EP3713555A1

EP3713555A1 - Quercetin-based composition for treating rhinosinusitis

Info

Publication number: EP3713555A1
Application number: EP18814794.6A
Authority: EP
Inventors: Ainura Chodoeva
Original assignee: Aphabio Therapeutics
Current assignee: Aphabio Therapeutics
Priority date: 2017-11-23
Filing date: 2018-11-22
Publication date: 2020-09-30
Also published as: FR3073737B1; CA3083179A1; CN111615384A; US20210000787A1; EA202090998A1; WO2019101867A1; FR3073737A1; US11351145B2; JP2021504325A

Abstract

The present invention concerns compositions for rinsing the nasal cavity and the sinus cavities for curative or prophylactic treatments of acute and/or chronic rhinitis, and acute and chronic sinusitis, comprising: sodium chloride and/or potassium chloride; quercetin, and at least one active agent of the polyphenolic group chosen from the compounds: quercetin-4'-glucoside, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone, protocatechuic acid; and wherein the quercetin is in the anhydrous form with an approximate molecular mass of 302 g/mol, or in the form of quercetin hydrate with an approximate molecular mass of 320 g/mol, or in the form of quercetin dihydrate with an approximate molecular mass of 338 g/mol. The present invention also relates to method for preparing these compositions and to the method for treating acute and chronic rhinosinusitis using these compositions.

Description

COMPOSITION BASED ON QUERCETIN FOR

TREATMENT OF RHINOSINUSITIS

The present invention relates to a particular composition and its use in the treatment of rhinosinusitis. The invention also relates to a process for obtaining such a composition.

Rhinosinusitis (RS) is an inflammatory disease of the nasal cavity and paranasal sinus cavities, characterized by obstruction or congestion and runny nose, pain and pressure in the face, reduction or loss of Smell and / or mucopurulent flow mainly from the middle meatus, edema and obstruction of the mucosa of the middle meatus area.

Rhinosinusitis is classified as follows: acute RS (RSA) with a duration of illness <12 weeks, chronic RS (CSR) with duration> 12 weeks, with polyp (RSCwNP) or without nasal polyp (RSCsNP).

In the context of this invention, rhinosinusitis terminology includes all forms of the disease, including acute and chronic rhinitis, acute and chronic sinusitis, with or without nasal polyp.

As a nosological unit, rhinosinusitis does not exist in the list of the International Classification of Diseases, published by the World Health Organization. In contrast, acute (J30) and chronic (J31), acute (J01) and chronic (J32) rhinitis were classified separately (CDI).

WHO.http: //apps.who.int/classifications/icdl0/browse/2016/en#/J30-J39) · Despite this, the European Academy of Allergology and Clinical Immunology, the European Society of Rhinology and The American Academy of Otolaryngology - Head and Neck Surgery Foundation, by publishing the guidelines for the management of rhinosinusitis, have agreed to use the terminology 'rhinosinusitis'. However, in most cases, rhinitis and sinusitis are concomitant diseases, always coexisting. "The adoption of the more common terminology 'rhinosinusitis', instead of 'sinusitis', indirectly supports the idea that foreign material introduced by air, or probably the nasopharynx, acts primarily on the nasal mucosa, then, secondly, acts directly or indirectly on the mucous membranes of the sinuses "(European Position Paper on Rhinosinusitis and Nasal Polyps 2012, Rhinology supplement 23: 1-298, 2012).

Rhinosinusitis is one of the most prevalent diseases in the world, with an average prevalence of more than 10% of the world's population.

In the United States, rhinosinusitis affects 1 in 8 people, with an annual health expenditure of more than $ 11 billion (Rosenfeld, RM et al., Clinical Practice Guideline (Update): Adult Sinusitis, Otolaryngology-Head and Neck Surgery 152 (2S), 2015, ppl-39). The total cost of treating a patient with chronic rhinosinusitis is $ 2609 per year in the United States; the total cost of treating a patient with chronic rhinosinusitis in a university hospital is $ 1861 per year in Europe. Rhinosinusitis is one of the most expensive diseases for employers in the United States. Among chronic diseases, rhinosinusitis is the disease that causes the most costs from treatment and home care. The bulk of indirect expenses include absenteeism (sick leave) and presenteeism (reduced productivity at work). The results of studies carried out by the National Health Surveillance Agency (NHIS, USA) between 1997 and 2006, comprising 315,000 people, showed that American citizens are on sick leave on average 5.7 days per year, at rhinosinusitis (Rhinosinusitis and Nasal Polyps 2012, Rhinology supplement 23: 1-298, 2012).

Currently, there is no specific treatment for rhinosinusitis. Some antibiotics have an indication for acute rhinosinusitis of bacterial origin; other groups of drugs, such as corticosteroids, antihistamines, alpha-adrenergic decongestants, have been recommended, but none of these drugs have been approved by the Food and Drug Administration (Guidance for Industry Sinusitis). Nonantimicrobial Drugs for Treatment US DHS FDA Center for Drug Evaluation and Research November 2006).

The treatment of rhinosinusitis is guided by the guidelines governed by international associations such as: the European Academy of Allergology and Clinical Immunology and the European Society of Rhinology. According to the protocol established by these associations (Rhinosinusitis and Nasal Polyps 2012), the different forms of rhinosinusitis are treated in the following way:

- For acute rhinosinusitis (duration <12 weeks): the first-line treatment is symptomatic therapy: analgesics, saline irrigation, antihistamines and decongestants may be offered, despite the lack of evidence of their effectiveness. If there has been no improvement in the patient's condition for 7 to 14 days, intra-nasal corticosteroids are recommended. In the case of a microbial complication of rhinosinusitis, first-line antibiotics (amoxicillin alone or with clavulanic acid) are prescribed. In parallel, radiological imaging should be performed to confirm the microbial cause of the disease and to verify the absence of a complication. If there is no improvement after the use of an antibiotic, it is recommended to change it (eg macrolides). If treatment fails, intra-nasal corticosteroid maintenance and / or surgery may be recommended.

- For chronic rhinosinusitis (duration> 12 weeks): despite the presence or absence of nasal polyp, the treatment protocol is as follows: after endoscopic examination and CT imaging of nasal cavities and sinuses, corticosteroids intravenously nasal are prescribed. If there has been no improvement in the condition of the patient for 3 months, long-term treatment with antibiotics may be added. If there is still no improvement, surgery is recommended.

The efficacy of intranasal corticosteroids is very limited: they are effective in one of 14 patients treated (Hayward, G et al., Intranasal Corticosteroids in Management of Acute Sinusitis:

A Systematic Review and Meta-Analysis. Ann Fam Med \> l. 10: 2012, pp241-249).

Evaluating the moderate effect versus side effects is especially recommended for treating physicians before prescribing antibiotics. The latest US guidelines recommend "monitoring on hold" before prescribing antibiotics, even though the bacterial origin of rhinosinusitis is confirmed, because the effectiveness of antibiotics in rhinosinusitis is very limited: they are effective in one person of 20 treated (Rosenfeld, RM et al., Clinical Practice Guideline (Update): Adult Sinusitis, Otolaryngology-Head and Neck Surgery Vol 152 (2S), 2015, ppl-39).

