RU2103024C1 - Method and device for activating biological substances - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves subjecting biological substance solution to be activated to action of high voltage electric discharges in the mode of electrohydraulic jet aerosol solution spray at potential difference being equal to 3-10 kV on discharge electrodes and discharge pulses burst frequency being equal to 1-70 Hz followed by biologically active substance solution cleaning and fractioning. The device has high voltage electric pulses generator, capacity with electrohydraulic unit, filtering member, transition pump, hydraulic system, hydraulic dispenser unit and measuring gage for biologically active substance solution to be activated. The electrohydraulic unit is set on the reservoir cover and is provided with discharge chamber of variable cross-section open at one side, separated with coaxial system of discharge electrodes on one side and with porous grid membrane covering nozzle opening of the discharge chamber on the other side. EFFECT: enhanced effectiveness of activation; controlled energy supply during the whole production cycle. 2 cl, 3 dwg, 1 tbl

Description

 The invention relates to medicine and biotechnology and can be used to obtain biologically active substances used, for example, in the treatment of pretumor and immunodeficiency states.

 A known method of activating biological substances by exposing them to a physical factor (evaporation under reduced pressure) to obtain a solution of a biologically active substance with an antitumor effect (see UK Patent N 1530846, MKI A 61 K 35/74, 1979). According to this method, after cultivation in a nutrient medium of a strain of Staphylococcus epidermidis and its identification, the biologically active substance is isolated by thermal treatment under reduced pressure (evaporation), followed by dissolution of the concentrate and fractionation of the aqueous solution to obtain fractions with a molecular weight of 1000-5000 and 5000-10000 in the form purified culture fluid that does not contain cells with anti-cancer activity.

 To implement the above method of activating an aqueous solution of biological substances, a device is used that contains a bacterial cell strain cultivation unit in a nutrient medium, a sealed container for the processed cell raw materials, a heater, a vacuum system, a centrifuge, a molecular sieve filter system, and a vessel for collecting finished products in the form of biologically active substance.

 However, this method and device do not provide a sufficient degree of activation of an aqueous solution of a biological substance, as evidenced by the presence at the final stage of fractionation in the culture fluid of a significant portion of fractions with a molecular weight of 5000-10000, with less antitumor activity.

 In addition, the implementation of the method requires the use of a complex device, significant time, material and energy costs for obtaining the finished product, and it is not connected in a single technological cycle of obtaining the finished product.

 Closest to the claimed subject matter of the invention is a method for activating biological substances by treating them with a physical factor - the energy of an alternating electromagnetic field (Aut. St. USSR N 639560, class A 61 N 1978. A method for activating biological substances).

 According to this method, the activation of aqueous solutions of biological substances is carried out using the energy of an alternating electromagnetic field at an intensity of 75-500 Oe for 50 to 70 minutes, 2-4 times with an interval between treatments of 23-25 hours

 To implement the above method of activation of biological substances, a device is used consisting of a sealed non-metallic container for the treated aqueous solution of a biological substance and a source of alternating electromagnetic field (inductor).

 The processing parameters of biological substances given in the above method indicate an insufficient degree of energy activation using an alternating electromagnetic field, which requires the use of the above long exposures of energy exposure, as well as the use of a multi-day technological cycle to obtain the final finished product.

