WO2014195741A1 - Probiotic formulation with apigenin and peppermint extract - Google Patents

Abstract

The present invention discloses improved formulation of probiotic cultures of lactic acid bacteria with apigenin (2) and peppermint (Mentha piperita L.) extract, process of its preparation, and use thereof. The formulation comprising: (i) one or more lyophilized cultures of probiotic bacteria selected from the groups consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum;- in -amount from 1»10⁷ to 5·10¹⁰ cfu/g or 0.01-50% w/w; (ii) apigenin (1), its O-glycosides, or dry herbal extract containing them, in amount from 10-50% w/w calculated to apigenin (1) content; (iii) dry extract of peppermint (Mentha piperita L.) leaf; in amount from 10-50% w/w and (iv) excipients required to- give final dosage forms to 100%- w/w cf the formulation. The formulation- is used as food, pharmaceutical: or veterinary product.

Description

PROBIOTIC FORMULATION WITH A IGENIN AND PEPPERMINT EXTRACT

DESCRIPTION

Field of the Invention

The present invention relates to an improved formulation of probiotic cultures of lactic acid bacteria with apigenin (1) and peppermint (Mentha piperita L.) extract, to process for its preparation, and use thereof.

Technical Problem

The present invention solves technical problem of simultaneous administration of probiotic bacteria (probiotics) and antimicrobial substances, apigenin (1) or its O-glycosides, as well as peppermint (Mentha piperita L.) extract, in order to achieve positive therapeutic effects on human or animal health.

Prior Art

Probiotics are live microorganisms, most often lactic acid bacteria (LAB) , which are normal inhabitants of human and animal gastrointestinal tract. They suppress population of pathogenic microorganisms by their presence themselves, acts like antiinflammatories, and generally provide several positive effects on human and animal health, see for instance the following literature references:

1. ) M. De Vrese et al.: Probiotics, prebiotics, and synbiotics,

Adv. Biochem. Eng. Biotechnol . Ill (2008) 1-66;

2. ) T. A. Oelschlaeger : Mechanisms of probiotic actions - A review,

Int. J. Med. Microbiol. 300 (2010) 57-62; 3. ) S. K. Hegazy et al.: Effect of probiotics on pro-inflammatory cytokines and NF-kappaB activation in ulcerative colitis, World J. Gastroenterol. 16 (2010) 4145-4151;

4. ) L. V. cFarland et al . : Pharmaceutical probiotic for the treatment of anaerobic and. other infections, Anaerobe 3 (1997) 73-78;

5. ) G. Perdigon et al.: Immune system stimulation by probiotics, J.

Diary Sci. 78 (1995) 1597-1606; te

6. ) R. Fuller: Probiotics in man and animals, J. Appl . Bacteriol.

66 (1989) 365-378.

Soluble fibers like inulin (polyfructose) , oligofructose, pectin (poly-D-galacturonic acid) and others, are essentially important as a food for probiotic bacteria. These compounds are called prebiotics. Synergistic role of prebiotics on the growth and development of probiotic cultures is described in the prior art in detail :

7. ) M. Roberfroid et al . : Prebiotic effects: metabolic and health benefits, Br. J. Nutr. 104 (2010) S1-S63;

8. ) G. R. Gibson: Dietary modulation of the human gut microflora using the prebiotics oligofructose and inulin, J. Nutr. 129 (1999) 1438S-1441S; te

9. ) R. Fuller et al.: Modification of the intestinal microflora using probiotics and prebiotics, Scand. J. Gastroenterol. Suppl. 222 (1997) 28-31.

Lactic acid bacteria (LAB) from the present invention: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii , Bifidobacterium animalis ssp. lactis and Bifidobacterium longum, are well known and widely used in production of fermented dairy products like various functional joghurts, cheeses, etc. There are several beneficial effects of LAB to human and animal gastrointestinal health:

(i) restoration and regulation of intestinal microflora;

(ii) can prevent gastrointestinal infections with pathogens like Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, Clostridium perfringens and Salmonella enteritidis;

(iii) can help in prevention of diarrhea, including also infective (Clostridium dificile, rotavirus) diarrhea which occurs during travel, and after the use of antibiotics;

(iv) can help in prevention and treatment of colon cancer;

(v) stabilize intestinal mucosal barrier what favours maintenance of bowel wall in a good condition;

(vi) can help at inflammatory gastrointestinal diseases, and alleviate allergic reactions on food;

(vii) enhance utility of lacose at subjects with diminished function of enzyme β-D-galactosidase; and

(viii) can help in strengthening immunity by activation of nonspecific (phagocytose function, activity of NK cells) and specific (antibody production, cytokinase, proliferation of lymphocytes) immunity.

Statements under (i)-(viii) are described in the following prior art :

10. ) K. Kailasapathy, J. Chin: Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp., Immunol. Cell Biol. 78 (2000) 80-88;

11. ) N. M. de Roos, M. B. Katan: Effects of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998, Am. J. Clin. Nutr. 71 (2000) 405-411; te

12. ) M. de Vrese, P. R. Marteau: Probiotics and Prebiotics: Effects on Diarrhea, J. Nutr. (2007) 803S-811S. Apigenin (1) or 4 ' , 5 , 7-t

1

is a naturally occuring compound from the class of flavonoids, with the following positive pharmacological effects on gastointestinal system: anti-inflammatory, carminative, spasmolytic, and preventive action on intestinal cancer. This is well-described in the prior art :

13. ) P. Gardiner: Chamomile (Matricaria recutita, Anthemis nobilis) ;

htt : //www . longwoodherbal . org/chamomile/chamomile . pdf;

14. ) M. Funakoshi-Tago, K. Nakamura, K. Tago, T. Mashino, T.

