 Shark cartilage extract
专利摘要:
The present invention relates to a cartilage extract and a production method thereof. Shark cartilage extracts with anti-angiogenic, anti-tumor, anti-inflammatory and anti-collagenolytic activity were obtained by improved methods. The process comprises obtaining a homogenate of cartilage in an aqueous solution, wherein the homogenate is separated into a solid fraction (solid extract) and a liquid fraction, the liquid fraction having molecules having a molecular weight comprised between 0 and 500 kDa And further fractionated to obtain liquid extract. Thereafter, the composition of the liquid extract was examined by various methods. The liquid extract was further fractionated to further characterize some of its active ingredients. Because of the diverse biological activity of the whole liquid extract, it can be used in a variety of diseases or conditions such as those selected from the group consisting of tumor growth, angiogenesis, inflammation and collagen decomposition. Some cosmetic applications based on the ability of the liquid extract to enhance skin barrier function are also an object of the present invention. The extracts do not have any aggressive effect on normal body function. Therefore, the shark cartilage extract has promising therapeutic value. The method of obtaining the cartilage extract is simple and efficient. Therefore, the highly valued product obtained by this process is an indication of a new, non-autonomous process. 公开号:KR19990067245A 申请号:KR1019980703204 申请日:1996-08-07 公开日:1999-08-16 发明作者:에릭 뒤뽕；뽈 브라조；크리스티나 쥐노；다니엘 에이치. 마에스；케네쓰 마레누스 申请人:에릭 듀퐁；레 라보라뚜와르 제떼르나 인코오포레이티드； IPC主号:
专利说明:
Shark cartilage extract The present invention provides a novel method of producing cartilage extracts having the advantage of containing various therapeutically important activities. Among these activities, anti-angiogenesis, anti-inflammation, anti-collagen degradation, in vivo anti-tumor growth and direct in vitro anti-tumor proliferative activities were found to be present in satisfactory concentrations in shark cartilage extract. Other activities are being detected or confirmed. All activities were obtained in liquid extracts of shark cartilage, some of which were obtained or identified as solid extracts of shark cartilage. The present invention relates to a novel process for obtaining a liquid extract of cartilage having a substantial portion of a water-soluble component of biological activity present in the natural cartilage, comprising the following steps: a) homogenizing the cartilage in an aqueous solution under conditions that preserve the biologically active ingredients until the size of the cartilage is reduced to particles equal to or less than about 500 [mu] m, resulting in a particle mixture with the biologically active ingredients Making a mixture of natural liquid extracts; b) centrifuging the homogenate to separate particles from the natural liquid extract; And c) further separating said natural liquid extract to obtain a final liquid extract containing cartilage molecules having a molecular weight equal to or lower than about 500 kDa. This novel process has the advantage of being easy to perform and effective. High yield cartilage extracts were obtained, and in particular extracts obtained from shark cartilage contain at least all of the above-mentioned biological activities. To maximize the likelihood of recovering a compound with unknown physico-chemical properties, the process is carried out at about neutral pH (about 5 to 8) at a cold temperature (about 0 to 20 DEG C) ). ≪ / RTI > According to this process, the cartilage component can be extracted after a short homogenization time (as short as 10 to 20 minutes) within a small volume of solution (as small as 1 L per kg of cartilage). The same method was used except that the supernatant was discarded for recovery of the solid extract, and the pellet was recovered and lyophilized. The present invention relates to extracts from cartilage extracts, in particular cartilaginous fish, more particularly from sharks. The solid extract shows activity. It may contain collagen and non-water soluble components. It may also contain residual activity extracted in the whole liquid extract. The whole liquid extract is very active. It can be used as is or concentrated. Concentration steps to promote retention of biological activity are special. The use of methods to degrade active ingredients such as heat-evaporation has been avoided with caution. Ultrafiltration on a membrane with a molecular weight cut-off value of about 1 kDa was used to concentrate the liquid extract of the present invention. Nanofiltration on a membrane with a molecular weight cut-off value of about 100 Da is better for concentrating the biological activity of the liquid extract (anti-angiogenesis, anti-collagen degradability). As a result, concentrated extracts containing molecules with molecular weights between about 0.1 and about 500 kDa were tested. The liquid extract (0-500 kDa) was further fractionated to characterize these active ingredients. Numerous active fractions were obtained in a variety of ways. Some of these tested for their anti-tumor activity on tumor cell lines have been highly characterized by their molecular weight and isoelectric point. Other methods have been found to be active, particularly anti-collagen degrading or anti-angiogenic. These fractions are under full characterization and validation. Therefore, important activities have been recovered in whole liquid extracts and fractions thereof, which can be advantageously used. Instead of supplying a large amount of powdered cartilage, more acceptable and rich extracts can now be supplied. The present invention also relates to a therapeutic or cosmetic composition comprising, as an active ingredient, an effective amount of one of said cartilage extracts. Much attention is drawn to the products for use in dermatology and cosmetology. This interest arises from the activity observed in the cartilage extract. In this respect, the anti-angiogenesis, anti-collagen degradation and anti-inflammatory activity observed and anti-cell differentiation mediated by the signal pathway such as Protein Kinase C in keratinocyte The effects are believed to be based on the use of shark cartilage extract in the composition and the reduction of inflammation or irritation, the control of wrinkles or skin atrophy, the delay of premature aging, the reduction of acne, the enhancement of skin barrier function, And reduction of venous outflow, as well as suppression and skin sedation. These methods are for the purposes of the present invention. In addition, since the shark cartilage liquid extract has been successfully tested in cancer, arthritis, psoriasis and acne, conditions or conditions having one or more components selected from the group consisting of tumor growth, angiogenesis, inflammation and collagenolysis Are also objects of the present invention. Cartilage has been studied as a potent candidate containing anti-angiogenic factor, which is avirulent tubular tissue. It is also a relatively resistant tissue for tumor development. Chondrosarcoma, a tumor associated with cartilage, is a minimally vascularized solid tumor. Angiogenesis is one of the important factors in tumor development. Individual fidelity tumor groups appear to be able to stimulate the adjacent vascular network as if the tumor cells meet their nutritional needs. Therefore, factors related to the stimulation of angiogenesis have been studied for its role in the development of tumors, and anti-angiogenic factors as well as drugs with anti-angiogenic activity have also been shown to modulate or inhibit tumor growth As a tool for providing It has been found that calf cartilage contains substances that inhibit the angiogenesis of solid tumors (Langer et al., 1976). Due to its potential action as an anti-tumor agent, a better source of cartilage has been expected. Sharks are an important source of this type of angiogenesis inhibitor, because their endoskeleton is entirely composed of cartilage (6% of total weight compared to 0.6% of calves). Sharks also have an interesting low tumor evolving tendency. Many hypotheses have been proposed to explain this low possibility of tumor spread in sharks. Marchalonis et al. (1990) suggested IgM antibodies that could easily attack any aggressive agent. Mckinney et al. (1990) found that sharks differentiate normal cells from neoplastic cells and have macrophages capable of destroying neoplastic cells. Rosen and Woodhead (1980) hypothesized that tumor sparing in cartilaginous fish (including shark and stingray) is due to the high ionic strength of these tissues, as well as high body temperature. Under these conditions, the authors determined that the immune system was close to 100% immunological survival. Moore et al. (1993) found that sharks produce aminosterols with the characteristics of anti-bacterial and anti-protozoan. Finally, Lee and Langer (1983) and Folkman and Klagsbrun (1987) found that sharks produce substances that inhibit neovascularization. Lee and Langer (op.cit) extracted and separated the material from shark cartilage under denaturing conditions (guanidine extract). However, this extraction process is very long (41 days) and can produce an extract having a denaturating factor, and the yield of the active ingredient is not excellent. The active substance isolated from the calf had a molecular weight of about 16 kilodaltons (Kd), whereas the same study group did not achieve the correct molecular weight for the shark. By definition, the material has a molecular weight of at least 3500 Da. Oikawa et al. (1990) applied the same extraction method as described by Lee and Langer, but took a shorter time (2 days instead of 41 days). Anti-angiogenic materials isolated from shark cartilage by Oikawa et al. Were restricted to molecules with molecular weights ranging from 1000 to 10,000 Da. Schinitsky (US Pat. No. 4,473,551) discloses a water extract of natural powdered shark cartilage in which fractions of 100,000 Da or more, alone or in combination with glucosamine, have anti-inflammatory activity. In this patent, anti-angiogenic or anti-tumor activity was not shown for the components of the extract. Kuetner et al. (US Pat. No. 4,746,729) isolated a polymorphonuclear neutrophil (PMN) elastase inhibitor from bovine cartilage. The inhibitors were obtained from aqueous extracts of cartilage, from which molecules having a molecular weight of 50,000 or less were obtained. Numerous fractions were obtained by fractionation on Sephacryl S-2000, from which the 10-40 kDa fraction was collected after exhibiting anti-elastase activity. The active ingredient has an isoelectric point of 9.5 and can have a molecular weight of about 15,000 Da. Kuetner et al. (U.S. Pat. No. 4,042,457) also showed that bovine cartilage has a molecular weight component of 50,000 Da or less, which has cell proliferation inhibitory activity without any activity on endothelial cell growth. Balassa et al. (US Pat. No. 4,822,607) obtained a cartilage extract having anti-tumor activity in aqueous solution. However, no anti-angiogenic activity is found in the extract obtained by re-engineering Balassa's method. Spilburg et al. (US Pat. No. 4,243,582) isolated two glycoproteins of molecular weight 65 kda and pI 3.8 from bovine cartilage (guanidine-extracted) exhibiting anti-trypsin activity and endothelial cell growth inhibitory activity . The calf and shark cartilage can be used for pro-inflammatory activity, anti-inflammatory activity, anti-angiogenic activity, lysozyme activity, cell growth-promoting activity, type I and IV collagenase, And inhibitory activity against other proteases such as trypsin, chymotrypsin and plasmin. However, cartilage extracts containing clinically valuable active groups have not yet been obtained. The shark cartilage anti-angiogenic component (s) have generally been tested with rabbit corneal pocket assay or chick chorioallantoic membrane (CAM) assay. Until recently, total powder cartilage has not only been tested by CAM testing for its anti-angiogenic effect, but also has been shown to be directly in vivo to tumors on human melanoma xenografts transplanted with nude mouse mice ≪ / RTI > Although the anti-tumor effect has been identified as a cartilage extract, this effect has often contributed to the anti-angiogenic component which inhibits blood supply to the tumor. So far, there is no evidence that shark cartilage has a direct effect on tumor cell proliferation. Several methods of obtaining shark cartilage extract and fractions are already known. Some of them are related to powder natural cartilage production without any extraction (U.S. Pat. No. 5,075,112). Others use a denaturant such as guanidine (US Pat. No. 4,243,582). Others pre-treat cartilage by enzymatic digestion to remove any muscle, nerve or vascular structures surrounding the cartilage and, after such pre-treatment step, remove the fat with organic solvent, (Balassa et al., U.S. Pat. Nos. 3,478,146, 4,350,682, 4,656,137 and 4,822,607). The pre-treatment effect to preserve the biologically active cartilage component intact is not known. If too much, the enzyme digestion can hydrolyze the active protein component. For example, the method of Balassa (US Pat. No. 4,822,607) yielded a liquid extract without anti-angiogenic activity; Such a mistake will be the result of the enzymatic degradation. The Balassa method does not include a fractionation step which further includes the active ingredient in the extract. Other methods obtain aqueous extracts of cartilage (water (US Pat. No. 4,473,551) or salt solution (US Pat. No. 4,746,729) by simply removing the undissolved material. Of the latter, certain fractions of specific molecular weights have been used specifically for additional research and purification methods (see above). The above cited methods have several disadvantages. They can break down some important components. Otherwise, they are too long and impractical. In addition, such an elongate method does not necessarily produce a sufficient amount of active ingredient, some of the recovered components are either not recovered at all or are recovered in an insufficient amount to show detectable activity, Focused on and ignored. Angiogenesis is not only involved in cancer development. Conditions that affect many diseases or other physiological systems (indicated by parentheses) are angiogenesis-dependent, including: arthritis and atherosclerotic plaques (bones and ligaments), diabetic retinas (Scleroderma), angiogenesis and hypertrophic scarring (skin), vasculopathy and angiogenesis, neovascular glaucoma, neovascular glaucoma, spot phase decomposition, ocular herpes, trachoma and corneal graft neovascularization Fibroids (blood system). Therefore, any novel and patentable anti-angiogenic " factor " can be used in the treatment of these diseases as well as in cancer therapy and other angiogenesis-dependent diseases. In addition, since many of the above-mentioned diseases and conditions also have an inflammatory component, any novel and unique anti-inflammatory " agent " can be used in the treatment of the above diseases and conditions as well as in other inflammatory diseases or conditions have. Furthermore, because of its collagenolytic activity, proteases such as collagenase are involved in a variety of diseases and conditions such as cancer and premature aging, the novel and patentable anti-collagenolytic " factor "Lt; / RTI > Because angiogenesis, inflammation, and protein hydrolysis can occur alone or in combination with various diseases or conditions, products that are capable of counteracting at least all of these activities that do not affect normal bodily function, will be. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be better understood by means of the method of the specific embodiments shown in the following drawings, which illustrate the invention without restricting the object of the invention. 