 Such a long and intermittent technological cycle of activation of biological substances in the known method is explained by insufficient energy exposure to an aqueous solution of a biologically active component performed by an alternating electromagnetic field, despite its relatively high intensity (up to 500 Oe). Apparently, the insufficiently powerful energy effect of an alternating electromagnetic field cannot cause disturbances in intermolecular forces and chemical bonds in the processed biological substance, leading, ultimately, to a directed change in its physicochemical and biological properties characterizing its activation. So, for example, the energy of some chemical bonds characteristic of the interaction of organic molecules are: H-S bond 3.58 eV; N-N bond 3.64 eV; H-C bond 3.74 eV; N-C bond 3.65 eV, etc. (see Aut. St. USSR N 886907, class A 61 N 5/00, 1981). Therefore, the energy of an alternating magnetic field is clearly not enough for a direct violation of these and similar bonds, because this field is only capable of influencing the orientation of macromolecules due to polarization (see Yu.A. Kholodov. The brain in electromagnetic fields. M: Nauka, 1982, pp. 108-109), and therefore, a long-term effect is necessary with the initiation of other mechanisms inside - and / or intermolecular interactions in an activated biological substance. At the same time, the insufficient energy capabilities of the method require the use of a powerful and overall inductor, as well as large time and energy costs. In addition, the known device does not allow you to control the energy input into the treated aqueous solution of the activated biological substance, the properties of which during the activation process may vary significantly with the processing time.

 All of the above reduces the effectiveness of the activation of biological substances and does not allow you to control the energy input into the activated substance during the entire technological process of its activation.

 The objective of the invention is: in terms of the method - increasing the efficiency of activation due to the intensification of the energy effect on the activated biological substance, and in the part of the device - providing control of energy input throughout the entire process of activation of the biological substance, as well as ensuring uniformity of energy input into the activated substance.

 The task in terms of the method is achieved by the fact that in addition to the aqueous solution of the activated substance, high-voltage pulsed electric discharges are applied in the mode of electro-hydraulic jet-aerosol spraying of the solution with a potential difference of 3 to 10 kV at the discharge electrodes and a discharge pulse repetition rate of 1 - 70 Hz, followed by cleaning and fractionation of the activated biological substance solution.

 The task in terms of the device is achieved by the fact that it is equipped with an electro-hydraulic unit mounted on a removable lid of the tank, containing a coaxial converter connected to a power source in the form of a high-voltage electric pulse generator (HVEI), oppositely mounted to the electro-hydraulic unit by a filter element, hermetically connected to the output a branch pipe at the bottom of the tank, communicating with the transfer pump, the output of which through the valve in the form of a regulating three of a water supply valve and a supply pipe and fitting are connected to a one-sided open variable section of a discharge chamber of an electro-hydraulic unit, on the one hand limited by a coaxial converter, and on the other hand, by a porous mesh membrane overlapping the nozzle opening of the discharge chamber, in addition, the device contains on the side wall of the container and outside the zone of power electro-hydraulic impact, the conductivity measurement sensor of the activated solution connected via a shielded cable to the measurement unit conductivity of the solution, which is connected through the control unit to the control input of the high-voltage electric pulse generator (HVEI), the output of which, in turn, is connected to the input of electric power to the coaxial converter of the electro-hydraulic unit.

 Additionally, the invention is illustrated by drawings, where in FIG. 1 shows a diagram of a General view and a block diagram of a device for activating biological substances; in FIG. 2 is a diagram of an electro-hydraulic unit with a section in its terminal working part; in FIG. 3 is a section in the region of a coaxial converter and a discharge chamber of an electro-hydraulic assembly.

 To implement the method of activation of biological substances, a device is used that contains a power source in the form of a high-voltage electric pulse generator (HVEI) 1, an electro-hydraulic unit 2 mounted on a removable roof 3 of a sealed container 4 for an activated solution 5 made of a polymeric material (food-grade polyethylene or polystyrene medical appointment). Opposite to the electro-hydraulic unit 2, a filter element 6 is installed at the bottom of the container 4, the inner cavity of which communicates with the cavity of the container 4 through the membrane of the filter cartridge 7, made of porous fluoroplastic F-4 with a nominal holding capacity of 5-10 microns. In this case, the internal cavity of the filter element 6, and through it the cavity of the container 4, is communicated through the outlet pipe 8 at the bottom of the container 4 with a closed hydraulic system 9 of withdrawing the activated solution 5 from the container 4 and supplying it through a pump 10 with adjustable capacity and a three-way control valve 11 either to the supply pipe 12 mounted on the removable cover 3, and from it through the supply fitting 13 to the discharge chamber 14 of the electro-hydraulic unit 2, or to a vessel (not shown) for collecting the finished product (activated solution biological substance) at the final stage of the technological process of solution activation 5.

