RU2344167C2 - Hop extract - Google Patents

Abstract

FIELD: chemistry, alcohol industry.

SUBSTANCE: invention refers to functional food industry. Hop extract is produced trough vibration of milled raw materials in extractant medium. For extraction oil is used in high-gradient mass exchange in raw materials and extractant ratio 1:10-1:20. Produced oily hop extract contains bitter acids at least 0.1 %, xanthohumole at least 0.15 %, and prenyl flavones at least 0.06 %. Hop extract is characterised with improved biological activity.

EFFECT: activation of innate immunity receptors and linked adaptation mechanism.

8 cl, 13 dwg, 4 ex

Description

The invention relates to the food industry, namely to the production of functional foods based on plant materials, and can find application in dietetics, preventive, adaptive, clinical medicine and cosmetology.

This product, in particular, can be used as a tool for the prevention and treatment of pathological conditions associated with impaired function of innate immunity receptors.

It is known that genetically programmed image-recognizing receptors are located on the surface of human cells that coordinate the mechanisms of an innate and adaptive immune response.

Toll-like receptors (hereinafter TLRs) recognize molecular structures characteristic of microorganisms. Biopathogens activate TLR and chemokine synthesis by epithelial cells, followed by migration to the focus of immune response effectors. The activation of the TLR of these cells causes the secretion of cytokines and the development of an immune response to biopathogens.

At the same time, innate immunity reactions develop rapidly - within one to two hours, while adaptive reactions require more than two days.

With a decrease in TLR activity, the microbial landscape (biocenosis) is disturbed, the number of opportunistic microorganisms increases, which causes an atypical course of inflammatory processes. The interaction of endotoxin with TLR leads to activation of innate immunity reactions.

One of the signal receptors of the innate immune system TLR4 plays an important role in the development of the innate immune response processes, suppresses the reaction of chronic inflammation. It acts like a inhibitory mechanism: in the absence of a TLR4 receptor (released brake), inflammatory processes and mortality from an infectious pathology increase. Such conclusions were made after experiments on genetically modified mice in the cells of which TLR4 is absent [1].

Endogenous activators of TLR4 innate immunity are heat shock proteins and uric acid, as well as products of degradation of hyaluronic acid, products of necrosis and apoptosis.

However, excessive TLR activity can lead to allergic, chronic inflammatory or autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus.

Violations of the regulatory function of TLR can cause pathologies of various organs and systems. It has been established that impaired function of innate immunity receptors plays an important role in the pathogenesis of diseases of the gastrointestinal tract. Their activation is necessary for the implementation of mechanisms of wound healing and tissue regeneration. TLRs regulate the sensitivity of adipose and muscle tissue to insulin.

It follows from the foregoing that in order to increase the effectiveness of the prophylaxis and treatment of infectious diseases, chronic inflammatory processes, allergic and autoimmune diseases, the development and production of drugs that block or activate TLR is an urgent task.

The claimed technical solution allows to obtain a product, namely an oil extract of hops with a high content of target compounds.

The use of such a product increases the effectiveness of the prevention and treatment of pathological conditions associated with impaired function of the innate immunity receptors.

The extracts are concentrated extracts from medicinal plant materials.

Known oil extract of plant materials obtained by heating the crushed raw materials in an oil medium at a temperature of up to 80-90 ° C and further insisting of the resulting mass [2].

This extract has low biological activity, since the heating of plant materials leads to a decrease in the activity of the target components.

Also known is a plant extract obtained by extraction in the cavitation mode by means of ultrasonic treatment on a homogeneous mixture of a liquid extractant with a crushed plant substrate [3].

This extract is also not biologically active enough, since cavitation treatment of the mixture leads to the development of uncontrolled oxidation of both the extractant and the extractable target compounds, which leads to a significant destruction of biologically active substances.

Known extract based on plant materials obtained by processing these materials with high-voltage pulsed discharges in the gap between the electrodes [4].

The disadvantage of this product is that the induction of high-voltage pulsed electric discharges of free-radical oxidation of the solvent and components of plant materials leads to the loss of the target properties of its components.

Also known is the extract of plant materials obtained by pulsed evacuation [5].

