HU231213B1 - Pharmaceutical composition comprising extracts of viola tricolor and common dandelion (taraxaum officinale) and process for producing thereof - Google Patents

Description

Pharmaceutical preparation containing extracts of tricolor (Viola tricolor) and dandelion (Taraxacum Officinale) and process for its preparation

The present invention relates to

The present invention relates to a pharmaceutical composition comprising extracts of Viola tricolor and Taraxacum officinale. The active ingredient of the pharmaceutical composition according to the invention is a mixture of active ingredients containing a solid or concentrated solution remaining after evaporation of part or all of the extracts of the extracellular part and / or root extract of Viola tricolor and Taraxacum officinale, which is suitable for mitochondrial activity (rotenone-sensitive NADH: Ubiquinone oxidoreductase activity) and for the prevention and treatment of diseases associated with mitochondrial dysfunction and / or impaired function of the complex I enzyme system. treatment. The invention further relates to the preparation of this composition.

Description of the prior art

Complex I (NADH: Ubiquinone oxidoreductase) is one of the largest membrane protein assemblages and plays a key role in the mitochondrial respiratory chain. Complex I-mediated electron transport provides 4040% of the proton driving force required for ATP synthesis. The molar mass of the complex I consists of 11 MDa and 44 subunits. Mitochondrial activity is dynamically regulated. More and more observations suggest that complex I regulates mitochondrial activity and metabolic-related cellular functions.

Complex I is a target for many signaling pathways and senses its own substrate availability. The mitochondrial oxidative phosphorylation system is associated with plasma membrane lipid grafts, which are also key players in cell signaling (Kim BW, Lee CS, Yi JS, Lee JH, Lee JW, Choo HJ, Jung SY, Kim MS, Lee SW, Lee MS, Yoon G, Ko YG. Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane (Expert Rev Proteomics. 2010 Dec; 7 (6): 849-866). Phosphorylation affects the function of complexes and enzymes in the electron transport chain, which are the basic elements of the metabolic signaling pathways (Deng N, Zhang J, Zong C, Wang Y, Lu H, Yang P, Wang W, Young GW, Wang Y, Korge P, Lotz C, Doran P, Liem DA, Apweiler R, Weiss JN, Duan H, Ping P. Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria.Mol Cell Proteomics.Feb 2011; 10 (2) Epub 2010 May 22). The cAMP-mediated intracellular signal results in the phosphorylation of the 18 kDa subunit of complex I, which modulates the activity of complex I (Papa S. The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade. Biochim Biophys Acta. 2002 Sep 10; 1555 (1-3): 147-153). In addition to the mentioned example, there is also a special active and inactive form: the transition to oxygenation, mitochondrial Ca ²⁺ , Mg ²⁺ levels, resp. it is also regulated by natural complex I Q site inhibitors (Maklashina E, Kotlyar AB, Cecchini G. Active / de-active transition of respiratory complex I in bacteria, fungi, and animals. Biochim Biophys Acta. 2003 Sep 30; 1606 (1-3) : 95-103 Review). Mitochondrial nitric oxide synthase (mtNOS) is functionally linked to complex I. Inactivation of complex I results in the production of superoxide by the enzyme mtNOS, consequently inactivation of mitochondrial complex I may contribute to oxidative stress (Parihar MS, Parihar A, Villamena FA, Vaccaro PS, Ghafourifar P. Inactivation of mitochondrial respiratory chain complex I leads to mitochondrial to become prooxidative Biochem Biophys Res Commun 2008 21 Mar;

367 (4): 761-767; Epub 2008 Jan. 11.) through proapoptotic stress-activated JNK and NF-κB transcription factor pathways, which are well known factors in the development of insulin resistance and inflammation. Mitochondria also regulate a number of cellular functions. Respiratory stress in the mitochondria activates Ca ²⁺ / Calcineurin-mediated signaling pathways, which activate and inhibit a number of nuclear genes. results in inhibition. This signal is propagated through the activation of regulatory proteins NFκB, c-Rel / p50, C / EBP, CREB and NFAT (M Guha, Molecular Biology of the Cell V: 21, 3578-3589, 2010).

The regulation of mitochondrial signaling pathways and their networks and their contribution to pathological processes is not known in detail. However, mitochondrial dysfunction has been shown to play a prominent role in the pathogenesis of mitochondrial, neurodegenerative diseases, and diabetes and obesity.

Mitochondrial diseases are a group of diseases with a heterogeneous pathology, typically with decreased cellular energy production. The defect in oxidative phosphorylation is most often associated with insufficient complex I activity, usually due to mutations in structural genes (Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS. of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy (Ann Neurol. 2002 Nov; 52 (5): 534-542). However, mutations in the complex I assembly factor genes may cause similarly isolated complex I deficiencies (Saat A, Vogel RO, Hoefs SJ, Wessels HJ, Willems PH, Venselaar H, Shaag A, Barghuti F, Reish O, Shohat M, Huynen MA, Smeitink JA, van den Heuvel LP,

Nijtmans LG. Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66) -interacting complex I assembly protein, cause fatal neonatal mitochondrial disease. Am J Hum Genet. 2009 Jun; 84 (6): 718-727. Epub 2009 May 21). Respiratory chain complex I dysfunction occurs in cases of hereditary mitochondrial encephalitis in ca. Responsible for 30%. Injury to complex I has also been demonstrated in other neurological diseases that may be associated with mitochondrial dysfunction induced by protein aggregates, such as e.g. the sporadic resp. familial Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob syndrome, hereditary bilateral paralysis, Friedrich's ataxia. Products of genes that may be associated with Parkinson's disease, such as alpha-synuclein, Parkin, PINK1, DJ-1, LRRK2, and HTR2A demonstrated mitochondrial localization and compromised mitochondrial function reduced neuronal cell survival. There is currently no effective treatment for mitochondrial diseases. Decreased complex I activity has been demonstrated in aging, asthma, Down syndrome (Janssen RJ, Nijtmans LG, van den Heuvel LP, Smeitink JA.

Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis. 2006 Aug; 29 (4): 499-515. Epub 2006 Jul 11. Review.). Mitochondrial dysfunction inhibits the level and function of the tumor suppressor p53, which contributes to the development of cancer (Compton S, Kim C, Griner NB, Potluri P, Scheffler IE, Sen S, Jerry DJ, Schneider S, Yadava N.

