RU2848613C1 - Feed antioxidant in the form of liposome for the preparation of wet and liquid feed for domestic and farm animals and poultry and method for obtaining it - Google Patents

Abstract

FIELD: feed antioxidant.

SUBSTANCE: feed antioxidant in the form of liposomes for the preparation of wet and liquid feeds for domestic and farm animals and birds, containing soy phosphatidylcholine and non-ionic surfactant Tween-20 in a molar ratio of 4:1, with bisphenol-5 included in the bilayer liposome in an amount of up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20, also relates to a method for producing a feed antioxidant in the form of liposomes, which consists in preparing a solution containing soy phosphatidylcholine and the non-ionic surfactant Tween-20 in a molar ratio of 4:1, as well as bisphenol-5 up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20, in chloroform, for which the specified reagents are mixed at room temperature until a homogeneous mass is obtained; remove the chloroform under vacuum on a rotary evaporator at a temperature of 25-35°C to obtain a lipid film; hydrate the lipid film with ultrapure water for 1 hour in a water bath at 60°C with constant stirring; a five-fold freeze-thaw cycle is performed using liquid nitrogen and a water bath to incorporate bisphenol-5 into the liposomes, yielding a liposomal dispersion; To homogenise the liposomal dispersion, the liposomal dispersion is extruded through polycarbonate membranes with a pore size of 100 nm, yielding a feed antioxidant in the form of liposomes no larger than 100 nm. EFFECT: group of inventions provides for the production of a feed antioxidant in the form of stable water-soluble liposomes from water-insoluble bisphenol-5, with incorporated molecules of the biologically active substance – the antioxidant bisphenol-5.

2 cl, 4 dwg, 1 tbl

Description

The group of inventions relates to medicine, veterinary science, the chemical and pharmaceutical industry, and agriculture, in particular to liposomes containing the antioxidant Bisphenol-5 and a method for producing them, and can be used as a feed antioxidant in the preparation of wet and liquid feed for domestic and farm animals and birds.

To improve metabolism and meat quality in domestic and farm animals and poultry raised for slaughter, various feed additives, including vitamins, antioxidants, and biologically active substances, are used. Increasing the bioavailability of medicinal substances by improving their water solubility is a commonly used approach in biomedicine.

Liposomes are microscopic, closed vesicles with an internal phase surrounded by one or more lipid bilayers. They can retain water-soluble material in the internal phase and oil-soluble material in the phospholipid bilayer. When encapsulating an active substance within a liposome and delivering it to target tissues, important objectives include highly efficient uptake of the active compound into the liposome and ensuring stable retention of the active compound within the liposome.

One of the advantages of liposomal dosage forms is the protection of healthy cells from the toxic effects of drugs, and the included substance from degradation in the body; prolongation of the action of drugs; selective accumulation in the affected area; the possibility of creating a water-soluble dosage form for a number of hydrophobic drugs and thereby increasing their bioavailability [Pharmaceutical development: concept and practical recommendations. Scientific and practical guide for the pharmaceutical industry. Edited by Bykovsky S.N. et al. - 2015. - 472 p.].

A very important property of liposomes has become the basis for the design of effective drugs. This relates to the ratio of their size to the capillary pore diameter. Since liposomes are larger than the capillary pore diameter, their volume of distribution is limited by the administration parameters. Liposomes will accumulate at sites of inflammation and deformation. This phenomenon is known as passive targeting. Thus, there are two reasons why liposomal drugs are more effective than standard ones: reduced toxicity and passive targeting [RU Patent No. 2462236, published September 27, 2012].

The creation of liposomal compositions is more promising compared to the production of micellar systems or microemulsions from a biomedical point of view, since vesicles are thermodynamically stable systems that can be obtained with given parameters for size and zeta potential, and are more stable in in vivo experiments.

Currently, there is a sufficient number of liposomal drugs on the pharmaceutical market, the composition of which is diverse, and the choice of one or another component is determined on the basis of the analysis of a large number of technological, physicochemical and biological factors.

The main structural components of liposomes are phospholipids. The phospholipid most commonly used in liposomal dosage forms is phosphatidylcholine, which has a hydrophilic polar group (phosphocholine) and hydrophobic acyl hydrocarbon chains. Phosphatidylcholine molecules are insoluble in water and aqueous media; they are oriented such that the fatty acid chains of the molecules face each other, while the polar heads are in the aqueous medium. This overcomes the instability of single molecules and leads to the formation of a bilayer. In addition to phosphatidylcholine, liposomes can be made of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, and sphingophospholipid.

