solution of guanidine derivatives, an aqueous solution of an acid salt of guanidine, 2- (N-morpholine Aqueous solution of ethanesulfonic acid, 2- (N-morpholinyl) ethanesulfonic acid derivative solution or 2- (N-morpholinyl) ethanesulfonic acid alkali metal salt solution, or mixture or composition thereof Extracting shark cartilage for 2 hours to 15 days, separating the solids to obtain a crude extract; and (b) separating the crude extract by chromatography with the effective molecular weight of 10 Kda and / or 14 KDa. By. 19. The composition as described in item 18 of the scope of the patent application, wherein the aqueous solution that does not degrade shark cartilage is guanidine or its derivative or salt, and 2- (N-morpholinyl) ethanesulfonic acid Or its derivatives or salts in water. 20. The composition according to item 19 of the scope of the patent application, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine and 2- (N-morpholinyl) ethanesulfonic acid. 21. The composition as described in item 20 of the scope of patent application, wherein the aqueous solution that does not degrade shark cartilage is 2- (N-morpholinyl) ethanesulfonate containing 0.1M or more guanidine and 0.005M to 0.1M Aqueous acid solution. 22 · The composition as described in the scope of application for patents No. 18, 19, 20, or 21 ', which removes the middle separation of the crude extract by ultrafiltration before chromatographic separation -18-(Please read the Please fill out this page again) t Order! !! ---- Line. The size of this paper applies to China National Standard (CNS) A4 (210 X 297 mm) 575425 A8 B8 C8 D8 November 2003 _1 £ 6. The scope of patent application. 23. The composition according to item 22 of the scope of application for patent, which is' acidified with an inorganic acid after ultrafiltration, and removes solids produced after acidification, and then neutralizes the acidified crude extract with an inorganic base, and The solids are removed to obtain a substantially neutral crude extract. 24. The composition as described in item 23 of the scope of the patent application, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt is MOH, where M is an alkali metal, and neutralization The subsequent pH 値 is 6.0 to 9.0. 25. The composition as described in item 24 of the scope of the patent application, which is a substantially neutral crude extract, and then adds an ammonium salt to make a low-saturation ammonium salt solution, and removes solids. 26. The composition as described in item 25 of the patent application, wherein the ammonium salt is ammonium sulfate or ammonium chloride, and the low saturation is 30% by weight or less. 27. The composition as described in item 26 of the scope of patent application, further adding the ammonium salt to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid, separating the solid, and This solid is dissolved in an aqueous solution which does not degrade shark cartilage. -19-This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) _ (Please read the precautions on the back before filling this page) # 」" --- ^ ---- line · economic Printed by the Consumer Cooperative of the Ministry of Intellectual Property Bureau 575425 A8 B8 C8 D8 Yuan iE in November 2003. 6. Application for patent scope 28. The composition described in item 27 of the scope of patent application, which should not cause the degeneration of shark cartilage The aqueous solution is an aqueous solution containing guanidine or a derivative thereof or a salt thereof. 29. The composition described in claim 18, 19, 20, or 21, prior to chromatographic separation, is first acidified with an inorganic acid, and the solid produced after acidification is removed, and then neutralized with an inorganic base After the acidified crude extract and the solids are removed, a substantially neutral crude extract is obtained. 30. The composition according to item 29 of the scope of the patent application, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt is MOH, where M is an alkali metal, and neutralization The subsequent pH 値 is 6.0 to 9.0. 31. The composition as described in item 30 of the scope of patent application, which is a substantially neutral crude extract, and then adds an ammonium salt to make a low-saturation ammonium salt solution, and removes solids. 32. The composition according to item 31 of the scope of the patent application, wherein the ammonium salt is ammonium sulfate or ammonium chloride, and the low saturation is 30% by weight or less. 33. The composition according to item 32 of the scope of the patent application, further adding the ammonium salt to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid, separating the solid, and Make this solid dissolve in -2 0-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) -------------------- -------- ^ (Please read the note on the back? Matters before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 575425 A8 B8 C8 D8-November 2003 An aqueous solution that causes degeneration of shark cartilage. 34. The composition as described in claim 33, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine or a derivative thereof or a salt thereof. 