Nasal decongestants may be recommended for palliative treatment, but continued administration (greater than 3 - 5 days) may cause rhinitis drug.

Isotonic or hypertonic saline solutions for nasal irrigation are widely used in the practice of daily hygiene. In general, saline is the most used. The volume of nasal solution varies from 5 mL to 500 mL. The effect of isotonic saline is explained by the ability of solutions to remove irritating foreign materials and mediators of inflammation of the nasal cavity. Currently, several formulations of nasal solutions, including different buffer solutions, are being developed. One of the formulations for nasal lavage was developed by Ted Keller (US2013 / 0156871, Keller Ted, June 20, 2013), where he discovers the nasal solution comprising sodium chloride and a buffer consisting of sodium ascorbate and sodium bicarbonate. sodium. After dissolution in water, this composition can be used to clean the cavities of the nose and sinuses. However, like the other existing solutions, such a composition is not satisfactory and does not make it possible to treat rhinosinusitis.

The present invention aims to overcome the problems of existing solutions and provide the compositions for cleaning the nose and sinuses cavity to effectively prevent and treat rhinosinusitis acute and chronic. For this purpose the invention relates to compositions comprising salt; and active agents of the polyphenolic group chosen from the compounds: quercetin, quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, protocatechic acid, which may be contain impurities derived from quercetin, comprising quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and quercetin triglucosides, quercetin dimers, in an amount of less than 5% by weight of the total amount of active agents.

According to the present invention, it is necessary to combine quercetin with quercetin derivatives such as quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) - benzofuranone, protocatechic acid. This combination provides a synergistic effect that increases the efficacy of rhinosinusitis treatment. The invention is therefore also directed to these compositions for their use in the prevention and treatment of rhinosinusitis.

Finally, another object of the invention is to provide a process for the preparation of these compositions.

The invention is now described in detail.

DEFINITIONS

By "active agent" within the meaning of the invention is meant the compound or the ingredient producing the desired therapeutic effect.

By "impurity" within the meaning of the invention is meant the compound or the ingredient without therapeutic effect, concomitant with the active agents, present in the composition of a drug in a minor amount.

For the purposes of the invention, the term "effective therapeutic quantity" is intended to mean the amount of active agent that can produce the desired therapeutic effect in the subjects treated.

By "patient" or "subject" within the meaning of the invention is meant a mammal, comprising the animal or the human, to whom the present invention may be applied.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a composition for rinsing the nasal cavity and sinus cavities for the prevention or the curative or prophylactic treatment of acute and chronic rhinitis, acute and chronic sinusitis, comprising:

sodium chloride and / or potassium chloride;

- quercetin, and

at least one active agent of the polyphenolic group chosen from the compounds: quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, protocatechic acid; and

wherein quercetin is in anhydrous form with an approximate molecular weight of 302 g / mol, or in the form of quercetin hydrate with a mass approximate molecular weight of 320 g / mol, or in the form of quercetin dihydrate with an approximate molecular weight of 338 g / mol.

The invention relates to nasal compositions containing active ingredients with proven efficacy for the treatment of rhinosinusitis.

The molecules contained in the composition according to the invention can be extracted from the onion peel. The efficacy of the crude extract and isolated compounds has been studied in rhinosinusite models developed in animals. The results of this invention are described in the examples. The results of this research have established the effectiveness of onion peel extract and compounds isolated from this extract: quercetin, quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2 , 4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid for rhinosinusitis, to develop formulations containing these agents, and to develop methods of treatment.

Depending on the extraction and preparation conditions, quercetin can be used in anhydrous form, with an approximate molecular weight of 302 g / mol, or in the form of quercetin hydrate with an approximate molecular weight of 320 g / mol, or form of quercetin dihydrate, with an approximate molecular weight of 338 g / mol.

Several variants of compositions according to the invention are conceivable.

Nasal composition, including salt and quercetin, quercetin-4'-glucoside 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and acid

According to one embodiment, the nasal formulation comprises salt and a plant extract containing flavonoids: quercetin, quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 ( 2H) -benzofuranone and protocatechic acid. The salt may be selected from sodium chloride or potassium chloride, or both, in an amount sufficient to produce the hypotonic, isotonic or hypertonic solution. The amount of salt may be from 83 to 99.77% by weight of the dry composition. The amount of quercetin in the dry composition may comprise 0.23 to 3% by weight. The amount of quercetin-4'-glucoside in the dry composition may be between 0.000001 - 3% by weight. The amount of 2- (3,4- dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in the dry composition may be between 0.000001 - 3% by weight. The amount of protocatechic acid in the dry composition may be between 0.000001 - 3% by weight.

According to one embodiment, the active agent comprising quercetin, quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and the acid protocatechic can be prepared by a process of extracting plants containing flavonoids, for example onion peel. The method of preparing the extract containing active agents may comprise the following steps:

- mix the ground onion peel with cold water in a proportion of 0.1-2 g / 50 mL (m / v);

bring the mixture to a boil, and simmer for 30 minutes at boiling point;

- Cool the mixture, filter and lyophilize.

This method makes it possible to obtain a crude extract composed of the active agents: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside, protocatechic acid . The crude extract may also contain other impurities derived from quercetin, for example, quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and triglucosides of quercetin, quercetin dimers, in an amount not exceeding 5% of the total mass of active agents.

Another extraction method consists in using organic solvents, preferably methanol or ethanol diluted with water, in a proportion of between 50/50 and 90/10 (v / v). According to this method, the defined amount of onion peel powder is mixed with the organic solvent in a proportion of 1-10 g / 30 mL (w / v), and stirred for 24 hours at room temperature. The solution obtained is filtered and then evaporated to dryness. The crude extract obtained can be separated by the method of high performance liquid chromatography.

The separation conditions can be as follows:

50 mg to 200 mg of crude extract are injected into a Phenomenex C18 column (21.2 mm x 150 mm, 5 μm, 100 A) of the HPLC apparatus. The following solvents may be used: Solvent A: 0.5% H2O / TFA (1 L / 5 mL); Solvent B: ACN. The flow rate can be: 15 mL / min. Qualitative and quantitative analyzes of the compositions can be performed by UPLC-DAD-MS techniques (Agilent Technologies, USA). The elution conditions may be as follows: 1 μl of solution is injected into the Agilent C18 column (2.1 mm, 1.8 μm) of UPLC at a temperature of 25 ° C. The solvents used can be: Solvent A: H ₂ 0 / 0.1% HCOH 0.1 and Solvent B: ACN / 0.1% HCOOH. The flow rate can be: 0.4 mL / min. Isolated compounds can be detected first by the diode array detector and then by the mass spectrometry analyzer. Mass Spectrometry analyzes can be performed in negative or positive mode. The separation and analysis techniques by the UPLC-DAD-MS method can be the same for all compositions.