At the same time, the disadvantages of liposomes as a dosage form are the difficulties associated not only with their production, but also those arising during long-term storage of the liposomal dispersion.

The disadvantage of liposomal compositions is that, once in the bloodstream, liposomes are absorbed by macrophages, which prevents the specific effect of the liposomal dosage form from being achieved.

Methods for increasing the stability of liposomes are known. For example, their stability is enhanced by the introduction of cholesterol into liposomes, which increases bilayer stability and reduces its permeability. Cholesterol can be incorporated into the liposomal membrane in fairly high concentrations (phospholipid/cholesterol molar ratios can reach 1:1 and even 1:2). The inclusion of polyethylene glycol in liposomes prevents their uptake by macrophages [Pharmaceutical Development: Concept and Practical Recommendations. Scientific and Practical Guide for the Pharmaceutical Industry. Edited by S.N. Bykovsky et al. - 2015. - 472 p.].

However, despite numerous developments of liposomal preparations, the problem of obtaining an acceptable and storage-stable liposomal form of the antioxidant Bisphenol-5 has not yet been solved.

A composition known from the prior art consists of an active substance - 6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone, a non-ionic surfactant, an antioxidant, a preservative, a fat-soluble emulsion stabilizer, water-soluble cellulose derivatives, a water-soluble emulsion stabilizer and water, which is characterized by high bioavailability and an extended shelf life. The method for producing the said composition consists of mixing certain quantities of a non-ionic surfactant and an antioxidant, heating them to 40-120°C, dissolving the required quantities of a fat-soluble emulsion stabilizer and 6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone in the resulting solution, cooling the resulting mixture to 30-60°C and, with vigorous stirring, pouring it into a mixed solution of water-soluble cellulose derivatives, a water-soluble emulsion stabilizer and a preservative in water, preheated to 30-60°C (RU Patent No. 2445079, publication date 03/20/2012).

The disadvantage of the known technical solution is the use of coenzyme Q ₁₀ in an oxidized, rather than reduced form, as well as the use of a conventional emulsion as the finished form, which is prone to rapid stratification and rapid rancidity.

A method is known for obtaining a water-soluble form of coenzyme Q ₁₀ , which is an inclusion complex of β-cyclodextrin with coenzyme Q ₁₀ , in which the molar ratio of β-cyclodextrin and coenzyme Q ₁₀ is approximately 1:1, which consists in the fact that β-cyclodextrin is dissolved in water at a temperature from 55°C to the boiling point, then coenzyme Q ₁₀ is added in solid form and stirring is continued at a temperature of 60-70°C, and then at room temperature (Patent of the Russian Federation No. 2375053, publication date 12/10/2009).

A drawback of the known technical solution is the impossibility of obtaining a reduced form of the coenzyme using this technology, as the complex production process occurs at elevated temperatures of 60-70°C, which will lead to oxidation of the product. Furthermore, the method is aimed at producing a food additive and does not disclose the specifics of producing pharmaceuticals based on the inclusion complex.

A composition of 6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone in the form of a transdermal, oral and injection agent containing a surfactant and presented in the form of liposomes is known (Patent WO 9835660 A1, publication date 08/20/1998).

However, the known technical solution only discloses the possibility of incorporating ubiquinone into liposomes, but does not describe the composition or technology for producing ubiquinone-based liposomes. A drawback of the composition is the use of coenzyme _Q10 in its oxidized, rather than reduced, form, which reduces the biological efficacy of the product.

The source [SHUQIN XIA et al. / Optimization in the Preparation of Coenzyme Q10 Nanoliposomes / Journal of Agriculture and Food Chemistry / 2006, Vol. 54, pp. 6358-6366] discloses a method for obtaining nanoliposomes containing ubiquinone-10 (coenzyme Q ₁₀ ), including dissolving coenzyme Q ₁₀ , phospholipid, cholesterol and Tween 80 in their various ratios in ethanol at a temperature of 55°C, adding the ethanol solution to a phosphate buffer (pH=7.4) at a temperature of 55°C and stirring, removing ethanol on a rotary evaporator, adding water, and treating the resulting suspension with ultrasound. It is noted that the optimal composition was obtained with a mass ratio of phospholipid/coenzyme Q ₁₀ /cholesterol/Tween 80 equal to 2.5:1.2:0.4:1.8, while the average size of liposomes was 68 nm.