Mitochondrial dysfunction impaired tumor suppressor p53 expression / function. J Biol Chem. 2011 Jun 10; 286 (23): 2029720312. Epub 2011 Apr 18).

A II. Decreased complex I activity and mitochondrial function are observed in type 1 diabetes mellitus (Ritov WC, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE. Deficiency of subsarcotence mitochondria in obesity and type 2 diabetes. Diabetes. 2005 Jan; ): 8-14). A II. The incidence of type 2 diabetes has increased significantly in many countries around the world. Obesity is strongly correlated with the risk of developing diabetes. Insulin resistance and β-cell dysfunction are listed in Appendix II. at the center of the pathomechanism of type 2 diabetes (Leahy JL, Hirsch IB, Peterson KA, Schneider D. Targeting beta-cell function early in the course of therapy for type 2 diabetes mellitus. J Clin Endocrinol Metab. 2010 Sep; 95 (9): 4206-4216 .Epub 2010 Aug 25 Review). A II. Current treatment protocols for type 2 diabetes include the use of oral antidiabetic agents, insulin, antihypertensives, and antihypertensive agents, or combinations thereof, in combination with lifestyle changes. Currently available drugs do not stop the II. development of type 2 diabetes, they only slow down the progression of certain complications, such as microangiopathy, while barely protecting against macrovascular disease. A II. Targeted mechanisms of type 2 diabetes include insulin resistance, metabolic overload, and increased free radical production - is likely to result in a change in the progression of the disease. Decreased mitochondrial function and increased mitochondrial free radical production, as well as the resulting oxidative stress, play a key role in the causal mechanisms of insulin resistance. Excess substrate is a major contributor to mitochondrial dysfunction. The skeletal muscle of obese and diabetic patients is characterized by a significant decrease in mitochondrial function (Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, Mandarino LJ Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF 1. Proc Natl Acad Sci U S A.

2003 Jul 8; 100 (14): 8466-71. Epub 2003 Jun 27), with decreased complex complex I activity in the background (Ritov WC, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE. Deficiency of subsarcolemic mitochondria in obesity and type 2 diabetes. Diabetes. 2005 Jan; 54 (1): 8-14) with decreased respiratory capacity in the presence of a smaller and less extensive mitochondrial network (Mogensen M, Sahlin K, Fernström M, Glintborg D, Vind BF, Beck-Nielsen H, H0jlund K. Mitochondrial respiration is reduced in skeletal muscle of patients with type 2 diabetes.Diabetes 2007 Jun; 56 (6): 1592-9 Epub 2007 Mar 9.). Significantly reduced oxidative phosphorylation and expression of genes involved in mitochondrial function in skeletal muscle of diabetic patients (Sreekumar R, Nair KS. Skeletal muscle mitochondrial dysfunction & diabetes. Indian J Med Res. 2007 Mar; 125 (3): 399-410. Review) . Downregulation of mitochondrial complex I gene transcription has been observed for three days on an isoenergetic-high-fat diet, suggesting a causal relationship to insulin resistance (Sparks LM, Xie H, Koza RA, Mynatt R, Hulver MW, Bray GA, Smith SR. fat diet coordinatively downregulates genes required for mitochondrial oxidative phosphorylation in skeletal muscle. Diabetes. 2005 Jul; 54 (7): 1926-1933). The above observations have led to the concept that decreased ability to oxidize fats due to acquired or hereditary mitochondrial insufficiency is a major cause of fat accumulation and insulin resistance (Loell BB, Shulman GI Mitochondrial Dysfunction and Type 2 Diabetes Science, 2005 Jan 21; 307 (5708): 384-387). Mitochondrial-derived oxidative stress affects itself in part, as mitochondrial free radicals from excessive substrate presence exacerbate the late stages of diabetes by activating stress-induced signaling pathways, thereby contributing to the development of the disease (Evans JL, Goldfine ID, Maddux BA,

Grodsky GM, Oxidative stress and stress-activated signaling patways: a unifying hypothesis of type 2 diabetes Endocr Rev 2002 Oct; 23 (5): 599-622).

The root drug (Taraxaci radix) is widely used in addition to the aboveground part (Taraxaci folium, Taraxaci herba) of the dandelion (Aaraacacum officinale) of the order Asteraceae. Substance-rich triterpenes (triterpene alcohols and esters), phytosterols, germacranolide-type sesquiterpene γ-lactones and their glycosides, caffeic acid derivatives, and polyfructosan inulin are significant. Dandelion has been considered a European herbicide for more than 700 years due to its diuretic effect, which stimulates digestion and stimulates liver and bile function. Taraxacum officinale is an herb commonly used for ocular purulent inflammation (panophthalmia), constipation, and diabetes (J Ethnopharmacol. 2011; 135 (1): 102-9). Taraxaci radix is well known and traditionally used in the prevention of diabetes and in the adjunctive therapy of diabetes (Rédei D., Szendrei K .: Pharmacy 49, 615-622, 2005; Önal S., Timur S., Okutucu B., Zihnioglu F .: Preparative Biochemistry and Biotechnology 35, 29-36, 2005; Funke I., Melzig M.F .: Zeitschrift für Phytotherapie 26, 271-274, 2005; Cicero A.F.G., Derosa G., Gaddi A. Acta Diabetol 41, 91-98, 2004 ).

Taraxaci radix is an outstanding component of many blood glucose-lowering tea blends together with other herbs (Béla Alró Varad: Medicinal effects of herbs: Description of our Hungarian herbs, their collection and use in everyday life in the service of health 1941; Dr. Augustin-Dr. herbs I-II Bp., 1948, Ministry of Agriculture;

Medicinal plants - 1969, Rápóti-Romváry, Medicine). Taraxaci radix is an important component of a number of pharmaceutical products that, although not classified as drugs, have been registered with the ATC group as oral antidiabetic agents. The license was confirmed by the National Institute of Pharmacy and Food Health (OGYI): Herbarium-Mecsek diet tea blend (001/86), Diabole filter tea blend (926/06), Herbarium food supplement filter tea blend (981/06) .