1 is a graph showing a specific amino acid composition of the liquid extract. Figure 2 is a graph showing the dose-response inhibitory activity of shark cartilage (solid extract) on cell lines ZR75-1 and MCF-7. FIG. 3 is a graph showing the dose-response of MCF-7 cell volume to an increase in estradiol concentration in the presence or absence of two concentrations of solid cartilage extract. Figures 4 a) and b) are comparisons of mammary gland tumor cross-sections administered by combination of gavage water (A) or solid and liquid cartilage extract (B). Figure 5 is a bar graph showing a 50% reduction in the angiogenic area of these tumors in cartilage treated rats. Figure 6 is a graph showing that liquid cartilage extract does not show any effect on fibroblast proliferation. 7 is a graph showing the dose-response inhibition curve of the liquid cartilage extract for HUVEC proliferation. 8 is a graph showing that liquid cartilage extract inhibits TPA-induced keratocyte differentiation. 9 is a graph showing the dose-response inhibition curve of a liquid cartilage extract against collagenase activity. 10 is a graph showing the dose-response inhibition curve of the liquid cartilage extract against the embryonic vascularization test (ex ovo). 11 is a graph showing the effect of varying amounts of liquid cartilage extract on tumor growth inhibition in mice. Figure 12 is a graph showing that the intraperitoneal injection of the liquid cartilage extract can significantly increase the efficacy of a substance that inhibits tumor growth. Figure 13 is an electrophoretic profile under non-denaturing conditions of a liquid fraction separated on Rotofor; Molecular weight markers are shown on the left, followed by samples of liquid extracts before fractionation to compare the separated fractions. 14 is a graph depicting the HPLC shift of the total liquid extract fraction of the present invention having a molecular weight lower than 10,000 Da, which fraction was concentrated and separated into five sub-fractions. 15 is a graph showing EVT results obtained in two fractions of a liquid extract of shark cartilage of the present invention (DUP), one having a molecular weight lower than 10,000 Da and the other having a molecular weight higher than 10,000 Da. 16 is a graph showing the FPLC movement pattern of three different extracts of shark cartilage. In panel A, DUP represents the cartilage liquid extract according to the invention. In panels B and C, BAL and OIK represent prior art extracts of Balassa et al. And Oikawa et al., Respectively. 17 is a graph showing the HPLC shift of the same extract as defined in Fig. 18 is a graph showing a comparison of CZE of the liquid extract of the present invention with the prior art. A = DUP; B = BAL; C = OIK. 19 is a graph showing EVT comparison of the liquid extract of the present invention with the prior art. 20 is a graph comparing amino acid contents of the above-described prior art and the present invention. Figure 21 is an illustration illustrating a significant improvement in the condition of two patients with psoriasis, with hyperkeratosis (22a and b), without hyperkeratosis (22c and d), an effective amount of concentrated liquid cartilage When they are treated with the topical composition containing the extract (bottom of the photograph), they are compared to their initial state (top of the picture). 22 is a graph showing the improvement of spider vein development in a human face treated with liquid cartilage extract. 23 is a graph showing an improvement in occurrence of dark circles around a human eye treated with liquid cartilage extract. 24 is a graph showing an improvement in the occurrence of a varicose vein of a human leg treated with a liquid cartilage extract. Figure 25 is a photograph illustrating a significant improvement in the condition of a patient with acne when treated with a topical composition containing an effective amount of a liquid cartilage extract (lower photo) compared to their initial condition (upper photo). 26 is a graph showing the anti-inflammatory power of the liquid cartilage extract against human skin. FIG. 27 is a graph showing an improvement in the blocking function of human skin treated with liquid cartilage extract. FIG. In certain embodiments, cartilage is obtained from healthy sharks Black Spiny Dog Fish and Common Spiny Dog Fish. Muscles and connective tissues were removed by disassembly with ethanol-treated surgical scalpel and scissors. The cartilage was then vacuum-packed into a plastic bag and frozen at -20 ° C for further use. In an embodiment of the method, any cartilage material may be used. The present invention has chosen shark cartilage for reasons described in the art. It was judged that a cartilaginous fish (including shark and stingray as an animal species of the above group) could be obtained from an almost equivalent product starting from cartilage. If mammalian cartilaginous materials are used, these products will almost all be different in their natural state and active elements. Any modification in the preparation of cartilage prior to extraction may be used as long as it does not substantially affect the activity of the subject product (e.g., the whole liquid extract or a particular fraction thereof). Some active ingredients can tolerate proteolytic digestion to remove any surrounding tissues from the cartilage, as indicated by Balassa et al. (US Pat. No. 4,822,607), while other ingredients can not withstand this treatment. One of the activities that does not appear to endure the pre-treatment is anti-angiogenic activity (Figure 19). Therefore, if it is desired to produce a liquid extract containing as much of the water-soluble active ingredients identified as independent activities as possible, the digestion step may be avoided during the extraction process, or careful to avoid strong hydrolysis or proteolysis Should be addressed. Isolation of cartilage extract Clean cartilage was used fresh, frozen or frozen at 4 ° C. The cartilage is treated with an appropriate volume of water (the same amount (weight / volume) is almost the minimum volume but can be increased without any effect on the yield of important components) over several hours (more specifically, 3 hours) And passed through a hole in a sterilized meat chopper. From a practical point of view, less volume is desirable because it is easier to work than a lot of unnecessary volumes. Practically, water was purified by reverse filtration and reverse osmosis of a 0.1 [mu] m filter. Many aqueous solutions (e.g., those containing salts) can be used instead of water. When the recovery of various water-soluble activities is contaminated, it is desirable to avoid degradation or denaturation of some of the cartilage active ingredients in operation at near-neutral pH (5.0 to 8.0) and non-denaturing conditions. The properties of unknown proteins in aqueous solvent are undetectable; Some at acidic pH, some at "basic" pH can be more "stable". In addition, some proteins may be extracted under weak denaturation conditions, provided that the denaturation does not adversely affect the regeneration of the proteins in aqueous solution. For the sake of clarity, any extraction conditions that can preserve the biologically active water soluble cartilage component intact are an object of the present invention. Therefore, taking all of the above factors into consideration, it has been shown that carrying out the extraction process of cartilage active ingredients in pure water is a viable option to recover components that have not yet been defined in structure and properties in very good yields. The cartilage / water mixture was then homogenized by stirring at full speed in a kitchen mixer at about 4 ° C for 20 minutes; The temperature rises to approximately 20 ° C during homogenization of the homogenate. Of course, the volume of the aqueous solution as well as the stirring speed can also affect the extraction time and yield. Therefore, a suitable range of time for homogenization (defined as a particle size smaller than 500 mu m) can be from the shortest of about 10 minutes to the longest of 24 hours, preferably between about 10 minutes and 60 minutes. The temperature should be maintained below about 10 ° C to avoid any degradation of the active ingredient by intracellular enzymes when no enzyme inhibitor is used. Ideally, a temperature close to 0 占 폚 is preferable. In general, the experiment is performed in a refrigerated room where the temperature can be maintained between 4 and 10 < 0 > C, which is suitable for application to the process of the present invention. For the sake of clarity and simplicity, the term "about 4 ° C" is used below to indicate the acceptable temperature range. If the mixer does not sufficiently reduce the size of the particles, the liquefaction of the homogenate can be further advanced by providing particles in a Polytron mill for about 10 minutes at about 4 ° C. Alternatively, it can be simply homogenized by further performing a mixer-grinder, which manually shortens the liquefaction step by 10 minutes. At the end of the complete homogenization step, the residual particle size is less than about 500 mu m. Of course, the same acceptable range of time and temperature contemplated to obtain the first comminuted cartilage is applied. The size of the particles after homogenization need not be very small. Therefore, it is not necessary to powder the cartilage before extraction. In fact, ballasting the cartilage in powder form prior to aqueous solution extraction can inversely denature important activities when the powdering is carried out, in particular in freeze-dried and / or heat-dried conditions. The homogenate was centrifuged at 13,600 x g for 15 minutes at 4 DEG C, which is one method for quickly and effectively separating the supernatant from the pellet. Variations and adjustments of these parameters are well known to those skilled in the art and depend only on the volume of device and homogenizer used. The resultant pellet was lyophilized for 24 to 48 hours. The first fraction will hereinafter be defined as a lyophilizate or a solid extract. The supernatant can be filtered on a 24 [mu] m Whatman filter if necessary to remove particles that may affect the performance of the ultrafilter column. The filtered material is then ultrafiltered on a tangential flow filter column having a pore of about 500 kDa at about 4 DEG C to form a water soluble molecule having a molecular weight comprised between about 0 and about 500 kDa . The natural permeate extract was filtered on a 0.22 mu m filter and divided into aseptic bottles for further use. Such fractions will hereinafter be referred to as natural permeate or liquid extract. The implementation of selective and higher centrifugation procedures was developed to obtain the pellets and supernatant. Approximate Filtering on a Whatman Filter The centrifugation at 13,600 x g for 15 minutes was replaced by centrifugation in a CEPA centrifuge equipped with a porous nylon bag at 3000-4000 x g to 1 μm. The 25 kg / 25 L preparation can be centrifuged in this manner within 30 minutes and can provide about 29 liters of supernatant. The volume of the aqueous solution obtained is greater than the volume of the initial water, suggesting that some of the water content of the cartilage itself has been obtained. The lyophilizate and the total liquid extract may have the following approximate composition, taking into account the changes observed when using other materials and over the batch: Solid extract: Lipid 7.35% 1 Protein 46.2% 2 Moisture 20.4% Sodium 4.16 mg / g 3 Potassium 2.64 mg / g Calcium 114 mg / g Magnesium 1.49 mg / g Zinc and Iron Trace Liquid extract: Lipid 0.10 - 0.20% 1 Protein 8 - 25 mg / ml 2 Dry weight 8 - 25 mg / ml Moisture 97 - 99% Sodium 30 - 220 mg / 100 g 3 Potassium 30 - 40 mg / 100 g Calcium 2.0 mg / 100 g Magnesium 1.1 mg / 100 g Zinc and Iron Trace 1 and 2 are numbers according to the disclosure in AOAC Offical (1994) 16.219-220 and 2.055, respectively. 3 is a measurement following the SAA procedure. The protein content is measured by the Kjeldahl method, which measures organic nitrogen (N) in nature. Organic nitrogen is converted to an equivalent amount of protein using the following equation: Protein content (mg / ml) = (% N x 6.25) / 100 Since no hydrocarbons are detected, it can be assumed that they are in the form of one or another extract, except in the form of proteoglycan and / or mucopolysaccharides. The compounds may be included as a measure of the moisture measured by the OH-group. The 20% water content is close to the percent of hydrocarbons normally recovered in the cartilage, while the moisture content of the lyophilizate should be close to 0%, and this hypothesis is under test. The liquid cartilage extract was analyzed for its amino acid content. The average amount of total amino acids was about 1.1 mg / ml, 0.67 mg (61%) of free amino acid and 0.44 mg (39%) of protein derived amino acid. The distribution of each amino acid is shown in Fig. A significant amount of taurine was also detected (not shown). The major amino acids present in the liquid extract are represented by proteins and peptides derived from cartilage. For example, lysine, glycine, aspartic acid and glutamic acid appear at a high ratio in the amino acid content of the liquid extract, and the N-telopeptide in the collagen ) Intermolecular cross-linking agent (Hanson et al. (1992) J. Bone & Min. Res. 7: 1251-1258). The microbial limit of the liquid extract was controlled by applying the USP XXIII < 61 > standard. Activity test Solid extract: In vitro assay: The assay was performed on hormone-dependent cancer cell lines MCF-7 and ZR75-1 (ATCC (R) 22-HTB and 1500-CRL, respectively). ZR75-1 cells: a. Basic RPMI medium: 52 g RPMI 1640 without phenol red (Sigma R8755), 17.875 g Hepes (free acid; Sigma H0763), 0.55 g sodium pyruvate (Sigma P5280) and 10 g NaHCO 3 Mixed with 5 L of pure water and made up to pH 7.40 with NaOH. If not used immediately, the solution should be shielded from light to preserve the photodegradable material. The solution was filtered and dispensed into a 500 ml sterile bottle, which is stored at 4 ° C for up to 3 months. b. Cell culture preservation culture medium: 10% (v / v) FBS (fetal bovine serum), 100 U penicillin G / 50 g streptomycin sulfate (Sigma P0906) / ml culture medium, 2 mM L- glutamine ) And 1 nM E 2 ( -Estradiol Sigma E8875). c. Experimental culture medium: 5% FBSA (fetal bovine serum adsorbed on dextran-charcoal), 2 mM L-glutamine, 100 U penicillin G / 50 g streptomycin sulfate / Sigma P0906 / Of insulin (Sigma). To this culture was added an increased concentration of lyophilisate as described above, as well as E 2 at different concentrations (10 -12 to -5 M). MCF-7 cells: a. Primary DME-F12 culture: DME-F12 medium (bicarbonate and no red phenol; Sigma) was reconstituted in pure water according to the manufacturer's instructions. For one liter, 1.2 g of sodium bicarbonate was added and the pH was adjusted to 7.40 with NaOH / HCl. The solution was filtered and dispensed into a 500 ml sterile bottle, which is stored at 4 [deg.] C for up to 3 months. b. Cell culture preservation culture medium: The basic DME-F12 culture was supplemented with 10% (v / v) FBS, 100 U penicillin G / 50 스트 streptomycin sulfate / ml culture medium, 2 mM L-glutamine (Sigma) and 1 mM E 2 . c. Experimental culture medium: The primary DME-F12 culture was supplemented with 5% FBSA, 2 mM L-glutamine (Sigma), 100 U penicillin G / 50 g streptomycin sulfate / ml culture and 50 ng / ml insulin (Sigma). As described for the ZR75-1 cells, lyophilizate and E 2 were added at the same concentration. d. Preparation of FBSA: Fetal bovine serum was mixed with 1% (w / v) charcoal (carbon decolorized with alkalin). Dextran T70 was added to the charcoal-serum solution to obtain a concentration of 0.1% (w / v). The mixture was stirred at 4 < 0 > C overnight. After centrifugation at 10,000 x g for 30 minutes at 4 ° C, the serum was transferred and again mixed with the same proportions of charcoal and dextran, stirred at room temperature for 3 hours, and re-centrifuged. The serum was then heat-inactivated at 56 ° C for 20 minutes, sterilized, and aliquoted into a sterile conical falcon