According to one embodiment, the composition may be prepared by mixing the commercially available compounds: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'- glucoside, protocatechic acid and salt selected from sodium chloride or potassium chloride.

The dry composition can be prepared by one of the following methods: Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside, protocatechic acid with salt in water water heated to 80 - 100 ° C. Then cooling the solution to room temperature, followed by freezing and lyophilization. b). Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside, protocatechic acid in heated water at 80 - 100 ° C. Then cooling the solution to room temperature, adding and dissolving the salt, followed by freezing and lyophilization. c). Quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside and protocatechic acid are dissolved in the organic solvent, preferably in ethanol or methanol diluted with water. The proportion of organic solvent / water can vary between 50/50 and 90/10 (v / v). Salt is dissolved in water. Then both solutions are mixed at room temperature, then frozen and lyophilized. d). The quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside and protocatechic acid powder is mixed with sodium or potassium chloride.

The composition can be sterilized or not, can be supplied in dry or liquid form, distributed in single-dose or multi-dose containers.

The composition may comprise one or more biologically active ingredients consistent with the therapy and additives selected from pharmaceutical carriers, stabilizers, surfactants, preservatives, and essential oils. The biologically active ingredients may be selected from antibiotics, corticosteroids, and / or copper, manganese and sulfur salts. Essential oils can be chosen from eucalyptus oil, rosemary oil, tea tree oil or menthol.

According to one embodiment, the dry composition can subsequently be mixed with water. The solution or suspension obtained comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023 - 0.03% by mass of quercetin, 0.00000001 - 0.03% by mass of quercetin 4'-glucoside, 0.00000001 - 0.03% by weight of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and 0.00000001 - 0.03% mass of protocatechic acid and other impurities derived from quercetin up to 0.5% by total weight of the active agents.

In another embodiment, the dry composition may be mixed with the solution containing the bicarbonate salt. The bicarbonate salt may be selected from sodium bicarbonate or potassium bicarbonate or both. The concentration of bicarbonate salt can vary up to 0.5%, preferably between 0.1% - 0.5% by weight, more preferably 0.25 - 0.5% by weight. The temperature of the water or bicarbonate solution can be between 30 ° C and 38 ° C. At this temperature, some compounds, for example quercetin, may remain in suspension, others can be completely dissolved. According to a particular variant, the water may be boiled and then cooled to 37 ° C. before mixing.

The water used for the preparation of the liquid composition can be distilled, deionized, purified and sterilized.

Although the use of preservatives and other excipients is not excluded, sterile preparations without preservatives can be made. This makes it possible to preserve the compositions under sterile conditions and to limit the undesirable effects due to the presence of excipients. The blow-fill-seal technique, using single-dose plastic containers or pre-filled syringes under aseptic conditions, may be preferred.

In one embodiment, the composition may be presented as follows:

Composition consisting of two parts:

Part A: containing the dry powder, including sodium chloride or potassium chloride, quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin 4'-glucoside and protocatechic acid.

- Part B: containing 20 ml of water or solution of sodium bicarbonate or potassium bicarbonate.

2. Nasal composition, including salt and quercetin, quercetin-4'-glucoside and protocatechic acid.

According to one embodiment, the nasal formulation comprises salt and a plant extract containing flavonoids: quercetin, quercetin-4'-glucoside and protocatechic acid. The salt may be selected from sodium chloride or potassium chloride, or both, in an amount sufficient to produce the hypotonic, isotonic or hypertonic solution. The amount of salt may be from 86 to 99.77% by weight of the dry composition. The amount of quercetin in the dry composition may comprise 0.23 to 3% by weight. The amount of quercetin-4'-glucoside in the dry composition may be between 0.000001 - 3% by weight. The amount of protocatechic acid in the dry composition may be between 0.000001 - 3% by weight. According to one embodiment, the active agent comprising quercetin, quercetin-4'-glucoside and protocatechic acid may be prepared by a process for extracting plants containing flavonoids, for example onion peel. The method of preparing the extract containing active agents may comprise the following steps:

- mix the ground onion peel with cold water in the proportion 0.1-2 g / 50 mL (m / v);

bring the mixture to a boil, and simmer for 30 minutes at boiling point;

- Cool the mixture, filter and lyophilize.

This method makes it possible to obtain a crude extract composed of the active agents: quercetin, quercetin-4'-glucoside, protocatechic acid. The crude extract may also contain other impurities derived from quercetin, for example, quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and triglucosides of quercetin, quercetin dimers, in an amount not exceeding 5% of the total mass of active agents.

The separation conditions can be as follows:

50 mg to 200 mg of crude extract are injected into a Phenomenex C18 column (21.2 mm x 150 mm, 5 μm, 100 A) of the HPLC apparatus. The following solvents may be used: Solvent A: 0.5% H2O / TFA (1 L / 5 mL); Solvent B: ACN. The flow rate can be: 15 mL / min.

Qualitative and quantitative analyzes of the compositions can be performed by UPLC-DAD-MS techniques (Agilent Technologies, USA). The elution conditions may be as follows: 1 μl of solution is injected into the Agilent C18 column (2.1 mm, 1.8 μm) of UPLC at a temperature of 25 ° C. The solvents used can be: Solvent A: H ₂ 0 / 0.1% HCOOH and Solvent B: ACN / 0.1% HCOOH. The flow rate can be: 0.4 mL / min. Isolated compounds can be detected first by the diode array detector and then by the mass spectrometry analyzer. Mass Spectrometry analyzes can be performed in negative or positive mode. The separation and analysis techniques by the UPLC-DAD-MS method can be the same for all compositions.

According to one embodiment, the composition may be prepared by mixing the commercially available compounds: quercetin, quercetin-4'-glucoside, protocatechic acid and salt selected from sodium chloride or potassium chloride.

The dry composition can be prepared by one of the following methods: Dissolution of quercetin, quercetin-4'-glucoside, protocatechic acid with salt in water heated to 80-100 ° C. Then cooling the solution to room temperature, followed by freezing and lyophilization. b). Dissolution of quercetin, quercetin-4'-glucoside, protocatechic acid in water heated to 80-100 ° C. Then cooling the solution to room temperature, adding and dissolving the salt, followed by freezing and lyophilization. c). Quercetin, quercetin-4'-glucoside and protocatechic acid are dissolved in the organic solvent, preferably in ethanol or methanol diluted with water. The proportion of organic solvent / water can vary between 50/50 and 90/10 (v / v). Salt is dissolved in water. Then both solutions are mixed at room temperature, then frozen and lyophilized. d). The quercetin, quercetin-4'-glucoside and protocatechic acid powder is mixed with sodium chloride or potassium chloride. The composition can be sterilized or not, can be supplied in dry or liquid form, distributed in single-dose or multi-dose containers.

According to one embodiment, the dry composition can subsequently be mixed with water. The solution or suspension obtained comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023 - 0.03% by mass of quercetin, 0.00000001 - 0.03% by mass of quercetin 4'-glucoside and 0.00000001 - 0.03% by weight of protocatechic acid, and other impurities derived from quercetin up to 0.5% by total weight of the active agents.

