LV14787B - An aerosol containing plant extracts for sublingual use and a method for preparation thereof - Google Patents

Abstract

Invention relates to the food supplement which is offered in the form of spray for sublingual application with antibacterial, anti-inflammatory and immunostimulatory effects. It contains 1.218 g of cinnamon bark (Cinnamomum Verum), 1.218 g of Echinacea (Echinacea Angustifolia), 1.218 g of grapefruit seeds (Citrus Paradisi), 0.468 g of propolis, 1.218 g of the peppermint (Mentha Piperita) ethanol extract, 10 g of glycerin, 1 g of beta-cyclodextrin, and 83.66 g of 70 % ethanol per 100 g of the product. The product is recommended for acute respiratory and throat infections, and for prophylaxis.

Description

DESCRIPTION OF THE INVENTION

Technical field

The present invention relates to the manufacture of a nutritional supplement, in particular to an aerosol dispenser with antimicrobial and immunostimulatory effects, and methods for its preparation.

State of the art

Acute respiratory and neck infections are among the most common diseases in the world.

Pathogenic and relatively pathogenic microorganisms are known that can persist in the neck of a healthy person because they inhibit their activity in normal microflora. Pathogenic microorganisms are always dangerous. In cases where the body's immune system is suppressed, for example due to viral infections, relatively pathogenic microorganisms turn into pathogens and "trigger" the infection process in the neck. Therefore, acute respiratory viral diseases, incl. influenza, very common causes of neck disease.

Neck diseases cannot be considered as a mere local process affecting the throat, tonsils, larynx. Rheumatoid arthritis, glomeruloneritis, meningitis and a number of other diseases could be the result of cervical infections. This means that even in cases where the infectious process predominates locally, i.e. in the throat, systemic strengthening of the body's defenses should be considered.

Quite a wide range of antiviral drugs and antibiotics are available to fight infections. However, in all countries alternatives to chemically synthesized drugs are sought among natural substances because:

- acute respiratory and neck infections are characterized by a very high prevalence and geographical spread,

- medicines are expensive,

- there are a large number of bacterial and viral strains which are rapidly altered by mutations,

- there is a well-known phenomenon of drug resistance (when the drug is inoperative),

- vaccination has an effect only after a reasonably long period of time.

Phytotherapy is capable of providing antimicrobial, antiviral and immunostimulatory effects simultaneously (Coutinho H.D. et al. BMC Complement Altem Med.

2009,9,13) with a lower risk of side effects than drugs (Vajdy M., Expert Opin Biol Ther. 2011,11,1501-13). It has a proven ability of acorn extracts to suppress Klebsiella pneumoniae Staphylococcu aureus, Pseudomonas aeroginosa (Mohebi R. et al. Roum. Arch. Microbiol. Immunol. 2011,70,4,149). Extracts of Pelargonium sidoides, Nigella sativa and Phyllanthus niruri are effective against tonsillopharyngitis (Bereznoy W.V. et al. Altem.Ther. Health. Med. 2003,9,5,68; Dirjomuljono M. et al. Int. J. Clin. . Pharmacol.Ther. 2008,46,6,295). Herbal substances also possess antiviral activity, for example, black elderberry fruit extract showed high efficacy in clinical conditions against influenza A and B viruses (Krawitz C. et al. BMC Complement. Altem. Med. 2011, Feb 25,11). Symptoms of colds and flu are rapidly relieved by oral administration of a liquid composition of extracts of black pepper, ginseng, cinnamon, saffron (WO2011034528 (A1) -20111-03-24). Patent applications include plant adaptogens such as ginseng and golden root (patent application AU20100310406 20101022), plants of the genus Andrographolide (patent application US2008011486320080505), and fungi such as Korean Phellinus linteus (patent application 2003200306). The composition of Chinese folk herbal extracts for the treatment of throat diseases contains 10 plants (patent application CN20101502288 20100928) and the anti-cold formula - 26 ingredients (patent application KR20020069954 20021112).