A disadvantage of the known method is that it produces liposomes containing coenzyme _Q10 in an oxidized, rather than reduced, form, which reduces the biological efficacy of the product. Furthermore, this method does not employ cryoprotectants such as trehalose, lactose, and others, preventing lyophilization and the production of a liposomal form of the active agent as a free-flowing powder.

A method for producing a storage-stable liposome solution containing a reduced form of coenzyme Q ₁₀ as the active substance is known. The method involves dissolving phospholipids, reduced coenzyme Q ₁₀ , in the presence or absence of a surfactant, in an organic solvent, removing the organic solvent to form a film and dispersing the resulting lipid film in an aqueous medium using ultrasound for 30 minutes at a temperature of 4°C, followed by the formation of a liposome solution (EP 1386905 A1, publication date 04.02.2004).

A disadvantage of the known method is that it requires ultrasonic dispersion to produce ubiquinol liposomes, which complicates the production of monodisperse systems. Furthermore, the size of the resulting liposomes is not specified, complicating their use in creating liquid dosage forms and precluding the targeted delivery of ubiquinol. When creating water-soluble liposomal formulations, it is important to consider that the optimal liposome size for selective accumulation of the biologically active substance at the affected area is 50 to 200 nm. Furthermore, the conditions and equipment for purifying the liposomes from ubiquinol not included in the liposomal composition are not specified, making it impossible to assess the degree of ubiquinol incorporation into the liposomal membrane during production and storage. Of note, the process specifications include the lack of instructions on the conditions for removing the organic solvent, which is important, as elevated temperatures can affect the quality of the finished product. The invention indicates that the content of reduced coenzyme in liposomes can be up to 50%, which does not take into account the solubility of the coenzyme and is technologically impossible to achieve even in the presence of 20% surfactant.

The technical result of the claimed technical solution is the production of a feed antioxidant from water-insoluble Bisphenol-5 in the form of stable water-soluble liposomes, with included molecules of a biologically active substance - the antioxidant Bisphenol-5.

The essence of the claimed technical solution is a feed antioxidant in the form of liposomes for the preparation of wet and liquid feed for domestic and farm animals and birds, containing soy phosphatidylcholine and a non-ionic surfactant Tween-20 at a molar ratio of 4:1, wherein Bisphenol-5 is included in the liposome bilayer in an amount of up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20. The method for producing a feed antioxidant in the form of liposomes according to claim 1, consisting in preparing a solution containing soy phosphatidylcholine and a non-ionic surfactant Tween-20 at a molar ratio of 4:1, as well as Bisphenol-5 up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20, in chloroform, for which purpose the said reagents are mixed at room temperature until a homogeneous mass is obtained; Chloroform is removed under vacuum in a rotary evaporator at a temperature of 25-35 °C, resulting in a lipid film; the lipid film is hydrated with ultrapure water for 1 hour in a water bath at 60 °C with constant stirring; a five-fold freeze-thaw cycle is carried out using liquid nitrogen and a water bath to incorporate Bisphenol-5 into liposomes, resulting in a liposomal dispersion; to homogenize the liposomal dispersion, the liposomal dispersion is extruded through polycarbonate membranes with a pore size of 100 nm, resulting in a feed antioxidant in the form of liposomes no larger than 100 nm.

The claimed technical solution is illustrated in Fig. 1 – Fig. 4.

Fig. 1 shows a cross-sectional schematic representation of the claimed liposomes formed by soy phosphatidylcholine, the surfactant Tween 20 with incorporated Bisphenol-5 molecules.

Fig. 2 shows a photo of the appearance of the claimed liposomes with the inclusion of BP-5 in the form of a dispersion.

Fig. 3 shows the spectrum of the claimed liposomes with the inclusion of BP-5 in ethanol, l = 1 cm, 25 °C.

Fig. 4 shows the extinction coefficient of the claimed liposomes with the inclusion of BP-5 in ethanol, λ = 267 nm, 25 °C.

The applicant then provides a description of the claimed technical solution.

The stated technical result is achieved by developing liposomes containing the biologically active substance Bisphenol-5 (hereinafter referred to as BF-5) and a method for its production.

Chemical formulas of the substances included in the declared liposomes with the inclusion of BP-5:

Below is a description of the starting reagents: Bisphenol-5, soy phosphatidylcholine, surfactant Tween-20.