Kasahara: Anti-inflammatory activity of structurally related flavonoids, Apigenin, Luteolin and Fisetin, Int. I munopharmacol . 11 (2011) 1150-1159;

15. ) E. M. Drummond, N. Harbourne, E. Marete, D. Martyn, J.

Jacquier, D. O'Riordan, E. R. Gibney: Inhibition of Proinflammatory Biomarkers in THP1 Macrophages by Polyphenols Derived from Chamomile, Meadowsweet and Willow bark, Phytother. Res. (2012) PMID: 22711544;

16. ) R. Lemmens-Gruber, E. Marchart, P. Rawnduzi, N. Engel, B.

Benedek, B. Kopp: Investigation of the spasmolytic activity of the flavonoid fraction of Achillea millefolium s.l. on isolated guinea-pig ilea, Arzneimittel . Forsch. 56 (2006) 582-588;

17. ) S. Shukla, S. Gupta: Apigenin: A Promising Molecule for Cancer

Prevention, Pharm. Res. 27 (2010) 962-978; ^"te

18. ) A. B. Ganjare, S. A. Nirmal, A. N. Patil: Use of apigenin from

Cordia dichotoma in the treatment of colitis, Fitoterapia 82 (2011) 1052-1056.

Peppermint [Mentha piperita L.) is a well-known healing herb whose dried leaves are used as herbal drug. The latter contains 1-1.5% of essential oil (volatile ingredients) with menthol (min. 44%) and menthone (15-32%), which is obtained by steam distillation of fresh herb. Dry extracts with predominant content of non-volatile ingredients are manufactured by extraction of dried leaves with aqueous alcohol followed by subsequent evaporation. Peppermint extract is of similar chemical composition like extracts of other healing herbs from the Lamiaceae family like Lemon Balm {Melissa officinalis L.), Rosemary (Rosmarinus officinalis L.), Sage {Salvia officinalis L.), and the like, which contain:

(i) polyphenolic acids: rosmarinic acid (Lamiaceae tannins), caffeic acid, carnosic acid, etc.

(ii) flavonoids: luteolin, acacetin, eriocitrin, hesperidin, etc.;

(iii) antraquinone polyphenols: emodin, crysophanol;

(iv) triterpenic acids: oleanolic and ursolic acids;

and other compounds, as a result of whose it exhibits strong antioxidative , anti-inflammatory, spasmolytic, carminative, immunomodulating, as well as radioprotective and enteroprotective actions. This is described in the following prior art documents:

19. ) D. Keifer, C. Ulbricht, T. R. Abrams, E. Basch, N. Giese, M.

Giles, C. DeFranco Kirkwood, M. Miranda, J. Woods: Peppermint (Mentha piperita) : an evidence-based systematic review by the Natural Standard Research Collaboration, J. Herb. Pharmacother . 7 (2007) 91-143;

20. ) D. L. McKay, J. B. Blumberg: A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.), Phytother. Res. 20 (2006) 619-633; te

21. ) M. S. Baliga, S. Rao: Radioprotective potential of mint: A brief review, J. Cancer. Res. Ther. 6 (2010) 255-262.

According to the prior art of the present invention, there is not mentioned any probiotic formulation of lyophilized lactic acid bacteria with apigenin (1) or any other flavonoid, and peppermint extract (Mentha piperita L.) . Despite the fact that positive effect of lyophilized lactic acid bacteria (e.g. Lactobacillus acidophilus) , apigenin (1) and peppermint extract on gastrointestinal (GI) tract can be expected, their combined use is unexpected, due to a well-known wide spectrum of antimicrobial actions of both apigenin (1) and particular ingredients of peppermint extract (rosmarinic acid, carnosol, carnosic acid) against different bacteria; see for example:_.

22. ) B. Ozcelik, M. Kartal, I. Orhan: Cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids, Pharm. Biol. 49 (2011) 396-402;

23. ) D. Wu, Y. Kong, C. Han, J. Chen, L. Hu, H. Jiang, X. Shen: D-

Alanine : D-alanine ligase as a new target for the flavonoids quercetin and apigenin, Int. J. Antimicrob . Agents 32 (2008) 421-426;

24. ) G. Eumkeb, S. Chukrathok: Synergistic activity and mechanism of action of ceftazidime and apigenin combination against ceftazidime-resistant Enterobacter cloacae, Phytomedicine 20 (2013) 262-269;

25. ) M. Petersen, _M. S. Simmonds: Rosmarinic acid, Phytochemistry 62

(2003) 121-125; te

26. ) S. Moreno, T. Scheyer, C. S. Romano, A. A. Vojnov: Antioxidant. and antimicrobial activities of rosemary extracts linked to their polyphenol composition, Free Radic. Res. 40 (2006) 223- 231. '

Due to this fact, combined use of probiotic bacterial cultures with antimicrobial substances such as apigenin (1) and peppermint extract is completely unexpected.

Unexpected microbiological stability of probiotic cultures from the present invention against the mixture of apigenin (1) and ingredients of peppermint (Mentha piperita L.) extract has been proved by the present invention.

The technical problem of production of improved. probiotic formulation which, beside lactic acid bacteria, also contains above- mentioned antimicrobial compounds which, together with positive therapeutic actions (spasmolytic and anti-inflammatory) on gastro- intestinal (GI) system, is unexpected and is not obvious from the prior art documents.

This is the reason why we think that the technical problem of production of improved probiotic product with the. content of both lactic acid bacteria (LAB) and antimicrobial substances (apigenin and peppermint extract) is solved in the present invention in a novel and inventive manner, as is disclosed in the detailed description of the invention.

Summary of the Invention

The present invention solves the technical problem of production of improved probiotic formulation comprising:

(i) one or more lyophilized cultures of probiotic bacteria selected from the groups consisting of:- Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum; in amount from 1·10⁷ to 5^»10^1ΰ cfu/g or 0.01-50% w/w; .