Extract of grapefruit seed, echinacea and propolis are the most commonly used in the fight against acute respiratory and neck infections. Grapefruit (Citrus paradise) extract has antibacterial and antifungal activity (Cyetnic Z. et al. Acta Pharm., 2004,54,3,243), which is particularly high for glycerol based extract (Giamperi L. et al., Fitoterapia, 2004,75, 2,221). Grapefruit seed extract exhibits antimicrobial activity against a wide range of Gram-negative and Gram-positive organisms in dilutions that are safe. This extract destroys the bacterial cell envelope after 15 minutes of contact (Heggers, J.P., et al., J. Altem. Complement Med., 2002,8,3,333).

Propolis is a complex resin or glue-like substance that helps protect the bee nest from harmful microorganisms. In medicine, propolis is known for its antimicrobial (Elbaz GA, Elsayad II Oral Health. Prev. Dent., 2012,10,2,155), anti-inflammatory (Abdulrahman M. et al. Pediatr. Hematol. Oncol., 2012,29,3,285) and immunostimulatory (Bratter C. et al. Forsch. Complementarmed. 1999,6,5,256). The antimicrobial and anti-inflammatory activity of propolis is coupled to polyphenols with which a particularly rich water-alcohol extract of propolis (Silva J.C. et al. Food Chem. Toxicol., 2012,50,5,1790).

Echinacea (Echinacea angustifolia) is undoubtedly the most commonly used plant with immunostimulatory properties. Echinacea extract is particularly effective in respiratory tract diseases (Tiralongo E. et al. Evid. Based. Complement. Altemat. Med. 2012,417267) in regulating haemokine and cytokine synthesis in leukocytes in accordance with current immune system status (Ritchie MR et al. Phytomedicine, 2011,18,10826). Immunostimulatory activity is associated with echinacea polysaccharides (heteroglycans) (Wagner H. et al. Arzneimittelforschung, 1985,35,7,1069). Echinacea preparations inhibit influenza A virus in vivo (Bodinet C. et al., Planta Med., 2002,68,1,896) and improve the immune response to the influenza vaccine; the role of echinacea preparations in the prevention of respiratory tract infections is emphasized (Di Pierro F. et al. Altem. Med. Rews. 2010,17,1,36). Immunomodulatory agents from echinacea extract-containing ice cream were absorbed through the oral mucosa (Guiotto P. et al. Phytomedicine, 2008,15,8,547). Immunostimulatory potential of Echinacea extract is increased in combination with Glycyrrhiza glabra extract (Wagner H., Jurcic

K. Phytomedicine, 2002,9,5,390). A synergistic anti-inflammatory effect of a combination of propolis and echinacea extracts has been described (Barak V. et al., IMAJ,

2002,4, suppl. 919). The combination of echinacea, propolis extracts and vitamin C in the prevention of respiratory tract infections in children has been demonstrated in randomized, double-blind, placebo-controlled clinical trials (Arsh. Pediatr. Adolesc. Med., 2004, 158,3,217).

As is known, phytopreparations often have immunostimulatory effects (nonspecific) as well as the ability to produce an adjuvant-like effect (Kumar, S. et al., J. Chin. Integr. Med., 2011,9,2,65). This aspect should be taken into account when formulating the formulation of the new preparation. The immune stimulatory effect of plants is related to the action of polyphenols (Feldman K. S, et al. Bioorg. Med. Chem. Lett. 1999,9,7,985; Hikosaka K. et al. Phytother. Res. 2007,21,2,120). It is the phenolic compounds that are responsible for the synergism that characterizes modern phytocellular preparations (Wagner H., Ulrich-Merzenich G. Phytomedicine, 2009,16,2-3,97).

