RU2706788C1 - Emulsion preparation of polyprenoles - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention refers to pharmaceutical industry, namely to a preparation in the form of an emulsion having high stability and retention of polyprenol activity during storage. An emulsion preparation of polyprenols containing polyprenols of Siberian fir, arabinogalactan, dihydroquercetin, water taken in certain proportions. Above described preparation of polyprenols is stable.EFFECT: disclosed is an emulsion preparation of polyprenols.1 cl, 4 dwg, 2 tbl, 10 ex

Description

The invention relates to the pharmaceutical industry, namely to preparations of polyprenols in aerosol form, which have high stability and preservation of the activity of polyprenols during storage.

A fungicidal agent is known from the prior art (RF Patent 2432744. Published on November 10, 2011, Bull. No. 31). This mixed composition contains Siberian fir essential oil (Abies Sibiricd), which is isolated by hydrothermal treatment or extraction from needles and woody greens, obtained in the form of a stable 2-5% aqueous emulsion containing, as an emulsifier, liquid soap and formed when high speed mixing components. The disadvantage of this tool is the low stability over time of the emulsion due to the weak emulsifying properties of liquid soap, which requires its preparation immediately before use.

A production method is proposed (RF Patent 2369383. Published 10.10.2009, Bull. No. 28) of a phospholipid emulsion containing the active form of dihydroquercetin, in particular a liposomal biologically active form of dihydroquercetin, and a fairly simple and economical method for producing such an emulsion is proposed. The phospholipid emulsion includes dihydroquercetin, a membrane-forming phospholipid, glycine, an aqueous solution of ethyl alcohol. The introduction of the amino acid - glycine into the emulsion, provides, in addition to the known positive effects of biological effects, the stability of the rheological parameters of the emulsion, preventing its separation into fractions. The method for producing a phospholipid emulsion is complicated by the reaction of esterification of glycine with ethanol with the reversible formation of aminoacetic ester. Unlike amino acids, their esters do not have a bipolar structure, and are volatile compounds, which leads to a change in the composition of the emulsion. The safety of the drug is several months.

Closest to the proposed one is (Patent 2189231. Published on September 20, 2002, Bull. No. 26) an emulsion preparation comprising polyisoprenoids, and fatty acid esters with polyethylene glycol or fatty acid esters with polyhydric alcohols (sorbitol) are used as a nonionic surfactant or block copolymers of ethylene and propylene, or ethoxylated castor oil, or higher fatty alcohols (C ₁₆ -C ₁₈ ). The advantage of this solution is the ability to significantly reduce the applied dose of polyprenols, as well as to provide selective activation of the respiratory tract immunity system in order to provide a therapeutic and preventive effect against respiratory infections.

Derewiaka D., Obiedzinrski M., Kania M, Danikiewicz W., Swiezewska E. (2015) Double bond stereochemistry influences the susceptibility of short chain isoprenoids and polyprenols to decomposition by thermo-oxidation. Lipids, V. 50(4), pp. 359-370.] и особенно старению вторичного органического аэрозоля [Rohr А.С.(2013) The health significance of gas- and particle-phase terpene oxidation products: a review. Environ Int. V. 60, pp 145-162; doi:10.1016/j.envint.2013.08.002.]. Подобное наблюдается при окислении других важных липидов к которым относятся ПП, например жирных кислот, стеринов, деградирующих до низкомолекулярных продуктов, например олефинов, спиртов, кетонов, альдегидов, кислот. Эти летучие продукты образуются при расщеплении эпоксидного кольца, [Frankel E.N. Chemistry of free radical and singlet oxidation of lipids. Prog. Lipid Res. 1984, V.23, pp.197-221].The disadvantage of the prototype is the sensitivity of polyesters of fatty acids to oxygen. In the presence of ultraviolet radiation, they form hydroperoxides with high reactivity, and the absence of an antioxidant leads to the decomposition of polyisoprenoids during storage of the drug with the formation of peroxides with subsequent decomposition of the latter and the formation of secondary oxidation products: epoxides and aldehydes. [Molinska E., Klimczak U.,