 The final working part 15 of the electro-hydraulic unit 2, all of whose elements are made of stainless steel, contains a coaxial converter (in the form of a coaxial electrode system of discharge electrodes), consisting of a ring electrode 16 with an inner cone and a rod electrode 17 with a flange 18 at its end part, separated dielectric insulator 19 (for example, ftoroplast F-4). The electrodes 16 and 17 form a small cone-shaped cavity 20 of a one-sided open variable section of the discharge chamber 14. At the same time, the flange 18 on the terminal part of the rod electrode 17 serves as a protective shield and a reflector of thermal and shock loads arising in the high-voltage discharge zone in the liquid and protects the insulator 19 from rapid destruction along with the gas-liquid barrier phase filling the small conical cavity 20 during discharge. The small conical cavity 20 of the discharge chamber 14 communicates through an annular gap between the times in-line electrodes 16 and 17 with a cone-shaped working cavity 21 of the discharge chamber 14 having a larger size and hydraulic cross section than the cavity 20. The working cavity 21 is made in a bearing nut 22 connected through a threaded connection to the housing 23 of the acoustic unit 2, and is limited on one side a coaxial converter made in the form of a system of discharge electrodes 16 and 17, and on the other hand, a porous mesh membrane 24 made of stainless steel with a holding capacity of 200 - 500 μm, which ensures the elimination of free leakage of solution and 5 (during pauses between discharge pulses) from the working cavity 21 of the discharge deck 14 filled into it to the container 4 by increasing the surface tension forces, depending on the size of the cells and capillaries of the porous mesh membrane 24. The porous mesh membrane 24 is installed between the outlet of the discharge chamber 14 and a nozzle cone hole 25 of the bearing washer 26, fixed on the bearing nut 22 with pins 27 and a supply fitting 13, which communicates the hydraulic system 9 for supplying the activated solution 5 with the working cavity 21 of the discharge chamber 14 in the middle stvom supply opening 28 provided in the bearing nut 22.

 A sensor 29 is installed on the side wall of the vessel 4 outside the zone of direct electro-hydraulic force (in the niche of the vessel wall) to measure the conductivity of the activated solution 5. Its installation is caused by the destruction of protein structures and molecules of biological substances by the electro-hydraulic effect and their saturation with an aqueous solution and a consequent change in its electrical resistance, permittivity and viscosity of the solution. A change in the indicated parameters of the activated solution due to physical, physicochemical, and biological processes occurring in the zone of electrohydraulic processing of solution 5 causes a destabilization of the energy parameters of initiation of the VER, and, consequently, uneven energy input into one or another volume of the solution located in the working cavity of the 21 discharge chamber 14 at the time of passage of the discharge pulses or their failure due to the impossibility of initiating a discharge. The sensor 29 consists of two electrodes isolated from each other 30, the working ends of which are in contact with the activated solution 5 in the tank, and the opposite ends connected through a shielded cable 31 (to reduce interference from the VER) to the block of conductivity change of the solution 32, equipped with an indicator 33 for visual assessment changes in the conductivity of the activated solution 5 in the process of the entire technological cycle of activation of a biological substance under the influence of energy VER.

 A signal about a change in the conductivity of the activated solution 5 is fed to the input of the conductivity measuring unit 32, which is made, for example, according to the principle of an ohmmeter. From the output of block 32 of the block, a signal proportional to conductivity is fed to indicator 33 and simultaneously to the input of control unit 34 by a high-voltage electric pulse generator (HVEI) 1, changing the level of the discharge voltage either by changing the moment of initiation of the discharge gap at a controlled discharge moment, or by changes in the magnitude of the generated discharge gap of the generator of high-voltage electric pulses (HVEI) 1 (see I.T.Vovk et al. Control of electrohydropulse processes. Kiev, 1986, p. 109).