Cinnamon preparations are widely used in folk medicine. The results of scientific studies show the antimicrobial activity of cinnamon (Keskin D., Toroglu S.J.Environment. Biol. 2011,32,2,251), antioxidant properties, especially ethanol extract (Yang C.H. et al. Molecules, 2012,17,6,7294). After antimicrobial activity against Streptococcus mutans, cinnamon oil is more commonly used than clove oil (Gupta C. et al. Acta Biomed. 2011,82,3,197). The immunomodulatory effect of cinnamon extract is mediated by promoting cytokine secretion in activated T lymphocytes (Lee B.J. et al. Immunopharmacol. Immunotoxicol. 2011,33,4,714). It is interesting to note that the cinnamic acid derivative was found in the rare form of propolis (Australia), which is famous for its extremely high antioxidant activity (Abu-Mellal A. et al. Phytochemistry, 2012,77,251).

For both medicaments and phytochemical supplements, the most important determinant of efficacy is the formulation and mode of administration. For acute respiratory and neck infections it would be logical to use a product that provides not only topical but also systemic exposure. Administration of herbal extracts in the form of drops, tablets, capsules will not always be effective for many reasons. Such finished forms provide only short-term contact of the active ingredients with oral tissue. Plant polyphenols are characterized by poor stability in the gastric acid environment and low bioavailability in the gastrointestinal tract (Schantz

M., et al. Biotechnol. J., 2010,5,10,1050). It should also be borne in mind that plant extracts can inhibit the intestinal absorption of drugs by inhibiting the function of the organic anion transporting polypeptide of epithelial cells involved in drug absorption (Fuchikami H. et al. Drug Metab. Dispos., 2006,34,4,577). On the other hand, saliva, mucin and erythrocytes significantly increase the antiradical activity of cinnamon extract lipophilic polyphenols (Ginsburg I. et al. Arch. Oral. Biol., May 26, 2012, Epub ahead of print). This phenomenon could be of particular importance in the suppression of inflammation of the mouth and throat.

With regard to increasing the bioavailability of plant extracts in the oral cavity, the development of ready-to-use formulations for sublingual and transbuccal administration has been rapidly developing in recent years. Uses films (AU2010279441 (Al) - 5/4/2012), aerosols (KR20040076005 (A) - 8/31/2004). How to ensure maximum local exposure of the active substances and their efficient transport in the blood stream?

Particularly promising are aerosol formulations of natural substances that provide mouth and throat disinfection, sublingual and transbuccal absorption and transport of biologically active substances to stimulate the body's immune system. The anatomy of the circulatory system of the cold and sublingual region is very "suitable" for the transfer of active substances from the oral cavity into the central circulatory circulation, so that biologically active substances are not destroyed by the action of digestive enzymes. The method of sublingual administration is considered as a novel approach to the problem of rapid and safe absorption of plant extracts which induces mucosal and systemic T cell and antibody response with a very wide distribution in the mucosa of various organs, incl. gastrointestinal, respiratory and reproductive systems (Czerkinsky C., Holmgren J. Curr. Top. Microbiol. Immunol., 2012,354,1).

On the one hand, oral mucosa is an attractive alternative to macromolecular administration. At the same time, in general, hydrophilic macromolecules are difficult to absorb in the oral cavity due to their size and hydrophilicity (Goswami T. et al. Arch. Oral Biol., 2009,54,6,577). Saliva, which rinses the active substances from the mucous membrane, also interferes with the process (Pademi C. et al. Oral Surg. Oral Med. Oral Pathol. Oral. Radiol. 2012, Jul.6, Epub ahead of print). These reasons may explain why there are few herbal extracts for oral health on the market.

An aerosol formulation for sublingual and transbuccal administration is more commonly used for drugs and predominantly those containing only one active ingredient. Mention may be made of a salicylic acid derivative (patent application US200905 84247 20090902), fentanyl (patent application GB2011001274320100630), sildenafil (patent application EP20110174378 20030827) and the like. A variety of technological techniques are used to increase bioavailability: nano-emulsification (patent application CN20021029357 20020906), inclusion of transcutol in a formulation for increasing tissue permeability (patent application NZ20070569949 20070125), sophisticated solvent systems (patent application GB20110015179 20090423); However, there are many unresolved technical problems in the development of plant extracts for transmucosal oral administration. Illustrative of this conclusion is the patent application US2003 / 0082107 A1 (published 1.05.2003), which provides a liquid variant multi-variant formulation of buccal aerosol for virtually all pharmacological grouping substances, but does not mention plant extract substances.