Derewiaka D., Obiedzinrski M., Kania M, Danikiewicz W., Swiezewska E. (2015) Double bond stereochemistry influences the susceptibility of short chain isoprenoids and polyprenols to decomposition by thermo-oxidation. Lipids, V. 50 (4), pp. 359-370.] And especially the aging of secondary organic aerosol [Rohr A.S. (2013) The health significance of gas- and particle-phase terpene oxidation products: a review. Environ Int. V. 60, pp 145-162; doi: 10.1016 / j.envint.2013.08.002.]. This is observed during the oxidation of other important lipids, which include PP, for example, fatty acids, sterols, degrading to low molecular weight products, such as olefins, alcohols, ketones, aldehydes, acids. These volatile products are formed by cleavage of the epoxy ring, [Frankel EN Chemistry of free radical and singlet oxidation of lipids. Prog. Lipid Res. 1984, V.23, pp. 177-221].

It should be noted that with the introduction of Tween 80 above 1% leads to thickening of the final product, high concentrations are usually used only for the use of solid products. The disadvantages of the prototype should also include that the proposed surfactants are of a synthetic nature and can be dangerous to human health [https://www.thevaccinereaction.org/2016/01/polysorbate-80-a-risky-vaccine-ingredient/].

The task of the invention is to develop the composition of the emulsion preparation to eliminate the disadvantages of the prototype - the instability of polyprenols to oxidation processes, which leads to a decrease in the therapeutic and prophylactic effect of the drug during prolonged storage, as well as to increase the safety of the emulsion product.

The technical result is an increase in the quality of the emulsion preparation of polyprenols.

The technical result is an increase in the stability of polyprenols (PP).

The specified technical result is achieved by the fact that a PP-based aqueous emulsion is proposed, where a natural nonionic surfactant, arabinogalactan (AG), is used as an emulsion stabilizer, while an antioxidant, dihydroquercetin (DQV), is additionally introduced into the emulsion in the following ratio of components, in weight. %:

Polyprenols of Siberian fir - 1-5.0

Nonionic surfactant - 2-5.0

Antioxidant - 0.02-0.2

Water - The rest is up to 100%

1. Polyprenols - natural isoprenoid alcohols containing more than 4 isoprenoid units are released from the greenery of conifers. Polyprenols of most organisms contain more than 10 isoprenoid units. Possessing structural similarities with dolichol, they take part in the restoration of damaged body cells, while developing pathological conditions, strengthening their cell membranes. [Sweiezewska E., Danikiewiez W. Polyisoprenoids: structure, biosynthesis and function // Progress in Lipid Research, 44 (2005), p. 235-250.]. The pure fraction of polyprenols - Ropren is obtained by the method described in the patent (RF Patent No. 2252026. Published on 05/20/2005. Bull. No. 14). The product exhibits hepatoprotective [Vyshlov E.V.,. Tsoi E.I., Sultanov V.S., Trusov V.B., Ryabov V.V. Hypolipidemic and hepatoprotective effect of the polyprenol-containing drug in patients with acute coronary syndrome. Bull. expert. biol. 2018.Vol. 168, No. 8. S. 289-291.], Immunomodulating (RF Patent 2137479 Published September 20, 1999). The drug is used as a tool for the treatment of patients with dementia syndrome (RF Patent 2327480. Published on June 27, 2008. Bull. No. 18). Polyprenols are involved in the biosynthesis of glycoproteins. The use of emulsions for PP transport opens up opportunities not only to increase the bioavailability of the latter, but also to ensure prolonged action of PP. Polyprenols are non-toxic and safe [Danilov L.L., Maltsev S.D., Deyeva A.V., Narovlyansky A.N., Sanin A.V., Ozherelkov S.V., Pronin A.V. Phosprenyl: A novel drug with antiviral and immunomodulatory activity. Arch. Immunol. Ther. Exp. 1995, 44, 395-400.]. We used the drug Ropren (registration certificate LSR-001521/07 of 07/12/2007) with a content of the sum of polyprenols of 95%.