Taraxacum officinale is also commonly used in inflammatory diseases (Seo SW, Koo HN, An HJ, Kwon KB, Lim BC, Seo EA, Ryu DG, Moon G, Kim HY, Kim HM, Hong SH. Taraxacum officinale protects against cholecystokinin -induced acute pancreatitis in rats World J Gastroenterol 2005 Jan 28; 11 (4): 597-599) - the advantageous property is attributed to the triterpene components (taraxsteol, arnidiol, faradiol) (Committee on Herbal Medicinal Products (HMPC), EMA / HMPC / 212895/2008, November 12, 2009); Czygan F.C .: Taraxacum officinale Zeitschrift für Phytotherapie 11, 99-102, 1990; Barnes J., Anderson A.L., Phillipson J.D .: Herbal Medicine: Dandelion, Pharmaceutical Press, London pp. 204-206, 1997).

The potential antidiabetic effect of Taraxacum officinale is likely in a number of reports, but the current state of science does not confirm this. Among the mechanisms of action revealed so far, the enhancement of insulin secretion may be outstanding: 40 μg / ml Taraxacum extract increased the in vitro insulin secretion at normal glucose levels using glibenclamide control (Hussain Z., Waheed A., Quereshi R.A., Kurdhan D.K., Verspokl ., Hasan M .: The effect of medicinal plants in Islamabad and Muree region of Pakistan on insulin secretion from INS-1 cells Phytotherapy Research, 18, 73-77, 2004; Schütz K.,

Carle R., Schieber A .: Taraxacum - A review on its phytochemical and pharmacological profile. Journal of Ethnopharmacology, 107, 313-323, 2006).

The aqueous extract of Taraxaci radix significantly reduced the activity of the α-glucosidase enzyme system by 21–27%, depending on the experimental conditions. Α-glucosidase, also known as maltase (EC 3.2.1.20), is a carbohydrate hydrolase that releases α-glucose terminally from the non-reducing α- (1 ^ 4) ends and α-D-glucose α- (1 ^ 4) ) hydrolyzes glycosides of glucose containing a hydroxyl group. Αglucosidase breaks down starch and disaccharides into glucose. The enzyme also hydrolyzes sucrose because they also contain α-glycosidic bonds. If it inhibits the intestinal tract, the starch does not release glucose, is not absorbed in the intestinal villi, and does not raise blood sugar levels. The inhibition was comparable to that of Glucobay®, gliclazide (Betanorm®), glimeride (Amaryl®) (Önal S., Timur S., Okutucu B., Zihnioglu F .: Inhibition of αglucosidase by aqueous extracts of some potent antidiabetic medicinal herbs). Preparative Biochemistry and Biotechnology 35, 29-36 (2005).

In addition, an aqueous extract of Taraxaci radix significantly reduced hepatic maldialdehyde (MDA) levels in the liver and blood glucose levels in STZ-treated diabetic rats. The MDA level indicates the degree of oxidative stress, so if something reduces this, it has a strong antioxidant effect or eliminated the cause of free radical production. (Cho S.Y., Park Y.J., Parl E.M., Choi M.S., Lee M.K., Jeon S.M., Jang M.K., Kim M.J., Park Y.B .: Alternation of hepatic antioxidant enzyme activities and lipid profile in streptozotocin-induced diabetic rat by supplementation of dandelion water extract. Clinica Chimica Acta 317, 109-117, 2002).

The hypoglycaemic effect of Taraxacum officinale has not been established in human studies. Only one case was described in 2010, according to which a II. A hypoglycaemic effect was observed in a diabetic female patient with type 2 diabetes mellitus (Goksu E., Eken C., Karadeniz O., Kumkyilmaz O. First report of hypoglycemia secondary to dandelion) (Taraxacum officinale). American Journal of Emergency Medicine 28, 111.e1-111.e2., 2010]).

Overall, Taraxacum officinale is known to treat certain diseases that are not associated with mitochondrial damage and / or decreased mitochondrial complex I activity.

Some herbs belonging to the genus Viola are widely used in folk medicine. The leaves and roots of Viola odorata have a mucolytic, antiperspirant, blood purifying effect and the flower also has a mucolytic, sedative and blood pressure lowering effect. Viola tricolor has expectorant, diuretic, hemostatic, and anti-inflammatory effects (Keville K., The Illustrated Encyclopedia of Herbs, Chancellor Press, 302 (1992); Hansel, R., Keller, K., Rimpler H., Schneider, G. 0 : Hager's Handbuch der Pharmazeutischen Praxis, Springer Verlag, 6th Band, 1141-1153). Both Viola odorata and Viola tricolor teas and extracts from the aboveground part are often used in colds, inflammation, skin and rheumatic diseases (Leporatti, M. L., Ivancheva, S .: Preliminary comparative analysis of medicinal plants used in traditional medicine of Bulgaria and Italy, J. Ethnopharm., 87, 123-142 (2003)). Certain parts of the species Viola odorata are used to treat chronic bronchitis, whooping cough and asthma due to its expectorant and antitussive effects. Viola odorata has also been shown to be effective in migraines. Viola odorata leaf is effective for hoarseness, sore throat, sleep disorders and neurotic disorders. The active ingredients in Viola tricolor are flavonoids such as rutin, quercetin, luteolin, luteolin-7-glycoside, scoparin, saponarin, saponaretin, violantin, orientin and isoorientin, vicenin-2 and vitexin (Hansel R et al., Supra). Violarvensin, a flavone di-C-glycoside isolated from the pansy Viola arvensis (Carnat, A. P., Carnat, A., Fraisse, D., Lamaison J. L. Violarvensin, New Flavone Di-C-glycoside from Viola arvensis, J. Nat. Prod., 61, 272-274 (1998)). The presence of triterpene saponins in Viola species, e.g. urzolic acid and the galactose containing it and its oxidized form, galacturonic acid as a sugar component, have been confirmed by several authors (Hansel et al., supra). However, other authors attribute the violapeptide I protein to hemolytic properties (Schopke, Th., Hasan Agha, M. I., Kraft, R., Otto, A., Hiller, K .: Haemolytic active components of Viola tricolor L. and viola arvensis Murray , Sci. Pharm., 61, 145-153 (1993)). According to literature data, carotenoids that can be isolated from Viola tricolor include violaxanthin and its derivatives (Hansmann, P., Kleinig, H .: Violaxanthin esters from Viola tricolor flowers, Phytochemistry, 21, 238-239 (1982); Molnár, P., Szabolcs, J .: Occurence of 15-cis-violaxanthin in Viola tricolor, Phytochemistry, 19, 623-627 (1980) Molnár, P., Szabolcs, J., Radics, L .: Naturally occuring di-cis -violaxanthins from Viola tricolor: Isolation and identification by 1H NMR spectroscopy of four di-cis-isomers, Phytochemistry, 25, 195-199 (1986); Radics, L., Molnar, P., Szabolcs, J .: 13C NMR evidence for the central mono-cis-stereochemistry of naturally occurring violaxanthin isomers, Phytochemistry, 22, 306 (1983)], violeoxanthine, lutein, luteinepoxide and neoxanthine (Hansel et al., supra). and violanin chloride are also found in Viola tricolor (Saito, N., Timberlake, C. F., Tucknott, O. G., Lewis , I. A. S .: Fast atom bombardment mass spectrometry of the anthocyanins violan and platyconin, Phytochemistry, 22, 1007-1009 (1983)). Phenolecarboxylic acid and its derivatives and compounds derived therefrom: trans and ciscoumaric acid, 5-hydroxysalicylic acid, p-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, trans-caffeic acid, 3,4-dihydroxybenzoic acid, 4-hydroxy-3 -methoxybenzoic acid, salicylic acid and its derivatives are important components of Viola tricolor extracts. Other ingredients available from Viola tricolor include glucose-based polysaccharides, galactose, arabinose, rhamnose, xylose, and glucuronic acid, as well as vitamins E and C, triacylglycerols, and fatty substances (Hansel R. et al., Supra).