This extract has a sufficient content of the target compounds, however, this method of extraction is applicable to obtain aqueous extracts, but unsuitable for obtaining oil extracts of plant materials.

From the analysis of the prior art it is seen that the existing methods for producing extracts from plant materials have certain disadvantages and do not allow to obtain a product with a high content of target compounds.

Hop raw materials are included in the European and British Pharmacopoeia, and also included in the I-III edition of the domestic Pharmacopoeia. Medicines from hop cones have versatile properties - soothing, analgesic, anti-inflammatory. The main substances that determine the biological activity of hop cones are bitterness and phenolic compounds, as well as essential oil.

The closest in technical essence to the claimed solution is a hop extract obtained by continuous extraction of crushed hops with ethyl alcohol [6].

The disadvantages of this solution are the low concentration of hop substances in the resulting extract, as well as the fact that alcohol extracts are not suitable for prolonged use in dietary and therapeutic nutrition due to the possibility of alcohol dependence.

The technical result of the present invention is to create a product with a high content of target substances of increased biological activity, capable of effectively regulating TLR and suitable for long-term use.

To achieve the technical result in a hop extract obtained by pulsating processing of raw materials in an extractant medium, according to the invention, the extraction is carried out with oil in a high gradient mass transfer field with a ratio between the feed and extractant of 1: 10-1: 20, the resulting hop oil containing bitter acids not less than 0.1%, xanthohumol not less than 0.15% and prenylflavones not less than 0.06%, suitable for activation of innate immunity receptors and the mechanisms of adaptation of the organism linked to them. In this case, a high-gradient field is created by a rotary-pulsating apparatus.

The presence of distinctive features, namely, oil extraction in a highly gradient mass transfer field with a ratio between raw materials and extractant of 1: 10-1: 20, obtaining an hop oil extract with a bitter acid content of at least 0.1%, xanthohumol at least 0.15%, prenylflavones of at least 0.06%, suitable for activation of innate immunity receptors and mechanisms of adaptation of the organism linked to them, testifies to the conformity of the claimed technical solution to the patentability condition “novelty”.

In the claimed technical solution to obtain a highly effective extract from plant materials, a method based on pulsation technologies was used.

Under the influence of pulsation, the extraction mass (liquid-solid) enters into reciprocating motion, which ensures uniform distribution of heterogeneous components in the volume of the extractant and intensive mass transfer processes. Due to this, the concentration of components in the entire volume of the extractant is equalized. The solid phase (raw materials) continuously fluctuates in the extractant, which leads to an increase in the efficiency of extraction processes.

A method of pulsed extraction of plant materials is known from the patent literature, where specific examples illustrate the preparation of aqueous or alcoholic extracts with an increased yield of dry soluble substances in them [7].

The experiments showed that only a combination in one technical solution of such components as the use of hop cones as a plant raw material, vegetable oil as an extractant, and a high-energy pulsed action method as an extraction method allowed us to obtain a unique product with a maximum content of target substances ( xanthohumol, isoxanthohumol, 8-prenylnaringenin) and their biological activity. This product allows you to activate innate immunity receptors and the mechanisms of adaptation of the body associated with them.

The mass ratio between hops and oil was selected in the range of 1: 10-1: 20.

The pulsed-periodic nature of the processing of the oil medium during pulsed extraction allowed not only to increase the yield of extractable substances, but also to prevent their free radical oxidation with loss of biological activity.

The high-energy short-term effect on the oil medium containing crushed vegetable raw materials allowed to significantly enhance the extraction of the target components and at the same time avoid their irreversible thermal destruction.

The study of oil extracts obtained by means of pulse-periodic effects showed that the content of extractives (xanthohumol, isoxanthohumol, 8-prenylnaringenin) in them is much higher than with other extraction methods (electrohydraulic shock, ultrasonic extraction,

light-hydraulic shock, boiling and infusion). Accordingly, their biological activity is much higher.

The product we obtained as a result of pulsed oil extraction of hops contains bitter acids of at least 0.1%, xanthohumol at least 0.15%, and prenylflavones (6-prenylnaringenin, 8-prenylnaringenin and isoxanthohumol) at least 0.06%.