In another embodiment, the dry composition may be mixed with the solution containing the bicarbonate salt. The bicarbonate salt may be selected from sodium bicarbonate or potassium bicarbonate or both. The concentration of bicarbonate salt can vary up to 0.5%, preferably between 0.1% - 0.5% by weight, more preferably 0.25 - 0.5% by weight. The temperature of the water or bicarbonate solution may be between 30 and 38 ° C. At this temperature, some compounds, for example quercetin, may remain in suspension, others may be completely dissolved. According to a particular variant, the water may be boiled and then cooled to 37 ° C. before mixing.

In one embodiment, the composition may be presented as follows: Composition consisting of two parts:

part A: the dry powder, comprising sodium chloride or potassium chloride, quercetin, quercetin-4'-glucoside and protocatechic acid.

3. Nasal composition, including salt and quercetin, 2- (3,4-dihydroxybenzoyl)

2,4,6-trihydroxy-3-benzofuranone and protocatechic acid.

In one embodiment, the nasal formulation comprises salt, quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, and protocatechic acid. The salt may be selected from sodium chloride or potassium chloride or both, in an amount sufficient to produce the hypotonic, isotonic or hypertonic solution. The amount of salt may comprise from 91 to 99.77% by weight of the dry composition. The amount of quercetin in the dry composition may comprise 0.23 to 3% by weight. The amount of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in the dry composition may comprise between 0.000001 - 3% by weight. The amount of protocatechic acid in the dry composition may comprise between 0.000001 - 3% by weight.

According to one embodiment, the active agent comprising quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid can be prepared by a method extracting plants containing flavonoids, for example onion peel. The method of preparing the extract containing active agents may comprise the following steps:

- mix the ground onion peel with cold water in a proportion of 0.1-2 g / 50 mL (m / v); bring the mixture to a boil, and simmer for 30 minutes at boiling point;

- Cool the mixture, filter and lyophilize.

This method makes it possible to obtain a crude extract composed of the active agents: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside, acid protocatechic. The crude extract may also contain other impurities derived from quercetin, for example, quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and triglucosides of quercetin, quercetin dimers, in an amount not exceeding 5% of the total mass of active agents.

Another method of extraction is to use organic solvents, preferably methanol or ethanol diluted with water in a proportion of between 50/50 and 90/10 (v / v). According to this method, the defined amount of onion peel powder is mixed with the organic solvent in a proportion of 1-10 g / 30 ml (w / v), and stirred for 24 hours at room temperature. The solution obtained is filtered and then evaporated to dryness. The crude extract obtained can be separated by the method of high performance liquid chromatography.

The separation conditions can be as follows:

According to one embodiment, the composition can be prepared by mixing the commercially available compounds: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) - benzofuranone, protocatechic acid and salt selected from sodium chloride or potassium chloride.

The dry composition can be prepared by one of the following methods: Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, protocatechic acid with salt in water heated to 80 - 100 ° C. Then cooling the solution to room temperature, followed by freezing and lyophilization. b). Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, protocatechic acid in water heated to 80-100 ° C. Then cooling the solution to room temperature, adding and dissolving the salt, followed by freezing and lyophilization. c). Quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid are dissolved in the organic solvent, preferably in diluted ethanol or methanol. with water. The proportion of organic solvent / water can vary between 50/50 and 90/10 (v / v). Salt is dissolved in water. Then, both solutions are mixed at room temperature, frozen and lyophilized. d). The quercetin powder of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid is mixed with sodium chloride or potassium chloride.

The composition may comprise one or more biologically active ingredients consistent with the therapy and additives selected from pharmaceutical carriers, stabilizers, surfactants, preservatives, and essential oils. The biologically active ingredients may be selected from antibiotics, corticosteroids, and / or copper salts, manganese and sulfur. Essential oils can be chosen from eucalyptus oil, rosemary oil, tea tree oil or menthol.

According to one embodiment, the dry composition can subsequently be mixed with water. The solution or suspension obtained comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023 - 0.03% by mass of quercetin, 0.00000001 - 0.03% by mass of 2 - (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and 0.00000001 - 0.03% by weight of protocatechic acid.

In another embodiment, the dry composition may be mixed with the solution containing the bicarbonate salt. The bicarbonate salt may be selected from sodium bicarbonate or potassium bicarbonate or both. The concentration of bicarbonate salt can vary up to 0.5%, preferably between 0.1% - 0.5% by weight, more preferably 0.25 - 0.5% by weight. The temperature of the water or bicarbonate solution may be between 30 and 38 ° C. At this temperature, certain compounds, for example quercetin, may remain in suspension; others can be completely dissolved. According to a particular variant, the water may be boiled and then cooled to 37 ° C. before mixing.

Although the use of preservatives and other excipients is not excluded, sterile preparations without preservatives can be manufactured. This makes it possible to preserve the compositions under sterile conditions and to limit the undesirable effects due to the presence of excipients. The blow-fill-seal technique, using single-dose plastic containers or pre-filled syringes under aseptic conditions, may be preferred. In one embodiment, the composition may be presented as follows:

Composition consisting of two parts:

- Part A: dry powder, including sodium chloride or potassium chloride, quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid . - Part B: containing 20 ml of water or solution of sodium bicarbonate or potassium bicarbonate.

4. Nasal composition, including salt and quercetin, quercetin-4'-glucoside and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3-benzofuranone.

According to one embodiment, the nasal formulation comprises salt and quercetin, quercetin-4'-glucoside and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone. The salt may be selected from sodium chloride or potassium chloride, or both, in an amount sufficient to produce the hypotonic, isotonic or hypertonic solution. The amount of salt may be from 91 to 99.99% by weight of the dry composition. The amount of quercetin in the dry composition can comprise between 0.000001 - 3% by weight. The amount of quercetin-4'-glucoside in the dry composition may be between 0.000001 - 3% by weight. The amount of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in the dry composition can be between 0.000001 - 3% by weight.

According to one embodiment, the active agent comprising quercetin, quercetin-4'-glucoside and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone can be prepared by a plant extraction process containing flavonoids, for example, onion peel. The method of preparing the extract containing active agents may comprise the following steps:

bring the mixture to a boil, and simmer for 30 minutes at boiling point;

- Cool the mixture, filter and lyophilize. This method makes it possible to obtain a crude extract composed of the active agents: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside, protocatechic acid . The crude extract may also contain other impurities derived from quercetin, for example, quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and triglycerides of quercetin, quercetin dimers, in an amount not exceeding 5% of the total mass of the active agents.

Another method of extraction is to use organic solvents, preferably methanol or ethanol diluted with water in a proportion between 50/50 and 90/10 (v / v). According to this method, the defined amount of onion peel powder is mixed with the organic solvent in a proportion of 1-10 g / 30 mL (w / v), and stirred for 24 hours at room temperature. The solution obtained is filtered and then evaporated to dryness. The crude extract obtained can be separated by the method of high performance liquid chromatography.