Cyclic peptides isolated from Viola tricolor, vitri A, vary A and E, have been shown to be cytotoxic in certain human tumor cell lines (Svangard, E., Goransson, U., Hocaoglu, Z., Gullbo, J., Larsson, R. , Claeson, P., Bohlin, L .: Cytotoxic Cyclotides from Viola tricolor, J: Nat. Prod., 67, 144-147 (2004). The antioxidant properties of Viola tricolor (Mantle, D., Eddeb, F., Pickering A. T.: Comparison of relative antioxidant activities of British medicinal plant species in vitro, J. Ethnopharm., 72, 47-51 (2000)) and significant antithrombin activity has been reported (Goun, E. A., Petrichenko, V. M., Solodnikov, S. U., Suhinina, T. V., Kline, M. A., Cunningham, G., Nguyen, C., Miles, H .: Anticancer and antithrombin activity of Russian plants, J. Ethnopharm., 81, 337-342 (2002)). The antimalarial property of Viola surinamensis has been described (Lopes, N. P., Kato, M.

J., de A. Andrade, E. H., Maia, J. G. S., Yosida, M., Planchart, A. R., Katzin, A. M .: Antimalarial use of volatile oil from leaves of Viola surinamensis (Rol.) Warb. by Waiipi Amazon Indians, J. Ethnopharm., 67, 313-319 (1999)). Data on the antipyretic effect of Viola odorata can also be found, which, like the anticoagulant effect, may be related to the salicylate content of Viola species (Khattak, S. G., Naeemuddin, G., Ikram, M .: Antipyretic studies on some indigenous Pakistani medicinal plants, J. Ethnopharm. 14, 24-51 (1985)).

Although some herbs belonging to the genus Viola have already been used in the use of certain diseases as herbal tea ingredients and extracts, no pharmaceutical preparation has yet been made from these herbs.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to prevent or control diseases associated with mitochondrial damage and / or impaired function of the complex I enzyme system. treatment with a pharmaceutical preparation based on herbal extracts.

The object of the present invention can be achieved with a pharmaceutical composition comprising appropriate extracts of Taraxacum officinale and Viola tricolor. The pharmaceutical composition is a mixture of active ingredients containing a solid remaining after evaporation of the extract from the aboveground parts and / or roots of said plants, optionally supplemented with one or more customary pharmaceutical excipients. The pharmaceutical composition of the present invention is useful for enhancing mitochondrial complex I activity (rotenone-sensitive NADH: Ubiquinone oxidoreductase activity) and for preventing and treating diseases associated with mitochondrial dysfunction and / or impaired function of the complex I enzyme system, as exemplified above. treatment

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical composition of the present invention comprises, on the one hand, one or more extracts of the species Taraxacum officinale. This herb belongs to the family Asteraceae, and both the above-ground parts (Taraxaci folium, Taraxaci herba) and the root (so-called root drug) (Taraxaci radix) can be used for the purposes of the present invention.

On the other hand, the pharmaceutical composition of the present invention contains one or more extracts of the species Viola tricolor. Both above-ground and root roots of this herb can be used for the purposes of the present invention.

The above-ground part of an herb is the leaf and / or stem and / or flower (inflorescence).

The extract according to the invention can be prepared (in other words, extraction or extraction) according to a method known per se. For this purpose, extraction is carried out from the above-ground part of the herb which can be used according to the present invention, optionally after drying and size reduction. This is done with water or an organic solvent (e.g. ethanol from alcohols) or an aqueous solution of organic solvents (e.g. aqueous ethanol), generally between 0 and 100 ^° C, but preferably at 20 ^° C. Extraction is performed with stirring in most cases, although ultrasonication can also be used. The extract is separated from the plant parts by sedimentation or by squeezing out the plant, filtering, centrifuging or a combination of the above. The resulting extract can then be formulated into a pharmaceutical composition such as an aqueous solution or syrup. In another embodiment, the solvent can be removed by evaporation, spray drying, or freeze-drying, and the remaining solid can be used as the active ingredient in the preparation of the pharmaceutical composition of the present invention.

As used herein, the term active ingredient refers to an aqueous solution, syrup or solid residue obtained as a result of the extraction, provided that it has previously been in solution in the extract and can be prepared from an herbal extract. Both the extract and the solid residue obtained from it are characterized by flavonoid and polyphenol content. For example, in a preferred embodiment, the solid residue has a flavonoid content of 2.8 to 3.4 g / 100 g and a total polyphenol content of 9.7 to 11.0 g / 100 g. It will be apparent to those skilled in the art that within the scope of the present invention based on herbs, the term active ingredient encompasses several compounds at the same time.