tube. Experimental Culture Assay and Results: ZR75-1 and MCF-7 cells were grown on a 24-well plate to a density of 20,000 cells / well or 150000 cells / well on a 6-well plate and the presence of different concentrations of lyophilisate as prepared ≪ / RTI > For this effect, the solid cartilage extract was resuspended in culture broth and sterile filtered so that their water-soluble components could be recovered and tested. All experiments were repeated three times. The cultures were harvested every 2 days and replaced with fresh culture medium. Cells were grown in an incubator for 17, 7, 3, or 3 days at 37 캜 in an atmosphere containing continuously humidified 5% CO 2, which contained first, second, third, It is the same as the fourth experiment. Cell growth inhibition was measured by measuring the total DNA content of the wells or by counting the number of cells. Concentration of lyophilizate Cell inhibition (%) MCF-7 ZR75-1First Experiment: 17 days 1 mg / ml 5 mg / ml 10 mg / ml 1.514.3362.66 2.033.690.8Second experiment: 7 days 1 mg / ml 5 mg / ml 10 mg / ml 3.7315.768.37 0.9729.066.0Third experiment: 3 days 50 mg / ml 100 mg / ml 95.894.6 95.098.0Fourth experiment: 3 days 10 mg / ml 20 mg / ml 50 mg / ml 100 mg / ml 34.462.595.894.6 51.570.59598 The percentage of cell growth inhibition indicates that the solid cartilage extract can inhibit the cell growth of the two cell lines in a dose-dependent manner. Figure 2 shows that the solid extracts of 50 and 100 mg / ml produced apparently abnormal growth in the cell lines after 3 days of treatment. Figure 3 shows that in the presence of 10 -12 to 10 -9 M of estradiol, treated cells responded like regulatory cells by not being stimulated by the hormone dose rate. However, the 1 nM regulatory cells were strongly stimulated and the DNA concentration reached 3.75 μg (0.69 μg in the control without estradiol) in the presence of 10 -7 M estradiol. In the cells treated with 30 and 50 mg / ml lyophilisate, the DNA measured at the maximum stimulus was 1.9 and 1.8 μg, respectively. Figure 3 shows that the affinity constants (Km) of the treated cells for estradiol were 3 and 16 times higher than the Km value (11.7 nM) of control cells in the presence of 30 and 50 mg / ml, respectively (31.3 nM and 174.0 nM). This means that a higher concentration of estradiol is essential for achieving the same growth of the cells when solid cartilage extract is present. Therefore, the extract reduces the maximal response (90% inhibition thereof) and increases the affinity constant of cells treated with estradiol. In vivo assay: DMBA induced mammary tumor model a. Description of the test system: 400 40-day-old female Sprague-Dawley rats (purchased from Charles River Co., St-Constant, Quebec) were adapted to their environment for 12 days. At that point, 20 mg DMBA / 1 ml corn oil (9,10-dimethyl-1,2-benzanthracene; purchased from Sigma Chemical Co.) was administered by gavage. At 3 months after the treatment, 240 rats with developed mammary gland cancer were selected and divided into two groups. The first group consisted of five sub-groups. The rats in the treated group were given a daily dose while increasing the concentration of the lyophilized extract in 3 ml of water for 8 weeks, while the control group was given the same volume of water. Group 2 consisted of 4 sub-group rats. The rats of the treated group also gave the lyophilisate a certain amount of the lyophilisate in 3 ml of water for 10 weeks in the presence or absence of the liquid extract, whereas the control group gave the same volume of water. Only one sub-group of the second group of rats treated with the lyophilisate of 3000 mg / Kg / day and 3 ml of the liquid extract and also a small amount of the liquid extract (about 8 mg protein in 1 ml of water) ip) injection. The weight of the rats was 151-175 g at the beginning of both experiments and gave ad libitum, the food and water, to rats. Group 1 rats have tumors with an average diameter of 0.9 cm whereas rats in group 2 have tumors with an average diameter of 0.6 cm. b. Anti-tumor activity: The results are summarized in the table below. Daily dose of cartilage extract administered by gavage % Tumor growth inhibition (decrease in tumor diameter vs. control)First experiment: 8 weeks placebo 500 mg / Kg / day 1000 mg / Kg / day 3000 mg / Kg / day 5000 mg / Kg / day 0241415Second experiment: 10 weeks placebo (Placebo) 3000 mg / Kg / day 3000 mg / Kg / day + 3ml liquid extract 3000 mg / Kg / day + 3ml liquid extract + 1ml injections. 0121820 These results indicate that the lyophilizate is absorbed in the gastrointestinal tract and contains the active ingredient which slows down the tumor spread. The inhibition may be a direct effect on the tumor cell or an anti-angiogenic mediator effect that interferes with tumor growth. The liquid extract also contains inhibitory activity because its incorporation causes an additional reduction in tumor size of about 6%. The results also suggest that the lyophilizate may contain the remaining water-soluble active ingredient and / or may contain the non-water soluble active ingredient. Thus, finally, incidentally, if the yield can still be improved, the pellets can be re-extracted in an aqueous solution to maximally recover the water-soluble component. c. Histopathology: To assess the no-toxicity of the solid cartilage extract, the animals used in the in vivo experiments were sacrificed by beheading and the following tissues were analyzed: liver, lung, kidney, heart, brain, muscle and mammary gland. After removing the fat from the tissues, they were fixed in Bouin liquid for 2 days. After dehydration in ethanol, the fixed tissue was inserted into paraffin. Their cross-sections were obtained, placed on glass slides, stained with haematoxylin and observed under a microscope. The histopathological examination revealed that no harmful effect was observed when using the solid extract of the largest dose alone or when using the solid extract mixed with the liquid extract (not shown). This suggests that the lyophilizate and liquid extract have selective tumor size inhibiting activity. In mammary tumors (see Figs. 4a and b), a significant reduction in the area of blood vessels (55%) was observed in the group of rats receiving solid and liquid cartilage extracts (Fig. 5). The reduction in tumor size can be attributed to a significant decrease in its vascularization, a direct effect on tumor cells, or a combination of positive phenomena. The anti-angiogenic effect of the extract is well illustrated above. The direct hypoplasia effect was observed in hormone-dependent cells in vitro, which was still confirmed in vivo. Since the above-mentioned results showed that the liquid extract had an increasing effect over the effect of the solid cartilage extract on ZR75-1 cells, these components were further investigated. Liquid extract In vitro assay: Tumor cell line: Several tumor cell lines were grown in the presence of liquid cartilage extract to check for the presence of the hypoactive activity observed with the solid extract (described above). Briefly, cells are plated in 96-well plates and grown in the presence or absence of various concentrations of liquid extracts in culture (specific for each cell type; for example, MCF-7 cells are grown as described above) . After 3 to 5 days of culture, cell proliferation was measured using MTT assay. The tumor cell line is as follows: CHANG: Tumor-bearing hepatocytes Hep-G2: Tumor-bearing hepatocytes A2780: ovarian lineage cells MCF-7: Adenocarcinoma cells (estrogen-dependent) MCF-7-ADR: adriamicin resistant cell adenocarcinoma cell The liquid cartilage extract showed antiproliferative activity in all tumor cell lines. 50 and 80%, respectively, at a concentration of 8.5 mg / ml (dry weight of liquid extract / ml of culture) on MCF-7 and A2780 cells, respectively. The non-lyophilized and freeze-dried liquid cartilage extracts are equivalent in terms of inhibiting tumor cell proliferation. This indicates that the inhibitory factor (s) is not denatured by the enrichment procedure. Primary cultured cells: a. Fibroblasts Obtained from New Vascular Glaucoma: In order to evaluate the specific activity on tumor cells, the passage obtained after the ultrafiltration method was tested on human TENON fibroblasts (HTF), which are normal human fibroblast mesenchymal cells. Only HTF was obtained from two patients (one with NVG, one with neovascular glaucoma and the other with POAG, the primary open angle glaucoma). Secondary culture and maintenance of HTF: Each over-culture was washed and detached with 0.5 ml of 0.05% trypsin / 0.5 mM EDTA (Gibco 610-5300 AG) at 37 ° C for 5-10 minutes. Thereafter, 1.5 ml DME / F-12 medium containing 15% fetal bovine serum (FBS) was added to neutralize trypsin / EDTA. Softened and transferred to a 25 cm 2 T-flask, and the cells were allowed to coalesce by the addition of additional culture medium containing 10% (FBS). After complete culture, the HTF was transferred to 75 cm 2 and consequently to a 180 cm 2 T-flask. When sufficient cells were obtained, some cells were used for the experiments described below, and other cells were instantaneously cooled to obtain the same results for additional experiments. EXPERIMENTAL METHOD: Cells obtained from one patient who had grown in two or three identical 180 cm 2 T-flasks upon complete culture were resolved by the method described above. After short and low speed centrifugation, they were counted with a ZMI Coulter Counter 216013 equipped with a 256-channel channerzer. For all in vitro experiments, approximately 50,000 cells were injected into 16-mm dishes and 12-well plates, respectively, in 1 ml of DME / F-12 medium containing 1% FBS. After 17 hours of feeding, 1 ml of fresh equal culture ("absolute" control) supplemented with 1% FBS was added. Depending on the experimental design (see above and below), the 1% FBS culture was sterile filtered without supplementing or supplementing GF (growth factor), or supplementing or not supplementing the liquid cartilage extract. At this point (day 0), a portion of the cell was also counted to determine the plate culture efficiency (which should be equal to or greater than 95%). Forty-eight hours after the start of the experiment, the cells were resuspended, digested, and counted. The number of cells was expressed as a percentage obtained in the " absolute " control. Each "absolute" control containing 1% or 5% FBS, respectively, and each experimental group was supplemented with 1% FBS and supplemented with liquid cartilage extract consisting of GF or three duplicate samples, respectively. At the same time, each experiment was performed on one or two patient cells and repeated at least twice. In this experiment, pigs platelet-derived growth factor (pPDGF) and recombinant human fibroblast growth factor (hr bFGF) (donated from P. Brazeau, Farmitalia Carlo Erba, Milan, Italy) Respectively, at a concentration of 10 to 100 ng / ml. Forty-eight hours after the start of the experiment, the cells were dispersed with trypsin-EDTA and counted in a Coulter counter. All three duplicate values (lengths 1, 2 and 3) shown below are one-twentieth the number of cells per well. Results: The results are summarized in FIG. HTF was obtained from a 53-year-old man with glaucoma. Growth factors such as PDGF and bFGF showed stimulating activity ( * P <0.02, ** P <0.01; measured by Student-Fisher test) on HTF while the cells were treated with liquid cartilage extract (1 Kg / There was no effect, negative or positive when grown in the presence of. This indicates that the hypoactive activity of the liquid cartilage extract on tumor cells is not universal and has no effect on fibroblast growth. The same cartilage extract also has no effect on another form of fibroblast HSF (fibroblast of human skin; not shown). Although not tested, it can be inferred that the solid extract also has no effect on normal cells. b. Endothelial cells from human veins (HUVEC): Collagenase-regulated digestive HUVECs as described in Jaffe et al. (1973) were extracted. Pure endothelial cells were used prior to the four-step procedure (trypsin-EDTA in each procedure). The quality of the cells was assayed for their ability to be labeled with Factor VIII and to insert di-aceyl LDL. Endothelial cells were plated at a density of 25000 cells / cm2 on a sterilized dish coated with gelatin. The culture medium was changed after 24 hours and every other day. After 168 hours of incubation, BrdU (10 mM final) was added to each culture and incubated for 2 hours at 37 ° C. The cells were then detached with a short trypsin-EDTA digestion and transferred to a 96-well plate to allow ELISA detection of BrdU. ELISA was performed using the Boehringer Mannheim kit and method. One control was performed without cells to determine the baseline level of background. Another control was performed by measuring the DNA content in the culture medium in the latter half of the incubation period to distinguish whether the liquid cartilage extract affects cell attachment. Cell proliferation was also assessed by the amount of DNA present in the petri dish. Each experiment was performed in duplicate three times, and statistical analysis was performed for comparison. The culture medium was changed every day. After 168 hours of incubation, the cells were lysed with Na-citrate-SDS solution and incubated with Hoescht 33358. Samples were read at 365 nm with a spectrofluorometer. Finally, the amount of cells present in the Petri dish was evaluated by measuring acid phosphatase activity. Each experiment was performed in duplicate three times, and statistical analysis was performed for comparison. The activity of the enzyme was strongly correlated with the number of endothelial cells in the Petri dish (BrdU insertion and Hoescht labeling; not shown). The acid phosphatase activity was measured with a kit purchased from Sigma Chemical Company (measured with slight modification of the preparation method). The results indicate that endothelial cell proliferation is inhibited by the liquid cartilage extract (Fig. 7). The ED50 obtained is about 90 μl of liquid extract (equivalent to about 1.5 mg dry weight present in the liquid extract). c. Corneal Cells: Liquid cartilage extracts were tested with corneal cells activated with triphorbol acetate (TPA), a protein kinase C (PKC), a known agonist of the cell's delivery pathway . Normal human epidermal keratocytes were formed from primary cultures (J. Invest. Dermatol. 99: 565-571 of Matsui et al. (1992)). Cultures were incubated with serum containing growth factor (10 ng / ml), insulin (5 μg / ml), hydrocortisone (0.5 μg / ml) and bovine pituitary extract (70 μg / ml) in the form of modified MCDB 153 - pre-defined culture medium (KGM). The corneal cells were grown up to 70% of the total culture and re-fed with fresh culture medium. After 48 hours, 200 ng / ml of TPA or 2 μl / ml of DMSO was treated without any additional re-feeding. Various concentrations of liquid cartilage extract were added to or not added to the culture. The results showed no effect of the liquid extract on corneal cell proliferation; And did not show any effect on TPA-induced inhibition of proliferation. However, the liquid cartilage extract could inhibit TPA-induced corneal differentiation (Figure 8). The level of differentiation of the corneal cells was increased 5-fold by TPA. Liquid cartilage extracts did not affect the formation of keratinized surfaces by themselves. However, its presence inhibited TPA-induced keratinized surface formation by about 60% or more. In recent publications it has been reported that PKC activation produces an increase in interleukin-8 (IL-8), the mediator of inflammation in normal corneal cells (J. Invest. Dermatol. 103: 509-515). In addition, psoriatic corneal cells produce a very large amount of IL-8, further promoting renal vascularization in psoriatic plaques (Nickoloff et al. (1994) Am. J. Pathol. 