 A change in the level of discharge voltage in HVEI 1 causes, in turn, a change in the operating mode of the discharge chamber of the electro-hydraulic unit 2 in the direction of maintaining the required processing mode of the activated solution 5 when its conductivity changes.

 The method of activation of biological substances is as follows.

 Initially, the initial solution of the biological substance 5 to be activated is filled into the container 4 and sealed with a removable cap 3 with an electro-hydraulic unit 2 (EG-unit) fixed on it with the exception of immersion in the solution 5. The distance between the nozzle exit (nozzle hole 25 and the carrier the washer 26) of the electro-hydraulic unit 2 and the surface of the solution 5 should be at least 20-30 mm to exclude its immersion in the volume of the solution 5 and the violation of the spray-spray mode (during activation) of solution 5. P This hydraulic system and must be completely filled with a solution of 5.

 After that, turn on the pump 10 and provide controlled circulation of the activated solution 5 by pumping it through a closed hydraulic system 9, an electro-hydraulic unit 2 and a tank 4. In this case, the solution 5 is initially filtered through the membrane of the filter cartridge 7 in the inner cavity of the filter element 6 through the membrane of the filter cartridge 7, fills the cavity of the filter element 6 and through the outlet pipe 8 at the bottom of the tank 4 gets into a closed hydraulic system 9 of removal of the activated solution 5.

 Next, the solution 5 is pumped through the pump 10 and enters the discharge line of the closed hydraulic system 9. Having passed a three-way control valve through passage 11, which opens the passage into the hydraulic system for supplying the solution to the electro-hydraulic unit 2, the activated solution 5 through the supply pipe 12 and the supply nozzle 13 is introduced into the discharge chamber 14 .

 Simultaneously with the beginning of the circulation of the activated solution 5 include a generator of high-voltage electrical pulses GVEI 1 (hereinafter referred to as the generator GVEI 1). When filling with a solution 5 of the discharge chamber 14, high-voltage pulsed electric discharges (hereinafter - VER) are initiated with the achievement of high-voltage pulsed electrical breakdowns of the activated solution 5.

 The parameters of the electro-hydraulic spray-aerosol spraying of the activated solution 5, at which the maximum energy input in the small volume of the discharge chamber 14 of the electro-hydraulic assembly 2 is achieved, is as follows: the potential difference on the discharge electrodes is 3-10 kV, the pulse repetition rate is 1-70 Hz.

 Moreover, more stringent parameters of the regime (U = 7-10 kV and F = 1-10 Hz) are used in the case of EG treatment of activated solutions with a high initial viscosity and resistivity). The sparing parameters of the EG treatment regime of activated solutions (U = 3-5 kV and F = 10-70 Hz) are used in cases where the activated solution has small and medium initial viscosity and specific resistance values. The total time of electro-hydraulic treatment of the activated saline solution is not more than 20-30 min / l of solution.

Electro-hydraulic treatment of the solution is accompanied by powerful shock waves, intense hydraulic pressures and flows, light, ultrasound and UV radiation, cavitation processes, pulsed electromagnetic field, ionization, temperature shock in the interelectrode gap, the formation of metal complexes and products of electroerosion of the electrode system, as well as decomposition products ( in the field of high-voltage pulsed electrical breakdown) of a gas-liquid system in the form of ozone (O ₃ ); various forms of nitrogen NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{2}}$ NO $\genfrac{}{}{0ex}{}{\text{-}}{\text{3}}$ NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ ; O, H atoms; atomic ions (H ⁺ , O ⁺ ); electrons - e, molecules H ₂ O, H ₂ , O ₂ , OH, molecular ions H $\genfrac{}{}{0ex}{}{\text{+}}{\text{2}}$ , O $\genfrac{}{}{0ex}{}{\text{+}}{\text{2}}$ active radicals, etc. (see L.A. Yutkin. Electro-hydraulic effect and its application in industry. L., 1986, pp. 10-85; I.A. Nugolny and others. Electric discharges in water. M.: Science , 1971, 197c; M.A. Greysukh et al. Ozone generation during pulsed breakdown of gas-liquid systems. - Electronic processing of materials. 1987, N 2, p. 48-50, etc.).