The term "pharmaceutical composition" refers to a formulation and dosing protocol suitable for the prevention or treatment of diseases and for oral, parenteral, rectal and subcutaneous administration or topical administration. The pharmaceutical composition of the present invention is in a solid or liquid state; in addition, it may optionally contain one or more pharmaceutical excipients in addition to the active ingredient. The pharmaceutical composition of the present invention generally contains 0.1 to 100%, preferably 1 to 50%, more preferably 5 to 30% of the active ingredient. It should be noted that a 100% active ingredient content is only possible in certain cases, e.g. in capsules where dilution or other excipients are not required. In most dosage forms, diluents and / or other excipients are required to prepare the pharmaceutical composition.

Solid pharmaceutical compositions suitable for oral administration include powders, capsules, tablets, film-coated tablets, microcapsules; which may also contain excipients as excipients, e.g. gelatin, sorbitol, polyvinylpyrrolidine; fillers such as lactose, glucose, starch, calcium phosphate; tableting excipients such as magnesium stearate, talc, polyethylene glycol, silica gel; wetting agents such as sodium lauryl sulfate and carriers.

Liquid pharmaceutical compositions suitable for oral administration may be in the form of a solution, suspension or emulsion; which may also contain suspending agents as excipients, e.g. gelatin, carboxymethylcellulose; emulsifying agents such as sorbitan, monooleate; solvents such as water, oils, glycerin, propylene glycol, ethanol; preservatives such as methyl or propyl hydroxybenzoate and carriers.

Parenteral pharmaceutical preparations are generally sterile solutions of the active ingredients. Pharmaceutical compositions suitable for topical administration include solutions, creams, ointments and other formulations known in the art.

The above dosage forms and other formulations are known, for example, from Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, USA (1990). Pharmaceutical compositions generally contain a unit dose. The daily dose may be administered in one or more portions. The actual dose depends on many factors and is determined by your doctor. In general, the typical dose for adult patients weighing 70 kg is 0.1 to 10 g, preferably 1 to 5 g of active ingredient per day.

In general, the pharmaceutical composition can be prepared by mixing the active ingredient and one or more carriers according to methods known in the art. Applicable methods are well known in the literature, e.g. from the Remington manual mentioned above.

It will be appreciated by those skilled in the art that the solid residue of the extract may be encapsulated directly or the extract itself may be converted into a liquid pharmaceutical composition using additional carriers if necessary.

In the case where the extract or active ingredient of the present invention is used for roborization or muscle building using induction of mitochondrial biogenesis, the composition is not for therapeutic purposes. In the context of the present invention, a composition useful for this purpose is also referred to as a pharmaceutical composition. In other words, the pharmaceutical composition of the present invention is not limited to pharmaceutical use within the scope of the invention.

Preparation of extracts and fractions from Taraxacum officinale and Viola Tricolor herbs

The extracellular portion of Viola tricolor was extracted with water, alcohol, and chloroform; the water was extracted with a mixture containing alcohol in various proportions. The extract was prepared by ultrasonic dispersion at 60 ^° C in a 1: 100 volume ratio based on the powdered plant. The resulting extract was pelleted, centrifuged, and the supernatant was freeze-dried and the extract powder was used for further analysis. The extraction agents used in the fractions were as follows: Fraction 1: 100% ethanol; Fraction 2: 100% water; Fraction 3: 100% chloroform; Fraction 4: 100% methanol; Fraction 5: 50% ethanol - 50% water; Fraction 6: 50% methanol to 50% water; Fraction 7: 50% ethanol - 50% chloroform.

During the fractionation of the roots of Taraxacum officinale, four fractions were prepared: Fraction 1 was prepared by aqueous extraction with shaking of the powdered roots at a room temperature of 1: 100 by volume. The resulting aqueous solution was freeze-dried. The composition of the remaining hydrophilic solid extract was determined: sesquiterpene lactone glycosides and some phenolic components became identifiable by thin layer chromatography. Fraction 2 was prepared by chloroform extraction in a similar procedure to that described above. Residual hydrophobic extract - thin layer chromatography using reference materials confirmed triterpenes (taraxasterols, α-, β-amirin, faradiol), triterpene esters (taraxasterol acetate) and phytosterol. For fraction 3, the starting material was an extract of fraction 2. The column was significantly enriched by column chromatography: it contained mainly faradiol (~ 80%) accompanied by a small amount of triterpene. In the case of fraction 4, the extract from fraction 2 was also started, but in this case saponification was carried out with alkali, followed by a repeated extraction step with chloroform. Triterpenes (α-, β-amirin, taraxasterol) and phytosterols (β-sitosterol, stigmasterol) were identified in this fraction.

Stock solutions were prepared from each fraction using DMSO to a final concentration of 20 mg / ml and used in various dilutions.

Extracts of Taraxacum officinale and Viola Tricolor were tested by the following biological tests:

High fat diet - mouse treatment

4-6 month old Balb / c male mice were treated with free access to water and food (21 wt% fat, 50 wt% carbohydrate, 20 wt% protein) for 3 months. The Balb / c control group of the same age and gender received a normal diet (4.5 wt% fat, 53 wt% carbohydrate, 23 wt% protein) for a similar period of time. Animals were ordered from Charles River and acclimatized to our animal housing conditions for 3 weeks prior to the start of the experiments.

Preparation of mitochondrial enriched fraction

Mice were anesthetized with ether and cervical dislocation was performed. After excision of the heart and gastrocnemius, they were minced and rinsed in the following digestion buffer: 250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 mM EGTA. The tissue was excised in 900 μl of explosive medium on ice with a Teflon potter ~12 uniformly. After homogenization, an additional 900 μl of digestion buffer was added and centrifuged (2500 rpm, 5 min, 4 ^° C). The supernatant was then aspirated with a 2 ml syringe, avoiding the fat layer and the pellet, and centrifuged again (2500 rpm, 5 min, 4 ^° C). The supernatant was carefully aspirated again and centrifuged (9500 rpm, 5 min, 4 ^° C). The mitochondrial pellet was taken up in 200 μl of uptake buffer (250 mM sucrose, 20 mM Tris-HCl, pH 7.4, 0.1 mM EGTA) for heart and 150 μl for gastrocnemius and stored at -80 ^° C until use. Mitochondrial protein concentration was determined with the BioRad Protein DC Assay Kit.