144: 820-828) . In addition, other growth factors and integrins are involved, which is important in expanding the target molecule family (Il-1, TNF, etc.) that may be involved. We do not know if TPA-derivatization mimics psoriatic corneal cells. If so, these results suggest that cartilage has no effect on normal corneal cells in vivo, but may affect psoriatic (or activated) corneal cells. Inhibition of the production of IL-8 by the liquid cartilage extract in TPA-active corneal cells as well as in psoriatic plaques or corneal cells is also being demonstrated. Reduced levels of IL-8 and / or other growth factors are of interest in explaining the anti-inflammatory and anti-angiogenic effects of the extract. Collagenase assay method: a. Test 1: This test is performed by Knight et al. (1992) FEBS Let. 296, 263-266. The method utilizes a metalloproteinase azepin resembles the active site of the (metalloproteinase) is fluorescent genetic peptide substrate (Mca-pro-leu-glu -leu-Dpa-ala-arg-NH 2). The substrate has a fluorescence group (Mca) at one end and a fluorescence interrupting group (Dpa) at the other end. In the present substrate, the interrupted group effectively blocks fluorescence. Fluorescence increases in the test tube upon enzyme cleavage of the substrate. Collagenase activation is described by Weingarten et al. (1985) in Biochemistry 24, 6730. 1㎍ dissolved in 50mM Tris-HCl, 10mM CaCl 2 100㎕. 1 [mu] l at 10 mg / ml of trypsin (in 1 mM HCl) solution was added and incubated at 20 [deg.] C for 15 minutes. Activation was stopped by adding 10 [mu] l of Soybean trypsin inhibitor (SBTT, 5 mg / ml). To each microcuvette the following were added: 25 or 50 [mu] l of inhibitor * (prepared up to 50 [mu] l in water); Of 40㎕ 50mM Tris-HCl, 200mM NaCl , 10mM CaCl 2, pH 7.5; 8 [mu] l of activated collagenase ** (final 67 ng); And 2 [mu] l of substrate (1 mM source solution in DMSO, final 20 [mu] M). Fluorescence was measured at Ex = 328 nm and Em = 393 nm. * : The inhibitor is defined as a control substance (such as EDTA, ortho-phenanthrolene) or a liquid cartilage extract. ** : The collagenase is defined as human type I, type IV and amphibian tadpole collagenase; Gelatinase was also used. b. Test 2: This test is described in Welgus et al. (1979) JBC 256, 9511-9516. The method uses SDS-PAGE to check for cleavage by collagenase type I (MMP1). Collagenase type 1 provides a single cut to the original collagen molecule to provide two fragments of 75% and 25% size of the original collagen. After cleavage for several hours, the reaction is observed by separating the product from the substrate by SDS-PAGE. The ratio of chopped to uncut chopped is assessed visually after staining the gel with Comassie blue (or silver stain). 21 ng of activated collagenase (see Assay 1) was added to 5 소 of bovine skin collagen (Worthington) inhibitor in a final volume of 20.. The reaction was incubated at 35 DEG C for 16 hours and then stopped by adding SDS-PAGE sample with 40 mM EDTA, heated and spotted onto 8% acrylamide gel. c. Concentration-response inhibition: The results obtained with liquid cartilage extract showed a dose-response inhibition of collagenase activity with the positive assay. Figure 9 shows the results obtained with Assay 1. The ED50 is obtained with 30 l of liquid extract (or 0.51 mg dry weight present in 30 l of liquid extract). In vivo assay: Fetal Angiogenesis Test (EVT): a. Definition of the test-system: The normal development of the chick embryo is related to the formation of an external vascular system located in the yolk sac that carries nutrients from the egg yolk (egg yolk) that develops the fetus. When placed on the yolk membrane, the anti-angiogenic substance can inhibit the blood vessel development that occurs in the yolk membrane. To facilitate access to the yolk membrane, the chick embryos were transferred to a sterile culture box (Petri dish) and placed in a humidity- and temperature-controlled incubator. The fetus can then develop for several days under the above ex ovo conditions. Liquid cartilage extract was mixed with methylcellulose solution and air-dried to make a thin disc. The endogenous NaCl present in the liquid cartilage extract during the process is concentrated and blocked with the EVT when the amount per disc is greater than 25 μg. Therefore, it may be necessary to desalinate the liquid extract; Dialysis or electrodialysis by a membrane cut to less than 100 Da is possible. Methylcellulose forms an inert matrix in which the liquid extract can be slowly dispersed. A methylcellulose disk containing the liquid extract is placed on the outer boundary of the blood vessel of the yolk sac, where the angiogenesis process is still active. The effect of disc-containing liquid cartilage extract on adjacent vascular development is always compared to the effect of disc-containing water plus equivalent NaCl. Placing the disk on the yolk membrane of the fetus at day 0 or day 1 of the ex ovo growth process; At this time, only the initial stage of the main blood vessel invades the egg yolk. The fetus is then placed under culture conditions until vascularization is evaluated (about 24 hours). Water- and liquid extract-containing discs are always added simultaneously on the yolk of the same embryo. When comparing the control and shark cartilage extracts, all disks are placed symmetrically about the cephalo-caudal axis of the fetus to minimize mutual-to-individual variation. b. Anti-angiogenic activity: EVT was carried out with different concentrations of protamine (protamine, 37, 75 and 150 μg) as a positive control or liquid cartilage extract. One day after incubation, the level of vascularization at the area covered by the disc was graded by a pair of scientists in the usual emotional form. To facilitate the placement of the disks, a black O-ring is placed around it immediately after deposition on the yolk sac. The evaluation range for the EVT-test is based on a 1-2-3 score: normal vasculization when compared to a matched quadrant of opposite quadrant or control fetus (score = 3); (Score = 2) blood vessels appear on the area covered by the disc, but disappear from the intermediate path. Except for that pathway, major blood vessels over the area covered by the disc are obviously affected or a decrease in side chain density is observed; (Score = 1) no blood vessels are observed in the area covered by the disc, or they are quickly disengaged in such a way that the path deviates from the area covered by the disc. The blood vessels do not grow beyond the area covered by the disc, except when they pass through or beyond the backside. Dose-response inhibition was obtained with protamine (not shown) and liquid cartilage extract (Fig. 10). The ED50 was obtained with approximately 170 g of dry liquid extract (dry weight present in the liquid extract). A Wilcoxon-signed rank statistical test was used to compare the meanings of the differences between two disks (water and cartilage extracts) placed in the same egg. Adenocarcinoma of the rat breast model: a. Description of the Test-System: The anti-tumor ability of the liquid cartilage extract was tested in a rat adenocarcinoma model (allograft). The test-system consists of inoculating 1 x 106 DA3 cells into BALB / C mice. These cells are derived from adenocarcinomas of the murine breast induced by 7,12-dimethylbenzanthracene (DMBA). The model was made by Daniel Medina (J. Natl. Cancer Inst. (1969) 42: 303-310; ibid. (1976) 57: 1185-1189). Inoculated cells grow slowly in vivo and form a solid tumor with a low metastatic prognosis. DA3 cells were cultured in RPMI 1640 medium supplemented with 1 mM maltitol ethanol, 1 M Hepes buffer, 100 mM Na pyruvate, 200 mM L-glutamine, 10 mM insoluble amino acid, 1 M vitamin, 10% fetal bovine serum, 1% penicillin-streptomycin at 37 ℃ was maintained with 5% CO 2. For tumor induction, the cells were grown up to a full 70% culture in culture medium and collected using trypsin-EDTA solution. Then, centrifuged the cells, washed three times with phosphate buffer and, 1 × 10 6 cells were resuspended in dilutions of /0.1ml. DA3-cell-inoculated mice (n = 15) received daily oral administration of shark cartilage liquid extract or placebo (saline solution). The treatment started at 7 days after DA3 cell inoculation. Various concentrations of liquid extracts were tested. The amount of liquid extract administered is expressed as the dry weight present in the liquid extract. The test product was prepared as described herein: The liquid extract was lyophilized and resuspended in water at various concentrations (0.2, 1.5, 3, 10 and 20 mg / 200 μl). The final dose administered daily was 10, 75, 150, 500 and 1000 mg / Kg. b. Anti-tumor activity: As a result, maximum inhibition of tumor development was obtained upon administration of a liquid extract of about 75 mg / Kg (FIG. 11). Interestingly, it showed smaller capabilities in larger quantities. This suggests that the liquid extract contains a substance capable of inhibiting tumor spread and other substances capable of inhibiting the activity of the tumor suppressor. This phenomenon has already been reported for biological drugs. Finally, intraperitoneal administration of the liquid extract significantly reduces the maximum effective dose for inhibition of tumor growth (x75) (Fig. 12). c. Toxicity: There was no weight loss or liquid extract-related lethality at all treatments. There were no symptoms or characteristic changes observed during the treatment period through day-to-day examination of the rats. At the end of the treatment, mice were sacrificed and the overall condition of all organs was analyzed by a qualified pathologist; No abnormality was observed. Blood analysis showed no abnormal signs. d. Histopathology: In tumor histopathology, there were no significant differences between the tumors obtained from the placebo- or liquid extract-treated rats. The survival rate of the tumor was very high in all groups. Analysis of many organs (lung, liver, kidney, pancreas, stomach, duodenum, ovaries, breast, brain and heart) did not show any unusual changes that could be associated with the liquid extract. Rat hypersensitivity model (CHS): a. Description of the Test System: Dinitrofluorobenzene (DNFB) is a potent skin irritant that can induce a strong inflammatory response in BALB / C rats. At day 0, 10 rats were stimulated by staining their abdomen with DNFB. After stimulation, the right ear of the rats was stimulated by applying 10 [mu] l of DNFB on the 5th day. As a list of tissue stimuli, ear swelling was measured over several hours after exposure. The liquid cartilage extract was tested to see if it could reduce the inflammatory response to DNFB in rats. The culture solution alone (0.2 ml saline solution: 5 rats) or the liquid extract (0.2 ml liquid extract containing 20 mg / ml dry weight: 5 rats) was orally administered for 3 consecutive days on the 4th day before stimulation . b. Anti-hypersensitivity activity: On day 1 after ear stimulation, rats treated with culture alone showed swelling of the ear to a thickness of 8.2 mm. Interestingly, rats treated with cartilage liquid extract showed only a 2.8 mm ear swelling. The statistical significance of these data is p value <0.001. These results indicate that liquid cartilage extracts are potent inflammation inhibitors. Obtaining liquid fractions containing active molecules In vitro assay: Tumor cell line: a. Test-System Preparation: The shark cartilage was collected and subjected to the same procedure as described above. After centrifugation, the pellet was discarded and the supernatant was ultra-filtered with a sterile filter on a 0.22 mu m filter as described above. The liquid extract thus obtained was further fractionated by another method. Tumor cell lines were grown as described above. b. FPLC conditions: column: Hiload 26 mm x 60 cm Sephacryl S-300. FPLC system: purchased from Pharmacia. All samples were passed through a 0.22 탆 filter before being placed on the column. The elution buffer was filtered and degassed phosphate buffer saline (PBS) for 15 minutes. The volume of the spotted sample was typically 3.2 ml (may be up to 13 ml). The flow rate was 1 ml / min. 10 ml fractions were collected. The eluted components are those of U.V. Absorbance (280 nm). A calibration chart was obtained using an MW-GF-1000 calibration kit purchased from Sigma, and the calibration sample had the same volume (3.2 ml) as the sample to be analyzed for analysis. The elution volume of the sample was estimated by plotting the elution volumes of these compounds minus the molecular weight of the compound of the calibration kit versus the void volume of the column. The void volume is obtained by injecting dextran blue (molecular weight = 2,000,000). The fractions were tested for their activity in ZR75-1 cells. Fractions of the subject group were identified and further investigated to confirm their characteristics (see below). Additional properties of the active component of the pass through are shown in Rotofor (Biorad 170-2950; see isoelectrofocalization) and other cut values of Amicon < (R) >Lt; / RTI > c. Equivalent Focusing Conditions: 4 liters of a 5% glycerin solution at 4 ° C using a Spectra Pore # 7 MWCO 3500 kDa membrane (spectrum 132110) in the preparation of shark cartilage liquid extract (46 ml of 1 Kg / And dialyzed against pure water overnight. The dialyzed solution was added to 2.75 ml of ampholyte (Pharmacia # 80-1125-87) pH 3.5-10.0 and 0.5 g CHAPS (Sigma C3023; 3 - [(3-Cholamidopropyl) -dimethylammonio] -1-propane-sulfonate ). The volume was filled up to 55 ml with pure water. The solution was spotted onto Rotofor. Isoelectric focusing was carried out with continuous water circulation for the maintenance of this temperature at a constant power of 12 watts (3000 xi power supply, Biorad 165-0554) at 4 ° C. At the beginning of the separation, the potential was 380 volts and the current was 31 mA. When the current stabilized (14 mA), the potential appeared to be 870 volts. The iso-focusing was stopped and 20 fractions were collected. Fraction volume pH One 3.7 3.56 2 2.1 4.01 3 2.2 4.18 4 2.3 4.31 5 2.2 4.63 6 2.1 5.03 7 2.5 5.30 8 2.1 5.50 9 2.4 5.81 10 2.5 6.26 11 2.3 7.00 12 2.4 7.29 13 2.4 7.64 14 2.5 7.94 15 2.3 8.32 16 2.5 8.62 17 2.4 8.94 18 2.9 9.30 19 3.1 9.88 20 3.6 10.71 Identification of the proteins was accomplished by estimating their molecular weight on an electrophoretic gel (Laemmli, U. K. (1970) Nature (Lond.) 227: 680). The fractions were diluted 4-fold with drip buffer (see Laemmli) and 8 ㎕ aliquots were subjected to electrophoresis under non-reducing conditions. Figure 13 shows the electrophoretic profile of each fraction and the isoelectric focusing material. All fractions were sterile-stored under a laminar flow hood by passing them through a sterile Millipack-60 filter with a 0.22 mu m aperture. d. Inhibitory Activity on Tumor Cells: The protein content of the fractions was assessed by the Lowry titration method. 