2. The source for the industrial production of arabinogalactan is larch wood containing up to 20% of this polysaccharide (RF Patent No. 2256668. Published July 20, 2005. Bull. No. 20). Arabinogalactan - a water-soluble highly branched polysaccharide with m.m. 9-18 kDa, consisting of D - galactopyranosyl units connected by β-bonds and side chains of D - galactopyranosyl and L - arabinofuranosyl residues. Such a structure ensures the formation of intermolecular complexes with hydrogen bonds of the side chains and double bonds of polyprenols. It is characterized by low toxicity (does not exhibit acute toxicity at a dose of 5000 mg / kg body weight) and high biological activity: prebiotic, gastro-and hepatoprotective, etc. [Medvedeva EN, Babkin VA, Ostroukhova L.A. Larch arabinogalactan - properties and prospects of use (review). // Chemistry of plant materials. 2003. No1. S. 27-37.]. The safety of AH from larch has been officially certified by the USA Food and Drag Administration (Agency Response Letter GRAS Notice No. GRN 000084 February 25, 1964, 30 FR 2430; 21 CFR 172. 610), and since 1965. It is used in the USA as a food supplement. It is registered under the code E409 in the Codex of the Food Committee of the World Health Organization (FAO / WHO) for food products as a stabilizer of emulsions and thickener. It is allowed in our country for use as an additive to food products by normative acts [SanPiN 2.3.2.1078-01 "Hygienic requirements for safety and nutritional value of food products"; Section 2.3.2. Food Raw Materials and Food Products, p. 120, E409]. According to the List approved by the Chief Sanitary Doctor of the Russian Federation, an adequate level of AH consumption is 10 g / day, and the upper allowable level is 20 g / day. [Balanced diet. Recommended levels of intake of food and biologically active substances. // Methodical recommendations MP 2.3.1. 19150-04. Approved July 2, 2004]. The most valuable properties of AG are its good solubility in cold water, low viscosity of concentrated solutions, high dispersing ability, lack of odor, and stability during heat treatment. The ability of AG to bind fat and retain moisture expands the prospect of using emulsions as a stabilizer. We used arabinogalactan FibrolarS approved as a raw material for the production of biologically active additives TU 9363-021-39094141-08. SanPin No. 77.99.03.003.T.001137.05.08.

The choice of DHQ, which is a compound of the flavonoid group, for the development of the composition is determined, on the one hand, by a wide spectrum of biological action: it has an angioprotective, regenerating, detoxifying, decongestant, antioxidant effect. The level of its antioxidant activity allows you to put it in the first position among substances of a similar spectrum of action. It was established that dihydroquercetin increases the shelf life of products by 1.5-3 times, interrupting the self-oxidation of fat components [Klebanov GI, Teselkin Yu.O., Babenkova IV, Zhambalova BA, Tyukavkina N. A., Rulenko I.A., Kolesnik Yu.A., Radaeva I.A., Shulkina S.N. Dihydroquercetin inhibition of free-radical oxidation of milk powder lipids. Biotechnology and management. - 1995. - No. 1. - from. 36-39.]. In addition, dihydroquercetin has the function of inhibiting the growth of microorganisms in products that are already susceptible to oxidation. Based on the test results of canned milk, the shelf life of canned milk with the addition of dihydroquercetin is up to 24 months [Guseva TB, Karanyan OM, Rassokha SN, Radaeva IA The use of the natural antioxidant dihydroquercetin to increase the shelf life of canned milk. Food industry №8 / 2017 С.54-56.]. DHQ inhibits premature cell aging and the development of various diseases; strengthens the walls of blood vessels, improves microcirculation; inhibits inflammatory processes, has a decongestant effect; favorably affects the skin, normalizes the synthesis of collagen / elastin in the skin. By Decree No. 36 of SanPiN 2.3.2.1078-01 dated November 14, 2001, it classifies dihydroquercetin as an antioxidant. According to the Methodological recommendations of the State Sanitary and Epidemiological Regulation of the Russian Federation No. 2.3.1.1915-04 of 2004 "Recommended levels of intake of food and biologically active substances" establish an adequate and upper allowable levels of dihydroquercetin consumption in the amount of 25 and 100 mg per day. Recommended as an antioxidant - GOST 33504-2015 Food additives. Dihydroquercetin. Technical conditions

We used Lavitol (dihydroquercetin) (TU 9325-001-70692152-07) products of Ametis CJSC, the content of dihydroquercetin was not less than 90%.

