RU2121844C1 - Method of preparing a carbohydrate-protein complex from japanese scallop - Google Patents

Abstract

FIELD: biotechnology, pharmacy. SUBSTANCE: Japanese scallop (Patinopecten yessoensis) is irradiated by ultraviolet rays at the dose 8.2-8.6 kJ/m² followed by their care in natural environment for 22-26 h. Then mollusk mantle is milled, homogenized and extracted with physiological solution, dialyzed and lyophilized. Method provides preparing a preparation showing antitumor and immunostimulating activity. EFFECT: improved method of complex preparing. 5 tbl, 1 ex

Description

 The invention relates to bioorganic chemistry and relates to the production of biologically active preparations from animal raw materials.

 It was found that most of the known low molecular weight antitumor drugs are highly toxic immunosuppressants, while the inhibitory immunogenesis of these agents is due to damage to the lymphoid tissue. In contrast, high molecular weight polysaccharides and glycoproteins obtained by extraction from higher plants, fungi, bacteria, algae, yeast, lichens and other natural objects have low toxicity and are active, especially against implanted solid tumors. However, the spectrum of their antitumor properties is narrow, and activity is induced by the body in response to the action of a biologically active substance. Most of these drugs do not show direct cytotoxicity against cultured tumor cells, which is one of the reasons why research in this area was not conducted as actively as it should have been [1].

 An ideal antitumor drug would have to have selective toxicity to tumor cells, but not damage the cells of the lymphoid system, which are necessary to mobilize the body's defenses, and also strengthen immunity, i.e. show immunostimulating activity.

 A known method of obtaining an antitumor drug from mollusks, which is a mixture of water-soluble macromolecular glycoproteins [2]. The essence of the method lies in the fact that from the liquid remaining during the cooking of mollusks, usually discarded as waste, a concentrate or dry powder is obtained and the target glycoprotein fraction is isolated from it. The drug obtained by this method does not exhibit immunostimulating activity.

As a prototype, a method for producing an antitumor substance from the scallop Patinopecten yessoensis was selected, which consists in removing the mollusk organs from the shell, boiling them for 30 minutes, grinding and homogenizing in water for 15 minutes at 0 ^° C, followed by grinding on an ultrasonic unit. The resulting mixture was centrifuged, then the supernatant was subjected to ultrafiltration and fractions with a molecular weight of more than 300,000 were collected. Subsequent ultrafiltration of the filtrate allowed to obtain fractions with a molecular weight of 10,000-300,000. After freeze-drying, a substance is obtained with a yield of 0.085% of the wet weight of the starting material. The resulting substance is a glycoprotein with antitumor activity against tumors implanted in mice.

 The disadvantage of this method is the almost complete absence of toxicity of the obtained drug against tumor cells and the absence of any noticeable effect on the immune system of the body, as a result of which it is recommended to increase the effectiveness of the drug while stimulating the immune activity by other means.

 The objective of the invention is to develop a method that would ensure the production of a carbohydrate-protein complex with higher antitumor activity, which also has immunostimulating activity.

The problem is solved in that in the method for producing a carbohydrate-protein complex from the scallop Patinopecten yessoensis, including the extraction of mollusk organs from the shell, grinding, homogenization, centrifugation and isolation of the target product, the mollusk is preliminarily irradiated with ultraviolet radiation at a dose of 8.2-8.6 kJ / m ² followed by its content in the natural habitat for 22-26 hours, and after homogenization, the target product is extracted with saline followed by its dialysis and lyophilization.

 The obtained preparation, according to chromatography and analysis, is a mixture of high molecular weight water-soluble glycoprotein compounds with a molecular weight of 10-100000 (according to polyacrylamide gel electrophoresis with sodium dodecyl sulfate), a carbohydrate content of 1-15%, which has high antitumor and immunostimulating activities.

 At a concentration of 0.05 mg / ml, it inhibits the growth of tumor cells of Ehrlich ascites carcinoma in vitro by 55% compared to 3% in the control (unirradiated scallop). At a concentration of 0.5 mg / ml, inhibition of tumor cell growth reaches 85%, and this type of activity is associated with lysis of tumor cells. When injected into the body of a mouse at a dose of 10 mg, the drug enhances the functional activity of macrophages, expressed in the inhibition of 95% growth of tumor cells of Ehrlich carcinoma, cultured in vitro together with the extracted macrophages. Moreover, the activity of the drug obtained from unirradiated mollusks, in similar trials, is 45% lower.