The content of xanthohumol and prenylated flavonoids (isoxanthohumol, 8-prenylnaringenin) was determined by reverse phase high performance liquid chromatography (HPLC) with photometric detection. Xantohumol standards (Alexis USA San Diego, USA), isoxanthumol (Alexis USA San Diego, USA), and 8-prenylnaringenin (Alexis USA San Diego, USA) were used.

To prepare working standard samples with a concentration of 0.1 mg / ml, 5 mg of xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin were placed in 50 ml volumetric flasks, bringing the volume to the mark with methanol. 25 g of hop oil extract (hereinafter MEX) was placed in a separatory funnel with a capacity of 100 cm ³ , extracted three times with 10 ml of ethyl acetate. The extract was combined and filtered.

NUCLEOSIL PR C18 column, 5 μm, 250 × 4.6 mm (Macherey Nagel), mobile phase: acetonitrile-methanol-1% formic acid (35:32:33), space velocity: 1.0 ml / min, column temperature : 25 ° C, detection: UV, 370 nm, the volume of the injected sample 10 μl.

The concentration of flavonoids was calculated according to the calibration graph or by the formula: X% = C × S1 × 30 × 100% / S2 × M, where C is the concentration of the standard solution, mg / ml; S1 is the peak area in the analyzed sample; S2 is the peak area in the standard sample; M - weight of the sample, mg.

The fat-soluble hop fraction containing xanthohumol and 8-prenylnaringenin, as well as naringin and naringenin, inhibits the secretion of pro-inflammatory cytokines that occurs when lipopolysaccharide (LPS) activates innate immunity receptors (TLRs). Activation of TLRs is accompanied by sensitization of their signal molecules linked to them, for example, peroxisome proliferative activated receptor (PPAR). The anti-inflammatory, antidiabetic and normalizing lipid metabolism effect of the isohumulones that are part of the hop extract is associated with the activation of PPAR.

Considering the high protective effect of fat-soluble hop fractions in various bacterial and viral infections, we conducted studies to study the protective effect of the obtained hop oil extract in experimental endotoxic shock and influenza infection and revealed the connection between the protective effect and activation of innate immunity receptors.

The immunogenicity of antiviral vaccines is usually weak. Activation of innate immunity receptors can be used to obtain the adjuvant effect of vaccination. The innate immunity receptors TLR2 and TLR4 are involved in the recognition of viral antigenic determinants.

It was found that MEX activates TLR and causes sensitization of receptors linked to TLR (to AFP, IL-1, IL-2, GMCSF, insulin). Therefore, it can be a promising tool for the prevention and treatment of pathological conditions associated with functional insufficiency of cell receptors linked to TLR: diabetes mellitus, metabolic syndrome, obesity, chronic infectious diseases, dishormonal and menopausal disorders, mastopathy, inflammatory lesions of the gastrointestinal tract .

MEC can be used for infectious diseases and septic conditions, especially during the recovery period, as well as an adjuvant of vaccines.

The effectiveness of intradermal use of bacterial toxins as adjuvants of antiviral vaccines has been experimentally established. This served as the basis for research on the adjuvant properties of hop oil extract and its combination with radio-modified LPS with the use of influenza vaccine.

Experiments have shown that the protective effect of MEX obtained using high-intensity rotor-pulsation effects is much more pronounced than MEX obtained by ultrasonic extraction. The severity of the protective effect also depends on the route of administration of MEX and reaches maximum values with inhalation in comparison with oral administration of MEX.

From the foregoing, it follows that the technical result of the invention is achieved by a new set of essential features, both newly introduced and known, therefore, the claimed method meets the condition of patentability "inventive step".