(ii) apigenin (1) , its O-glycosides , or dry herbal extract v. containing them:

1

in amount from 10-50% w/w calculated to apigenin (1) content;

(iii) dry extract of peppermint (Mentha piperita L.) leaf; in amount from 10-50% w/w; and (iv) excipients required to give final dosage form; to 100% w/w of the formulation.

The formulation enables simultaneous administration of probiotic cultures of lactic acid bacteria together with antimicrobial substances: apigenin (1) and pepermint (Mentha piperita L.) extract.

The formulation is used as food (food supplement), pharmaceutical or veterinary product for: restoration and regulation of intestinal microflora; strengthening immunity; alleviation of intestinal spasms; against flatulence and meteorism; prevention and treatment of diarrhea and intestinal infections, inflammatory diseases of gastrointestinal system and food allergies; stabilization of mucosal intestinal membrane; as adjuvant in prevention and treatment of cancer of various organs of gastrointestinal system; and as adjuvant for alleviation of lactose intolerance.

Detailed Description of the Invention

Composition of the formulation from the present invention

The present invention includes improved probiotic formulation which is used as. food (food supplement), medicinal or veterinary product, process for its preparation, and use thereof.

The product formulation comprising:

(i) one or more lyophilized culture of probiotic bacteria selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus,

Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum; in amount from 1·10⁷ to 5·10¹⁰ cfu/g or •0.01-50% w/w; (ii) apigenin (1), its O-glycosides or dry extracts containing them;

in amount from 10-50% w/w calculated on the content of apigenin (1) ;

(iii) dry extract of peppermint {Mentha piperita L. ) leaf; in amount from 10-50% w/w; and

(iv) excipients required to give final dosage form; to 100% w/w of the formulation.

Lyophilized cultures of probiotic lactic acid bacteria (LAB) are selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus ¹ bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum.

Due to objective and expected variability of industrial processes for manufacturing and isolatio of lyophilized bacterial cultures, the latter commonly contain"- *^!eirtain_:— -"amount of inert filler (excipient) . Its role is to standardize the content of live probiotic bacteria in the product. Usual content of such inert filler in lyophilized cultures of LAB is 20-50% w/w.:

The following inert filler (excipient) in commercially available lyophilized cultures are used: maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic fillers.

As apigenin (1), here can be used synthetic substance or herbal extracts with at least 25% w/w of apigenin. Alternatively, pure O-glycosides ^' (la) of apigenin like apiin (2), apigetrin (3) or rhoifolin (4), or dry herbal extracts containing them, can be employed. These O-glycosides of apigenin undergo smooth hydrolysis under acidic condition of gastric juice or under influence of microbial enzymes in gastrointestinal system releasing their common

Examples of typical herbal extract which contains apigenin (1) or their O-glycosides are as follows: dry extract of Chamomile {Matricaria recutita L.) with apigenin (1), dry extract of Parsley (Petroselinum crispum L.) with apiin (2), dry extract of Dandelion {Taraxacum officinalis L.) with apigetrin (3), and dry extract of Grapefruit (Citrus x paradisi L.) containing rhoifolin . ( 4) .

As dry extract of peppermint {Mentha piperita L.) leaf, there can be used milled dry herbal material of leaves of peppermint or products whose production involves extraction of the latter with aqueous ethanol as solvent, subsequent concentration and spray drying of thus obtained liquid extract to dry powderous extract. Such extracts do not contain essential oils of peppermint at all or its content is negligible. The strength of extracts which are used in the preparation of the formulation of the present invention is as follows: plant dr g (dried- leaves) : extract = 1:1 to 20:1. Excipients for preparation of formulation from the present invention are one or more common auxiliary substances used in production of food supplements, pharmaceutical and veterinary products of various final dosage forms: tablets, capsules, powder, granules, oral suspensions, etc.

Excipients are selected from the group comprising: fillers/diluents, binders, disintegrants, anti-caking agents, tensides, antioxidants, sweeteners, flavourings, and other classes of pharmaceutically acceptable excipients.

Fillers are selected from the group comprising: inulin, oligofructose, pectin, modified pectins, macrocrystalline cellulose, lactose, starch, maltodextrin, saccharose, glucose, fructose, mannitol, xylitol, non-crystallizing sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharmacologically acceptable fillers, and mixtures of these substances.

At dosage form of oral suspension, fillers or diluents are selected from the group comprising: vegetable oil, oleic acid, oleyl alcohol, liquid polyethylene glycol, other pharmacologically acceptable inert liquids, or mixtures of these substances.

Binders in solid · dosage forms like tablets are: lactose, starch, modified starches, hydroxypropyl methylcellulose (HPMC) , other water soluble^' cellulose , ethers, polyvinylpyrrolidone (PVP), cross-linked povidon, · polyethylene glycol, ■ sorbitol, maltitoL, xylitol, other suitable pharmacologically- acceptable binders, or mixtures of these substances .

As disintegrants^' in solid dosage forms like tablet^'s,' the following substances are used: cross-linked polyvinyl pyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, other suitable pharmacologically acceptable disintegrant or their mixtures. As anti-caking agents in solid dosage forms like tablets, capsules, or powders, the following are used: magnesium stearate, colloidal silicon dioxide, talc, other pharmacologically acceptable anti- caking agents, or their mixtures.

Solid dosage forms of the formulation can be enterically coated to ensure intact passage through a strong acidic medium of gastric juice (pH around 1), and which allow release in duodenum or ileum (small intestine) . In this case, granules for capsules filling or already prepared tablets are coated with common enteric coating agents .