The development of a new effective plant extract for sublingual and transbuccal administration would be successful if two main tasks were pursued: the appropriate combination of active and excipients, which would provide high local antibacterial activity and systemic immunostimulatory effect, as well as rapid systemic absorption.

As noted above, an important problem in terms of the efficacy of sublingual and oromucosal plant extracts is the enhancement of the mucoadhesiveness and bioavailability of the administered substances. To improve mucoadhesion, the formulations include polymeric nanofibres (WO2012097763 (A2) -2012-07-26), microparticles containing the active ingredient (Pitarresi G. et al. Macromol. Biosci., 2012,12,6,770). Pectin (Sriamomsak P. Expert Opin. Drug. Beliv. 2011,8,8,1009) could also be used, but it is difficult to ensure standardization and stability of the formulations. Cyclodextrins, cyclic oligomers of glucose, are most suitable. Cyclodextrins not only promote sublingual absorption but also improve the taste of the formulations (Kaartama R. et al. Eur. J. Pharm. Biopharm. 2012, 81, 11, 178).

Close to the optimum aerosol for sublingual use, it looks like the product described in patent application KR20050059630 20050704 (published 11.01.2007). To obtain a stable composition of plant extracts, the extracts were filtered through a microfilter with a pore diameter of 0.5-2.0 µm, cyclodextrin was added at a concentration of 0.01-2.0%, diluted at 80-100 ° C and water was added to the desired volume. In that patent application, microfiltration was used to improve the stability of the composition.

Filtration is quite widely used in the production of plant extracts. Filtration methods are classified according to their ability to separate liquid components as follows: clarification up to 50 pm; microfiltration 50 pm - 0.1 pm; ultrafiltration 0.1 pm - 0.001 pm; reverse osmosis <0.001 pm (http://wwwwagag.com/Filtering-Through-the-Claims-oi-Varying-WaterTreatment-Methods-article 668). The assumption that plants contain not only activators of mammalian biochemical and physiological processes (vitamins, phenolic compounds, etc.) but also inhibitors suggests that ultrafiltration can affect the functional properties of plant products. The hypothesis of our experimental research work was related to the idea of using ultrafiltration to enhance the antibacterial activity of plant extracts.

SUMMARY OF THE INVENTION The object of the present invention is to provide a composition of plant extracts for sublingual aerosol administration with topical antibacterial activity and systemic immunostimulatory activity.

In our previous studies, 16 plant extracts (marigold, chamomile, sage, cinnamon, calendula, licorice, lavender, oregano, marigold, marigold, cocoa extract, grapefruit, stone fruit, , green tea), 6 juices (chokeberry, apple, quince, lemon, cranberry, black elderberry), as well as propolis extract, menthol and glycerin. Combinations of these plant extracts (total 71 variants) were also tested for Streptococcus mutans, Staphylocoecus aureus and Streptococcus ppogcnes by agar diffusion. Minimum inhibitory concentrations of active extracts and combinations thereof were determined. As a result of research, extracts of echinacea, cinnamon, propolis, grapefruit, peppermint, glycerin were selected. It should be noted that clove extract was not included in the list of selected ingredients as it did not improve the efficacy of the given composition.

In the final stage of our experiments we used the following substances:

- ethanol extract in a builder,

- ethanol extract of propolis,

- ethanol extract of grapefruit stone,

- ethanol extract of cinnamon bark,

- peppermint ethanol extract,

- glycerol.

Acute respiratory disease is more often caused by viruses, but relatively pathogenic microorganisms are involved in the development of the disease. Therefore, relatively strains of Streptoc & ccus mutans, Streptococcus pyogenes and Stapkylococcus aureus strains were used as test subjects for the study. Cultures were obtained from the University of Latvia Collection of Microorganisms.

For the preparation of the new product, mix all extracts (70% ethanol) in the required proportions (tab.), Add 10% of the total amount of glycerol, mix well.

