The method of obtaining funds from adaptogenic and radioprotective action

 

(57) Abstract:

The invention relates to the field of medicine and is concerned with the obtaining medicines from animal waste materials. The method includes grinding the spleen, the influence of water-extracting agent, and the specified effects complement the simultaneous effect of low-frequency ultrasonic radiation, the separation of the extract is carried out by centrifugation followed by dialysis and lyophilization. The weight ratio of spleen and water can be 1:6 to 1:3, weight 25000-50000. The ultrasound frequency is chosen from the interval 20 - 70 kHz, and its intensity is 0.1 to 2.3 W/cm2. Effect: method provides increased product yield while reducing the time of its receipt. 4 C.p. f-crystals, 5 PL.

The invention relates to the field of medicine and is concerned with the obtaining medicines from animal waste materials.

A method of obtaining biologically active substances from animal waste materials in which of milled tissue of the spleen and water to form a mash, it through various stages of deposition and extraction have free proteins from aqueous solution, from which then lidelraclio biologically active substances and due to this low degree of extraction.

A method of obtaining gonadotropic tools [2], similar starting materials and technical nature. However gonadotrope action received the drug has not been studied.

The closest to the destination is the drug splenin, manufactured by the company "Darnitsa", , Kiev [3-5]. The technology of its receipt has much in common with the proposed technical solution that allows you to choose a prototype of the invention.

According to this method, the spleen is crushed, act on it extracting agent (dichloroethane) at a ratio of 1:2 (one part meat and two parts of extractant), extracted with biologically active substances, after which separate the extract removed from dichloromethane and filtered. Drug (splenin) are obtained in the form of an aqueous extract, and 552 kg of meat of bovine spleen receive 3,60,1 kg BW of an aqueous extract of splenin, which in terms of the raw material is 0.04%.

The disadvantages of this method are:

- low yield of extractive substances having biological activity;

- the long duration of the extraction process;

- the use of environmentally harmful solvents (dichloromethane, petroleum ether).

And radioprotective [9] activity. Splenin used for medical purpose in the treatment of toxicosis of pregnancy. In the State Pharmacopoeia splenin attributed to biogenic stimulators [6]. Splenin has imunomoduliruyuschee action [6,7]. Currently splenin applies when ionizing breast cancer therapy.

The objective of the invention is to provide a method of obtaining funds with adaptogenic and radioprotective activity, with increased yield, reduced duration of its receipt.

In accordance with the task proposed method includes grinding the spleen, the influence of water-extracting agent, and the specified effects complement the simultaneous effect of low-frequency ultrasonic radiation, the separation of the extract is carried out by centrifugation, and then dialysis and lyophilization.

Unlike the prototype is that the extract is obtained by exposure to aqueous extractant under simultaneous exposure to ultrasonic radiation, and separation of the extract is carried out by centrifugation.

In the preferred implementation of the method, the weight ratio of spleen and water choose what and extraction ceteris paribus. The selection of the weight ratio greater than 1/3, leads to decreasing product yield. When the weight ratio of spleen to the extractant < 1/6, increasing the initial volume, the duration of the removal process extractant increases and, consequently, expressnet method generally decreases.

Increasing the concentration of the final product also contributes to the implementation of the dialysis of an aqueous extract using membranes with the size of the passage for the molecular weight of up to 5000.

Preferably the frequency specified low-frequency ultrasound to choose between 18-70 kHz. In this frequency range under other equal conditions is the most complete and rapid extraction ceteris paribus. Outside the specified frequency range efficiency of ultrasonic influence is reduced, which leads to an unacceptable reduction in product yield or increase the duration of extraction.

The intensity of ultrasonic vibrations, it is advisable to choose from the range of 0.1 to 2.3 W/cm2within which the extraction efficiency is maximum. When the intensity, the smaller of 0.1 W/cm2the extraction efficiency decreases dramatically. Increase intensiveclusters.

Below is an example of a preferred embodiment of the proposed method to obtain funds with adaptogenic and radioprotective activity of the spleen.

Fresh or frozen spleen clear of a lipid film and crushed, for example, with the grinder. To the resulting mass is added to five parts by weight of water, the resulting mixture was placed in an ultrasonic bath. The mixture is then exposed to continuous ultrasound radiation with a frequency of 18 kHz and an intensity of 2.0 W/cm2. After 45 minutes the contents of the ultrasonic bath is placed in a centrifuge where they separate the extract from the pulp of raw materials. Next product centrifugation is passed through a semi-permeable cellulose membrane with the size of the passage for the molecular weight of 25000-50000. The filtrate is tested for the absence of pyrogenic substances, lyophilized. The obtained dry powder with a yield of biologically active substances of about 5.4% by weight of the feedstock can be used to obtain the dosage form. The product in powder form for easy storage and transportation.

Examples of the proposed method with different modes and conditions are given in table. 1.