Enteric coating is prepared from pharmaceutically acceptable excipients selected from the group comprising: waxes or wax-like > substances such as beeswax, stearic acid, higher fatty alcohols like cetyl or stearyl alcohol, solid paraffin, earnauba wax, glycerol monostearate, glycerol distearate, and others; cellulose derivatives like hydroxypropyl methylcellulose (HPMC) phthaiate or succinate; synthetic polymers, and cc-polymers like polyvinylacetate phthaiate (PVAP); co-polymers of methyl acrylate-metacrylic acid; co-polymers of methyl metacrylate-metacrylic acid; and other pharmaceutically acceptable compounds which enable enteric resistance; or their combinations .

Alternatively, in . the" case of capsules, homogeneous mixture is filled into gastro-resistant capsules obtained from modified gelatin or hydroxypropyl methycellulose (HPMC) modified with . gellan gum, or other kinds of enteric coatings .

Except auxiliary' substances from the class of excipients, the formulation from the present invention can- contain ^■ other pharmacologically^' active or nutritive substances selected from the group comprising: ' .

(1) vitamins A (retinol) , Bl (thiamine), B2. (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine) , B9 .(folic ■. acid), . B12 (eyanocobalamin) , B15 (para-aminobenzoic acid), C (ascorbic acid), D2 (ergocalciferol ) , D3 (cholecalciferol) , Έ (tocopherol), F (essential higher fatty acids), H (biotin) , K (phylloquinone ) ; in the pharmaceutically acceptable chemical forms, salts or derivatives;

(2) minerals: potassium (K⁺) , calcium (Ca²⁺) , magnesium (Mg²⁺) , iron (Fe, Fe²⁺, Fe³⁺) , zinc (Zn²⁺) , manganese (Mn²⁺) , copper (Cu²⁺) , selenium, chromium (Cr³⁺) , molybdenum, fluor (F^~) , iodine (I^"), boron (H₃B0₃) , silicon, and phosphorus, in the form of pharmaceutically and nutritive acceptable chemical forms ;

(3) L-amino acids and related compounds: alanine, arginine, aspartic acid, citrulline, cysteine, cystine, histidine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, ornitine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine, carnitine and taurine; and also

(4) other., nutritive and pharmaceutically active substances with actions on gastrointestinal (GI) tract.

Preparation of the formulation from the present invention

The formulation from the present invention involves final dosage forms selected from the group comprising: tablets, capsules, powder, granules, oral suspension, and other, pharmaceutical final dosage forms suitable for oral use.

The process for preparation . of ^'solid dosage forms of the formulation includes homogenization of:

(1) one or more lyophilized cultures of probiotic bacteria selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus, rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii , Lactobacillus delbrueckii,

Bifidobacterium animalis ssp. lactis and- Bifidobacterium longum; in amount from 1·10⁷ to 5·10¹⁰ cfu/g or 0.01-50% w/w; (2) apigenin (1) or extract with >25% apigenin or equivalent amount of its O-glycosides; and

(3) dry extract of peppermint {Mentha piperita L,);

(4) with one or more excipients to form homogeneous mixture which is, according to requirements, subjected to lubrication with magnesium stearate or other lubricants yielding final dosage form of powder .

Such homogeneous powder is filled into ordinary gelatin capsules or vegetable capsules, or, alternatively, into gastro-resistant capsules .

Tablets are manufactured by direct compression! of homogeneous mixture of active substances of the formulation and suitable excipients such as anhydrous lactose, non-crystallizing sorbitol, and others; or by tablet processing of the mixture in granulated form.

Granules are prepared by granulation process of active ingredients of the formulation with suitable fillers, binders, disintegrants , and small amount ' of purified water. Such prepared granules are sieved and dried in a lyophilizator until the water content of <1% w/w.

The process for preparation of liquid dosage forms (e.g. oral suspension) involves homogenization _. of active substances of the formulation:

(i) one or more> lyophilized cultures of probiotic bacteria selected from , the group consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus ^■rhamnos^'us, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, . Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum; in amount from 1·10⁷ to 5·10¹⁰ cfu/g or 0.01-50% w/w;.

(ii) apigenin (1) or its extracts with >50% apigenin or. equivalent amounts of its O-glycosides; and (iii) dry extract of peppermint {Mentha piperita L.);

in an inert liquid diluent (filler) such as: various vegetable oils like sunflower, soybean or olive oil; oleic acid; oleyl alcohol; liquid polyethylene glycols like PEG 200, PEG 400 or PEG 600; or other inert pharmacologically acceptable liquids.

The process further involves treatment of homogeneous mixture with one or more processes selected from the group comprising:

(i) stabilization of the formulation; by addition and homogenization of suspension stabilizers like beeswax, colloidal silicon dioxide, etc.;

(ii) sweetening of the formulation; by addition and homogenization of sweetener;

(iii) flavouring of the formulation; by addition and homogenization of flavouring.

Such forms of the formulation can contain also other excipients like antioxidants, suspension stabilizers, sweeteners, flavourings, and other kinds of ingredients, usually employed in the art.

Study of microbiological stability of the formulation from the present invention

The formulation of the present invention in the form of capsules, whose preparation is disclosed i Example 3., was subjected to microbiological testing of viability of lyophilized lactic acid bacteria (LAB) .

The group of LAB according tc the invention includes: Lactobacillus acidophilus,- . Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricUs, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum; as representative bacterial cultures Lactobacillus acidophilus LA3, Bifidobacterium animalis ssp. lactis BLC1 and Lactobacillus casei BGP93 are selected. In this experiment the formulation of the present invention in the form of capsules based on Lactobacillus acidophilus LA3, Bifidobacterium animalis ssp. lactis BLC1 and Lactobacillus casei BGP93 was used, under, for them, drastic environmental conditions at 40 °C during 28 days.

The capsules from Example 3. were packed in brown glass bottles and closed with plastic caps. Filled bottles were placed into thermostat at 40 °C. Samples are analyzed against viability of probiotic bacteria at 7., 14., 21., and 32. day.