Preparation of intact mitochondria

Preparation of intact mitochondria was described by El-Mir et al. (El-Mir MY et al. J Biol Chem. (2000) 275: 223-228). Mice were anesthetized with ether and cervical dislocation was performed. After excision, the heart was minced and rinsed in ice-cold PBS buffer (pH 7.4). Tissue was resuspended in 2.9 ml ice-cold buffer (250 mM sucrose, 20 mM Tris, pH 7.4, 1 mM EGTA) at 4 ^° C using a glass / Teflon pot (25 μm spacing) using 12 strokes. The homogenate was centrifuged at 600 g for 10 minutes at 4 ^° C. The 600 g supernatant was centrifuged again at 10,000 g for 10 minutes at 4 ^° C. The supernatant was carefully removed and the mitochondrial precipitate was resuspended in 200 μl of ice-cold buffer (250 mM sucrose, 20 mM Tris, pH 7.4, 0.1 mM EGTA). Mitochondrial protein concentration was determined with the BioRad Protein DC Assay Kit.

Determination of complex I and II as well as HAR activity

Complex I, II, and HAR activity was performed on a mouse heart-prepared mitoplast using a specific electron acceptor, DCIP (2,6-dichloroindophenol), and NADH or a succinate electron donor.

Rotenone-sensitive complex I activity was determined spectrophotometrically at 600 nm in the following test solution: 25 mM K3PO4, 3.5 g / L BSA, 60 μM DCIP, 70 μΜ decyl ubiquinone, 1.0 μΜ antimycine-A, and 0.1 mM NADH, pH 7.8.

Complex II activity was determined in the following solution: 80 mM K3PO4, 1 g / L BSA, 2 mM EDTA, 0.2 mM ATP, 10 mM succinate, 0.3 mM KCN, 80 μM DCIP, 50 pM decylubiquinone, 1 pM antimycin-A and 3 pM rotenone, pH 7.8.

The amount of complex I present in the mitoplast was determined using HAR (hexaamine-ruthenium (III) chloride) activity. This data is essential to characterize the rate of specific complex I activity. NADH: HAR oxidoreductase activity was measured by oxidation of NADH in the presence of HAR artificial electron acceptor at 460 nm using a Polar Galaxy reader.

Determination of succinate-induced mitochondrial free radical production and NADH levels

H2O2 production was assayed on intact mouse heart mitochondria using the Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) fluorescent probe in the presence of horseradish peroxidase (HRP). H2O2 production was determined indirectly by time-resolved fluorescence measurement by detecting the accumulation of resorphine product. Excitation was in the range of 530 ± 10 nm, while emission was in the range of 600 ± 10 nm. NADH levels were measured in the 460 ± 20 nm range during the parallel reading. The so-called succinate was used as the electron donor to study electron reflux through complex I. Measurements were performed with a POLARstar (BMG Labtechnologies, Germany) fluorescent plate reader.

Glucose tolerance was studied in male Balb / c mice weighing 30 and 30 g, respectively.

At a dose of 100 mg / kg with the active ingredient prepared from Taraxacum officinale using method 'A' in Example 1, in which each test group consisted of 5 animals. On day 5, 1 hour after treatment, the animals were anesthetized by pentobarbital injection and blood was drawn from the eye socket, and the glucose value was determined with an AccuCheck blood glucose meter. Subsequently, 2 g of glucose per kg of body weight was administered intraperitoneally and blood glucose levels were determined at 30, 60, 90 and 30 mg, respectively. 120 minutes after glucose administration. Animals in the control group received the same amount of vehicle fluid (distilled water).

The results of treatment with Taraxacum officinale using the method ‘A’ given in Example 1 are shown in Table 1 (see below). A ‘B’, resp. The active substances obtained from Taraxacum officinale by method 'C' showed quite similar results, therefore these data are not reported.

The following results were obtained in the above tests:

# 1 Direct effect of herbal extracts on specific complex I activity - in the presence of palmitate - on mouse liver and heart mitoplast

Specific complex I activity accounts for 20% of total NADH consumption. Direct treatment with mitoplast with herbal extract increased specific NADH: Ubiquinone oxidido reductase complex I activity (complex I). A significant increase was observed for all herbal extracts tested. Similar results were obtained with the fractions of Taraxacum officinale and Viola Tricolor and their combinations. Fraction combinations showed the strongest increase. (No9 Taraxacum- # 3 & Viola- # 2 1.66-fold; No10 Taraxacum- # 3 & Viola- # 3 1.61-fold; Table 1).

Extracts Specific Complex I activity Liver mitoplast Heart mitoplast Control Control Palmitate, 80uM average scatter average scatter average Scattering 1. Control 1 0.1 1 0.1 0.26 0.02 2. Viola tricolor, 12 ug / ml 1.37 0.1 1.31 0.07 0.39 0.02 3. Taraxacum, 12 ug / ml 1.46 0.18 1.22 0.03 0.35 0.04 4. Tarax. + Viola (1: 1), 12 ug / ml 1.48 0.24 1.34 0.1 0.41 0.03 5. Taraxacum - # 3, 12 ug / ml 1.56 0.11 1.34 0.06 0.45 0.06 6. Taraxacum- # 4, 12 ug / ml 1.42 0.14 1.24 0.04 0.38 0.04 7. Viola- # 2, 12 ug / ml 1.45 0.1 1.35 0.09 0.42 0.03 8. Viola - # 3, 12 ug / ml 1.39 0.1 1.41 0.11 0.51 0.07 9. Taraxacum - # 3, 6 ug / ml + Viola- # 2.6 ug / ml 1, 66 0.11 1.41 0.12 0, 61 0.03 10. Taraxacum - # 3.6 ug / ml + Viola- # 3.6 ug / ml 1, 61 0.12 1.52 0.21 0, 65 0.06

11. Taraxacum - # 4.6 ug / ml + Viola- # 2.6 ug / ml 1.45 0.13 1.35 0.17 0.51 0.04

Table 1: Effect of herbal extracts on specific complex

I activity

Specific complex I activity accounts for 80% of total NADH intake in the heart. The inhibitory effect of palmitate direct complex I (NADH: ubiquinone oxidido reductase) can be observed and can be related to the active-deactive transition of the enzyme (Loskovich MV, Grivennikova VG, Cecchini G, Vinogradov AD. Inhibitory effect of palmitate on the mitochondrial NADH: (complex I) as related to the active-de-active enzyme transition Biochem J. 2005 May 1; 387 (Pt 3): 677-683). Palmitate treatment dose-dependently reduced complex I activity on cardiac mitoplasts in line with literature data. Combinations of fractions of the studied herbal extracts significantly reduced palmitate-induced inhibition (Table 1). These data support that the appropriate combinations of fractions of Taraxacum officinale and Viola tricolor herbal extracts synergistically increase specific complex I activity even in the presence of the inhibitory palmitate.