The properties of the obtained hop oil extract are illustrated by experimental data, where Fig. 1 is a diagram of the effect of LPS and modified endotoxin (LPSm) on ex vivo lymphocyte migration; figure 2 presents a diagram of the effect of modified LPS on the activation of neutrophils and monocytes caused by unmodified LPS; figure 3 presents a table showing the effect of the duration of use of hop oil extract on the survival of white outbred and linear mice (n = 20) C3H / HeJ in experimental endotoxic shock caused by the administration of LD ₅₀ prodigiosan; figure 4 presents a table showing the effect of the use of hop oil extract in combination with radio-modified LPS on the survival of white outbred mice (n = 20) in experimental endotoxic shock; figure 5 presents a diagram of changes in the migration of leukocytes in the blood of mice ex vivo in response to the introduction of alpha-fetoprotein (AFP); figure 6 presents a diagram of changes in the migration of leukocytes in the blood of mice ex vivo in response to the introduction of interleukin 1 (IL-1); 7 is a graph of changes in leukocyte migration in the blood of mice ex vivo in response to the introduction of interleukin 2 (IL-2); on Fig presents a diagram of changes in the migration of leukocytes in the blood of mice ex vivo in response to the introduction of granulocyte macrophage colony stimulating factor (GMCSF); figure 9 presents a diagram of changes in the migration of leukocytes in the blood of ex vivo mice in response to insulin administration; figure 10 presents a table with indicators of cellular immunity at certain intervals after vaccination; figure 11 shows changes in humoral immunity with the combined administration of the vaccine Waxigripe with oral administration of an oil extract of hops; on Fig presents a table with the content of the target substances in the oil extracts of hops, which were obtained in various ways; on Fig presents a table with data on the protective effect of MEX against lethal influenza H3N2 infection in mice.

Get hop oil extract using a rotary pulsation apparatus (RPA).

In the technological scheme RPA is installed below the bottom of the extractor. Pre-wash the hop cones in running water. Washed raw materials are loaded onto the false bottom of the extractor and poured with vegetable oil. Oil enters the RPA through the fittings, and hop cones through the screw.

During RPA operation, mechanical grinding of particles occurs, intense turbulization and pulsation of the mixture occur.

The oil, which is supplied under pressure or by gravity through the inlet pipe into the rotor cavity, passes through the rotor channels, stator channels, the working chamber and leaves the apparatus through the outlet pipe. When the rotor rotates, its channels periodically coincide with the stator channels. Coming out of the stator channel, the oil is collected in the working chamber and advances to the outlet pipe. During the period when the rotor channels are blocked by the stator wall, the pressure in the rotor cavity increases, and when the rotor channel is combined with the stator channel, the pressure is released in a short period of time and, as a result, a pressure pulse propagates into the stator channel.

The oil speed in the stator channel is a variable. When an overpressure pulse propagates in the stator channel, a short-term impulse of reduced ("negative") pressure arises after it, since the combination of the rotor and stator channels is completed, and oil is supplied to the stator channel only due to the transit flow from the radial clearance between the rotor and stator . The volume of oil entering the stator channel tends to exit the channel, and the inertial forces create tensile stresses in the liquid, which causes cavitation. Cavitation bubbles grow under the influence of a pulse of reduced pressure and collapse or pulsate with increasing pressure in the stator channel. Part of the cavitation bubbles is carried out into the working chamber.

Due to the fact that the oil flow rate in the stator channel is high and pulsating, the flow is turbulent. When the rotor rotates in the gap between the rotor and the stator, large shear stresses occur. The working surfaces of the rotor and stator act on the heterogeneous oil medium due to mechanical contact, creating high shearing and shear forces.

From the RPA, the pulp, that is, a mixture of crushed material and extractant, rises up and through the nozzle enters the extractor with a mixer. The method is based on multiple circulation of raw materials and extractant. The process is repeated until a concentrated extract is obtained. In this case, extraction and grinding occur simultaneously.

Thus, to obtain the claimed composition, hop cones are ground on an electric cutting machine TU-37-53, type 622-1M. The crushed raw materials are sieved through a sieve according to GOST 21.483 with a hole size of 1-2 mm. The extraction is carried out with sunflower oil in a rotary pulsation apparatus (RPA-4) with a ratio between plant material and extractant of 1:10. Before the extraction, the chopped hops are impregnated with 70 ° ethanol with a mass ratio between hops and ethyl alcohol of 2: 1. The extraction time was 4-8 minutes. After extraction is completed, insoluble hop particles are filtered on a nylon filter.