In the same manner, as a control, capsules of commercially available product containing L. acidophilus and B. animalis ssp. lactis of concentration of 2.1·10⁹ cfu/capsule were also subjected to viability testing. Other ingredients are: prebiotics inulin and ol,igofructose, potato starch, anhydrous lactose, microcrystalline cellulose, and lubricant magnesium stearate. One can conclude that the control sample does not contain ingredients of antimicrobial action.

Results of the study are presented in Table 1.

For comparison, paralel analysis of viability of probiotic cultures (cfu/capsule) were carried out for samples packed in the same kind of packaging at normal conditions (25 °C) during 35 days.

Results are shown in Table 2.

Table 1. Results of microbiological stability of the formulation from the present ■ invention: determination of the. count of live probiotic bacteria (cfu/capsule; cfu/cps) in the sample (product from Example 3) after storage under stress conditions at 40 °C during.32 days. Sample to 7 days 14 days 32 days

(cfu/cps) (cfu/cps) (cfu/cps) (cfu/cps)

(%)² (%)² ( )² (%>²

Control ¹ 2.1·10⁹ 1.1· 10* 8.0·10^Β 6.6·10^{Β •}(100%) (52:4%) (38.1%) (31.4%)

Formulation . 2.3^«10^y. 3.7·10⁸ 1.4«10^s... 1.4Ί0⁷ from Example 3 (100%) .(16%) (6.1%) . (0.6%)

.1 Commercially available product containing probiotic culture of L. acidophilus and B. animalis ssp. lactis; other ingredients in the capsule: prebiotics inulin and oligofructose, potato starch, anhydrous lactose, microcrystalline cellulose and lubricant magnesium stearate.

2 Degree of survival expressed as a percentage to starting concentration of live probiotic bacteria (cfu/capsule) , determined in the moment t₀.

Table 2. Results of microbiological stability of the formulation frpm .the present invention: determination of_. a count of live probiotic bacteria (cfu/capsule; cfu/cps) in the sample (product from Example 3) after storage at normal conditions (25 °C) during 35 days .

¹ Commercially available product containing probiotic culture of L. acidophilus and B. animalis ssp. lactis; other ingredients in a capsule: prebiotics inulin and oligofructose, potato starch, anhydrous lactose, microcrystalline cellulose and lubricant magnesium stearate.

² Degree of survival expressed as a percentage to starting concentration of live probiotic bacteria (cfu/capsule), determined in. the moment t₀. From the results of microbiological study,, it can be seen that the control sample which contains only live liophilized probiotic bacteria of. L. acidophilus, and B. animalis, ssp. lactis, without presence of antimicrobial substances, is characterized by very good degree of survival under very harsch conditions, where the observed degree of survival was 31.4% after 32 days at 40 °C.

In the case of the formulation from the present invention (the product from Example 3), significantly lower degree of survival of 0.6% was observed, what was generally expected, due to known antimicrobial effect of apigenin (1) and components of peppermint (Mentha piperita L.) extract, on the basis of literature references: 22.)-26.) .

However, such level of survival in the sense of total (absolute) concentration is completely unexpectable, since, even after 32 days, the number of live probiotic LAB was still >1·10⁷ cfu/capsule. This represents, in absolute sense, definitely very high concentration. Such degree of survival is still good enough and industrially applicable as a base for development of probiotic formulation with apigenin (1) and. extract of peppermint with sufficient level of live probiotic bacteria.

Results of the control of live probiotic bacteria '(cfu/capsule) for the same sample which were kept at normal conditions (25 °C) during 35 days, show that the degree of survival of 57.1% at the control sample, and 73.9% at the sample of the formulation from Example 3. The. duration of the study at normal conditions (25 °C) was relatively short (35 days) . This is the reason why the absolute conclusions about relative stability of the formulation from Example 3 against control formulation cannot be made.

Despite that the observed degree of survival at the control . sample was even lower, we think that these results at normal conditions (25 °C) are approximately equal (comparable) , because of accuracy of microbiological methods of determination of cfu/capsule.

It is well known that the probiotic formulations always has to be "overloaded" with certain excess of lyophilized cultures of probiotic bacteria in order to ensure declared concentration of live cells til the end of declared shelf life, e.g. 2 years at 25 °C. Even such relatively low level of survival represents just sufficient framework for good . and . industrially applicable formulation based on a combination of:

(a) probiotic bacterial cultures of L. acidophilus , B. animalis ssp. lactis, L. casei, and related bacterial cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, and Bifidobacterium longum; as well as

(b) antimicrobial substances such as apigenin (1) and peppermint extract .

In fact, by the. use of above-mentioned adequate . excess of live lyophilized cultures of probiotic bacteria, sufficient degree of survival til the end of shelf life of finished product, e.g. during 2· years under normal conditions (under max. 25 °C) , can be achieved.

Also,. · from the results .of this study, wherein, observed degree of survival was 0.6% ' despite the weight precentage of antimicrobial substance (Chamomile extract with ^'50% apigenin and peppermint extract) of 47% w/w, one can substantially claim that the degree of survival in any other variant of the formulation with lower percentage of antimicrobial substances will _.be even higher.