A method with the claimed distinguishing features in the analysis of patent and scientific literature is not found.

The method is as follows. A 20% AG gel is prepared by dissolving in distilled water at 50-70 ° C for 15-20 minutes. In a separate tank receive 20% concentrate DHQ in ethanol at 20-25 ° C. In a separate container, 1: 1 is dissolved in PP pentane at a temperature of no higher than 30 ° C for 15-20 minutes. A solution of DHQ in PP is prepared by dissolving a “fat” phase in a water bath by adding dropwise an alcoholic solution of DHQ to the PP solution for 5-10 min at a temperature of 30 ± 2 ° C and rotor speeds of 1000-1500 rpm until a homogeneous solution is obtained . The primary emulsion is obtained by adding to the AG solution, a PP solution with DHQ at a rotor speed of 1.0-1.5 thousand rpm and a temperature of 35 ± 2 ° C until a homogeneous emulsion is obtained, pentane is evaporated and a homogeneous emulsion is formed. The emulsion is stabilized by sonication for at least 15 minutes at an ultrasonic frequency of 22 kHz ± 1.65. The size of the globules is controlled using a hemovisor, the detection limit is 0.4 μm, irradiation with a monochromatic green LED, the wavelength is 0.528 nm, the resolution in the preparation field is 4 pixels / micron.

The present invention is illustrated by the following examples.

Example 1. The determination of the content of polyprenols during storage in the emulsion by HPLC. 10 ml of an emulsion containing from 2 to 10% PP are placed in a 20 ml centrifuge tube, 10 ml of hexane are added, shaken for 1 min, treated for 1 min in an ultrasonic bath, centrifuged for 3 min. 0.8 ml of the hexane layer was taken off, transferred to another tube and evaporated. The residue was redissolved in 0.5 ml of the mobile phase, and chromatographed. Chromatographic analysis conditions: 5 μm reverse phase column, C18, mobile phase: 99: 1 hexane / isopropyl alcohol mixture, UV detector, 210 nm wavelength. The HPLC chromatogram contains a set of peaks typical of polyprenols that correspond to individual isoprenologists from C 50 to C 100. The standard is a polyprenol substance containing 1 mg / ml (accurately weighed) of a standard polyprenol sample. The quantitative content of PP is determined by the external standard method by the sum of the areas of all peaks.

Example 2. Determination of dihydroquercetin content during storage in an emulsion by HPLC. 10 ml of an emulsion containing 0.01-0.2% DHQ was placed in a flask, 10 ml of ethanol was added to precipitate AH and shaken. 1.0 ml of water-ethanol solution is transferred by micropipette to a 2.0 ml centrifuge tube, 1.0 ml of petroleum ether is added to release the lipid fraction, shaken well for 3 minutes, centrifuged for 3 minutes and 0.8 ml of ethanol layer is transferred to another test tube and evaporate. The residue is redissolved in 0.5 ml of the mobile phase and chromatographed. Conditions for chromatographic analysis: Column with a reversed phase of 5 μm, C18, mobile phase: acetonitrile - 0.03% trifluoroacetic acid in a volume ratio of 30:70, UV detector , wavelength 290 nm. The standard is used (GSO State standard sample “Dihydroquercetin” Certificate for GSO-DKV-Ametis No. GSO 10766-2016) dihydroquercetin with a content of 1 mg / ml. The quantitative content of DHQ is determined by the external standard method in accordance with GOST 33504-2015 Food additives. Dihydroquercetin. Technical conditions Date of introduction 2017-01-01.

Example 3. A control solution of PP (using the Ropren preparation, registration certificate LSR-001521/07 of 12.07.2007, with a total polyprenol content of 95%>) is obtained by dissolving 2.5 g of PP in 97.5 g of pentane (154.8 ml) at a temperature of not more than 30 ± 2 ° C solution (stability control PP, see Table 1).