 Preparations obtained from irradiated and control animals exhibit exactly the opposite properties in this test: the first, immunostimulants that activate macrophages, and the second, immunosuppressants that inhibit the activity of macrophages in relation to tumor cells. At a concentration of 1-5 mg / ml, preparations obtained from irradiated animals exhibit mitogenic activity, which is manifested in the stimulation of lymphocytes in the blast transformation reaction, the average stimulation index is 1.9. Drugs from control non-irradiated animals inhibit lymphocyte proliferation (average stimulation index 0.3).

 In the indicated range of radiation doses, the biological activity of the preparations obtained reaches its greatest value. The timing of incubation of irradiated animals was dictated by the same reasons.

 Unlike the way in which the search for anticancer agents is reduced to extensive screening of numerous classes of substances of synthetic and natural origin, the proposed method uses the compounds formed in the process of the body's natural protection from the damaging effects of radiation for the same purposes.

 The proposed method allows to obtain from available types of marine raw materials - not used for food organs of commercial mollusks (mantle) - a drug with antitumor and immunostimulating activities, which can be used to stimulate the body's defenses in neoplasms.

 A significant difference of the method consists in irradiating the mollusk and ensuring its vital activity for several hours. Irradiation of isolated mollusk organs does not increase the activity of the resulting glycoprotein preparation.

 The method is illustrated by the following example.

 Example.

The shell flaps of the seaside scallop Patinopecten yessoensis are fixed in a half-open state and the body of the animal is irradiated with the light of a bactericidal lamp with a radiation wavelength of 254 nm at a distance of 15 cm from the light source in air for 5 min, which corresponds to an irradiation dose of 8.4 kJ / m ² . The irradiated and control non-irradiated animals were kept in their natural habitat for 24 hours, after which they opened their shells, removed mantles, washed successively with fresh and distilled water, crushed, weighed (1.0 kg) and homogenized with a 0.9% chilled solution sodium chloride (ratio of extractable material: extractant = 1: 5) for 5 minutes, then add a 0.9% solution of sodium chloride in the ratio of starting material: extractant = 1: 5. Extraction is carried out at a temperature of 4-6 ^o C in for 9-12 hours, periodically ne stirring the mixture, after which the solids are separated by centrifugation for 30-40 minutes at 20,000 rpm. The insoluble material is discarded, and the clarified supernatant is dialyzed through cellophane against distilled water at a temperature of 4-6 ^° C. until the chloride ion is completely removed. The dialysate is centrifuged at 20,000 rpm and dried by freeze drying. The resulting carbohydrate-protein preparation is a light, slightly colored, readily soluble powder, buffer and salt solutions. The yield is 1.2% by weight of the starting material.

 The preparation isolated from the control sample in a similar way served as a comparison base.

 The chemical composition of the experimental and control samples of the drug are presented in table. one.

 Significant differences in the composition of the total extracts are revealed in the study on an analytical centrifuge. So, with ultracentrifugation for 18 min at 50,000 rpm in saline solution, a clearly recorded peak in the resulting preparation is detected in the absence of such in the control.

 However, the biggest differences are found in determining physiological activity. The antitumor activity of the obtained preparations was determined by inhibiting the growth of tumor cells of Ehrlich ascites carcinoma in experiments in vitro and in vivo.

Ehrlich ascites carcinoma tumor cells were removed by syringe from the peritoneal cavity of a tumor-bearing mouse and suspended in RPMJ 1640 culture medium supplemented with 10% fetal calf serum, penicillin (100 u / ml) and streptomycin (100 μg / ml). Cell viability was determined using trypan blue and in all experiments was at least 90%. 1 • 10 ⁵ tumor cells in 1 ml of culture medium were introduced into sterile tubes, solutions of the tested substances and ¹²⁵ J-deoxyuridine, 0.04MBK (specific activity of the preparation 40,000 TBA / mol) were added, after which the samples were incubated for 4 hours at 37 ^o C in an atmosphere of moist air with 5% CO ₂ . At the end of the incubation, the unincorporated radioactivity was washed off with chilled 0.9% NaCl, the mixture was centrifuged at 900 rpm for 10 min, the supernatant was removed, and the radioactivity of the sediments was counted on a γ counter.

 The level of radioactivity of the pellet served as a measure of tumor cell growth. The results of the determinations are presented in table. 2.

The study of the cytostatic activity of the drug depending on the radiation dose during incubation of animals for 24 hours showed that the maximum inhibition of tumor growth is observed in the dose range of 8.2-8.6 kJ / m ² , which corresponds to 4.5-5 under experimental conditions, 5 minute exposure time. With an increase in the radiation dose, a significant decrease in cytostatic activity is observed up to its almost complete loss. At doses below 8.2 kJ / m ² was observed a similar pattern (Table. 3).