Bisphenol-5 is a synthetic fat-soluble antioxidant. It is a white or yellowish crystalline powder. It is soluble in fats and alcohol, but is practically insoluble in water. It is classified as a Class 4 hazard (low hazard) substance. Bisphenol-5 is used in animal husbandry to normalize metabolism, enhance growth, and improve meat quality in poultry, pigs, and young cattle.

Soy phosphatidylcholine is produced from purified soybean oil using low-temperature processing. It is the most widely used emulsifier in the modern food industry. It works well at the interface between various substances. In aqueous solution, lecithin phospholipids can form liposomes, bilayer membranes, micelles, or lamellar structures, depending on hydration and temperature. Lecithin allows for the formation of stable emulsions in oil-water systems and is a food additive (E322 and E476). It is widely used in the food industry for the production of chocolate and chocolate coating, confectionery, bakery products, pasta, margarine, mayonnaise, and baked goods. Products such as Essentiale Forte, Essliver Forte, Nash Lecithin, and others are based on phospholipids.

The claimed technical solution used soy phosphatidylcholine (S75, 70%, Lipoid GmbH , Germany).

The surfactant Tween-20 (Polysorbate-20) is used as an emulsifier in food products and as a wetting agent, for example, in flavored mouth drops like Ice Drops , helping other ingredients, such as SD alcohol and mint flavoring, disperse. The World Health Organization has proposed an acceptable daily intake of up to 25 mg per kilogram of body weight.

Tween 20 and Tween 80 surfactants are known to be used for modifying liposomes; they are non-toxic and approved for use in the food and pharmaceutical industries. Tween 80 is more widely used for modifying liposomes [Chaurasia & Lariya, 2020, https://doi.org/10.22159/ijap.2020v12i6.39391; Nurfitriyana et al., 2020, https://doi.org/10.22159/ijap.2020.v12s1.FF052], which is due to the longer hydrocarbon radical of Tween 80 (C18) compared to Tween 20 (C10) and the hydrophilic-lipophilic balance (15 for Tween 80 and 16.7 for Tween 20), which allows Tween 80 to better integrate into the lipid bilayer of vesicles. However, the length of the hydrocarbon tail of the surfactant directly affects the physicochemical parameters of the vesicles and, consequently, their encapsulation efficiency. When encapsulating hydrophobic substrates, a surfactant with a long hydrocarbon tail can compete with the substrate and reduce encapsulation efficiency [Bnyan et al., 2018, https://doi.org/10.1016/j.xphs.2018.01.005]. Since the present invention is aimed at increasing the water solubility of the hydrophobic antioxidant BP-5, Tween 20 was selected to achieve higher encapsulation efficiency values.

Below is a description of the claimed liposomes containing BF-5.

The claimed liposomes with the inclusion of BP-5 are vesicular nanocontainers formed on the basis of completely biocompatible components: phospholipid, specifically soy phosphatidylcholine, non-ionic surfactant Tween 20 at their molar ratio of 4:1, with the inclusion of BP-5 in an amount of up to 0.3% of the total mass of the lipid and surfactant, with BP-5 enclosed in a lipid bilayer.

The invention increases the bioavailability of the medicinal (active) substance, the antioxidant BP-5, by including it in the composition of liposomes.

A schematic representation of the claimed liposomes with the inclusion of BF-5 is presented in Fig. 1.

The following is the claimed method for obtaining the claimed liposomes with the inclusion of BF-5:

– prepare a solution containing soy phosphatidylcholine (SP) and non-ionic surfactant (surfactant) Tween-20 at their molar ratio of 4:1, respectively, as well as biologically active substance BP-5 up to 0.3% of the total mass of lipid and surfactant, in chloroform (for example, 0.5 ml), for which the specified reagents are mixed at room temperature until a homogeneous mass is obtained;

– chloroform is removed under vacuum on a rotary evaporator at a temperature of 25-35 °C, a lipid film is obtained;

– the lipid film is hydrated with ultrapure water for 1 hour in a water bath at 60 °C with constant stirring;

– a five-fold cycle of freezing and thawing is carried out using liquid nitrogen and a water bath to incorporate BP-5 into liposomes, and a liposomal dispersion is obtained;

– to homogenize the liposomal dispersion, the process of extrusion of the liposomal dispersion is carried out through polycarbonate membranes with a pore size of 100 nm, for example, using an automatic extruder.