# 2 Direct effect of herbal extracts on free radical production in the presence of palmitate in intact mouse heart mitochondria

In the presence of palmitate, succinate-induced free radical production was significantly increased in the intact mouse heart mitochondria. The studied herbal extracts, fractions and their combinations synergistically significantly reduced succinate-induced increased free radical production in both control and palmitate-treated mitochondria (Control vs. 9. Taraxacum- # 3 & Viola- # 2, 6.3 vs. 3.4; Control vs. 10. Taraxacum- # 3 & Viola- # 3, 6.3 vs. 3.8; Palm Control vs. 9. Taraxacum- # 3 & Viola- # 2, 8.2 vs.

4.4 vs .; Palm. control vs. 10. Taraxacum- # 3 & Viola- # 3, 8.2 vs. 4.7). The results are shown in Table 2.

The results support that the above fraction combinations are effective against succinate-induced backflow electron current generation with increased free radical production.

H2O2 production Control 40 μM Palmitate Basis 2.5 mM succinate Basis 2.5 mM s succinate average scatter average scatter average scatter average scatter 1. Control 1 0.07 6.35 0.09 0.54 0.05 8.27 0.07 2. Viola tricolor, 12 ug / ml 0, 62 0.11 5.03 0.14 0.31 0.03 5.33 0.08 3. Taraxacum, 12 ug / ml 1, 97 0.07 5.32 0.03 2.22 0.02 6, 08 0.04 4. Tarax. 6 ug / ml + Viola 6 ug / ml 1.42 0.13 5.13 0.34 1.33 0.23 5, 88 0.12 5. Tarax. - # 3, 12 ug / ml 2, 61 0.34 4.82 0.41 2.21 0.21 5.22 0.34 6. Tarax. - # 4, 12 ug / ml 1, 61 0.21 5.12 0.22 1.43 0.19 5.41 0.23 7. Viola- # 2, 12 ug / ml 0.34 0.07 4.13 0.04 0.42 0.07 4.56 0.31 8. Viola - 0.45 0.04 4.61 0.08 0.56 0.06 4.81 0.41

# 3, 12 ug / ml 9. Tarax. # 3, 6 ug / ml + Viola- # 2.6 ug / ml 2.12 0.45 3.47 0.21 2.35 0.56 4.41 0.35 10. Tarax. # 3, 6 ug / ml + Viola- # 3.6 ug / ml 2.34 0.38 3.89 0.31 2, 65 0.23 4.78 0.45 11. Tarax. # 4, 6 ug / ml + Viola- # 2.6 ug / ml 1.23 0.12 4.81 0.42 1.57 0.31 4, 65 0.78 12. Tarax. # 4, 6 ug / ml + Viola- # 3.6 ug / ml 1.32 0.18 4.17 0.21 1, 68 0.32 4.89 0.19

Table 2: Effect of herbal extracts on mitochondrial free radical production # 3 Effect of Taraxacum officinale, Viola tricolor and their fractions on mitochondrial function in mice on a high fat diet Mitochondrial complex I, HAR, complex II activity, basal and succinate-induced free radical production were examined. The high-fat diet decreased HRT activity and normalized complex I activity compared to the normal diet (~ 7%). HAR activity alone was 10% lower, which also confirmed a decrease in the absolute amount of complex I. In parallel, a decrease in complex II activity was also observed. Treatment with Taraxacum officinale and Viola tricolor in the heart mitochondria of mice fed a high-fat diet did not result in a change in HAR activity compared to controls. However, HAR activity, normalized complex I, resp. complex II activity was significantly increased in the case of herbal extracts, their fractions and in the presence of combinations thereof in a synergistic manner (see Table 3). These data support that both herbs and their fractions and compositions are suitable for the restoration of normal mitochondrial function - even under lipotoxic conditions modeling the pathological situation.

Medicines Electron Transport Chain Complex I / HAR activity Complex II activity HAR activity average scatter average scatter average scatter Control 1.00 0.25 1 0.11 1.00 0.07 Mag.Fat.D. 0, 93 0.11 0, 95 0.11 0, 91 0.12 M.Zs.D. + Tarax. 1.57 0.14 1.37 0.24 0.88 0.05 M.Zs.D. + Viol 1.76 0.27 1.53 0.34 0.87 0.04 M.Zs.D. + Tarax. # 3, Viola- # 2, 1, 92 0.37 1.73 0.22 0, 93 0.03 M.Zs.D. + Tarax. # 3, Viola # 3, 1.88 0.31 1.73 0.41 0.89 0.10

Table 3: Effect of herbal extracts on mitochondrial function

Another important aspect is the evolution of mitochondrial free radical production, which well reflects mitochondrial function (see Table 4). No significant difference in basal free radical production was found with the 9-day high-fat diet, but an increase of about 2323% compared to the normal diet was observed with succinate treatment. In the presence of Taraxacum officinale, the basal free radical production did not change in the case of the high-fat diet, but in the presence of succinate, the free radical production decreased significantly: it was lower than in the normal diet (Tarax. Offic. + M.Zs.D. vs. M.Zs.D. 1.47 ± 0.06 vs. 1.92 ± 0.05). Similar values were obtained for Viola tricolor (Viola. Tricol. + M.Zs.D. vs. M.Zs.D. 1.62 ± 0.14 vs. 1.92 ± 0.05). Combinations of fractions were also excellent, although here the basic free radical emission was higher.