Pharmacological effects of the formulation from the present invention'

Concerning the content of beneficial probiotic lactic acid bacteria, apigenin (1) and dry peppermint {Mentha piperita L.) extract, and their known pharmacological effects, the formulation is characterized by the following pharmacological effects:

(i) restoration and regulation of intestinal microflora; thus healthy microbial flora is ensured as v/ell as prevention of infections of gastrointestinal (GI) tract with pathogens such as Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica , Clostridium perf ingens and Salmonella enteritidis and others; in the case of already existing infections, formulation from the present invention also helps in therapy of infections providing high intake of live probiotic bacteria which suppress population of pathogenic microorganisms in a step-wise manner; apigenin (1) and peppermint extract help with their antiinflammatory actions by alleviating inflammatory condition (due to infection) of mucous wall of GI tract;

(ii) ' can help at digestive disorders like digestive spasms

. (spasmolytic action), . and at flatulence ■ and meteorism (carminative action)

(iii) can help in prevention and treatment of inflammatory- diseases of gastrointestinal (GI) tract;

(iv) can help in prevention of diarrhea, including infective one (Clostridium dificile, rotavirus), and diarrhea which occurs at traveling or after the use of antibiotics;

(v) can help in prevention and treatment of certain organs of „ gastrointestinal tract as adjuvant; ^-:

(vi) can help in . stabilization of mucosal intestinal barrier what is beneficial for maintenance of intestinal wall in a good condition;

(vii) can help in prevention and treatment of allergic reaction on foods;

(viii) increases utilization of lactose at persons with -diminished function of β-D-galactosidase, thus diminished intolerance to lactose taken by food; and

(ix) ^■ helps to boost immunity. The formulation of the invention is administered orally in dosages from 0.1-1 g daily which contain:

(1) _. 1·10⁶ to 5-10¹⁰ cfu of live lyophilized probiotic lactic acid bacteria of Lactobacillus acidophilus , Bifidobacterium animalis ssp. lactis and Lactobacillus casei;

(2) 10-500 mg of apigenin (1) ; and

(3) 10-500 mg of dry peppermint (Mentha piperita L.) extract (5:1 to 20 : 1)

wherein the administration can involve a single or several portions during the day.

At animals, the formulation of the present invention is used orally, in dosages of 1.5-15 mg / kg of body weight, wherein a dosing regimen also includes wither single or several portions during the day.

Conclusion

The formulation from the present invention provides simultaneous administration of:

(a) live lyophilized probiotic cultures of Lactobacillus acidophilus , Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei , Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis, Bifidobacterium longum; and

(b)_ _. apigenih (1) and peppermint; (Mentha piperita L.) extract,

• which are, beside other actions, known ,in the prior art as antimicrobial substances.

Concerning the antimicrobial action of apigenin (1) and peppermint extract, such simultaneous use together with lyophilized probiotic lactic acid bacteria (LAB) is completely unexpected to the person skilled in the art. The person skilled in the art would expect complete death of probiotic bacteria under normal conditions at room temperature (about 25 °C). during certain period of time..

The study of . microbiological stability of the formulation from the present invention showed significant fall of number of live probiotic bacteria, but, the level of this fall ensures preparation of combined formulation according to the invention. Precisely, with adequately increased weight percentage of live probiotic bacteria which die during the shelf life of the product (at temperatures up to 25 °C) for e.g. 2 years, the product can maintain declared number of live probiotic bacteria in a unit of the product: tablets, capsules, 1 ml of oral suspension or 1 g of oral powder.

Experimental Examples

General notices

The abbreviation cfu refers a number of units (bacterial cells) capable for life: colony forming units.

Example 1. Analytical methods used for analysis of^' the formulation from the present invention

The method for determination of. a number of live probiotic bacteria (cfu) in the formulation from the present invention is based on a method- of direct inoculation.

Sample preparation: in a test tube with thread, 5 g od the sample of the formulation from the present invention was weighted. Then, the dilution of 1:10 was · prepared by suspending 5 g of the sample in 45 ml of. Mitsuoka buffer. The sample was homogenized by mixing in a test tube shaker at the highest speed for 120 seconds. The suspension was left to stand for 30 minutes. Then,-, the mixture was homogenized for 120 seconds once again. Afterwards, decade dilutions were prepared by placing 1 ml of the suspension into 9 ml of Mitsuoka buffer. Each dilution was homogenized for at least 5 seconds. The time between the preparation of the dilutions til the inoculation must not be longer than 60 minutes .

Procedure :

Sample testing: in three Petri dishes per 1 ml of each of dilutions were placed. To each of Petri dish 12-15 ml of Mann-Rogosa-Sharpe (MRS) agar, previously dissolved and cooled to approximately 45 °C, was added. The plates were incubated at temperature of 37 °C for 3 days in anaerobic conditions^'. Afterwards, growed colonies were counted.

Negative control: in paralel analysis, per 1 ml of Mitsuoka buffer was placed into Petri dishes. To each of Petri dish, 12-15 ml of MRS agar, previously dissolved and cooled to approximately 45 °C, was added. The plates were incubated at 37 °C during 3 days in anaerobic conditions. The analysis is acceptable if the result in negative control plates were negative (absence of growth of colonies).

Results interpretation: after the incubation, grown colonies on all plates were counted. The acceptable plates are those with the number of colonies between 25 and 250 (Ph.Eur.). Mean value was calculated, and the results were expressed as a number of live cells (cfu) per' one gram of the tested sample (cfu/g) :

CFU

Number - = ∑ CFU / number of plates x 10

g whetein: x = decade dilution.

The number of live cells in capsule or tablet was calculated from theoretical mean weight of the capsule content or average weight of tablet. Alternatively, at oral powder, the content is expressed per 1 g of the product. At oral suspension, the content was expressed per 1 ml of .the product, taking into account the densit of the suspension. Method for determination of apigenin (1) content: the following HPLC method was used: mobile phase: water : acetonitrile = 50:50 V/V; flow: 1.0 ml/min.; volume: 10 μΐ; wavelength: 270 nm; column temperature: 35 °C; autosampler temperature: 25 °C elutioh: isocratic mode; screening duration: 4 minutes.