The claimed liposomes are obtained with the inclusion of Bisphenol-5 with a size of no more than 100 nm, containing soy phosphatidylcholine and non-ionic surfactant Tween-20 in their molar ratio of 4:1, while Bisphenol-5 is included in the liposome bilayer in an amount of up to 0.3% of the total mass of the lipid and Tween-20.

The hydrodynamic diameter and electrokinetic potential of the obtained liposomes with the inclusion of BF-5 are determined using the method of dynamic and electrophoretic light scattering;

The encapsulation efficiency of the obtained liposomes is assessed using spectrophotometry.

The resulting liposomes containing BP-5 can be included in wet and liquid feeds for domestic and farm animals and birds.

Fig. 2 shows a photo of the appearance of the claimed liposomes with the inclusion of BP-5 in the form of a dispersion.

Below are examples of the implementation of the declared technical solution.

Example 1. Obtaining classical liposomes with BP-5 without Tween 20 at a loading of 0.3% by weight of lipid (system No. 1).

– prepare a solution containing soy phosphatidylcholine (S75, 70%, Lipoid GmbH , Germany)) 30 mM and the biologically active substance BP-5 up to 0.3% of the lipid mass, in chloroform (for example, 0.5 ml), for which the specified reagents are mixed at room temperature until a homogeneous mass is obtained;

– chloroform is removed under vacuum on a rotary evaporator at a temperature of 25-35 °C, a lipid film is obtained;

– the lipid film is hydrated with ultrapure water obtained using a Simplicity®UV unit ( Millipore SAS , France) for 1 hour in a water bath at 60 °C with constant stirring (250 rpm) using an MR Hei-Tec magnetic stirrer ( Heidolph Instruments , Germany);

– a five-fold cycle of freezing and thawing is carried out using liquid nitrogen and a water bath to incorporate BP-5 into liposomes, and a liposomal dispersion is obtained;

– to homogenize the liposomal dispersion, the process of extrusion of the liposomal dispersion (for example, 21 times) is carried out through polycarbonate membranes with a pore size of 100 nm ( Whatman® Nuclepore™ Track-Etched Membranes, Cytiva , Poland), for example, using an automatic extruder LiposoFast LF-50 ( Avestin , Canada), liposomes with the inclusion of BP-5 with a size of no more than 100 nm are obtained.

The resulting classic liposomes are packaged in vials.

System No. 1 was obtained: soy phosphatidylcholine (30 mM) _{_} BP-5 (0.3% by weight of lipid);

Example 2. Obtaining the declared liposomes with BP-5 and Tween 20 at a loading of 0.3% of the total mass of lipid and surfactant (system No. 2).

The sequence of actions according to Example 1 is carried out, characterized in that at the first stage a solution is prepared containing soy phosphatidylcholine (S75, 70%, Lipoid GmbH, Germany)) 24 mM, non-ionic surfactant Tween 20 (Ferak Berlin GmbH, Germany) 6 mM and biologically active substance BP-5 up to 0.3% of the total mass of lipid and surfactant, in chloroform (for example, 0.5 ml), for which the specified reagents are mixed at room temperature until a homogeneous mass is obtained.

Next, carry out the sequence of actions according to Example 1.

System No. 2 was obtained: soy phosphatidylcholine (24 mM)_Tween-20 (6 mM)_BP-5 (0.3% of the total mass of lipid and surfactant).

Example 3. Obtaining liposomes with BP-5 and Tween 20 at a loading of 0.5% of the total mass of lipid and surfactant (system No. 3).

The sequence of actions according to Example 2 is carried out, differing in that the biologically active substance BF-5 is taken up to 0.5% of the total mass of lipid and surfactant.

System No. 3 was obtained: soy phosphatidylcholine (24 mM)_Tween-20 (6 mM)_BP-5 (0.5% of the total mass of lipid and surfactant);

Thus, all three systems obtained in Examples 1–3 contain BP-5, but differ in the concentration of BP-5 or the presence of Tween 20:

1. System 1: soy phosphatidylcholine (30 mM)_BP-5 (0.3% of lipid weight);

2. System 2: soy phosphatidylcholine (24 mM)_Tween-20 (6 mM)_BP-5 (0.3% of the total mass of lipid and surfactant);

3. System 3: soy phosphatidylcholine (24 mM)_Tween-20 (6 mM)_BP-5 (0.5% of the total mass of lipid and surfactant).

Example 4. Evaluation of the hydrodynamic diameter and electrokinetic potential of liposomes containing BF-5.