The invention is illustrated by examples.

Example 1

Studies were performed on outbred white male mice (20 animals in a group) and genetically modified mice of the C3H / HeJ line (20 animals in a group), in the cells of which there are no TLR4 innate immunity receptors. Only in mice without genetic modifications of TLR4, LPS at a dose of 20-100 mg / kg activates the secretion of cytokines by lymphocytes (according to RTML) and an “oxygen explosion” in mouse neutrophils and blood monocytes (application drawings Figs. 1, 2).

This suggests that the action of LPS mediates TLR4. The animals were supplemented with hop oil oil, in which bitter acids were 0.15%, xanthohumol - 0.18%, prenylflavones - 0.08%, in a volume of 2-4 ml per day for 4-12 weeks.

Feeding mice with an oil extract of hops (hereinafter referred to as MEX) led to sensitization of TLR4 (according to RTML with LPS). The dose-dependent nature of the protective effect of MEX in endotoxic shock caused by the introduction of 2 LD ₅₀ LPS was established. In the group of genetically modified mice, animal death under the action of LPS at a dose of 2 LD _{50 was} not detected, which indicates the role of TLR receptors in the development of endotoxic shock and the role of TLR in the implementation of the protective effect of MEX. The results of studies to determine the protective effect of the MEC with the introduction of 2 LD ₅₀ LPS are presented in the application materials in the table in figure 3.

Thus, the protective effect of MEX in experimental endotoxic shock was observed only in white mice and was not observed in C3H / HeJ mice that lack TLR4 innate immunity receptors. This indicates the role of TLR in the implementation of the protective effect of MEX.

The combined use of MEX with radio-modified LPS significantly enhanced its protective effect. The results are presented in the table in figure 4.

The composition of the hop oil extract indicated by composition was used to activate immune defense mechanisms for infectious diseases accompanied by impaired TLR functions.

MEC caused cross-sensitization of TLR4-linked receptors to alpha-fetoprotein (diagram in Fig. 5), interleukin 1 (Fig. 6), interleukin 2 (Fig. 7), granulocyte macrophage colony stimulating factor (Fig. 8), insulin ( Fig.9), which was most pronounced 4 weeks after the start of the experiment. This is the molecular mechanism for the implementation of the protective effect of MEX.

Example 2

The studies were performed on outbred white male mice, which were injected with 50 μl of the vaccine Vaksigripp Aventis company intradermally into the ventral surface of the auricle.

Hop oil extract, in which bitter acids amounted to 0.15%, xanthohumol - 0.19%, prenylflavones - 0.09%, was administered orally at a dose of 2 ml per day for 14 days before vaccination and 14 days after it.

Criteria for assessing the effectiveness of the immune response to the prophylactic administration of the vaccine Waxigripe: anti-influenza antibody titer 28 days after vaccination - log2 (M ± 2m), T-cell immunity indicators - RTML with common: concanavalin A, phytohemagglutinin, regulatory (alpha-fetoprotein) and specific vaccine antigens (Waxigrip).

Indicators of cellular immunity were determined 2, 7, 14, 21, and 28 days after vaccination. The research results are presented in the table in figure 10.

An analysis of the research results indicates that the use of hop oil extract increases the effectiveness of the vaccine vaccine Vaksigripp. In this case, activation of cellular immunity of both antigen-specific (RTML with ConA) and antigen-specific (RTML with vaccine) occurs. The maximum adjuvant effect was obtained with the combined administration of a vaccine, hop oil extract and radio-modified LPS.

Changes in humoral immunity with the combined administration of the vaccine Waxigripe with oral administration of the hop oil extract are presented in the table in FIG. 11.

Analysis of the research results suggests that the use of hop oil extract increased the effectiveness of the humoral response to the vaccine.

Example 3

Patient P., 39 years old, has been suffering from chronic persistent hepatitis B, HBsAg +, PCR DNA of hepatitis B + for 5 years.