The separation conditions can be as follows:

According to one embodiment, the composition may be prepared by mixing the commercially available compounds: quercetin, quercetin-4'-glucoside and salt selected from sodium chloride or potassium chloride.

The dry composition can be prepared by one of the following methods: at). Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside with salt in water heated to 80 - 100 ° C. Then cooling the solution to room temperature, followed by freezing and lyophilization. b). Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside in water heated to 80 - 100 ° C. Then cooling the solution to room temperature, adding and dissolving the salt, followed by freezing and lyophilization. c). Quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and quercetin-4'-glucoside are dissolved in the organic solvent, preferably in ethanol or methanol diluted with water. The proportion of organic solvent / water can vary between 50/50 and 90/10 (v / v). Salt is dissolved in water. Then both solutions are mixed at room temperature, then frozen and lyophilized. d). The quercetin powder of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin-4'-glucoside is mixed with sodium chloride or potassium chloride.

The composition may comprise one or more biologically active ingredients consistent with the therapy and additives selected from pharmaceutical carriers, stabilizers, surfactants, preservatives, and essential oils. The biologically active ingredients may be selected from antibiotics, corticosteroids, and / or copper, manganese and sulfur salts. Essential oils can be chosen from eucalyptus oil, rosemary oil, tea tree oil or menthol. According to one embodiment, the dry composition can subsequently be mixed with water. The solution or suspension obtained comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.00000001 - 0.03% by mass of quercetin, 0.00000001 - 0.03% by mass of 2 - (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and 0.00000001 - 0.03% by weight of quercetin-4'-glucoside.

In another embodiment, the dry composition may be mixed with the solution containing the bicarbonate salt. The bicarbonate salt may be selected from sodium bicarbonate or potassium bicarbonate or both. The concentration of bicarbonate salt can vary up to 0.5%, preferably between 0.1% - 0.5% by weight, more preferably 0.25 - 0.5% by weight. The temperature of the water or bicarbonate solution can be between

30 and 38 ° C. At this temperature, some compounds, for example quercetin, may remain in suspension, others may be completely dissolved. According to a particular variant, the water may be boiled and then cooled to 37 ° C. before mixing.

In one embodiment, the composition may be presented as follows:

Composition consisting of two parts:

Part A: the dry powder, comprising sodium chloride or potassium chloride, quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and quercetin-4 glucoside.

- Part B: containing 20 ml of water or solution of sodium bicarbonate or potassium bicarbonate. 5. The nasal composition, including salt, quercetin and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone.

According to one embodiment, the nasal formulation comprises salt, quercetin and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone. The salt may be selected from sodium chloride or potassium chloride, or both, in an amount sufficient to produce the hypotonic, isotonic or hypertonic solution. The amount of salt may be from 94 to 99.99% by weight of the dry composition. The amount of quercetin in the dry composition can comprise between 0.000001 - 3% by weight. The amount of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in the dry composition may be between 0.000001 - 3% by weight.

According to one embodiment, the active agent comprising quercetin and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone can be prepared by a plant extraction process. containing flavonoids, for example, onion peel. The method of preparing the extract containing active agents may comprise the following steps:

bring the mixture to a boil, and simmer for 30 minutes at boiling point;

- Cool the mixture, filter and lyophilize.

The separation conditions can be as follows:

According to one embodiment, the composition may be prepared by mixing the commercially available compounds: quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and salt selected from sodium chloride or potassium chloride.

The dry composition can be prepared by one of the following methods: Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone with salt in water heated to 80-100 ° C. Then cooling the solution to room temperature, followed by freezing and lyophilization. b). Dissolution of quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in water heated to 80-100 ° C. Then cooling the solution to room temperature, adding and dissolving the salt, followed by freezing and lyophilization. c). Quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone are dissolved in the organic solvent, preferably in ethanol or methanol diluted with water. . The proportion of organic solvent / water can vary between 50/50 and 90/10 (v / v). Salt is dissolved in water. Then both solutions are mixed at room temperature, then frozen and lyophilized. d). The quercetin powder of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone is mixed with sodium chloride or potassium chloride.

According to one embodiment, the dry composition can subsequently be mixed with water. The solution or suspension obtained comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.00000001 - 0.03% by weight of quercetin and 0.00000001 - 0.03% by weight of 2 - (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone.

In another embodiment, the dry composition may be mixed with the solution containing the bicarbonate salt. The bicarbonate salt may be selected from sodium bicarbonate or potassium bicarbonate or both. The salt concentration of bicarbonate can vary up to 0.5%, preferably between 0.1% - 0.5% by weight, more preferably 0.25 - 0.5% by weight. The temperature of the water or bicarbonate solution may be between 30 and 38 ° C. At this temperature, some compounds, for example quercetin, may remain in suspension, others may be completely dissolved. According to a particular variant, the water may be boiled and then cooled to 37 ° C. before mixing.

Part A: the dry powder, comprising sodium chloride or potassium chloride, quercetin and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone.

The invention is now illustrated by examples and tests demonstrating the effectiveness of the invention in the treatment of rhinosinusitis.

EXAMPLES

Example 1

Extraction of the onion peel.

40g ground onion peel was mixed with 2L cold water; boiled, and allowed to simmer for 30 minutes at boiling temperature. The resulting liquid is cooled, filtered and lyophilized. 5 g of dry extract are obtained. Ethanol extraction.

200 g of onion peel was extracted with 6L of an ethanol / water mixture in proportion 70/30 (v / v) for 24 hours at room temperature with stirring. The solution obtained is filtered and then evaporated to dryness. 25.5 g of dry extract are obtained.

The qualitative and quantitative analyzes of the two extracts were performed by UPLC-DAD-MS techniques (Agilent Technologies, USA). The elution conditions are as follows: 1 μL of solution is injected into the Agilent C18 column (2.1 mm, 1.8 μm) of UPLC at a temperature of 25 ° C. The solvents used are: Solvent A: H ₂ O / 0.1% HCOOH and Solvent B: ACN / 0.1% HCOOH. Flow rate: 0.4 mL / min. The isolated compounds are first detected by the diode array detector and then by the mass spectrometry analyzer. Mass spectrometry analyzes were performed in negative and positive modes.

The spectra of LC-MAS are acquired in "Full Scan" over the entire mass range (m / z) ranging from 100 to 1400. The data set is then collected and processed by the Hystar software version 3.0 .

The results of qualitative and quantitative analyzes of the alcoholic and water extracts obtained from onion peel are presented in Tables 1 and 2.

Table 1. Qualitative and Quantitative Characteristics of Onion Peel Water Extract

Table 2. Qualitative and quantitative characteristics of alcoholic extract of onion peel.

Example 2

Isolation of pure compounds.

The alcoholic crude extract was separated by the HPLC technique.