Example 4. A control solution of DHQ 0.2% in ethyl alcohol (used "Lavitol (dihydroquercetin)" (TU 9325-001-70692152-07) products of ZAO "Ametis", the content of dihydroquercetin is not less than 90%.) Is obtained by dissolving 0.2 g of DHQ in 98.8 ml of water with vigorous shaking (control of the stability of the DHQ, see Table 2).

Example 5. The emulsion form of PP is prepared using a surfactant - AG 1 g and PP -1 g (use arabinogalactan Fibrolar TU 9363-021-39094141-08.) In distilled water (see Table 1). Particle size less than 1 micron.

The percentage of components: weight. %:

Siberian fir polyprenols -1.0

Surfactant AG - 1.0

The rest is water up to 100%. Preparation of emulsion:

I. At the same time in a water bath in different containers dissolve the "fat" and "water" phases. PP is readily soluble in pentane. AG from larch is readily soluble in both hot and cold water. The solubility of arabinogalactan in water is more than 15 g / 100 ml. Within 15 minutes, 14 to 50 grams of arabinogalactan can be dissolved in 100 ml of water at a temperature of 20 to 90 ° C;

II. Emulsification. The oil phase is added dropwise to the aqueous method of rotor-stator homogenization for 10-20 minutes at a temperature of 30-35 ° C and rotor speeds of 1000-1500 rpm until the pentane evaporates and a homogeneous primary dispersion is obtained.

III. Dispersion Considering that ultrasound does not violate the biological activity of most pharmacological preparations, ultrasonic generators can be used to obtain homogeneous emulsions. The emulsion is processed at a frequency of ultrasonic exposure of 22 kHz ± 1.65 for 15-20 minutes The microemulsion PP reached an average micelle size of not more than 1 μm.

IV. The emulsion cools naturally, with constant continuous stirring.

Example 6. The emulsion form of PP is prepared using a surfactant - AG 5 g and PP -1 g in distilled water. The emulsion is obtained under the conditions of Example 5 (see Table 1). Particle size less than 1 micron.

The percentage of components: weight. %:

Siberian fir polyprenols - 1.0

Surfactant AG - 5.0

The rest is water up to 100%

Example 7. The emulsion form of PP is prepared using a surfactant - AG 2 g, PP -5 g and DHQ 0.5 g in distilled water. For a more uniform distribution of DQV by weight of PP, DQV was introduced as a solution in ethanol. Preparation of a solution of DHQ in PP. A water bath is obtained in a container of the "fat" phase by adding dropwise a 20% alcohol solution of DHQ to the oil phase for 5-10 minutes at a temperature of 30-35 ° C and rotor speeds of 1000-1500 rpm until a homogeneous solution is obtained. Emulsification is carried out under the conditions of example 5, to achieve a uniform distribution of particles with a size of 10 to 15 microns (Fig. 1). Dispersion is carried out under the conditions of example 5 (see Tables 1 and 2). The emulsion PP reached an average micelle size of 1-3 μm (Fig. 2).

The percentage of components: weight. %:

Siberian fir polyprenols - 5.0

Arabinogalactan from 2.0

Dihydroquercetin - 0.5

The rest is water up to 100%)

Example 8. The emulsion form of PP is prepared using a surfactant - AG 5 g, PP -2 g and DHQ 0.2 g in distilled water. Emulsification under the conditions of example 5, to achieve a uniform distribution of particles with a size of from 10 to 15 microns (Fig. 1). Dispersion is carried out under the conditions of example 5 (see Tables 1 and 2). The emulsion PP reached an average micelle size of not more than 1 μm (Fig. 3). View of the emulsion after 24 months (Fig. 4.) obtained in the conditions of example 7.

The percentage of components: weight. %:

Polyprenols of Siberian fir - 2.0

Arabinogalactan from 5.0

Dihydroquercetin - 0.2

The rest is water up to 100%

Example 9. The emulsion form of PP is prepared using a surfactant - AG 5 g, PP -2 g and DHQ 0.08 g in distilled water. The emulsion is obtained under the conditions of example 7 (see Tables 1 and 2).