The hydrodynamic diameter (D _h ), polydispersity index (PdI) and zeta potential (ζ) of three dispersions of liposomes with BP-5 according to Examples 1, 2, 3, diluted to 1 mM, were determined by dynamic and electrophoretic light scattering using a U-shaped cuvette on a Malvern ZetaSizer Nano instrument (Malvern Instruments Ltd., UK), equipped with a helium-neon laser with a wavelength of 633 nm, a power of 10 mW and a light scattering angle of 173°. The hydrodynamic diameter of the particles was calculated from the diffusion coefficient of the liquid, which was obtained on the basis of the correlation function of fluctuations in the intensity of scattered light using the Stokes-Einstein equation:

D = kT/3πηD _{h ,}

where D is the diffusion coefficient, k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the solvent.

The zeta potential was calculated based on the electrophoretic mobility of particles using the Smoluchowski equation:

ζ= (µ×π)/ε

where ε is the dielectric constant of the solvent, η is the dynamic viscosity of the liquid, μ is the electrophoretic mobility of the particles.

The results of the measurements are presented in the Table.

Example 5. Determination of the efficiency of encapsulation of BP-5 in liposomesaccording to Examples 1, 2, 3.

Because BP-5 is poorly soluble in water, some of the substrate not loaded into the liposomes remains on the polycarbonate membranes of the extruder. The membranes were immersed in ethanol to dissolve BP-5. After complete dissolution of the substrate in ethanol, the concentration was determined using a 0.2 cm cuvette on a Specord 250 Plus spectrophotometer (Analytik Jena AG, Germany). The maximum absorption of BP-5 was recorded at 267 nm (Figure 3). Determination of the extinction coefficient of BP-5 in ethyl alcohol: a 0.0033 g sample of BP-5 was dissolved in 0.809 ml of ethyl alcohol to obtain C _BP-5 = 0.01 M. This solution was diluted 10 times to a BP-5 concentration of 0.001 M. 2 ml of alcohol were placed in a cuvette (l = 1 cm) and 0.02 ml of a BP-5 solution with a concentration of 0.001 M were dosed. The extinction coefficient of BP-5 in ethanol was determined from the slope of the dependence of the optical density of the substrate solution on concentration (Fig. 4) and was equal to 17942 l/mol cm.

The encapsulation efficiency (EE) was calculated using the following equation:

The results of the measurements are presented in the Table.

Table. Physicochemical characteristics of liposomes based on phosphatidylcholine, Tween 20 and BP-5, 25°C

System Diameter, nm PdI Zeta potential, mV EI,% Mass of loaded substance in 10 ml of liposomes, mg 1 316 ± 14 0.504 ± 0.069 -64.9 ± 0.6 49.8 1.5 2 102 ± 3 0.116 ± 0.015 -26 ± 2 98.9 2.97 3 259 ± 4 0.661 ± 0.106 -50 ± 2 73.96 3.7

The values presented show that only system 2 contains liposomes with a hydrodynamic diameter close to 100 nm. This system is characterized by a low polydispersity index (0.116), which allows it to be considered monodisperse, and high encapsulation efficiency (EE), close to 100%. Therefore, this system was selected for further animal testing.

Example 6. Study of toxicity and cumulative properties of the declared liposomes with the inclusion of BP-5 in rats.

The cumulative properties of liposomes containing BP-5 were studied using the subchronic toxicity method [Guide to the experimental (preclinical) study of new pharmacological substances. Ed. by R. Yu. Khabriev. – M: Medicine, 2005 – 829 p.] on white rats by repeated administration of the drug.

For the experiments, 20 white rats with a live weight of 150-250 g were used. The animals were divided into experimental and control groups of 10 animals each, including 5 females and 5 males.

According to the guidelines for conducting preclinical studies of drugs [Mironov, A.N. Guidelines for conducting preclinical studies of drugs / A.N. Mironov, N.D. Bunyatyan, A.N. Vasiliev et al. - Grif i K, Moscow 2012. - ISBN 978-5 - 944 p.], if due to the low toxicity of the pharmacological substance it is impossible to determine LD50, the maximum dose that was administered to animals should be indicated, but not less than 2 g/kg.