50 mg to 200 mg of crude extract are injected into a Phenomenex C18 column (21.2 mm x 150 mm, 5 μm, 100 A) of the HPLC apparatus. The solvents used are: Solvent A: H ₂ O / 0.5% TFA (1 L / 5 mL); Solvent B: ACN. The flow rate is: 15 mL / min.

Three major compounds have been isolated:

1. protocatechic acid (purity - 98.6%), 92.5 mg;

2. quercetin (purity - 98.9%) 295 mg;

3. quercetin-4'-glucoside (purity - 96.5%), 143 mg.

Processes for preparing the compositions

1.

40 mg of quercetin and 3 g of NaCl were mixed with 150 mL of boiling water. Then, the solution was cooled, frozen and lyophilized. The powder obtained was dispensed in 197.6 mg in single-dose containers under sterile conditions.

2.

Quercetin 40 mg was mixed with 150 mL of boiling water. Then, the solution was cooled, and 3 g of NaCl was added and dissolved. The resulting solution was frozen and lyophilized. The powder obtained was dispensed in 197.6 mg in single-dose containers under sterile conditions.

3.

50 mg quercetin was dissolved in 50 mL MeOH / H ₂ O (50/50: v / v). 3.75 g of NaCl were dissolved in 150 ml of H ₂ O. The two solutions were mixed at room temperature, then frozen and lyophilized. The resulting powder was dispensed by 200 mg in single-dose containers under sterile conditions.

4.

40 mg quercetin was mixed with 3 g NaCl. The resulting powder was dispensed by 200 mg in single-dose containers under sterile conditions.

Example 3

Preparation of solvents

Three different solvents were prepared:

0.625 g of NaCl was dissolved in 250 ml of distilled water. The resulting solution was sterilized by filtration through a filter with a pore diameter of 0.22 μm and dispensed in single-dose containers under sterile conditions.

2. 0.625 g of NaHCO ₃ was dissolved in 250 ml of distilled water. The resulting solution was sterilized by filtration through a filter with a pore diameter of 0.22 μm and dispensed in single-dose containers under sterile conditions.

3. Distilled water was dispensed in single-dose containers under sterile conditions.

Formulations.

Based on the experimental results described in Examples 1 and 2, the initial amounts of the active ingredients in 20 mL (the volume used for the one-time treatment) of the onion peel water extract were were determined as: onion peel extract - 10 mg / 20 mL;

quercetin - 0.485 mg / 20 mL;

quercetin-4'-glucoside - 0.283 mg / 20 mL; protocatechic acid - 0.722 mg / 20 mL.

Various pharmaceutical compositions have been formulated based on water, NaCl or NaCl + NaHCO ₃ and active agents (onion peel extract, quercetin, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy- 3 (2H) -benzofuranone, quercetin-4'-glucoside or protocatechic acid).

The qualitative and quantitative characteristics of Quercetin used for this experiment are as follows:

quercetin - 98%

2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone - 0.5%

Quercetin-4'-glucoside - 0.1%.

TEST RESULTS DEMONSTRATING EFFECTIVENESS OF THE INVENTION

Treatment of rhinosinusitis.

Materials and methods.

Design of the study.

42 male rabbits were used in this study. The animals were separated into seven groups, each containing six rabbits. The first group served as an untreated control, the 2nd group was treated with a 0.9% NaCl solution, and five experimental groups were used to test the different formulations: onion peel extract, quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, quercetin, quercetin-4'-glucoside and protocatechic acid.

Induction of rhinosinusitis.

Rhinosinusitis was induced by the introduction of 100 μL of 1% lipopolysaccharide (LPS) into each nostril of rabbits for three days.

Compounds.

The compounds have been used in the following concentrations: onion peel water extract - 1 mg / mL;

the mixture of quercetin - 0.009 mg / mL and 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-

3 (2H) -benzofuranone - 0.001 mg / mL;

quercetin - 0.01 mg / mL

quercetin-4'-glucoside - 0.25 mg / mL;

protocatechic acid - 0.25 mg / mL;

The compounds were diluted in solutions of 0.9-1% sodium chloride or 0.75% NaCl + 0.25% NaHCO3 solution heated to 37 ° C prior to administration. With the exception of quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, which is a suspension, the compounds formed low opalescent color solutions.

Treatment regime.

The solutions were administered intranasally at a dose of 2 mL in each nasal cavity once a day for seven days. In order to achieve maximum contact of the tested products with the surface of the nasal cavity, the rabbits were kept in the "head down" position during administration.

The examination after the treatment.

The day after the last treatment, the animals were sacrificed by overdose of general anesthetics: Zoletil (Virbac, France) and Rometar (Bioveta, Czech Republic).

The nasal cavities of the animals were visually examined immediately after euthanasia.

After the macroscopic examination, the nasal parts of the rabbit heads were fixed in the 10% formaldehyde solution for 24 hours and then de-calcined for 14 days in the hydrochloric acid / water solution (25: 1). . Then, the animals' noses were sectioned transversely on four levels: A - at the incisor teeth, B - at the posterior incisors, C - at the second crest of the palatal bone, and D - at the level of the teeth. first premolars. Then, the tissues were cleaned with water for 12 hours and incorporated into the paraffin wax by the conventional method. The histological sections with a thickness of 3-5 μm were prepared using a microtome. To examine the goblet cells and their glucosamine content, the tissue sections were stained with blue alcian at pH 2.5. Subsequently, some tissue sections were re-stained with hematoxylin-eosin for subsequent histological examinations.

The morphological study of the histological preparations was carried out using CarIZeissAxioScopeAl (Germany) optical microscope at 25X, 50X, 100X, 200X and 400X magnifications. The microphotography of the samples was carried out using the AxioCamICc digital camera 1 and the AxioVisionRel.4.8 software (Germany). Morphometric measurements were performed using the AxioVisionRel.4.8 software (Germany).

The number of goblet cells was counted in 1 mm of the respiratory zone of the epithelium. The appearance, severity of acute catarrhal rhinosinusitis, acute purulent rhinosinusitis and the occurrence of post-inflammatory goblet cell hyperplasia were assessed by assessing the degree of inflammatory lesions in different areas: vestibule, the respiratory and olfactory regions, and the paranasal sinuses.

The degree of epithelial desquamation and lesions of the mucociliary glands was evaluated on a scale of 0 to 3, or:

0 - no lesion;

1 - weak lesion;

2 - moderate lesion;

3 - strong lesions.

The degree of leukocyte and mononuclear infiltration was evaluated on a scale of 0 to 3, or:

0 - the absent cells;

1 - the number of mononuclear cells present in the examination zone is between 1 and 5;

2 - the number of mononuclear cells present in the examination zone is between 6 and 10; 3 - the number of mononuclear cells present in the examination zone is greater than 10.

Statistical analyzes.