Agar diffusion method and determination of minimum inhibitory concentration were used to evaluate the antibacterial efficacy of the extract composition in vitro. The specific activity of the plant extracts was evaluated for both individual extracts and combinations thereof. Figure 1 shows the antimicrobial activity of the individual ingredients and combinations thereof against S. mans in vitro. The composition of the ingredients in the base version of the composition is given in Table 1 below.

Antimicrobial activity of the individual ingredients and the novel product against S. mutans in vitro.

.Table. The natural substance content of the base version of the new product.

ingredients

Concentration, g / 100 g

Cinnamon Extract (CAnnamonum verum. Bark) 1,218 Echinacea extract (Echinacea angustifolia} 1,218 Grapefruit stone extract (Citrus varadisi '} 1,218 Ethanol extract of Propolis 0.468 Peppermint extract (Mentha piperita) 1,218

As can be seen in Figure 1, the base product developed by the new product has a higher antimicrobial activity than certain extracts and preparations popular on the market with analogous indications.

Ultrafiltration was used to further increase the antimicrobial activity of the composition. After mixing the ingredients of the new product (plant ethanol extracts, glycerol), it was passed through fabric microfilters "Amafiltergroup" in the following order: 5-lane filter, 1 pm filter and ceramic ultrafilter with a cut-off point of 30 KDa. To the resulting ultrafiltrate was added beta-cyclodextrin at a final concentration of 1%.

Table 2 shows the antimicrobial activity dependence of the new product on the aforementioned technological manipulations.

Table 2. Antimicrobial (S.aureus) activity of the new product in vitro. Agar diffusion method.

Test product version Diameter of inhibition zone, mm 1. In the basic version, the ingredients were taken at the optimum concentration (see Table 1) 30 ± 3 2. Basic version without ultrafiltration 37 ± 1 3. Final version after ultrafiltration and addition of beta-cyclodextrin 41 ± 2

Note: Differences between versions 1-2.2-3 are statistically significant (p <0.05)

The results of the experiment (Table 2) show that ultrafiltration of the new product increased the antimicrobial activity of the composition by 23%. The exact mechanism of this phenomenon is unclear, but it may be related to the release of a solution of extracts from some of the high molecular weight compounds that play an inhibitory role in the activity of active substances in terms of antimicrobial activity.

Additional beta-cyclodextrin increased the antimicrobial activity of the new product by 11% in vitro (Table 2). There is reason to believe that cyclodextrin enhanced the antimicrobial activity of plant extracts by increasing the permeability of microbial cell envelopes to the active ingredients of the product.

Based on the researches, the final formulation of the new product was developed (Table 3).

Table 3. Ingredients for the final version of the new product.

Ingredients Concentration, g / 100g Quantity of ingredients alone per dose (2 acne) - 0.3 g Cinnamon Extract (Cinnamonum verum, bark) 1,218 3.65 mg Echinacea extract (Echinacea angustifolia) 1,218 3.65 mg Grapefruit stone extract (Citrus varadisi) 1,218 3.65 mg Propolis extract 0.468 1.4 mg Peppermint extract (Mentha piverita) 1,218 3.65 mg Glycerine, 99.5% 10.00 30.00 mg Beta-cyclodextrin 1.00 3.00 mg Ethanol 70% 83.66 0.25 g

The results of the comparison of the effectiveness of the product on the market with Tanna are presented in Figure 2.

Newprodulfts C-t'ftiritO'feSoSiiteipi'oilulitE)

2. Antimicrobial activity of a new and existing product in vitro.

To evaluate the ability of the final product to modulate the immune system in vivo, experiments were performed on laboratory rats: the ability of the new product to prevent or reduce inflammatory events using a local inflammation model was investigated.

Wistar * Kyoto male rats weighing 190 ± 11 g were fed standard food and watered ad libitum. Animals were randomized into 3 groups (n = 9):

Group 1 - Control,

Group 2 - Within 7 days the animals were administered the new product by spraying it (0, 1g, 3 times daily) in an open and fixed mouth for 2 min; On day 8, rats were injected with 50 ml of a 1% solution of carrageenan in saline, and in the right hind leg, in a pad of SOmkl saline; Animals were not allowed to eat or drink for 30 min after spraying to minimize the likelihood of entry into the stomach,

Group 3 - animals were dosed with saline (placebo) by spraying (0.1 g, 3 times daily) in an open and fixed mouth for 2 minutes for 7 days; further manipulations were similar to 2 animals in the group.