1 Kg / 2 L of solution (expressed as natural cartilage weight per liter of passage) was tested on ZR75-1 cells at different concentrations in culture. The results are summarized below: The first test was carried out on the Rotofor fraction (the passage is concentrated by evaporation). Identification of proteins: Identified fractions Isoelectric point Median Molecular Weight 7-8-9-10 5.30 to 6.26 5.78 29 +/- 1 kDa 7-8-9 5.30 to 6.26 5.68 60 +/- 1 kDa 12-13-14 7.29 to 7.94 7.62 48 +/- 1 kDa 13-14 7.64 to 7.94 7.79 35 +/- 1 kDa A second test was performed on the FPLC fraction (the passages were concentrated by evaporation): Fraction Molecular Weight 6 and 7 0.1 - 2.5 kDa A third test was performed on 100 mu l fractions obtained on an Amicon molecular filter: Tested concentration Molecular Weight Inhibition of ZR75-1 cell culture 100 [mu] g / ml MW> 100 kDa 64% 100 [mu] g / ml 30 kDa <MW <30 kDa 114% 100 [mu] g / ml 10 kDa < MW < 30 kDa 127% 400 / / ml MW <10 kDa 149% FPLC fractions 6 and 7 contain very small molecular weight active ingredients: 0.1 to 2.5 kDa. The negative effects of forming the fractions may be up to 33,000 times greater than that observed with the lyophilizate. e. Confirmation of the active ingredient of the eluate: The active fractions (tested for ZR75-1 cells) were subjected to the same run (1 kg / L) on a 100 mm diameter x 30 cm long Superose-12 column using FPLC and the Rotofor method described above, ≪ / RTI > in the following molecular weight ranges, as determined by another type of purification starting with < RTI ID = 0.0 > A flow rate of 1 ml / min was selected. Forty fractions of 1 ml were collected. Fraction 20-21 Activity in the fraction corresponding to a molecular weight of 70 to 120 kDa Fraction 22 Activity in fractions consistent with a molecular weight of 60-70 kDa Fraction 29-32 Activity in the fraction corresponding to a molecular weight of 35 to 46 kDa Fraction 34-35 Activity in the fraction corresponding to a molecular mass of 29 kDa Fraction 38-39 Lt; RTI ID = 0.0 > 0 < / RTI > to 2.5 kDa Collagenase assay method: a. HPLC Chromatography: 980 ml liquid extract samples (DUP) were filtered through a 10 kDa cut membrane in a tangential flow ultrafiltration unit (PELLICON, Millipore). The unit is first immersed in 1 L of water. The final product was 480 ml of > 10 kDa fraction and 1.8 L of < 10 kDa fraction. The <10 kDa was concentrated by cold-finger evaporation to 180 ml (<10-10X). <100㎕ equal parts mixture of 8 times the CDC-S Hexyl 10-10X (hexyl), 5㎛ was added dropwise onto the HPLC column (25 × 0.94cm), and then first eluted at 4ml / min in 100% H 2 O, 8.5 ml / min with 100% MeOH. Fractions corresponding to the OD 214 peak were collected. Five fractions were collected (Figure 14): Fraction 1, Fraction 2, Fraction 3, Fraction 4 and Fraction 5. The first three fractions include at least the main peak. b. Anti-collagenolytic activity: From the results, it can be seen that fraction 1 is the most active fraction inhibiting collagenase; There is a low level of activity in all active fractions: Assay 1 is shown in Figure 14; Test 2 is shown in the following table: sample Collagen staining Collagen short staining Collagen alone (C) ++++ - C + Enzyme + +++ C + Enzyme + EDTA ++++ - C + Enzyme + DUP + ++ C + Enzyme + Fraction 1 ++++ - C + Enzyme + Fraction 2 +++ + C + Enzyme + Fraction 3 +++ + C + Enzyme + Fraction 4 +++ + C + Enzyme + Fraction 5 +++ + C + enzyme + > 10 kDa + +++ EDTA 40 mM inhibited collagenase. Total liquid extract DUP showed low anti-collagenolytic activity. Fractions 1 to 5 were active; Fraction 1 was the most active. Fractions of molecular weights greater than 10 kDa did not exhibit any significant inhibitory activity. c. Confirmation of anti-collagen degrading factors: Liquid cartilage extracts were fractionated using a tangential flow filter method and a 10 kDa filter (Pellicon, Millipore). The filter material (<10 kDa fraction) was found to contain all anti-collagenase activity and was further characterized as follows: The <10 kDa fraction was separated on a 100 Da nominal molecular weight cleavage membrane (Spectra / 0.0 > MWCO: 100 < / RTI > Da, Cat # 131015). Anti-collagenolytic activity was recovered in the filter material (< 100 Da fraction). The <100 Da fraction was applied to a C8 reverse phase column (EM Science Lichroprep_RP-8, Cat # 9242), eluted with H 2 O, then eluted with 60% ethanol and finally 100% methanol. Most of the anti-collagenolytic activity (98%) was recovered in the H 2 O eluate and 2% activity was eluted with 60% methanol. In summary, the anti-collagenolytic activity in the extract can be dialyzed or passed through a Spectra / Por_CE (Cellulose Ester) MWCO: 100 membrane, and when applied to H 2 O, low (not adsorbed to C8 reverse phase column matrix (Lichroprep) It is due to molecular compounds. In vivo assay: Fetal Angiogenesis Test (EVT): The liquid cartilage extract was fractionated using a tangential flow filter method and a 10 kDa filter (Pellicon, Millipore). More than 10 kDa of the liquid cartilage extract and the following fractions were tested under the same conditions. They showed the same ability in inhibiting neovascularization (Fig. 15). This is in contrast to anti-collagenolytic activity that is not present in fractions above 10 kDa. a. ≪ 10 kDa Fraction: Anti-angiogenic factors in the fraction acted like anti-collagenolytic factors during the purification step described above. b. > 10 kDa fraction: The fraction was chromatographed on a gel permeation chromatography column (Sephacryl S-300, Pharmacia). The fraction with anti-angiogenic activity (S300-4) was characterized on SDS-PAGE. The active fraction (S300-4) exhibited some protein bands with molecular weights between about 8 and 18 kDa (as compared to BioRad SDS-PAGE labeled proteins). The fractions were further fractionated using anion exchange chromatography (Mono-Q, Pharmacia) using 25 mM Tris-HCl pH 8.0 and 0-1.0 M NaCl gradient. The fractions eluted from NaCl between 0.8-1.0M showed high anti-angiogenic activity. Fractions eluted at between 0.3-0.6 M and 0.08-0.2 M NaCl showed smaller anti-angiogenic activity. Comparison with prior art products Definition of Prior Art Since the present invention is not the first to have a great interest in cartilage extracts, the present invention is based on the prior art, that is to say, derived from products manufactured by the method of Balassa (U.S. Patent No. 4,822,607) and Oikawa et al. In comparative tests with two possible or described products, the unique properties of the shark cartilage liquid extracts produced by the method of the present invention were demonstrated. Oikawa et al. Describe a method for obtaining two main fractions, one having a molecular weight comprised between 1 and 10 kDa, and the second having a component heavier than 10 kDa. They were found to have anti-angiogenic properties only in the first fraction, while the other fractions showed no anti-angiogenic activity in the CAM test. For a proper comparison with the product of Oikawa, we divided the total liquid extract of the present invention into two corresponding fractions and retained one with 0 to 10 kDa. Since we describe a method for extracting whole liquid extract of Balassasms, we have used the whole liquid cartilage extract of the present invention (0-500 kDa) and Balassa's method as a starting material, replacing bovine cartilage with shark cartilage The products were compared. We hypothesized that if the Balassa and Oikawa describe the same way as we, the patterns obtained with FPLC, HPLC and CZE should be substantially duplicated and that the same anti-angiogenic activity should appear on the ECT. Final concentrations of all samples were made to 12 μg / μl (dry weight / volume of solution) prior to FPLC and HPLC chromatography. Oikawa's product was centrifuged and filtered prior to chromatography because it contained insoluble materials. Sample preparation The shark cartilage samples extracted by the above three methods were labeled (expressed as estimated dry weight per volume of solution) as follows: 1) DUP is the product of the invention (12 μg / μl) fractionated to contain between 0 and 500 kDa molecules; 2) BAL is the preparation according to the method of Balaassa et al. (12 μg / μl); 3) OIK is the product of fraction 3 (12 占 퐂 / 占 퐇) according to Oikawa et al. All samples were allowed to have a final concentration of 12 μg / μl prior to analysis. The OIK sample had a large amount of insoluble material, which can be easily deposited by centrifugation at 13,200 RPM or by filtering through a 0.2 mu m membrane. Removal of the insoluble material by a filter is essential prior to FPLC, HPLC and CZE (Figs. 16, 17, 18). FPLC comparison Condition: Superose 12 (Pharmacia); Gel permeation column. Samples were analyzed on a Superose 12 (10/30) gel permeation column using phosphate buffer saline (PBS) as eluent at 0.5 ml / min (chart speed = 0.25 cm / min). 100 μl aliquots of the sample with the concentration adjusted were filtered through a 0.2 μm membrane before injection. I read the OD 280 . The column was calibrated with the following standards (MW in Da): catalase (232,000), aldolase (158,000), albumin (56,000), ovalbumin (44,000), chymotrypsin (25,700), ribonuclease (13,700), insulin (5,700), insulin B chain (3,500), insulin A chain (2,500), bacitracin (1,450) and vitamin B-12 (1,355). The molecular weight of the main peak was calculated by the following formula: Log 10 MW = 7.52 - 0.212 x RT, where RT = elution volume in ml. R 2 = 0.976. The total column volume (V t ) was 21.93 ml as measured using cytidine (246 Da). The void volume (V o ) was determined to be 8.38 using blue dextran (2 x 10 6 da). Summary of results: In Figure 16A, the sample DUP of the present invention exhibited a first main peak (1) eluting at 18.76 ml giving a molecular weight of about 3500 Da. Continuous peaks at 22.7 (2) and 27.3 ml (3) deviated from the total column volume (21.93 ml, measured by cytidine). The peaks appear to have some affinity for the column matrix. In FIG. 16B, the sample BAL of Balassa was measured at a small peak (1) eluting near V o (8.4 ml), peak (2) at 18.5 ml (4000 da) and V t ml (4) showed two peaks. In Fig. 16c, Oikawa's sample OIK also exhibits (1) small peaks at V o , (2) peaks at 18.9 ml (3300 Da), (3) peaks at 21.5 ml ml. < / RTI > By comparison of the samples, it can be seen that the major band of the DUP sample, except for the 3500 Da peak, was not observed at the same intensity in the other samples. The OIK sample did not appear to have a small amount of the 27.3 ml peak. The BAL sample had a moving peak at 28.2 ml, which could be related to one of the minor peaks in the DUP sample. HPLC comparison Condition: CS-S-hexyl column 5 占 퐉, 25 占 0.94 cm, CSC # 059-085; Reversed column. Summary of results: For HPLC on a hexyl-reversed column, OD 210 and OD 280 were monitored simultaneously. Centrifuged and the separated sample (both 12㎍ / ㎕) dropwise a mixture of equal parts 50㎕, and eluted with 100% H 2 O. The peak for each chromatogram labeled according to OD 210 (eg 1) and the corresponding OD 280 peak are indicated by (eg. 1). The V o of the column was 5.5 ml (1.4 min). In Figure 17A, DUP had three main peaks and two minor peaks (4,5) observed through OD 210 (1, 2, 3). Two sub-peaks of peak 1 were observed, designated 1a and 1b. Significant OD 280 absorbance is associated with peaks 1, 1a, 1b and 3. By comparison, the corresponding OD 280 absorbance for peak 2 is significantly smaller than for OD 210 . In Figure 17B, BAL showed more OD 210 peaks, but its intensity was lower compared to the DUP peak. Peaks 3 and 7 in the Balassa sample appear to be related to the retention times of the peaks in the DUP sample (peak 1a or 1b and peak 4, respectively) as long as the overlap of the peaks can indicate the identity of the molecules. In Figure 17c, only three main peaks were observed in the OIK extract (1, 2, 3). Peaks 1 and 3 may be associated with peaks 1 and 3 of the DUP sample, but no negative peak of 1 was observed in the OIK chromatogram. The peak height in the OIK sample is lower than that of the DUP. Therefore, FPLC and HPLC patterns are characteristic of a prominent product. CZE comparison Condition: Devices: Beckman system (p / ace system 2050) using geol software (version 7.11 U); Capillary: Silice (TSPO 50375), 50 [mu] m x 97 cm; Buffer: 2M formic acid; Coated solution, 5% p / v hexadimethrene bromide and 2% v / v ethylene glycol aqueous solution; Detector: UV (200 nm); Current: -30 kV; Injection: 0.5 psi, 20 sec; Temperature: 22 ° C. The capillary was conditioned with 1 M NaOH (20 psi, 20 min), water (20 psi, 10 min), coated solution (20 psi, 20 min) and buffer (20 psi, 10 min). (20 psi, 2 min), sample injection (0.5 psi, 20 sec), run (-30 kV, 45 min), 1 M NaOH (20 psi, 4 min ) And buffer (20 psi, 4 min). Each sample (BAL, DUP, OIK fraction 3) was resuspended at 16.5 mg / ml. The pH of each solution in BAL, DUP and OIK was 7.1, 6.8 and 8.2, respectively. In BAL, DUP and OIK, NaCl concentrations of each solution were 2.08, 4.37 and 0.71 mg / ml, respectively. Summary of results: The molecular profiles of the respective samples (BAL, DUP and OIK-3) are shown in Fig. Comparing the DUP and BAL samples, it was found that the BAL samples contained a larger percentage of peaks with less than one percent area. BAL and DUP divide the peaks by MT / EOF = 1.06, 1.54, 1.59, 1.66 and 3.22. The peaks with the ratios of 1.06, 1.54 and 3.22 have a similar% area in BAL and DUP, while the peaks at ratio 1.59 are 8 times stronger than in BAL and vice versa at a ratio of 1.66. DUP and OIK samples show very different electrical chromatograms. The OIK has one main peak with several minor peaks. None of the peaks can be associated with one of the DUP samples. EVT comparison The anti-angiogenic potential of DUP, BAL and OIK was analyzed on EVT (Fig. 19). The extract of Balassa showed no anti-angiogenic activity. The DUP natural extract was compared to fraction 3 of Oikawa OIK. Nevertheless, Oikawa et al. Are not related to the present invention because they mention that they can not observe any activity in a fraction having a molecular weight of 10 kDa or more, which is contrary to the results of the present invention in Fig. Therefore, despite the similarities between the Balassa method and the method of the present invention, the products obtained by both methods are not quite the same. Amino acid content comparison The protein content of the BAL, DUP and OIK samples (all 16.5 mg / ml dry weight) was determined by the Lowry method; As a result, values of 3.31, 0.27 and 4.15 mg / ml are shown for BAL, DUP and OIK samples, respectively. Comparing the DUP sample with the BAL and OIK samples, the ratio of protein / dry weight is very different. A further assay was performed to further analyze the amino acid content of each liquid cartilage preparation. Figure 20 shows the ratio of each amino acid in the BAL, DUP and OIK samples. The proportion of free amino acids is very different between each cartilage preparation: 23%, 73% and 4% in the BAL, DUP and OIK samples, respectively. Obviously the ratio of amino acids from the original protein is also very different between each cartilage preparation: 77%, 27% and 86% in the BAL, DUP and OIK samples, respectively. See the following table for raw data: Cartilage product Free amino acid content (/ / ml) The amino acid (/ / ml) obtained from the original protein BAL 675 2314 DUP 604 223 OIK 181 3910 conclusion Two prior art products (BAL, OIK) compared to the present invention (DUP) are still considered as traditional processes for producing cartilage extracts. As a result, it can be seen that the process (DUP) of the present invention provides a product with unexpectedly excellent activity as far as anti-angiogenic, anti-tumor, anti-inflammatory and anti-collagenolytic activities are involved. We can infer that the process of the present invention is successful in recovering various water soluble inhibitors as an extract. Direct comparisons of BAL, DUP and OIK molecular profiles and protein content suggest that each cartilage product has unique properties. Although they seem to share some elements, it is clear that the ratio of one to the other is different. It is very important to consider that the DUP is anti-tumor when dosed orally in the dose range of about 75 mg / Kg or less, and gradually decreases the effect at higher doses. These results suggest that a greater amount of one or more factors is insufficient for the DUP liquid cartilage extract. Therefore, other cartilage preparations such as BAL, DUP, and OIK can exhibit very different biological properties because of their varying proportions of each component. Clinical experiment Preparation of liquid extracts for clinical trials Preliminary clinical trials were conducted with the shark cartilage liquid extract of the present invention. The liquid extract obtained after the ultrafiltering method was filtered with a 0.22 μm porous millipore filter. The microbial limit of the liquid extract was adjusted according to USP XXIII < 61 > The liquid extract was dispensed into sterile flasks in 7 ml aliquots (approximately 85 mg of protein), frozen overnight at -60 ° C and stored further until use at -20 ° C. Anti-angiogenic effect The liquid cartilage extract was used to treat angiogenesis-dependent diseases. Three different types of representative