All data were analyzed using descriptive statistics. The normality of the data distribution was verified by the Shapiro-Wilks test. In case of normal data distribution, the mean and standard error of the mean were calculated and presented together in a table with n (number of values). In the case where the data do not correspond to the normality criteria, the median and the 1st and 3rd interquartiles were calculated. The differences between the groups were analyzed by parametric or non-parametric tests, depending on the type of distribution. The analysis of the data following the normal distribution was performed by the single-factor dispersion test (ANOVA), then the additional post-hoc analysis was performed by the Tukey method. Data that did not follow the normal distribution were analyzed by the nonparametric Kruskal-Wallis test, and the differences between the groups were analyzed by the Mann-Whitney test. The difference was determined at a significant level of 0.05.

Statistical analyzes were performed using the Statistica 10.0 software (StatSoftlnc, USA).

Results.

The results of the visual examination.

All groups of rabbits showed the same signs of rhinitis after the first administration of LPS: sneezing and scratching of the nose, which is the first sign of rhinosinusitis. On the third day of administration, LPS triggered nasal congestion and edema of the mucous membrane in visible areas of the nasal cavity. After the first administration of the compounds under study, edema and nasal discharge were observed. On the 9th and 10th days of study, rare cases (1 out of 6) of dryness of the nasal cavity and sneezing without discharge were observed in the treated groups. The results of the histological examination.

The mucous membrane of the nasal vestibule presented the typical histological structure, represented by a stratified squamous epithelium, gradually transformed into single respiratory epithelium.

The inner nose was lined by two types of mucous membranes corresponding to the respiratory and olfactory regions. The mucosa of the respiratory tract consisted of ciliated prismatic pseudo-stratified epithelium, basement membrane, and underlying loose connective tissue. The epithelium of the respiratory tract was composed of ciliated epitheliocytes, basal cells and goblet cells. Histochemical staining with the blue alcian revealed the presence of large vesicles containing glucosamines in most goblet cells, which refers to the increased functional activity of these cells. The numerous sero-mucinous glands have been observed in the basement membrane.

The olfactory region was located on the dorsal areas of the horns and on the roof of the nasal cavity. The olfactory epithelium consisted of support cells, olfactory cells and basal cells.

Table 3 presents the morphometric data of the nasal mucosa.

Table 3. Results of the morphometric study of goblet cells (M ± m, n = 6).

* - Statistically significant difference, p <0.05; Tukey test.

Primary data followed the normal distribution criterion. The analysis performed by the uniforal dispersion test (ANOVA) and the additional post-hoc analysis carried out by the Tukey method revealed the following differences (Table 3): reduction of the proportion of cells positive for staining by the alcian relative to total number of goblet cells in rabbits after administration of onion peel extract and quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone in comparison with the control. Increased number of goblet cells in 1 mm epithelium in rabbits after administration of quercetin-4'-glucoside in comparison with control. Table 4. Morphometry and degree of inflammation. Evaluation in scale (0 - 3) n = 6.

* - Statistically significant difference, p <0.05; Mann-Whitney test.

Table 5. Results of the study of sino-nasal inflammation of rabbits.

The non-parametric analysis of the degree of pathological inflammation on the scale from 0 to 3, using the Kruskal-Wallis and Mann-Whitney tests, revealed the following differences between the groups:

in groups receiving onion peel extract, quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and protocatechic acid, the infiltration of leukocyte cells in the mucous membrane was lower than in the control group;

epithelial desquamation was not detected in the group receiving quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and quercetin-4'-glucoside;

the degree of inflammatory injury in the respiratory tract was significantly low in the group receiving the onion peel extract;

no sign of post-inflammatory hyperplasia of goblet cells and acute purulent rhinosinusitis in the group receiving quercetin;

- the frequency of acute catarrhal rhinosinusitis was lower in the group receiving quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and the peel extract. 'onion;

- no difference observed among the control and treated groups in the occurrence of acute purulent rhinosinusitis.

All goblet cells contain mucopolysaccharides that are stained by blue alcian. The number of goblet cells is increased during inflammation and should decrease after treatment. From the data obtained, it can be concluded that the proportion of these cells has increased drastically after the induction of inflammation. In general, this process is accompanied by infiltration of the mucosa. If there is no infiltration, but the number of goblet cells is high, this is considered post-inflammatory hyperplasia, which is a sign of improvement.

Control group. No macroscopic changes were observed in the control group. Microscopic examination revealed that administration of LPS triggered inflammation in the respiratory and olfactory areas. In the vestibular area, edema and leukocyte infiltration into the mucous membrane and underlying tissue were observed. In the respiratory tract, epithelial desquamation and atrophy of the mucous glands have been observed. Staining with the blue alcian revealed areas of clustered hyperplasia of goblet cells in the respiratory tract. Morphometric evaluation of the epithelium showed significant increase in goblet cells containing glucosamines and mucopolysaccharides relative to the total number of goblet cells. Moderate infiltration of lymphocytes and macrophages into the mucous membrane and underlying tissue was also observed. Moderate inflammatory changes were observed in the vomeronasal organ. In total, following LPS administration of the pro-inflammatory agent in rabbits, acute catarrhal rhinosinusitis was observed (Table 5).

Animals in the second group treated with 0.9% sodium chloride solution showed no sign of macroscopic change during visual observation.

Microscopic studies revealed some evidence of epithelial desquamation in the respiratory and olfactory areas of the mucous membrane, moderate or significant leukocyte infiltration, the presence of exudate with high levels of neutrophils and cellular debris. The number of blue alcian-positive cells in 1 mm epithelium, and the number of goblet cells were lower than those of untreated animals. This is due to necrosis of the mucous membrane and inflammatory lesions of the epithelium. In general, signs of acute catarrhal rhinosinusitis and purulent rhinosinusitis have been observed in this group.

Animals on the 3rd and 4th groups receiving onion peel extract and quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone did not show the signs of the disease during macroscopic examination.

Microscopic examination showed very insignificant signs of inflammation compared to the untreated group. We observed a very weak infiltration of lymphocytes into the membrane mucosa and no leukocyte cells in the underlying tissue. No evidence of epithelial desquamation was observed in either group. Signs of regeneration have been observed in the respiratory and olfactory regions and in mucous glands. Some animals showed post-inflammatory goblet cell hyperplasia. No cases of acute purulent rhinosinusitis were observed in the quercetin + 2- (3,4-dihydroxybenzoyl) group -

2,4,6-trihydroxy-3 (2H) -benzofuranone.

No external changes were observed in animals of the 5th, 6th and 7th groups receiving quercetin, quercetin-4'-glucoside and protocatechic acid respectively, during macroscopic examination.

Microscopic examination revealed signs of an inflammatory process, which was stronger than that of animals receiving onion peel extract and quercetin + 2- (3,4-dihydroxybenzoyl) -

2.4.6-trihydroxy-3 (2H) -benzofuranone, but lower than that of untreated animals. No cases of acute purulent rhinosinusitis were observed in these groups.

Conclusion.