Solution of the standard: in a 10 ml measuring flask, and 5.00 mg of apigenin (1) standard substance was weighted, 5 ml of 96% ethanol was added, and then dissolved by ultrasonic bath for 15 minutes. After the dissolution, the solution was filled with 96% ethanol up to the mark of 10 ml and mixed well. 5 ml of thus prepared solution was transferred into the 50 ml measuring flask. The solution of the standard was prepared in duplicate. The concentration of apigenin (1) in thus prepared solution was around 0.05 mg/ml.

Sample solution: the contents of 10 capsules were quantitatively emptied, combined, and homogenized. In a' 100 ml measuring flask, 460:;0 mg of homogenized capsules content was weighted, 50 ml of 96% ethanol was. added, and dissolved in an ultrasonic bath for 30 minutes. After dissolution, the flask was filled with 96% ethanol up to the mark of 50 ml, and shaked well. Aliquot of thus prepared solution was centrifuged for 5 minutes at 5.000 rpm. 5 ml of supernatant was transferred into a 50 ml measuring flask and filled with 96% ethanol up to the mark of 50 ml. The concentration of apigenin (1) in thus prepared solution was around 0.-05 mg/ml.

Example 2. Preparation of the formulation from the present invention in the ' form of tablets

Composition (1 kg batch) :

•1. Lyophilized culture Lactobacillus acidophilus LA.3, 50 3

1·10 cfu/g

2. Lyophilized culture Bifidobacterium longum, 1.5Ί0¹¹ 25 g cfu/g

3. Lyophilized culture Lactobacillus plantarum, 2Ί0¹¹ 25 g cfu/g

4. Apigenin (1) 100 g

5. Peppermint ( Mentha piperita L.) extract (10:1) 100 g

6. Lactose 655 g 7. Polyvinylpyrrolidone K30 20 g

8. Sodium starch glycolate 15 g

9. Magnesium stearate 10 g

Preparation: A mixture of powderous ingredients 1.-8. was homogenized for 15 minutes. To thus obtained mixture, ingredient 9. was added and homogenized during 2 minutes, yielding fine white to white-yellowish odourless powder. Thus obtained mixture was processed by direct compression giving approx. 2.000 tablets. Tablet weight was approx.^' 500 mg.

Assay:

(1) Total live probiotic bacteria: 6.9Ί0⁹ cfu/tablet;

(2) Apigenin (1) : 50 mg/tablet;

(3) Peppermint [Mentha piperita L.) extract (10:1): 50 mg/tablet;

Example 3. Preparation of the formulation from the present invention in the form of capsules

Composition (1 kg batch) :

Preparation: A mixture of 1.-8. was homogenized for 20 minutes. To this mixture, ingredient 9. was added and additionally homogenized f,or 2 minutes furnishing homogeneous fine pale brown powder of slight "herbal" scent. Thus obtained mixture was filled into vegetable (HPMC) . capsules of size "0" using manual capsule filling mashine .. Approx . _: 2.000. capsules were prepared. The weight of capsule was 550 mg, whilst the weight of capsule content was 460 mg. Assay:

(1) Total live probiotic bacteria: 2.3·10⁹ cfu/capsule;

(2) Apigenin (1): 50 mg/capsule;

(3) Peppermint (Mentha piperita L.) extract (10:1): 100 mg/capsule.

Example 4. Preparation of the formulation from the present invention in the form of powder for oral use

Composition (1 kg batch) :

1. Lyophilized culture Lactobacillus rhamnosus, 1·10^π 1 0 g cfu/g

2. Lyophilized culture Lactobacillus bulgaricus, 0 7 g 1.5·10^η cfu/g

3. Lyophilized culture Lactobacillus paracasei, 2Ί0¹¹ 0 5 g cfu/g

4. Apigetrin (3) 250 0 g

5.. Peppermint (Mentha piperita L.) extract (20:1) 500 0 g

6. Inulin (Frutafit TEX) 100 0 g

7.^' . Xylitol 127 8 g

8.. Colloidal silicon dioxide (Aerosil 200F) 20 0 g

Preparation: A mixture of ingredients 1.-8. was homogenized for 15 minutes yielding fine ^' homogeneous pale brown powder of slight characteristic odour. The mixture was filled into sachets per 1.0 g yielding approx. 990 sachets.

Assay:

(i) Total live probiotic bacteria: 3·10⁸ cfu/g of powder;

(ii) Apigetrin (3): equivalent of 250 mg of apigenin (l)/g of : powder

(iii)' Peppermint (Mentha piperita L.) extract (20:1): 500 mg/g of powder .

Example 5. Preparation of the formulation from the present invention in the form of oral suspension

Composition (1 kg batch) :

1. Lycphilized culture Lactobacillus acidophilus LA3, 10^" 0 g 1·10^η cfu/g

2. Apigenin (1) 100 0 g

3. Peppermint Mentha piperita L.) extract (10:1) 100 0 g 4. Rebaudiozid A 5 0 g

5. Beeswax, white 20 0 g

6. DL- -Tocopheryl acetate 1 0 g

7.·^■ Sunflower . oil 764 0 g

Preparation: To sunflower oil (7.) heated to 50 °C, white beeswax (5.)· was added.■ The. mixture was stirred for 15 minutes til yellowish clear oil liquid was formed. Then ingredients 2., 3., 4. and 6. were added and the mixture was homogenized for 15 minutes. Then the suspensio was cooled to 40-42 °C when 1. was added. The suspension was homogenized by stirring at 40 °C to room temperature (20-23 °C) during 30 minutes. The product in the form of a thick fine suspension of pale brown to yellowish precipitate in a clear yellow supernatant. Stability of the suspension against precipitation is satisfactory. The product was filled into 10 ml bottles. It was marked with the notice: "Shake well before use".

Assay:

(1) /Total■ live probiotic bacteria : 1·10⁹ cfu/mL of suspension;

(2) apigenin (1) : 100 mg/mL of suspension;

(3) Peppermint {Mentha piperita L.) extract (10:1): 100 mg/mL of suspension.