The claimed liposomes with the inclusion of BP-5 were administered directly into the drinking water of animals daily for 28 days. On the first day, liposomes with the inclusion of BP-5 were administered at a dose of 200 mg/kg (1/10 of LD50), then the dosage was increased by 1.5 times every four days [Lim R.K., Rink K.G., Glass H.G., Soaje–Ehagye E.A. Method for the evaluation of cumulation and tolerance by the determination of acute and subchronic median effective doses // Arch. Intern. Pharm. Ther. – 1961 – V. 130 – P. 336 – 352.].

Throughout the entire period, the general condition and behavior of the animals, food and drink intake, visible physiological functions, the condition of the coat, possible death, etc. were observed. Reweighing was carried out every four days.

At the end of the experiment, one day after the last administration of liposomes containing BF-5, the animals were slaughtered. The weight of the main organs was determined and mass coefficients were calculated. The functional state of the central nervous system was assessed by visual observations of motor activity and responses to external stimuli. The cumulation coefficient was determined by the formula: K = LD50(n)/LD50(1), [Guide to Experimental (Preclinical) Study of New Pharmacological Substances. Ed. by Khabriev R. Yu. - M: Medicine, 2005 - 829 p.] where K is the cumulation coefficient; LD50 (n) is the dosage for n-fold administration of the substance; LD50(1) is the dosage for a single administration of the substance. If the cumulation coefficient is greater than one, this indicates the absence of cumulative properties of the study drug [Tretyakov A.D. et al. Veterinary Drugs. - M., 1988 - P. 201-216, Tritek V. S., Gulyaev A. E. Method for determining the cumulation coefficient in a toxicological study // Experimental and clinical pharmacology. - 2011 - No. 3 - P. 35-36.].

The obtained results indicate that daily administration of the claimed liposomes containing BP-5 at a dose of 2000 mg/kg body weight did not result in changes in the general condition or behavior of the test rats. Administration of the claimed liposomes containing BP-5 did not have a statistically significant effect on current growth rates.

Due to the absence of animal mortality, it was not possible to determine the average lethal dose of the declared liposomes with the inclusion of BP-5, therefore, as a result of studying acute toxicity in experiments on laboratory animals, it can be concluded that the declared liposomes with the inclusion of BP-5 belong to hazard class IV - a non-hazardous compound.

Results of experiments studying the cumulative effect showed a cumulative coefficient of 3.96. This indicates that the claimed liposomes containing BP-5 are nontoxic and do not exhibit cumulative properties. Daily administration of the claimed liposomes containing BP-5 did not result in changes in relative organ weight in rats receiving a dose of 2000 mg/kg body weight.

From the above, it can be concluded that the applicant has solved the identified technical problem and achieved the claimed technical result : a feed antioxidant in the form of stable water-soluble liposomes was obtained from water-insoluble Bisphenol-5, with the direct inclusion of Bisphenol-5 in the inner shell of the latter, consisting of soy phosphatidylcholine and the non-ionic surfactant Tween-20.

The feed antioxidant can be used in the preparation of wet and liquid feed for domestic and farm animals and birds.

Claims (6)

1. A feed antioxidant in the form of liposomes for the preparation of wet and liquid feed for domestic and farm animals and birds, containing soy phosphatidylcholine and non-ionic surfactant Tween-20 in a molar ratio of 4:1, while Bisphenol-5 is included in the liposome bilayer in an amount of up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20. 2. A method for producing a feed antioxidant in the form of liposomes according to claim 1, which consists in preparing a solution containing soy phosphatidylcholine and a non-ionic surfactant Tween-20 in a molar ratio of 4:1, as well as Bisphenol-5 up to 0.3% of the total mass of soy phosphatidylcholine and Tween-20, in chloroform, for which purpose the said reagents are mixed at room temperature until a homogeneous mass is obtained; chloroform is removed under vacuum on a rotary evaporator at a temperature of 25-35 °C, resulting in a lipid film; the lipid film is hydrated with ultrapure water for 1 hour in a water bath at 60 °C with constant stirring; a five-fold freezing and thawing cycle is carried out using liquid nitrogen and a water bath to incorporate Bisphenol-5 into liposomes, and a liposomal dispersion is obtained; To homogenize the liposomal dispersion, the process of extrusion of the liposomal dispersion is carried out through polycarbonate membranes with a pore size of 100 nm, obtaining a feed antioxidant in the form of liposomes with a size of no more than 100 nm.