35. A composition for anti-neovascular effect and / or tumor inhibition, which contains an effective amount of a shark cartilage extract composition, characterized in that the shark cartilage extract composition (a) is substantially 10 ° C At a temperature of ~ 40 ° C, an aqueous solution containing guanidine, an aqueous solution of guanidine derivatives, an aqueous solution of guanidine acid salt, an aqueous solution of 2- (N-morpholinyl) ethanesulfonic acid, 2- (N-? Aqueous solution of phorolinyl) ethanesulfonic acid derivative or 2- (N-morpholinyl) ethanesulfonic acid alkali metal salt solution, or a mixture or composition thereof, the shark cartilage is extracted for 2 to 15 days, and separated Solid matter to obtain a crude extract; and (b) a chromatographic method for separating the crude extract from the active ingredient having a molecular weight of 10 Kda and / or 14 KDa. 36. The composition as described in claim 35, wherein the aqueous solution that does not degrade shark cartilage is guanidine or its derivative or salt, and 2- (N-morpholinyl) ethanesulfonic acid Or its derivatives or salts in water. 37. The composition as described in claim 36, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine and 2- (N · morpholinyl) ethanesulfonic acid. -21-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ---------------------------- -^ (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 575425 A8 B8 C8 D8 November 2003 Amendment VI. Patent Application Scope (Please read the back Note: Please fill in this page again.) 38. The composition as described in item 37 of the scope of patent application, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing more than 0.1M guanidine and 0.005M to 0.1M EMS. 39. The composition as described in the scope of patent application Nos. 35, 36, 37, or 38, before the chromatographic separation, the middle isolate of the crude extract is removed by ultrafiltration. 40. The composition as described in item 39 of the scope of patent application, which is acidified with an inorganic acid before the chromatographic separation, removes the solids produced after acidification, and then neutralizes the acidified crude extract with an inorganic base. The solids are removed to obtain a substantially neutral crude extract. 41. The composition according to item 40 of the scope of application for patent, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt is MOH, where M is an alkali metal, and neutralization The subsequent pH 値 is 6.0 to 9.0. Printed clothing for employees' cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Salt solution and remove solids. 43. The composition according to item 42 of the scope of application, wherein the ammonium salt is ammonium sulfate or ammonium chloride, and the low saturation is 30% by weight or less. -2 2-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm): 575425 A8 B8 C8 D8 'Amended in November 2003 6. Patent Application Scope Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 44. The composition as described in item 43 of the scope of the patent application, further adding the ammonium salt to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid, separating the solid, and This solid is dissolved in an aqueous solution which does not degrade shark cartilage. 45. The composition according to item 44 of the scope of the patent application, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine or a derivative thereof or a salt thereof. 46. The composition described in the scope of application for patents No. 35, 36, 37, or 38, which is acidified with an inorganic acid before the chromatographic separation, and the solid produced after the acidification is removed, and then neutralized with an inorganic base After the acidified crude extract and the solids are removed, a substantially neutral crude extract is obtained. 47. The composition as described in item 46 of the patent application, wherein the inorganic acid is sulfuric acid is hydrochloric acid, the pH after acidification is 1.0 to 3.0, and the inorganic salt is MOH, where M is an alkali metal, and neutralization The subsequent pH 値 is 6.0 to 9.0. 48. The composition described in item 47 of the scope of the patent application, which is a substantially neutral crude extract, and then adds an ammonium salt to make it a low-saturation ammonium salt solution, and removes solids. -23-This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) # Order ---- ^ ---- line · 575425 A8 B8 C8 D8 --- Revised in November 2003 i. Application scope of patent 49. The composition as described in item 48 of the scope of patent application, wherein the ammonium salt is ammonium sulfate or ammonium chloride and the low saturation is 30wt %the following. 50. The composition according to item 49 of the scope of the patent application, which is further added to a low-saturation ammonium salt solution to form a high-saturation ammonium salt solution and a solid substance, and the solid substance is separated, and This solid is dissolved in an aqueous solution which does not degrade shark cartilage. 51. The composition according to item 50 of the scope of application for a patent, wherein the aqueous solution that does not degrade shark cartilage is an aqueous solution containing guanidine or a derivative thereof or a salt thereof. ------------- 41 ^ -------- IT --------- il # (Please read the notes on the back before filling this page) Ministry of Economic Affairs Printed by the Intellectual Property Bureau Staff Consumer Cooperatives This paper is sized for China National Standard (CNS) A4 (210 X 297 mm)