Example 6. The study of microbiological stability of the formulation from the.^' present invention

The formulation from the ^' present- invention in the form of^' capsules, whose preparation ^' was dls^:c b^sse¹d^v,,inⁱ' .'Example ^■ 3. , was subjected to testing of microbiological stability of live lyophilized probiotic lactic acid bacteria (LAB): Lactobacillus acidophilus LA3, Bifidobacterium animalis ssp. lactis' BLCl and Lactobacillus casei BGP93 at 40 °C during 28 days.

Capsules from Example 3. were packed into brown glass bottles and closed with plastic caps. The filled bottles were placed into a thermostat at 40 °C The samples were analysed against the number of live probiotic bacteria at 7. , 14., 21., and 32. day. The method of analysis was described in Example 1. As the control, in the same manner, the_. capsules of commercially available product containing only L . acidophilus and B. animalis ssp. lactis, as well as prebiotics inulin and oligofructose, were tested on microbiological stability. Results are presented in Table 1.

Parallelly with the microbiological stability testing under stress conditions (40 °C) , analysis under normal conditions (25 °C) during 35 days was also performed. Results are given in Table 2.

Industrial applicability

Therefore, the invention as described hereby solves technical problem of simultaneous administration of probiotic bacteria (probiotics) and antimicrobial substances, apigenin (1) or its 0- glycpsides, as well as peppermint (Mentha piperita L. ) extract, in order to achieve positive therapeutic effects on human or animal health. The industrial applicability is therefore obvious.

Claims

1. Probiotic formulation with apigenin and peppermint extract comprising:

(i) one or more lyophilized cultures of probiotic bacteria selected from the groups consisting of: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricusj Lactobacillus johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis i Bifidobacterium longum; in amount from 1·10⁷ to 5·10¹⁰ cfu/g or 0.01-50% w/w;

(ii) apigenin (1), its O-glycosides, or dry herbal extract containing there

1

in amount from 10-50% w/w calculated to apigenin (1) content;

(iii) dr extract of peppermint {Mentha piperita L.) leaf; in amount from 10-50% w/w; and

(iv) excipients required to give final dosage forms; to 100% w/w of the formulation.

2 . A formulation according to the claim 1, wherein lyophilized cultures are selected from the group consisting of: Lactobacillus acidophilus, Bifidobacterium animalis ssp. lactis and Lactobacillus casei.

3. A formulation according to the claim 1, wherein, lyophilized cultures are selected from the group consisting of: Lactobacillus acidophilus LA3, Bifidobacterium animalis ssp. lactis BLC1 i- Lactobacillus■ casei BGP93. A formulation according to any of claims 1-3, wherein instead of pure apigenin (1) , dry extracts—with >25% w/w of ' apigenin, or its O-glycosides in equivalent amount calculated on the apigenin content, are used.

A formulation according to any of claims 1-4, wherein as dry peppermint extract, dry plant material of peppermint leaf or its extracts of the following strenght: "dry leaf of peppermint" to "extract" from 1:1 to 20:1 is used.

A formulation according to any of previous claims, wherein excipients are one or more substances selected from the group comprising: fillers, diluents, binders, disintegrants , anti- caking agents, tensides, antioxidants, sweeteners, flavourings, and. other classes of pharmaceutically-acceptable excipients.

^■"A process for preparation of solid final dosage forms of the formulation according to any of claims 1-6, wherein the process involves homogenization of:

(i) one or more lyophilized culture of Lactobacillus acidophilus, Lactobacillus plantarum, ^' Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus johnsonii, Lactobacillus , deibrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium lcngum;

(ii) apigenin ,. (1), its O-glycosides or dry herbal extracts containing them; and

(iii) dry peppermint extract; with one or more excipients yielding homogeneous mixture.

A process for preparation of solid dosage forms of the formulation according to claim 7, wherein said homogeneous mixture represents a dosage form of powder. A ^' process ^' for preparation of solid dosage forms of the formulation according to claim 7, wherein said homogeneous mixture is subjected to filling into common or gastro-resistant capsules .

A process for preparation of solid^' dosage forms of the formulation according to claim 7, wherein said homogeneous mixture is subjected to direct compressing into tablets.

A process for preparation of. liquid final dosage forms of the formulation, according to any of claims 1-6, wherein the process involves homogenization of:

(i) one or more lyophilized culture of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus . bulgaricus, Lactobacillus _, johnsonii, Lactobacillus delbrueckii, Bifidobacterium animalis ssp. lactis and Bifidobacterium longum;

(ii) apigenin (1) , its O-glycosides or dry herbal extracts containing them; and

(.iii) dry. peppermint extract with one .or more substances selected from the group of liquid inert diluents yielding homogeneous mixture .

A process for preparation of liquid final dosage form of the formulation according to claim. 11, wherein the pr.ocess further includes treatment ^' ,o^'"f 'homogeneotas 'ml ^'ture with . one or more processes selected from the group comprising: stabilization, sweetening, and- aromatization of the formulation.

A formulation- according to any of claims 1-6 for use as a medicament -for: restoration and regulation, of intestinal microflora, strengthening immunity, alleviation of digestive spasms, ^'■ against' .flatulence and meteorism, prevention and treatment of diarrhea, prevention and treatment of infective diseases- of ..gastrointestinal tract., stabilization of mucosal intestinal^' barrier, prevention and treatment ^' of inflammatory diseases of gastrointestinal tract, prevention and treatment of food allergies..

Formulation according to any of claims 1-6, for use as an adjuvant for: prevention and treatment of cancer . of gastrointestinal tract organs, and alleviation of lactose ■intolerance .

Formulation according to any _; of claims 13 or 14 used as a veterinary product.