angiogenesis-dependent diseases have been substantially tested in humans; The first type is cancer (prostate cancer), the second type is skin disease (psoriasis), and the third type is arthritis (rheumatoid arthritis and osteoarthritis). The following samples will illustrate and direct the anti-angiogenic activity of at least the liquid extract. The results shown below are very promising and make it possible not only to be specially tested, but also to be useful for the treatment of all angiogenesis-dependent diseases, as well as for their natural penetration and their fractions. As long as the disease has an angiogenic component, the cartilage extract of the present invention is expected to be effective assuming that they are the beginning of a composition comprising an effective amount thereof and that the composition is a suitable form for proper administration. Therefore, the present invention will not be limited to the following specific compositions for use in the treatment of angiogenic diseases, as those skilled in the art can derive many compositions selected by the target disease tissue and their mode of administration. The compositions can be administered by a variety of routes such as, for example, topical, oral, sublingual, rectal, intravenous, intramuscular, intraocular, intraperitoneal, Because of the malodorous taste and odor of the cartilage, seasonings and perfumes can be added or other gallenic compositions (liposomes, encapsulation, routes, etc.) can be designed to facilitate patient compliance. The term " patient " refers to a human or animal patient. cancer: One of the patients suffering from prostate cancer has added the liquid cartilage extract to the food and has shown significant health improvement since then. Adenocarcinoma was diagnosed in 1986. At that time, radiation therapy was performed. In 1991, when the acceptable upper limit of normal was 4 / / L, the PSA (prostate serum antigen) level was 138 / / L. Thereafter, the patient experienced a completely different treatment by castration combined with anti-androgen therapy (EUFLEX). The treatment was effective for three years, after which the PSA level began to rise again. Since June 1994, the patient has added the cartilage extract as a food (the oral dose per day is about 75 mg dry weight / 7 ml of extract, about 1-1.5 mg / Kg in weight / day). The PSA level gradually decreased from 12 to 4.0 μg / ml (normal limit) and the final result was obtained in April 1996. The dose regimen should be modified according to the route of administration, the physical availability of the active ingredients, and the desired aggressiveness to control histopathology. In this case, the liquid extract may be absorbed in a substantial proportion to the gastro-duodenal track. One can rely on the results obtained in DMBA-treated mice and inoculated mice (see above). Now, no-toxicity has been demonstrated in rats and mice (see above examples), and monkeys (not shown). Oral administration of the liquid extract in DMBA-treated and DA3-transplanted mice suggests a dose rate between 1 and 300 mg / Kg of weight, which is presumably responsible for the inhibition of tumor development and tumor vascularization in an animal model Contributing. Intraperitoneal administration of the liquid extract in mice (DA3-model) indicates that the route of administration is important in obtaining an effective amount to inhibit mid-dilatation. This can be as low as 0.01 mg / Kg if a parenteral route of administration is selected, which is an effective dose of 1 mg / Kg for prostate cancer. Therefore, a daily dosage weight from about 0.01 to about 200mg / Kg (ED 50) is substantially an appropriate range of medical doses for treating cancer by reducing or eliminating the increase angiogenesis, at least in part. Some other patients have added liquid cartilage extracts to their diet with more conventional treatments (surgical treatment, chemotherapy, antihormonal therapy, etc.) (daily oral doses include about 75 mg dry weight / 7 ml extract, about 1 -1.5 mg / Kg). A summary of some medical cases is provided in the table below. These results suggest that combined therapies with liquid cartilage extracts increase survival and quality of life of patients with fidelity tumors. Female type Medical record bladder A 70-year-old man's bladder; several lesions were resected (2 cm and 1.5 cm), liquid cartilage extract was added to the food, no residual tumor was seen after 9/94 Ovarian adenocarcinoma A 47-year-old middle-aged woman (female), 15 cm (right), 11 cm (left) and several 2 cm lesions; surgical experience and chemotherapy experience in 1991; recurred and treated by chemotherapy in 1992; Treated with chemotherapy; added liquid cartilage extract to the food in 1994; thereafter reduced the mass of malignant neoplasm Rhabdomyosarcoma A 63-year-old man aged 11 cm (450 g) in diameter invaded the tumor, undergoing surgery and chemotherapy, treated with radiotherapy, and added liquid cartilage extract to the food, then the tumor showed necrotic tissue and was stable. Metastases in the liver, 45 years old (female); pancreatic lesion (9 cm) + liver metastases; chemotherapy and then liquid cartilage extract added to the food; 80% tumor suppression in 1994; Mammary gland tumor 67 years old woman (female); 1978 surgery; Recurrence and pulmonary metastases (1994); addition of liquid cartilage extracts to Megace and food, followed by partial inhibition of tumor size (1.5 cm -> 1 cm) and number (12 -> 6). psoriasis: The following skin compositions were made and tested to demonstrate their efficacy in patients with psoriasis: -EMULGADE CLB 29% (W / W) -20X natural passing water 69.5% (W / W) -GERMABEN II 1% (W / W), and -Lavandula Angustifolia 0.5% (W / W) EMULGADE CLB, a mixture of stearate ester, fatty alcohol and nonionic emulsifier (purchased from Henkel Canada Ltd.), was heated to 65-70 ° C with stirring. Heating was discontinued and the mixture was stirred continuously. Lavandula Augustifolia, an essential oil, and GERMABEN II (30% diazonidyl urea, 11% methylparaben, 3% propylparaben and 56% propylene glycol) when the mixture was at a temperature of 45 &; Purchased from Sutton Laboratories, NJ, USA). When the temperature of the mixture reached 30 캜, the liquid cartilage extract was added. The composition so obtained was a smooth, greasy cream; By varying the percentage of EMULGADE, other forms with varying viscosities of the composition depending on the intention of the manufacturer (milk, lotion, ointment) can be obtained. Other carriers or excipients may be used to obtain a paste, a gel and any other form of surface preparation. The formulation phase was performed twice daily (topical application) for 12 weeks on a panel of 9 patients with psoriasis who responded to conventional treatments but were subsequently immunized against them. For this study, patients with similar and symmetric psoriasis on both sides were selected. The experiment was performed in a double-blind fashion in which both the dermatologists and patients were treated with a composition containing the cartilage extract, the affected side, and one was unaware that it had been treated with the control-composition. Significant improvements were observed in five patients in whom psoriasis was not amenable to hydrocephalus; For the groups with the hyperkeratosis, the results were moderately good. A photograph of a part of the body of two patients is shown in FIG. In Figures 21a and b, nonetheless, patients affected with psoriasis accompanied by hyperkeratosis showed a very significant reduction in erythema and did not accompany any itching after only one month of treatment. However, the hyperkeratosis is still important. Photographs of a second patient (Fig. 21c and d) suffering from psoriasis not associated with hyperkeratosis showed a very significant improvement after treatment-3 months. Since psoriasis appears to be a multifactorial disease, the response of patients depends on the extent to which components such as angiogenesis and inflammation affect the establishment and persistence of these conditions. As shown in the DMBA-treated mice (Figure 5), endothelial cell proliferation (Figure 7) and EVT (Figure 10), anti-angiogenic activity actually exists in the extract of the present invention. Anti-inflammatory activity was also confirmed (CHS model of mouse). Better results may be obtained if this type of formulation is supplemented with other therapeutic agents (such as excipients, additional anti-inflammatory agents, antihistamines, immunosuppressants, etc.) and delivered to other relevant factors. For example, forms for modifying the formation to include an effective amount of each layer release agent can be supplemented. It may also be achieved by the selective administration of the complementary therapeutic agent, or simultaneously with the application of the localized formation of the present invention. Also, supplemental drug therapy need not be administered in the same route. Despite the high proportion of liquid cartilage extract, the formulation did not show any overall effect (the effect was limited to the area of treatment) and no secondary effect. arthritis: Patients with arthritis were tested for volunteers with two units of total liquid extract of 7 ml per day for several months. The patients showed progressively improved status with restoration of joint function, pain and decreased inflammation (up to about 60%). Because arthritis has angiogenic and inflammatory components, the effects can be influenced by anti-angiogenic and anti-inflammatory activities of cartilage extracts. Preliminary clinical studies were carried out by Ryumati expert. Seven volunteers and aged subjects aged between 39 and 60 years of age with rheumatoid arthritis were enrolled in the present invention. Diagnosis was based on the traditional method in the re-edited edition of the Americal Rheumatism Association (Arthritis & Rheumatism, 31, 315-325 by Arnett, F.C. et al. (1988)). The treatment lasted for 30 days and 21 ml of liquid shark cartilage extract (12 mg / ml of dry weight) was daily dosed. The effect of the treatment was determined as a list of literature on the evaluation of tenderness (Quaterly J. Med, New Series, XXXVII, vol. 147, 393-406, Ritchie, D. M. et al. The list is based on the sum of the number of quantitative assessments of pain suffered by the patients when the joints are pressed momentarily or when the joints are moved over the joint space. From the results, it can be seen that four of the seven patients improved upon treatment with liquid cartilage extract (see table below), which could be used to treat other conditions complicated by rheumatoid arthritis or chronic inflammation . Patient (number) Age (years) List of Ritchie Enhancement 0 days 30 days One 60 30 22 Available 2 43 8 8 None 3 52 12 12 None 4 41 15 19 None 5 46 5 3 Available 6 39 6 2 Available 7 55 14 7 Available Spider veins: A total of 16 panelists were recruited for this study. The participants showed telangiectasia on the face but not severe. The participants were divided into two groups of 8 persons each. One group A is provided as a cholesterol lipocyte base containing 5% liquid cartilage extract, while the second group is provided as a cholesterol lipocomposite base alone. The product was used on the face twice a day for three months. A fiber optic surface microscope was used to obtain at least four sections of the face representing the spider veins. The images were analyzed for gray values through a Zeiss Ibas Image Analyzer. The integrated optical density (IOD) was calculated for each part of each participant. Four parts of each participant were averaged over each time point. As a result, there was a 35% reduction in the IOD after 4 weeks, and the effect was maintained during the course of the study (FIG. 22). The cholesterol lipoic acid base showed a background improvement of 5% and 8% after 8 and 12 weeks of use, respectively. Petiorbital dark ring: Skin pigmentation is not only due to the presence of melanin, but also to blood supply and plasma content. When blood flow is slowed and a great deal of oxygen is consumed in metabolism, the skin appears blue. These color differences are maximized around the eyes due to the thickness of the skin (Oresajo et al. (1987) Cosmetics & Toiletries 102: 29-34). Vessel changes in the septa present under the eyes also exacerbate the occurrence of dark circles. The dark circles around the eyes also appear to be due to fat accumulation, edema under the eyelids, and leakage of blood vessels around the eyes. Clinical studies were designed to evaluate the effects of shark cartilage liquid extracts on reducing the occurrence of orbital periosteal dark circles by modulating angiogenesis around the eye. A total of 18 female volunteers aged 18-65 years were included in the study. The participants showed a distinct dark ring around the eyes. All participants were healthy without any symptoms of acute or chronic disease, including skin or eye problems. Subjects who showed epidemics, rashes, scars, and burn marks that could interfere with the evaluation of test results were excluded from this study. Pregnant or lactating women were also excluded. There were no warts, dots, smears, tanning, scars, or active skin lesions observed at the test site. Participants were divided into two groups, Group A and Group B, each containing 10% liquid cartilage extract or carrier alone. Participants were provided with enough products to be applied around the eyes at least twice a day for 12 weeks. Measurements were taken at baseline, and at 4, 8, and 12 weeks. At each time point, the photographs were taken and analyzed using Image Analysis. The photographs were analyzed for the gray values showing the degree of skin cancer / person. The group treated with liquid cartilage extract showed a favorable increase in gray value (indicating the degree of lightness of dark coloring). After 4, 8 and 12 weeks, the lightness of the skin under the eyes of the group treated with liquid cartilage extracts was 11%, 21% and 14%. The group treated with the carrier only showed no change (Fig. 23). Varicose veins: A total of 20 participants participated in this study. The participants showed capillary dilatation seen on the leg but not excessive. The participants were divided into two groups. Group A (n = 9) received liquid cartilage extracts containing cream, while Group B (n = 11) only provided carrier creams for all legs, twice daily for three months. A fiber optic microscope was used to obtain images of the 2-4 sites of the legs representing the varicose veins. The phases were analyzed for gray values using a Zeiss Ibas analyzer. For each participant, the integrated optical density (IOD) for each region was calculated. All areas on each participant were averaged over each time point. The results are shown in Fig. There was a 21%, 17% and 26% reduction in IOD after 4, 8 and 12 weeks, respectively. The control group showed the basic improvement values of 5%, 0% and 0% after 4, 8 and 12 weeks, respectively. Other possible clinical and livestock treatment applications: Ophthalmology: Reduced vision or blindness can be caused by a number of conditions characterized by abnormal blood vessel growth or neovascularization. These include corneal neovascularization (caused by chemical and physical stimuli), corneal infections, corneal transplant rejection, neovascular glaucoma, spotting degeneration, herpes zoster keratitis and diabetic retinopathy. The liquid cartilage extract may act by inhibiting the formation of renal vessels and decreasing capillary dilatation in these clinical conditions. Wound therapy: Wound therapy involves the complex action of cells, biochemical messengers, extracellular matrix molecules and the microenvironment of the cells. After severe wound healing, the granulation tissue (fibroblasts, capillaries and inflammatory cells) first grows from the edge of the wound to a specific sequence. Fibroblasts begin to migrate from the connective tissue to the wound space at the wound edge within 24 hours. Fibroblasts produce matrix molecules (collagen