The energy parameters of the water component of the VIEER plasma at discharge powers of 10 ⁷ -10 ⁹ W are: pulse pressure P = 10 ⁸ -10 ⁹ Pa; T = 2000-60000 K, while only UV radiation has a quantum energy of the order of 10 ² eV. This powerful energy effect significantly exceeds the discharge energy of the chemical bonds of atoms and molecules of the substance of the activated solution 5, the energy of their electronic transitions, dissociation, coordination interaction, the vibrational energy of molecules, hydrogen bonds, etc., the interaction energy of which ranges from 10 ^-3 to 1-20 eV. In this case, the activated solution 5 decomposed into the VER region decomposes, the molecules constituting it break and decompose, the active chain fragments of molecules and radicals form, the chain fragments are modified by metal complexes, etc. In addition, ozone (O ₃ ) formed during the VIR to double bonds of high molecular weight compounds of a biological substance, "cuts" them into low molecular weight fragments in the form of fragments of macromolecules, molecules and polar functional groups that dramatically change their original physical and physico-chemical and biological properties.

 By adjusting the flow rate of the activated solution 5, carried out by a pumping pump 10 and a control valve through passage, supplied to the discharge chamber 14 of the electro-hydraulic unit 2, the volume of the solution participating in the act of initiating VER, and, therefore, perceiving a different level of energy impact and generating aerosol, is changed in it spray-jet or jet streams of the activated solution 5, flying out of the working cavity 21 through the porous mesh membrane 24 and the nozzle hole 25 of the bearing washer 26 in the form of a torch of activated fine particles having a large surface activation energy (Gibbs energy).

 The newly formed substance in its properties differs significantly from the original substance before its activation (both physicochemical and biological properties). In this case, its viscosity, conductivity and dielectric constant change sharply. Knowing in advance their final values for the newly obtained final product, they vary by the energy input into the initial activated solution until they are achieved by repeatedly pumping the activated solution 5 through the discharge chamber 14 of the electro-hydraulic unit 2 (with the passing control of achieving the necessary parameters of the activated solution 5, characterizing indirectly the depth of its activation). In this case, the speed and volume of the pumped solution 5 is controlled by the speed of the pump 10 and the volume of the solution passed into the discharge chamber 14 by a control valve 11. At the same time, the activated solution 5 is filtered from the secondary products of electroerosion of the electrodes of the discharge system of the electro-hydraulic unit 2 formed by plasma-chemical electrochemical - and hydroimpulse processes that accompany VER in a liquid.

 After reaching the required degree of activation of the activated solution 5, the HVEI generator 1 is turned off. The activated solution 5 from the tank 4 is finally filtered through the filter element 6 and the solution 5 through the pump 10 and a three-way control valve 11 are fed into the collection vessel of the finished product (not shown). The resulting solution after additional filtration by centrifugation and sterilization can be used later in pharmaceutical and biotechnological processes in both liquid and dry (in the form of powder) forms.

 The proposed method and device for its implementation can dramatically increase the efficiency of activation of solutions of biological substances due to the controlled high-energy exposure to them by a complex of physical and physico-chemical factors, the energy of which is an order of magnitude higher than the intra- and intermolecular bonds in the activated substance, which allows for a short time ( no more than 20-30 min / 1 liter of solution) to obtain biological substances with new bio- and pharmacological properties, bypassing the purely chemical methods of their modification and acti ation.