The results of the histological examination of the animals, after treatment with the onion peel extract and the compounds isolated from this extract, revealed that onion peel extract and quercetin + 2- (3,4 -dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone showed the best therapeutic effect, compared to untreated animals or animals receiving placebo. The best therapeutic effect of onion peel extract could be explained by the presence of several compounds, each with more or less anti-inflammatory effect and probably the potentiating capacity. Unexpectedly, quercetin + 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone showed the best therapeutic effect compared with that of quercetin. This could be explained by two reasons. 1st, by the presence of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone. The second, quercetin-shaped suspension could have a long-lasting effect compared to quercetin in solution. Quercetin -4'-glucoside (CGA) and protocatechic acid showed moderate activity against rhinosinusitis. By comparing the doses of each test substance and the therapeutic effect obtained, we concluded that quercetin-like suspension in NaCl or NaCl / NaHCO _{3 solution} could be a preferred drug for the treatment of acute and chronic rhinosinusitis.

Claims

A composition for rinsing the nasal cavity and sinus cavities for curative or prophylactic treatments of acute and chronic rhinitis and / or acute and chronic sinusitis, comprising:

sodium chloride and / or potassium chloride; quercetin, and at least one active agent of the polyphenolic group chosen from the compounds: quercetin-4'-glucoside, 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone, protocatechic acid; and wherein quercetin is in anhydrous form with an approximate molecular weight of 302 g / mol, or in the form of quercetin hydrates with an approximate molecular weight of 320 g / mol, or in the form of quercetin dihydrate with an approximate molecular weight of 338 g / mol.

2. Composition for a use according to claim 1, characterized in that it further comprises quercetin-derived impurities, chosen from quercetin-3'-glucoside, quercetin-7'-glucoside, diglucosides and quercetin triglucosides. Quercetin dimers in an amount of less than 5% of the total amount of the active agents.

Dry composition for use according to claim 1, comprising

94 - 99.99% by weight of sodium chloride and / or potassium chloride, 0.000001 - 3% by weight of quercetin and 0.000001 - 3% by weight of 2- (3,4-dihydroxybenzoyl) -2 , 4,6-trihydroxy-3 (2H) -benzofuranone.

Dry composition for use according to claim 1, comprising

91 - 99.99% by weight of sodium chloride and / or potassium chloride, 0.000001 - 3% by weight of quercetin, 0.000001 - 3% by weight of 2- (3,4-dihydroxybenzoyl) -2 , 4,6-trihydroxy-3 (2H) -benzofuranone and 0.000001 - 3% by weight of quercetin-4'-glucoside.

Dry composition for use according to claim 1, comprising

91 - 99.77% by weight of sodium chloride and / or potassium chloride, 0.23 - 3% by mass of quercetin, 0.000001 - 3% by weight of 2- (3,4-dihydroxybenzoyl) -2 , 4,6-trihydroxy-3 (2H) -benzofuranone and 0.000001 - 3% by weight of protocatechic acid.

6. Dry composition for use according to claim 2, comprising 86 - 99.77% by weight of sodium chloride and / or potassium chloride and of the extract of the plants containing flavonoids: 0.23 - 3% by weight of quercetin, 0.000001 - 3% by mass of quercetin-4'-glucoside and 0.000001 - 3% by weight of protocatechic acid, and impurities, derived from quercetin in an amount up to 5% by total mass of active agents.

7. Dry composition for use according to claim 2, comprising 83 - 99.77% by weight of sodium chloride and / or potassium chloride and of the extract of the plants containing the flavonoids: 0.23 - 3% by weight quercetin, 0.000001 - 3% by weight of quercetin-4'-glucoside, 0.000001 - 3% by weight of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) benzofuranone and 0.000001-3% by weight of protocatechic acid, and impurities derived from quercetin in an amount up to 5% by total weight of the active agents.

A water-based liquid composition comprising a dry composition according to claim 3, wherein the solution or suspension comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.00000001-0, 03% by weight of quercetin and 0.00000001 - 0.03% by weight of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone.

A water-based liquid composition comprising a dry composition according to claim 4, wherein the solution or suspension comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.00000001-0, 03% by weight of quercetin, 0.00000001 - 0.03% by weight of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -benzofuranone and 0.00000001 - 0.03 % by weight of quercetin-4'-glucoside.

A water-based liquid composition comprising a dry composition according to claim 5, wherein the solution or suspension comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023-0, 03% by weight of quercetin, 0.00000001 - 0.03% by weight of 2- (3,4-dihydroxybenzoyl) -2,4,6-trihydroxy-3 (2H) -enzofuranone and 0.00000001 - 0.03 % by weight of protocatechic acid.

A water-based liquid composition comprising a dry composition according to claim 6, wherein the solution or suspension comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023-0, 03% by mass of quercetin, 0.00000001 - 0.03% by mass of quercetin-4'-glucoside and 0.00000001 - 0.03% by weight of protocatechic acid, and impurities, derived from quercetin up to 0.5% by total mass active agents.

A water-based liquid composition comprising a dry composition according to claim 7, wherein the solution or suspension comprises 0.4 - 2% by weight of sodium chloride and / or potassium chloride, 0.0023-0, 03% by mass of quercetin, 0.00000001 - 0.03% by weight of quercetin-4'-glucoside, 0.00000001 - 0.03% by weight of 2- (3,4-dihydroxybenzoyl) -2,4, 6-trihydroxy-3 (2H) -benzofuranone and 0.00000001 - 0.03% by weight of protocatechic acid and impurities derived from quercetin up to 0.5% by total weight of the active agents.

The liquid composition according to any one of claims 8 to 12, wherein the solution or suspension further contains up to 0.5% by weight of sodium bicarbonate or potassium bicarbonate or a mixture of both.

The liquid composition according to any one of claims 8 to 13, which further comprises one or more therapeutic ingredients selected from antibiotics, corticosteroids and / or salts of copper, manganese and sulfur, essential oils and the like. excipients selected from pharmaceutical carriers, stabilizers, surfactants, preservatives.

15. Liquid composition according to one of claims 8 to 13 for use in the curative or prophylactic treatment of acute and chronic rhinitis and / or acute and chronic sinusitis.

16. Process for the preparation of dry compositions according to any one of Claims 1 to 7, comprising:

at). The dissolution of the active agent (s) and sodium chloride or potassium chloride in water heated to 80 - 100 ° C. Then cooling of the solution followed by freezing and lyophilization; or

b). The dissolution of the active agent (s) in the heated water up to 80 - 100 ° C. Then cooling the solution, adding sodium chloride or potassium chloride, followed by freezing and lyophilization; or c). The active agent (s) are dissolved in an organic solvent, preferably in ethanol or methanol diluted with water. The organic solvent / water ratio being between 50/50 and 90/10. Sodium chloride or potassium chloride is dissolved in water. Both solutions are mixed at room temperature, then frozen and lyophilized; or d). The powder of active agents is mixed with sodium chloride or potassium chloride.

17. Process for the preparation of dry compositions according to claim 16, characterized in that the active agent (s) is (are) derived from an extract which is prepared using ground onion peel, which is mixed with cold water in proportion 0.1-2 g / 50 mL; boiled, and allowed to simmer for 30 minutes at boiling temperature. The liquid is cooled, filtered and lyophilized.