The percentage of components: weight. %:

Polyprenols of Siberian fir - 2.0

Arabinogalactan from 5.0

Dihydroquercetin - 0.08

The rest is water up to 100%

Example 10. The emulsion form of PP is prepared using a surfactant - AG 5 g, PP -2 g and DHQ 0.02 g in distilled water. The emulsion is obtained under the conditions of example 7 (see Tables 1 and 2).

The percentage of components: weight. %:

Polyprenols of Siberian fir - 2.0

Arabinogalactan from 5.0

Dihydroquercetin - 0.02

The rest is water up to 100%

Table 1 shows the results of monitoring the content of PP in emulsions with arabinogalactan. Emulsification allows you to achieve a uniform distribution of particles with a size of from 10 to 15 microns (Fig. 1). Emulsions with a PP content of 5% and higher (FIG. 2) increase the micelle size and decrease the stability of the emulsion (FIG. 2). The stability of the emulsion was evaluated during storage in vivo (20 ° C) for 24 months. The emulsion remained transparent during the experiment and did not delaminate. The formation of individual drops of PP with a size of no more than 5 μm, after storage for 24 months (Fig. 4), shows the possibility of long-term storage of PP in emulsions of stabilized AGs, and allows us to conclude that long-term dynamic stability of the emulsion.

The stability of the emulsion, as in the conditions of the prototype when the AG content of 2-10%, mainly 5%> (see example 8, Fig. 3), showing good solubility, low viscosity of concentrated solutions, high dispersing ability, the absence of hazardous decomposition products, availability of all components of the emulsion on an industrial scale, allows us to recommend AG as a nonionic surfactant in the production of PP emulsions.

The preservation of PP up to 90% in the presence of DHQ, characterized by a wide spectrum of pharmacological activity, which is a natural antioxidant and produced on an industrial scale, expands the scope of application of such emulsions. Dihydroquercetin is a direct-acting antioxidant that directly binds free radicals, breaking the branching of the free radical chain. In this case, dihydroquercetin can function as a trap of reactive oxygen species. In this sense, it is a reference product in comparison with all known, including synthetic direct-acting antioxidants. It was found that dihydroquercetin inhibits free radical oxidation of both water-soluble and fat-soluble substrates. (Kruglova O.G., Dorovskikh V.A., Tikhanov V.I., Kruglova T.G., Bocharnikova I.A. Effect of dihydroquercetin on lipid peroxidation products under cold exposure. Far Eastern Medical Journal. 2011.-N 3 .-S. 90-92).

The most effective combination of antioxidant in the emulsion is achieved with a content of DHQ from 0.02 to 0.2%. A dosage of DHQ 0.5% does not significantly increase the preservation of PP, and a concentration below 0.02% (Example 10, Table 2) will be insufficient to reduce the products of oxidative degradation. In the course of the research, the following was established: as the duration of storage of emulsions increases, the PP content decreases by 10-15%> from the initial month of storage, then the rate of decline slows down and does not increase by more than 20-25% even by the end of the 24-month storage period . Losses during storage during stabilization of PP in the presence of DHQ did not exceed 4.0% (see Table 1).

The proposed composition allows to obtain an effective biologically active emulsion of PP according to the simplified technology in the absence of side effects. This tool is characterized by ease of manufacture, increased stability, convenient and safe to use.

These examples indicate the undoubted advantages of the proposed emulsion using DHQ. The obtained experimental data showed that the antioxidant effect of DHQ is a necessary condition for maintaining a stable PP form in the emulsion.

Claims (2)

An emulsion preparation of polyprenols, including polyprenols of woody Siberian fir (Abies sibirica), a nonionic surfactant and water, characterized in that, arabinogalactan is used as a nonionic surfactant, and dihydroquercetin is added to the emulsion with the following content of components, weight.%: Siberian fir polyprenols 1-5.0 Arabinogalactan from 2 to 5 Dihydroquercetin 0.02 to 0.2 Rest Water up to 100%

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