The amount of ingredient (0.1 g) administered to rats in the new product per dose was as follows: Propolis extract 0.47; grapefruit stone extract 1.22; ehinaoejas extract i, 22; glycerol 10; cinnamon extract 1.22; peppermint extract 1.22; beta-cyclodextrim 1.00; as well as ethanol 70% - 0.08 g.

Animal experiment approved by the Animal Welfare Board.

Prior to initiation and at the end of the experiment, rats were weighed, 24 hours after carrageenan or placebo, venous blood samples were repeated, rats were euthanized by cervical translocation. Both foot pads were prepared, tissue quantitated, and placed in tris-buffer, homogenized by sonication to obtain a homogeneous fluid. The homogenate was centrifuged at 14,000 x g for 30 min, aliquoted, and stored at -70 °. Samples were detected by ELISA for prostaglandin E2 (PGE2) using the Cayman Chemicals Reagent Kit and Nerve Growth Factor (NAF) with Promega reagents. Biochemical endpoints were calculated based on tissue mass obtained.

In group 2 rats (receiving the new product), the tissue mass of the pads at 24 hours after carrageenan was found to be 2.2 times less than that of the group 3 animals, namely the pads were less bloated due to less inflammation. PGE2 concentrations were 65.5 ng / g for tissue homogenate group 2 and 88.7 ng / g for placebo (p <0.01). The NAF content in the homogenate increased by 22% in group 3 rats receiving an inflammatory inducer (carrageenan) and saline (placebo). No differences in body mass dynamics between the groups were observed during the experiment.

Experimental in vivo data suggest that carrageenan promoted local inflammation in the hind paws, but was significantly (about 50%) reduced by sublingual prophylactic administration of the new product. Decreased concentrations of tissue in the affected pads of nerve growth factor inflammation indicate a reduction in discomfort and pain in Group 2 animals. It should also be emphasized that the new aerosol was injected into the oral cavity for a short period of time but provided a systemic effect. This is due to the rapid transmucosal absorption of the formulation ingredients.

Thus, the prophylactic use of the new product reduces the severity of the induced local inflammation. This fact gives rise to the expectation of an analogous prophylactic effect in acute respiratory and neck infections, characterized by a local inflammatory process, a response to microbial invasion. Considering the local antibacterial activity of the new product, it can be concluded that the new aerosol for plant extracts for sublingual administration will be able to provide the corresponding oral effect as well as systemic immunostimulatory effects. Such effects could be based on the antioxidant, antibacterial, anti-inflammatory, immunostimulatory properties of plant extracts and propolis, as well as the techniques used to enhance the functionality of the product.

Based on the results of the study, it can be concluded that the new formulation of herbal extracts in aerosol form has high local antimicrobial efficacy, rapid transmucosal absorption and systemic immunostimulatory effect. The product is indicated for acute respiratory and cervical infections, incl. cases of influenza. The composition can be used prophylactically. Recommended - 3 times a day for two doses (fluff).

Claims (5)

Claims 1. A composition comprising, per 100g of product, 218g of cinnamon bark (Cinnamonum verum), 1.218g of echinacea angustifolia, 1.218g of grapefruit (Citrus paradise), 0.468g of propolis, 1.218g of peppermint (Mentha piperita) , 10g glycerol, 1g beta-cyclodextrin, 41.83g 35% ethanol. The composition according to claim 1, for use in acute respiratory and neck infections. The composition of claim 1 is for systemic stimulation of the immune system. The composition according to claims 1, 2 and 3 for prophylactic use against colds. A process for preparing a composition according to claim 1, characterized in that a mixture of plant and propolis ethanol extracts is prepared and filtered through a ceramic ultrafilter with a cut-off point of 30 KDa.