 Using the above advantages of the proposed method and device for its implementation, positive experience has been accumulated in the treatment of background and precancerous diseases of the female genitalia (cervical leukoplakia, cervical erosion, leukoplakia and vulvar kraurosis), as well as superficial precancerous skin conditions such as melanoma.

 The advantages of the proposed method and device for its implementation allowed us to successfully conduct experimental clinical trials of new types of medicinal substances of biological origin with antitumor properties on the basis of the All-Russian Scientific Center for Medical Sciences of the USSR.

 Example. Evaluation of the effectiveness of improving the immunological and therapeutic properties obtained using the proposed method and device for its implementation of activated biological substances (biological products) was carried out in 1990 on the basis of the Research Institute of ED and Maintenance Center of the Central Academy of Medical Sciences of the USSR (Study of the antitumor activity of six drugs of natural origin: Research Report N 10 (stage 2) from 1.01.90. Research Institute of ED and Maintenance of the All-Union Scientific Research Center of the USSR and NPO Biomed. Perm, 1990, 47 p.).

где К - контроль(продолжительность жизни нелеченных животных), сут;
О - продолжительность жизни леченных животных, сут.The study of antitumor properties was carried out on a test model of lymphocytic leukemia P-388 mice. The effectiveness of the biological product (staphylococcal toxoid obtained by activation of the initial staphylococcal toxin with the energy of VER at the above parameters and modes of electro-hydraulic treatment) was studied with various intraperitoneal administration options - 1.2 and 5-fold administration. The therapeutic effect was evaluated by the increase in life expectancy (UP) of treated animals (O) compared with untreated animals (K):

where K is the control (life expectancy of untreated animals), days;
О - life expectancy of treated animals, days.

 For the minimum criterion of antitumor activity of the drug UPZH (for model P-388), an indicator was taken, the value of which should be at least 25% compared with the control.

 The results of experiments to evaluate the antitumor activity of the obtained energy-activated VIER biological product in comparison with the prototype method are shown in the table.

 The data presented indicate that the biopreparation activated by energy VER has a high antitumor activity, which is expressed by the average life expectancy in 35.6% of animals, with only 1-time administration of the drug in a volume of 1 ml.

 The claimed invention will improve the development of new bio- and medical technologies for obtaining more advanced biological products with pronounced immunological properties, as well as the development and implementation of new treatment methods (upon receipt of the relevant permissions of the Pharmaceutical Committee of the Russian Federation) with their help, the most dangerous viral infections, immunodeficiency states, tumor formations, AIDS and others

Claims (2)

 1. The method of activation of biological substances, mainly in the form of aqueous solutions by treating them with a physical factor, characterized in that it is additionally affected by high-voltage pulsed electric discharges in the electrohydraulic spray-aerosol solution spraying mode with a potential difference of 3 to 10 kV discharge electrodes and a repetition rate discharge pulses of 1 70 Hz, followed by purification and fractionation of the solution of the activated biological substance.  2. A device for activating biological substances, containing a sealed non-metallic container for an activated biological substance and a power source, characterized in that it is equipped with an electro-hydraulic unit mounted on a removable cover of the container, containing a coaxial converter connected to the power source in the form of a generator of high-voltage electrical pulses, opposite a filter element mounted to the electro-hydraulic unit; hermetically connected with the outlet pipe at the bottom a tank in communication with a transfer pump, the output of which through a directional control valve in the form of a three-way inlet valve and a supply pipe and fitting is connected to a one-sided open variable section of the discharge chamber of the electro-hydraulic unit, on the one hand delimited by a coaxial converter, and on the other a porous mesh membrane overlapping the nozzle opening the discharge chamber, in addition, the device contains on the side wall of the tank and outside the zone of direct electro-hydraulic power action, the conductivity measurement sensor of the activated solution connected via a shielded cable to the solution conductivity measuring unit, which is connected through the control unit to the control input of the high-voltage electric pulse generator, the output of which, in turn, is connected to the electric power input of the coaxial converter of the electro-hydraulic unit.

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