For 4 weeks I used MEC, in which the bitter acids were 0.11%, xanthohumol - 0.16%, prenylflavones - 0.07%, 30 ml per day orally in combination with traditional therapy. 4 weeks after initiation of therapy, hepatitis B DNA and HBsAg were not detected. Control examination after 3 and 6 months without negative dynamics. An immunogram showed inhibition of leukocyte migration (46-61%) due to ex vivo administration of alpha-fetoprotein (AFP), modified LPS and interleukins 1 (IL-1) and 2 (IL-2), which indicates the sensitization of TLR4 and linked to them regulatory molecules - receptors for AFP, IL-1, IL-2.

Example 4

Patient M., 52 years old, fibrocystic mastopathy. The size of the node according to the ultrasound data was 4 cm. For 4 weeks I received MEC, in which bitter acids were 0.2%, xanthohumol - 0.19%, prenylflavones - 0.07%, orally at a dose of 30 ml per day. At the end of taking the product on an ultrasound, there is no evidence of the presence of a fibrous bond. An immunogram shows inhibition of leukocyte migration (38-64%) to AFP, IL-1, IL-2, granulocyte macrophage-colony stimulating factor (GMCSF).

An increase in the migratory activity of leukocytes in the treatment of MEX reflects an increase in the functional activity of TLR4 and indicates the prospects of its use.

From the above it follows that the claimed product provides a technical result, obtaining it does not cause difficulties, involves the use of developed materials and standard equipment, which indicates the compliance of the claimed technical solution with the patentability condition "industrial applicability".

Hop oil extract, including an effective amount of the target components (isoxanthohumol, xanthohumol, 8-prenylnaringine) can be used in any convenient form. These can be oil-in-water or water-in-oil emulsions, or complexes of an oil extract with silicon carriers, or ointments, or creams, or gels, or oil sprays, or suppositories, or gelatin capsules, or mayonnaises, or sauces, or ice cream.

Information sources

1. Jeyaseelan S., Chu N.W., Young S.K., Freeman M.W., Worthen G.S. - Distinct roles of pattern recognition receptors CD 14 and Toll-like receptor 4 in acute lung iniury. // Infect Immun. 2006. Vol.73, No. 3. P.1754-1763;

2. RF patent No. 2280660, C09B 61/00, 2006;

3. RF patent No. 2279283, A61K 36/00, 2006;

4. RF patent No. 2191520, A23F 3/36, A 61K 35/78, 2002;

5. RF patent No. 2163827, B01D 11/02, 2001;

6. RF patent No. 2062785, C12C 3/08, 1996;

7 RF Patent No. 225751, А61К 35/78, 2005;

8. RF patent No. 2271245, B01F 11/00, B01F 7/28, 2006.

Claims (8)

1. Hop extract obtained by pulsating processing of raw materials in an extractant medium, characterized in that the extraction is carried out with oil in a high gradient mass transfer field with a ratio between raw materials and extractant of 1: 10-1: 20, and the resulting hop oil extract containing not less than bitter acids 0.1%, xanthohumol at least 0.15%, and prenylflavones at least 0.06%, suitable for activation of innate immunity receptors and the mechanisms of adaptation of the organism linked to them. 2. Hop extract according to claim 1, characterized in that the high-gradient field is created by a rotary pulsation apparatus. 3. Hop extract according to claim 1, characterized in that it activates the innate immunity receptors TLR4 and / or TLR2 4. Hop extract according to claim 1, characterized in that the activation of adaptation mechanisms is associated with the sensitization of regulatory molecules linked to TLR4 — receptors for alpha-fetoprotein. 5. Hop extract according to claim 1, characterized in that the activation of adaptation mechanisms is associated with the sensitization of regulatory molecules linked to TLR4 - receptors for interleukin 1. 6. Hop extract according to claim 1, characterized in that the activation of adaptation mechanisms is associated with the sensitization of regulatory molecules linked to TLR4 - receptors for interleukin 2. 7. Hop extract according to claim 1, characterized in that the activation of adaptation mechanisms is associated with sensitization of regulatory molecules linked to TLR4 — receptors for granulocyte macrophage colony-stimulating factor. 8. Hop extract according to claim 1, characterized in that the activation of adaptation mechanisms is associated with the sensitization of regulatory molecules linked to TLR4 - insulin receptors.

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