and glycosaminoglycan) that form an extracellular matrix when they migrate. The first capillary process can be observed in the microcirculation perfused at the wound edge as fast as 18 hours after wounding. The protrusions grow into the wound space and provide a new capillary network to the wound connective tissue. Fibroblast proliferation and migration and capillary growth lead to one unit until the wound space is filled with completely new tissue. Some wound treatment conditions that are complicated by over-expression of the granulation factors such as hypertrophic scarring and treatment of the skin of severely burned patients may be beneficial for the administration of the liquid cartilage extract (oral or topical administration) Reducing the process is slowing the wound healing process. Gum Raised Skin Disease: The excellent effect of liquid cartilage extracts on psoriatic lesions suggests that other diseases with a common characteristic may also be suitable for partial or total administration of the liquid extract. Glaucoma impression skin diseases are caused by endothelial hyperplasia and / or underlying skin inflammation and are characterized by the presence of redness, including psoriasis, Reiter syndrome, rose intolerance, squamous cell carcinoma, pemphigus vulgaris, secondary syphilis, Characterized by color or purple acne and plaque. Hair Loss: Ligation of small side camellia that directs blood flow to the scalp has been successful in inhibiting hair damage caused by androgen and exposure. Partial application of the liquid cartilage extract on some areas of the scalp may reduce scalp loss by reducing subsequent exposure to the vasculature and hormones. Application of livestock therapy: Solid and / or liquid cartilage extracts can be administered to animals for the same therapeutic and cosmetic applications described for humans. Non-anti-angiogenic effect acne: Although the acne is not classified as a disease with angiogenic components in the inventor's knowledge, it is nevertheless possible to test the cartilage extract in affected patients based on the fact that the liquid cartilage extract is also anti-inflammatory Attracted attention. To test these liquid extracts on acne patients, a gel composition was prepared as follows: CARBOPOL 1.2% Distilled water 77.2% NaOH 0.3% PHENOXETOL 0.3% TWEEN 80 0.3% Liquid cartilage extract 20.0% 40X Aloe extract 0.5% The liquid cartilage extract contains a dry weight of 9-12 mg / ml. The formation shows a noticeable improvement in the skin condition of some more serious acne patients (inflammatory acne and vaginitis acne). Figure 25 shows a significant improvement in the condition of the acne patient when treating the topical liquid extract containing the carrier for 12 weeks. The results may be due to anti-angiogenic effects (consequently exposing angiogenic factors in acne), or they may be caused by active ingredients with anti-angiogenic and other effects (e.g. anti-inflammatory effects) . All the results obtained in the clinical trials show a very potent effect of the liquid cartilage extracts in the treatment of angiogenesis-dependent and / or inflammatory diseases. The amount of cartilage extract as well as their formulation can be varied according to their willingness to meet a particular need. Based on the protein content, the topical and all other compositions may contain a wide range of cartilage extract quantities. Between the three specific ranges tested, very different dosages and / or formulations were used. Skin irritation: Since angiogenesis is often associated with inflammation in many diseases, it is desirable to independently ascertain each activity in the cartilage extract. To this effect, a skin irritation model which is not suspected of causing any angiogenic symptoms was selected for testing the extracts for anti-inflammatory and soothing activity. Nine volunteers with skin sensitivity to Balsam in Peru were selected for this study. The test compound is as follows: 1. D-MEN Median 1X Shark Cartilage 50% 2. D-MEN Median 1X Shark Cartilage 20% 3. D-MEN Medial 1X Shark Cartilage 10% 4. Cola nitida (Indena) 10% hydro-alcohol 1: 1 The four test compounds were applied on the ventral forearm of participants. After the material was absorbed for 20 minutes, Peruvian balsam, a stimulant, was applied to the test site. Skin irritation was measured as an increase in skin safflower. The degree of safflower was measured with Minolta Chromameter and compared with positive and negative control. The positive control was skin color treated with Peru balsam only, and the negative control was treated with cola solution and tried skin area like the test product. Statistical significance was calculated by the likelihood of two tail tails being tested. Figure 26 shows that 10% cola is 70% active. Shark cartilage was 58% and 60% as anti-irritant at 20% and 10% concentrations, respectively. There was no dose-response effect. The results suggest that the cartilage extract contains sedative and anti-inflammatory activities far from the anti-angiogenic effect. cancer: A 53-year-old woman was diagnosed with non-Hodgkin's lymphoma of type B non-large cell. The CAT scan method reveals a large volume of stenosis on the right parotid gland and on the right parotid gland around the carotid and jugular veins (2.5 cm in diameter). The patient refused the chemotherapy and added the liquid cartilage extract to her diet (Oct. 1993) (about 75 mg dry weight / 7 ml extract, daily oral dose, about 1-1.5 mg / Kg weight / day, Three months later (January 1994), the CAT scan method revealed neuropathy in a perfectly absorbed neck. By November 1994, abdominal pain had decreased by 75%. The disease has since stabilized and the patient has felt healthy. The results suggest that some non-fidelity cancers may also respond to anti-tumor activity of liquid cartilage extracts. Skin protection: Six healthy volunteers were included in the study. The participants received a cream containing the liquid cartilage extract for application on the left front forearm, twice daily for four weeks, and for application on the right forearm. The participants were women with no signs of acute or chronic disease, including 21-45-year-old skin or oculomotor problems. Subjects who showed severe grieving, warts, and traces of burning that interfered with the evaluation of test results were excluded from this study. There were no warts, spots, dots, smears, tanning, scars and active skin lesions observed at the test site. On test day 1, the participants were instructed to prohibit the use of any lotion, cream or other product on their face. During the measurement procedure, participants were equilibrated for at least 30 minutes before testing in a controlled environment at 20-22 < 0 > C temperature and 40% relative humidity. The test sites were the right and left palms of the forearms. A small area (3.5 cm x 7 cm) was displayed on each arm and the base surface water loss (TEWL) was determined from three sites within the area (Contact Dermatitis 22: 164-178; Grove (1994) Edited by Squier & Hill, CRC Publication, pp. 124-125). Tuck tapes were used to cover the test area, stuck firmly in both directions and then peeled off (Elias (1993) J. Invest. Dermatol. 80: 044s-049s). A total of 5 fragments were obtained. TEWL was recorded again. In five groups, exfoliation continued before the TEWL measurement. The exfoliation was stopped when the TEWL reached 18G / M 2 / Hr. The TEWL was again measured in the latter half of the final stripping sequence. The number of fragments required to damage the skin boundary was calculated by recognizing the maximum exfoliation number for each arm at each time point showing a TEWL of 18 G / M 2 / Hr or more. The results were analyzed for statistical significance between treatments at various times versus baseline using the one tailed rank constant Z test. Carrier treated arms did not appear to show any improvement as only 26% and 21% of the exfoliation was required to damage the skin after 2 weeks and 4 weeks treatment, respectively. There was a significant improvement (p < 0.05) in the marginal state of each participant after treatment with liquid cartilage extract product for 2 and 4 weeks when 60% and 55% of the exfoliation was required to peel the skin boundary ). Therefore, liquid cartilage extracts have proven useful for strengthening skin boundaries against physical damage. eczema: Liquid cartilage extracts were tested in beauty salons for their ability to reduce inflammatory lesions caused by eczema. Hairdressers applying the cream containing the liquid extract have chronic eczema in their hands for years. Interestingly, the use of cream containing cartilage significantly reduced the expression of eczema in the hands. Hairdressers are now using cream that successfully contains cartilage to stop the development of eczema. mantis: A 36-year-old middle-aged woman with plantar warts was treated by a dermatologist for almost three years to control warts development and accompanying pain. Among the treatments were liquid nitrogen, salicilic acid (40%), microbial methods, nitric acid, and sulfuric acid. These treatments were conducted weekly for a period of typically three months, with little results. In March 1996, she applied daily shark cartilage liquid extract directly to the warts (5 minutes); Two weeks later, a pink area of a new endothelium was created around the warts; The next week the warts disappeared. Therefore, the above results suggest that the liquid cartilage extract can help wart treatment. Other Potential Clinical and Livestock Therapeutic Applications: Implant rejection: Inflammation is one of the major factors associated with the death of transplanted cells. Therefore, tissue grafting can be advantageous as an anti-inflammatory component present in the shark cartilage liquid extract of the present invention. Multiple sclerosis: The cause of multiple sclerosis is unknown. Tissue reaction has a single nuclear cell infiltration around the vein and is characterized by an immunopathologic process with no apparent pathological evidence of infection. Matrix metalloproteinase is an important factor in the inflammatory response. Since liquid cartilage extract is a potent inhibitor of matrix metalloproteinase, it may be useful in the treatment of multiple sclerosis. Fibrosis: Current concepts suggest that fibrosis is similar to normal wound healing, but fails to finish, resulting in scarring instead of normal tissue. Most fibrosis reactions appear to be indirect to mental trauma, infection, inflammation, but may have genetic components, for unknown reasons. Typically, TGF-β is hyperplasia, leading to proliferation of fibroblasts and overproduction of collagen. Since the deposition of excess collagen is a hallmark of fibroids, we suggest that liquid shark cartilage extracts, which are capable of geothermal formation of granulation tissue, may be beneficial for a long period of time in suppressing fibrosis response. Inflammatory Bowel Disease: The etiology of inflammatory bowel disease is unknown, but abdominal intestinal immunity is associated with Crohn's disease and ulcerative colitis. Mucosal mononuclear cells express altered antibody production, proliferation, cytotoxicity and synthesis of cytokines (FGF, PDGF, EGF, TNF). Liquid cartilage extracts exhibit anti-inflammatory activity, and then oral administration may be helpful in the treatment of inflammatory bowel disease. Heart Disease: Endothelial dysfunction of the coronary arteries may be due to microvascular anomalies of the coronary arteries and possibly myocardial reduction anemia and heart pain. At the cellular level, endothelial dysfunction is associated with reduced expression of nitric oxide (NO), an endothelium-induced relaxant. No synthesis can keep the vascular system in a vasodilator state to control arterial pressure. The adverse conditions in the internal synthesis of NO contribute to conditions such as arterial hypertension, pulmonary hypertension and heart disease. We have the dominant result that the liquid cartilage extract from cultured endothelial cells increases NO production. Therefore, it can be judged that the liquid extract can be helpful for some heart diseases not only in pediatric patients having congenital heart disease combined with pulmonary arterial hypertension through NO. In addition, liquid cartilage extracts can help to reduce inflammation-related complications in athelemia. Scleroderma: Scleroderma (hard skin) is a rare disease caused by an increase in the skin and often fibrous connective tissue of the internal organs. This often manifests as hyperkeratinization of partial skin patches. And keratinization are cellular processes in which the corneal cells of the skin differentiate and accumulate rigid keratinous fibers. If skin around the joints is affected, skin conditions can result in limited joint fluidity. When added to an experimental system in which keratinocyte differentiation is inspected, the liquid cartilage extract partially obstructs the differentiation or keratinization process. Therefore, the liquid extract can act favorably on the skin condition by inhibiting the accumulation of keratinocytes as a whole. Application of livestock treatments: Solid and / or liquid cartilage extracts can be administered to animals for the same therapeutic and cosmetic applications as described for humans. Cosmetic applications and compositions: The tests and experiments show that the cartilage extract of the present invention can find various medical applications. Of the various activities recovered in the extract of the present invention, the inhibitory effect on anti-angiogenic, anti-collagenolytic, anti-inflammatory and PKC-induced differentiation is particularly desirable for cosmetic applications. Since the cartilage extract of the present invention exhibits an adverse effect on the progress of cells mediated by PKC and the opposite effect is described in the literature to improve the skin barrier function, the cartilage extract and the pharmaceutically acceptable carrier And the composition is an object of the present invention. ≪ Desc / Clms Page number 2 > A different or similar composition may also be considered to be used in the method of reducing the inflammation of the mammalian skin or soothing the skin. Inflammation can be caused by various reagents such as chemical stimuli, physical abrasion, and exposure to ultraviolet light. Methods and compositions for inhibiting collagenase in the skin may also be considered. Collagenase and inflammation are associated with premature aging (degradation of collagen), and therefore counteractivity from the cartilage extracts can also contribute to methods and compositions for calming premature aging and controlling wrinkles or atrophy in mammals . Causes of wrinkling or atrophy include, for example, exposure to aging, ultraviolet radiation or pollution environments. The topical composition may contain an effective amount of shark cartilage determined for a particular application. Generally, the compositions may contain about 0.1 to 75% by weight of the liquid cartilage extract and about 25 to 99.9% by weight of the pharmaceutically acceptable carrier. The compositions may contain anti-oxidants such as reagents that interfere with the formation of lipid peroxides in the skin. Examples of such anti-oxidants are tocopherol, tocopherol derivatives, ascorbic acid, ascorbic acid derivatives and BHF. The composition may be supplemented with an anti-inflammatory agent such as a phospholipase A2 inhibitor or an anti-irritant cola and a green tea extract derived from wild type. The topical compositions may take a variety of forms such as solutions, suspensions, lotions, tinctures, gels, creams, sprays, emulsions, adhesives, ointments or liposomes (at least part of liquid cartilage extract). Other cosmetic applications include dark circles around the eyes and skin barrier function. conclusion It has been argued that the methods of the present invention provide for the production of cartilage extracts with excellent clinical value. The shark cartilage