In in vivo experiments, macrophage-mediated cytostatic activity of drugs with respect to tumor cells was determined. CBA mice were injected into the abdominal cavity with a known amount of the test substance in 2 ml of physiological saline, and in the control, 2 ml of saline. At certain intervals after the injection, the mice were sacrificed by cervical dislocation, fixed on the plaque, and 3 ml of culture medium 199 with 100 u / ml heparin, 100 u / ml penicillin and 100 μg / ml streptomycin, as well as air, were introduced into the peritoneal cavity. After massage of the abdominal cavity for 2-3 minutes, peritoneal exudates were taken with a syringe, transferred to Petri dishes, where they were incubated at 37 ^° C for 1 h in an atmosphere of moist air containing 5% CO ₂ . Non-adherent cells were removed from Petri dishes by washing three times with culture medium. A population of adherent macrophages was removed with a rubber scraper and used in the experiment. The following were introduced into sterile tubes, divided into three series (a, b, c): a) 1 • 10 ⁵ tumor cells and 2 • 10 ⁶ activated macrophage preparations, b) 1 • 10 ⁵ tumor cells, c) 2 • 10 ⁶ activated macrophages. Samples containing: a) 1 • 10 ⁵ tumor cells and 2 • 10 ⁶ intact (not activated) macrophages, b) 2 • 10 ⁶ intact macrophages served as a control. The volume of the mixture in each tube was 1 ml. Samples were incubated in a culture medium supplemented with 10% fetal calf serum at 37 ^o C for 18-20 hours in a humid atmosphere with CO ₂ . 4 hours before the end of the incubation, 0.15 MBC ³ H-thymidine was introduced into the tubes. The inclusion of the label in the samples was interrupted by placing them on ice and adding 5 ml of chilled physiological saline, the cells were resuspended and precipitated by centrifugation at 900 rpm for 10 min. The supernatants were discharged, after which 0.2 ml of 0.3N NaOH was added to each sample, the mixture was heated at 70 ^° C for 1.5 hours. After cooling, aliquots (50-200 μl) of the liquid from each tube were applied to numbered filter paper disks, the disks were dried, then sequentially passed through a chilled 5% trichloroacetic acid solution, washed twice with ethyl alcohol, ether, dried and placed in a toluene scintillator to calculate the radioactivity on a β-counter. Inhibition of tumor cell proliferation was determined by the formula:
% inhibition = (N _op.cl + N _m -N _cm / N _op.cl ) 100,
Where
N _op.cl - the count rate of tumor cells, imp / min,
N _m - macrophage count rate, imp / min,
N _cm - the count rate of tumor cells with macrophages, imp / min

 The experimental results are presented in table. 4.

 As can be seen from the data table. 4, macrophages activated by the irradiated scallop extract almost completely inhibit tumor growth. Extracts of unirradiated scallop have an immunosuppressive effect and inhibit the activity of macrophages in antitumor protection. Thus, this experiment proves the immunostimulating properties of the drug obtained by the proposed method, and its significant differences from the prototype.

 Stimulation of lymphocytes was evaluated in the reaction of blast transformation according to the generally accepted method [3]. The drug obtained by the proposed method has a mitogenic effect and at a concentration of 1-5 mg / ml stimulates lymphocytes. The stimulation index was 1.9 compared to 0.3 by a known method.

 The cytotoxic activity of the drugs was evaluated by trypan blue staining of Ehrlich ascites carcinoma cells. These definitions are presented in table. 5.

Bibliography
1. Proceeding of the thirol Annual MYT Sea Grant College. Program Zecture and Seminar (ed.by Rita R. et al, WwJZ, 1985, p. 377-388.

 2. US patent N 4390468, A 23 J 1/04, C 07 C 7/00, publ. 06/28/83, "A method for producing an antitumor preparation from mollusks."

 3. Japan Patent N 8088/82, A 61 K 35/56, "Antitumor substance and antitumor agent, the effective component of which is this substance.

Claims (1)

A method of producing a carbohydrate-protein complex from the scallop Patinopecten yessoensis, including extraction of mollusk organs from a shell, grinding, homogenization, centrifugation and isolation of the target product, characterized in that the mollusk is preliminarily irradiated with ultraviolet radiation at a dose of 8.2-8.6 kJ / m ² followed by its content in the natural habitat for 22 to 24 hours, and after homogenization, the target product is extracted with saline followed by its dialysis and lyophilization.

Images