Bring the influence of ultrasound. The exception is the example 7, in which the period of extraction has been extended to 70 minutes As seen from the above examples, within the optimal range of the weight ratio of spleen to the extractant (1/6 - 1/3), and frequency of the radiation (18-70 kHz) and intensity of radiation (0.1 to 2.3 W/cm2the proposed method provides more than 5% of the yield of dry product from the weight of the feedstock. Outside these intervals, the product yield relative to the source of raw materials is less than 5%, although in this case exceeds the output method prototype much more. The use of the claimed invention will:

to increase the yield of the target product;

to increase saving;

- to improve the environmental situation in obtaining the product.

In table. 2 - 4 shows experimental data for the study of x-ray irradiation, which confirm the radioprotective effect of extract obtained by our proposed method.

In recent decades have been extensively studied biologically active substances produced by cells of various organs and regulate many body functions and, above all, proliferation and differentiation of cleto modify the metabolism of cells critical organs and contribute to the recovery of the body after radiation damage.

In our experiments obtained by the proposed method extracts of spleen (ES) was introduced white outbred mice, males Mature age daily intraperitoneally at a dose of 10 mg/kg for 14 days. For evaluating the effect of the extract used is common in radiobiological research methods:

- determination of the mass of the thymus and spleen, characterizing the state of the lymphoid system;

- definition of micronucleus frequency in erythrocytes, characterize the mutagenic and carcinogenic;

- determination of the activity of the 5*-nucleotidase-(membrane-associated enzyme), for which changes in serum characteristic damage of the plasma membrane of cells.

Due to interphase death of lymphoid cells after irradiation of animals in the dose of 0.25 Gy to decrease the weight of lymphoid organs. The spleen extract, administered to mice after x-ray irradiation at a dose of 0.5 Gy, contributed to the conservation of mass of the thymus and spleen (table. 2). So, on the 14th day thymus of mice treated with the drug, 3.5 times the weight of the thymus control irradiated animals that are in a similar situation was injected with saline.

When radiationhardened chromosomes and the formation of microkernels. Micronuclear test is considered to be quite informative in identifying as radiation damage, and when detecting mutagenic and carcinogenic actions. In our experiments, the introduction of ES did not affect micronucleus frequency in erythrocytes in non-irradiated animals, i.e., the extract is not a mutagen or carcinogen. After irradiation, the micronuclei frequency was increased compared to the norm, and ES decreased during all periods of observation (table. 3).

In the literature there is evidence that after irradiation increases the activity of the 5*-nucleotidase, a marker enzyme of the plasma membrane of cells. Increased activity of serum 5*-nucleotidase is due to the destruction of the membranes of lymphoid cells and release of the enzyme into the blood. Us reliably shown that ES reduces the activity of the 5*-nucleotidase increased after irradiation of mice (table. 4). We believe that the radioprotective effect of the drug is associated with stabilization of plasma membranes of lymphoid cells, damaged by x-ray irradiation.

Adaptogenic properties have been studied in models of acute stress on Yu GI Dobryakova sent (1978). The obtained results were compared with similar "splenin" (PL. 5) and show the R>
1. Khavinson C. H., Morozov, C. G., Sidorov N. D., Konstantinov, C. L., O. Chaika Century, Towarowa N. In. Auth.St. USSR N 1228776, publ. 30.04.86, BI N6, 1986.

2. Andras Bales, Mihai Saigo, Lajos Kisfaludy and other Patent Hungary N 162447, CL Y 01 N 13/00, 1967.

3. Industrial regulations on "Splenin" (substance) N 64-04-34-36, 1984, Kiev Darnitsa.

4. Pharmacopeia 42-1255-90. Splenin, the USSR Ministry of health, Formamide, 10.07.91.

5. State Pharmacopoeia of the USSR. The USSR Ministry of health, 11, Product - M.: Medicine, 1990.

6. Mashkovsky M. D. Medicines. - M.: Medicine, 1986, so 2, S. 157.

7. Yearbook "pharmacies", N 27, 1998, Kiev, Ukraine.

8. Radioprotective funds. The Handbook. Ed. by L. A. Tiunova and other M-L. , 1964, S. 158.

9. Balitsky K. P., Wexler, I. Reactivity of the organism and chemotherapy of tumors. - Kiev, Naukova Dumka, 1975, S. 151-152.

1. The method of obtaining funds from adaptogenic and radioprotective activity, including grinding the spleen, the influence of water-extracting agent, subsequent separation of the extract, dialysis and lyophilization, characterized in that the impact of water extracting agent is complemented by simultaneous exposure to low-frequency ultrasound, and separating the extract of spleen and extractant selected from a range of 1:6 - 1:3.

3. The method according to PP.1 and 2, characterized in that in it the dialysis water-extracting agent is performed with the value of the molecular weight of 25,000 to 50,000.

4. The method according to PP.1 to 3, characterized in that it is the frequency of the ultrasonic vibrations is chosen from the interval 18 - 70 kHz.

5. The method according to PP.1 to 4, characterized in that it specified the impact of ultrasound exercise intensity of 0.1 to 2.3 W/cm2.

 

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