extract produced by the novel method includes various activities recovered in excellent yield. The cartilage extracts, especially the liquid extracts and their functions have a strong potential since they are non-toxic to normal cells, while being effective in a wide variety of diseases or conditions. For all virtual applications (from ophthalmic drugs to the manufacture of skin and cancer treatments), the minimum final protein concentration of the total liquid extract is estimated to be very low (from about 0.01 mg / ml). This low-range dosage is dependent on accessibility and depends on the permeability of the active ingredient to the active site as well as the tissue's sensitivity and response to effective capture of the components and angiogenesis inhibitors. The upper limit of the final protein concentration in manufacture for some applications is currently unknown. The highest final concentration tested was about 9 mg / ml of protein at the time of topical administration in the preparation for the treatment of psoriasis, about 12 mg / ml for oral administration, 7 ml for cancer, and 21 ml for arthritis . The shark cartilage liquid extract may lose some of its activity during lyophilization. However, the addition of known stabilizers or preservatives in the prior art for lyophilisation can preserve the sensitive activity and enable high doses of the cartilage extract to be administered in the dry state. Materials needed: - coolant - Surgical instruments - Chopping meat - Plastic bags - Industrial mixers (3-speed mixer products purchased from Fisher Scientific) - Water purification system (Reverse osmosis and 0.1Em filter method; Continental Water System, Model PRE 2202, Product number 91089, Modulab Bioscience RQ / Polishing System purchased from Fisher Scientific, Montreal, Quebec). The system provides a non-volatile material of high quality. - Precision balance Mettler, AE series purchased from Fisher Scientific - Centrifuge purchased from DuPont Canada Sorvall RC-285 - Centrifuge CEPA - Nylon pouch with 1 μM hole - Sterilizer * Automatic steam sterilizer Sanyo, model MAC 350P) - Nalgene 500 ml container which is sterilized at 132 ° C for 10 minutes and dried for 35 minutes. - Conical filter with 24 μm holes Whatman Reeve Angel - Ultra filter column (molecular weight cut-off: 500 kDa and 1 kDa at application; surface: 25 square feet; flow: 130 L / min; internal pressure: 30 psi; external pressure: 5 psi; Koch Membrane System Inc., Wilmington, USA) - A sterile centrifugal pump (Manrch Industries, model ACE-S100, Type A) to provide a flow of 130 L / - sterile hut (laminar flow hut NuAire purchased from Ingram & Bell) - Millipack-60 0.22 μm sterile filter - sterile transparent or amber glass bottles - Concentrator DC-10 Amicon - Rotofor Biorad 170-2950 - Amicon filters SIOY10, SIOY30 and SIOY100 with cut-off values of 10, 30 and 100 kDa, respectively - FPLC Pharmacia 216007 (Computer Pharmacia 216014) - Hilstand S-300 26mm / 60cm (Pharmacia) - Superose S-12 10 mm / 30 cm (Pharmacia) - Freeze-drying agent Labconco 10273 A
权利要求:
Claims (44) [1" claim-type="Currently amended] A method of obtaining a liquid extract of cartilage having a substantial portion of a biologically active water-soluble component present in natural cartilage, characterized by comprising the steps of: a) homogenizing the cartilage in an aqueous solution on condition that the biologically active ingredients are preserved intact until the cartilage is reduced to particles equal to or less than 500 탆, resulting in a natural Making a mixture of liquid extract and particles; b) centrifuging the homogenate to separate particles from the natural liquid extract; c) further separating said natural liquid extract to obtain a final liquid extract containing cartilage molecules having a molecular weight equal to or less than 500 kilodaltons (kDa); And d) concentrating said final liquid extract to prevent any substantial damage to said biologically active water soluble component of said final liquid extract. [2" claim-type="Currently amended] 2. Process according to claim 1, characterized in that the conditions comprise a working temperature comprised between 0 and 20 DEG C for step a) and between 0 and 10 DEG C for steps b), c) and d). [3" claim-type="Currently amended] The method of claim 1, wherein said condition comprises a working pH in the range of 6 to 8 for said aqueous solution. [4" claim-type="Currently amended] The method of claim 1, wherein step a) is performed within 15 minutes to 24 hours. [5" claim-type="Currently amended] 5. The method according to claim 4, wherein step a) is carried out within 15 minutes to 1 hour. [6" claim-type="Currently amended] 2. The method of claim 1, wherein the cartilage and aqueous solution are at a rate of one kilogram for at least one liter. [7" claim-type="Currently amended] The method according to claim 1, wherein said concentration step d) is to filter out said final liquid extract on a membrane having a molecular weight cut-off value of 0.1 kDa, thereby removing molecules having a molecular weight lower than said cut-off value . [8" claim-type="Currently amended] The method of claim 1, wherein said concentration step (d) comprises lyophilization of said final liquid extract. [9" claim-type="Currently amended] 9. The method of claim 8, wherein the lyophilization is performed in the presence of a stabilizer or preservative added to the liquid extract in an amount sufficient to enhance preservation of the biologically active ingredient. [10" claim-type="Currently amended] 10. A method according to any one of claims 8 and 9, wherein the cartilage is obtained from a shark. [11" claim-type="Currently amended] A method for obtaining a liquid extract of shark cartilage having the anti-collagen degrading and anti-angiogenic water-soluble components present in natural cartilage. a) homogenizing the cartilage in an aqueous solution, with the conditions that the biologically active ingredients can be preserved intact, until the cartilage is reduced to particles equal to or less than 500 < RTI ID = 0.0 > Obtaining a mixture of liquid extract and particles; b) centrifuging the homogenate to separate particles from the natural liquid extract; c) further separating said natural liquid extract to obtain a final liquid extract containing cartilage molecules having a molecular weight equal to or less than 500 kilodaltons (kDa); d) filtering said final liquid extract on a membrane having a molecular weight cut-off value of 100 Daltons; And e) recovering a fraction comprising molecules having a molecular weight between 0 and 0.1 kDa. [12" claim-type="Currently amended] A method for obtaining a liquid extract of shark cartilage having a water-soluble component of anti-angiogenesis present in natural cartilage comprising the steps of: a) homogenizing the cartilage in an aqueous solution on condition that the biologically active ingredients are preserved intact until the cartilage is reduced to particles equal to or less than 500 탆, resulting in a natural liquid having the biologically active ingredient Obtaining a mixture of extract and particles; b) centrifuging the homogenate to separate particles from the natural liquid extract; c) further separating said natural liquid extract to obtain a final liquid extract containing cartilage molecules having a molecular weight equal to or less than 500 kilodaltons (kDa); d) filtering said final liquid extract on a membrane having a molecular weight cut-off value of 10 kDa; e) recovering a fraction comprising a molecule having a molecular weight of 10 kDa or greater; f) fractionating said fraction on an anion exchange chromatographic column; And g) recovering a fraction eluting between 0.8-1.0 M NaCl. [13" claim-type="Currently amended] A cartilage extract obtained from a shark, which is produced by the method of any one of claims 1 to 6. [14" claim-type="Currently amended] Wherein the extract is obtained from a shark and is produced by the method of claim 7. [15" claim-type="Currently amended] A cartilage extract produced by the method of claim 10. [16" claim-type="Currently amended] 11. An anti-collagen degradable and anti-angiogenic cartilage extract which is produced by the method of claim 11. [17" claim-type="Currently amended] 12. An anti-angiogenic cartilage extract which is produced according to the method of claim 12. [18" claim-type="Currently amended] Comprising an effective amount of a cartilage extract obtained from cartilage and produced according to the method of any one of claims 13, 14 and 15, and a pharmaceutically acceptable carrier. Inflammatory and collagenolysis. ≪ RTI ID = 0.0 > 11. < / RTI > [19" claim-type="Currently amended] A composition for treating a disease or condition having collagen degradability characterized by comprising an effective amount of cartilage extract as defined in claim 16. [20" claim-type="Currently amended] Comprising an effective amount of a cartilage extract as defined in claim 16 or 17 or a mixture of cartilage extracts as defined in claims 16 and 17 for the treatment of a disease or condition having an angiogenic component. Composition. [21" claim-type="Currently amended] 19. The composition of claim 18, wherein the composition is for intraperitoneal, subcutaneous, ocular, non-nasal, oral, rectal, sublingual, intramuscular, intradermal or intravaginal use. [22" claim-type="Currently amended] 19. The composition of claim 18, wherein the composition is a topical composition. [23" claim-type="Currently amended] 23. The composition of claim 22 wherein said cartilage extract is added such that the final solids weight content of the composition is between 1 and 50 mg / ml. [24" claim-type="Currently amended] Characterized in that it comprises a solids weight content of 2 mg of cartilage extract in the cartilage extract defined in any one of claims 7, 8 and 9 per ml of the composition as therapeutically effective ingredient and a pharmaceutically suitable carrier A topical composition for the treatment of acne. [25" claim-type="Currently amended] Characterized in that it comprises, as a therapeutically active ingredient, a solid weight content of the cartilage extract of from 30 to 50 mg of the cartilage extract defined in any one of claims 7, 8 and 9 per ml of composition and a pharmaceutically suitable carrier Lt; RTI ID = 0.0 > psoriasis. ≪ / RTI > [26" claim-type="Currently amended] 9. A composition for treating cancer, comprising a therapeutically effective amount of a cartilage extract as defined in any one of claims 7, 8 and 9, and a pharmaceutically acceptable carrier. [27" claim-type="Currently amended] 27. The composition of claim 26, wherein the cartilage extract is administered so that the total dry weight of the extract per kg of patient weight is from 0.01 to 200 mg of the total dry weight of the extract. [28" claim-type="Currently amended] 28. The composition of claim 27, wherein the composition is administered orally or sublingually to a patient in need of such treatment. [29" claim-type="Currently amended] Comprising administering to a patient in need thereof an effective amount of an anti-inflammatory cartilage extract as defined in any one of claims 13,14 and 15 and an acceptable pharmaceutical carrier. How to Treat Inflammation in. [30" claim-type="Currently amended] 15. A method for treating angiogenesis, comprising the step of orally administering an effective amount of an anti-angiogenic cartilage extract as defined in any one of claims 13,14 and 15 and an acceptable pharmaceutical carrier. To inhibit angiogenesis in a patient in need thereof. [31" claim-type="Currently amended] 22. A method of enhancing skin barrier function in mammalian skin, comprising applying to a skin composition as defined in claim 22. [32" claim-type="Currently amended] 22. A method for calming mammalian skin, comprising applying the composition as defined in claim 22 to the skin. [33" claim-type="Currently amended] A method of reducing inflammation in mammalian skin, comprising applying the composition as defined in claim 22 to the skin. [34" claim-type="Currently amended] 34. The method of claim 33, wherein the inflammation is caused by physical friction. [35" claim-type="Currently amended] 34. The method of claim 33, wherein said inflammation is caused by chemical stimulation. [36" claim-type="Currently amended] A method of inhibiting collagenase activity in mammalian skin, comprising applying the composition as defined in claim 22 to the skin. [37" claim-type="Currently amended] A method of controlling wrinkles or atrophies in mammalian skin, comprising applying the composition as defined in claim 22 to the skin. [38" claim-type="Currently amended] A method of reducing acne in a mammalian skin, comprising applying the composition as defined in claim 24 to the skin. [39" claim-type="Currently amended] 26. A method of treating psoriasis in a mammalian skin, comprising applying the composition as defined in claim 25 to the skin. [40" claim-type="Currently amended] A method for delaying premature aging in a mammalian skin, comprising applying to a skin composition as defined in claim 22. [41" claim-type="Currently amended] Comprising administering a composition as defined in claim 18 to a patient in need of treating one or more diseases or conditions selected from the group consisting of tumor growth, angiogenesis, inflammation and collagenolysis. . [42" claim-type="Currently amended] 42. The method of claim 41, wherein said disease or condition is selected from the group consisting of cancer, pap smear, skin disease, arthritis, acne, spider veins, varicose veins, orbital periosteum dark ring, hypertrophic scar, alopecia, eczema, warts, A corneal neovascularization, corneal infections, neovascular glaucoma, herpes zoster virus keratitis, diabetic retinopathy, and transplant rejection. [43" claim-type="Currently amended] 15. A method of inhibiting endothelial cell proliferation comprising contacting endothelial cells with a composition comprising a cartilage extract as defined in any one of claims 13, 14 and 15. [44" claim-type="Currently amended] 15. A method for inhibiting activated-corneal cell differentiation comprising contacting a corneal cell with a composition comprising a cartilage extract as defined in any one of claims 13, 14 and 15.
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同族专利:
公开号 | 公开日 DE69625698D1|2003-02-13| HU225490B1|2006-12-28| IN188198B|2002-08-31| CN1206348A|1999-01-27| RU2181292C2|2002-04-20| BG102419A|1999-01-29| BR9611507A|1999-12-28| NO981781D0|1998-04-21| CO4750839A1|1999-03-31| CA2236021C|2002-07-30| IS2017B|2005-06-15| JPH11513990A|1999-11-30| DE69625698T2|2003-10-16| US6025334A|2000-02-15| EP0859622B1|2003-01-08| NO981781L|1998-06-19| RO118567B1|2003-07-30| PL187989B1|2004-11-30| HU9802604A3|2001-06-28| ES2190475T3|2003-08-01| NZ313957A|2000-05-26| ZA9606726B|1997-02-18| TW503108B|2002-09-21| WO1997016197A1|1997-05-09| EP0859622A1|1998-08-26| HU9802604A2|1999-10-28| CN1187060C|2005-02-02| CA2236021A1|1997-05-09| PL326888A1|1998-10-26| JP2003246740A|2003-09-02| IL119030D0|1996-11-14| DK859622T3| BG63907B1|2003-06-30| HK1015683A1|2003-04-17| DK0859622T3|2003-05-05| IL119030A|2000-08-13| MX9803419A|1998-10-31| CZ129398A3|1998-12-16| AT230604T|2003-01-15| IS4728A|1998-04-29|
引用文献:
公开号 | 申请日 | 公开日 | 申请人 | 专利标题
法律状态:
1995-10-30|Priority to US8/550003 1995-10-30|Priority to US08/550,003 1995-10-30|Priority to US08/550003 1996-08-07|Application filed by 에릭 듀퐁, 레 라보라뚜와르 제떼르나 인코오포레이티드 1996-08-07|Priority to PCT/CA1996/000549 1999-08-16|Publication of KR19990067245A 2001-10-26|Application granted 2001-10-26|Publication of KR100296016B1
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申请号 | 申请日 | 专利标题 US8/550003|1995-10-30| US08/550,003|US6025334A|1994-04-28|1995-10-30|Extracts of shark cartilage having anti-collagenolytic, anti-inflammatory, anti-angiogenic and anti-tumoral activities; process of making, methods of using and compositions thereof| US08/550003|1995-10-30| PCT/CA1996/000549|WO1997016197A1|1995-10-30|1996-08-07|Extracts of shark cartilage| 相关专利
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