These data support that a high-fat diet does not increase basal free radical production, whereas increased succinate-induced free radical production, a sign of mitochondrial dysfunction, confirms the dysfunction of the complex I enzyme system. Both herbs and their fractions and compositions are suitable for the restoration of normal mitochondrial function, reducing the differences in mitochondrial complex I and II even with lipotoxicity.

average scatter average scatter Normal Diet 1.00 0.15 1.56 0.08 High Fat Diet 1.06 0.17 1, 92 0.05 M.Zs.D. + Tarax. 1.13 0.17 1.47 0.06 M.Zs.D. + Viola 1.21 0.15 1, 62 0.14 M.Zs.D. + Tarax. # 3, 1.32 0.14 1.58 0.17

Viola- # 2, M.Zs.D. + Tarax. - # 3, Viola- # 3, 1.22 0.11 1.48 0.29

Table 4: Effect of herbal extracts on mitochondrial free radical production # 4 Effect of Taraxacum officinale, Viola tricolor fractions and combinations on glucose tolerance in mice on a high fat diet The consequences of a high fat diet were studied. The use of the pharmaceutical composition of the present invention significantly improved glucose tolerance in mice. In the table below, blood glucose values are given in [mM], measured before oral administration of 2 g sugar / kg body weight (0 min), respectively. They occurred 30, 60 and 120 minutes after administration. The treatment protocol for the animals is shown in Table 5. Animals in the high fat diet group i.p. received a dose of 120 mg active substance / kg body weight, corresponding to Taraxacum officinale or Viola tricolor extract, resp. they consisted of their respective factions.

Elapsed time after administration of 2 g / kg glucose [min] Blood glucose level [mM] Standard Diet, High Fat. Diet, 21 days Mag. Diet. 21 days + 5 days 120 mg / kg Tarax. offic. Mag. Diet. 21 days + 5 days 120 mg / kg Viola tricolor Mag. Diet. 21 days + 5 days 60 mg / kg Tarax- # 3 + 60 mg / kg Viola- # 2 Mag. Fat. Diet. 21 days + 5 days 60 mg / kg Tarax- # 3 + 60 mg / kg Viola- # 3

0 6, 6 0, 8 8.5 0, 6 8.3 0.4 8.9 0.7 7.2 1.1 6.7 0.5 30 15.4 1.2 25.1 1.1 20.6 1.3 22.3 0, 9 18.2 1.2 16.2 1.4 60 10.1 0, 6 24.3 1.5 21.7 0.9 21.2 1.2 18.3 0.7 17.4 1.2 120 7.1 0.5 16, 6 1.2 12.3 1.5 13.3 0.7 11.3 1.4 12.1 1.3 AUC 1228 56 2472 53 2088 53 2155 55 1816 54 1732 32

Table 5: Effect of herbal extracts on OGTT values in mice on a high fat diet

It can be seen from Table 5 that after intraperitoneal administration of 2 g glucose / kg body weight, blood glucose levels increased significantly in the standard / normal diet group: the initial 6.6 mM (animals were fasted before the start of the test) increased by 15.4 mM- 30 minutes after treatment; After 120 minutes, glucose levels returned to baseline (AUC = 1228 ± 56). The group on a high-fat diet (21 days) had a baseline blood glucose level of 8.5 mM (these animals were also fasted before the start of the test). The glucose concentration rose to 25.1 mM 30 minutes after the start of the test and the glucose concentration was 16.6 mM at 120 minutes - well above baseline (AUC = 2472 ± 53). In the high-fat diet group treated with Taraxacum officinale for the last 5 days, the increase in blood glucose was slow and did not exceed a maximum of 22 mM (AUC = 2088 ± 53). A similar pattern of increase in blood glucose was observed after Viola tricolor treatment (AUC = 2155 ± 55). The effect of the increase in synergistic complex I activity observed during the combination of fractions was studied in the oral glucose tolerance test (OGTT). The increase in blood glucose was very slow in animals receiving 60 mg / kg Taraxacum officinale # 3 and 60 mg / kg Viola tricolor # 2 for 5 days, with not even the highest blood glucose (AUC) = 1816 ± 54).

Interestingly, these results were slightly better than those obtained from animals receiving Taraxacum officinale # 2 & Viola tricolor # 3 fraction (AUC = 1732 ± 32).

In summary, extracts of both herbs significantly reduced blood glucose levels following glucose bolus administration. A synergistic effect was observed in mice fed a high-fat diet after administration of combinations of the above fractions of Taraxacum officinale and Viola tricolor: the maximal effect was seen with equal proportions of Taraxacum officinale- # 2 & Viola tricolor- # 3 fractions, and - Also a combination of # 3 & Viola tricolor # 2.

# 5 Toxicity Test Male Balb / c white mice were treated i.p. 500 mg / kg for 4 weeks in the 4 groups mentioned in the glucose tolerance test. This was 4 times the daily dose used for treatment. We examined the total extract from both herbs and the combination of their selected fractions. The behavior and body weight of the animals were checked on a daily basis, no changes were observed during the 1 week examined. Based on these results, it was concluded that the 500 mg / kg dose had no acute toxic effects in mice in any combination of fractions or in the total extract.

Utilization of the invention

The specific combination of extract fractions from Taraxacum officinale and Viola tricolor, as previously defined, may be particularly effective in the following conditions:

I. Use through action on mitochondrial function and oxidative phosphorylation

(a) Conditions and diseases requiring rapid and improving mitochondrial status:

- conditions associated with prolonged bed rest, weight-related illnesses;

- recovery phase following anorexia.

b) Conditions requiring increased mitochondrial performance:

- to support muscle building training (especially striated muscle);

- to support increased activities and subsequent conditions that require high muscle performance (especially striated muscle);

- to relieve muscle spasms and cramps;

- to facilitate altitude adaptation.

(c) Conditions associated with increased mitochondrial weight loss and damage:

- oxidative damage;

- Acute and chronic myocardial and peripheral nerve damage associated with hypoxia-reoxygenation.

C1) Diseases associated with mitochondrial DNA damage:

- MERRF syndrome;

- Leber's hereditary optic neuropathy;

- MELAS;

- Leigh disease;

- Kern-Sayre syndrome;

- chronic progressive ophthalmoplegia;

- Alper syndrome;

- Down syndrome;

- Multiple Sclerosis.

C2) Diseases not associated with mitochondrial DNA damage:

- ALS (Amiotrophic Lateral Sclerosis);

- Huntington's disease;

- Alzheimer's disease;

- Parkinson's disease.

d) Exogenous conditions causing mitochondrial damage: - Skin damage after UV radiation (sunburn, pre-cancerous condition).

e) Myopathies with special regard to myocardial infarction.

II. Treatment of metabolic diseases due to insufficient mitochondrial function: - diabetes;

- insulin resistance;

- metabolic syndrome;

- obesity.

III. Slowing the aging process and the development of cancer associated with mitochondrial dysfunction.

ARC. Other applications in which the recovery of complex I activity is accompanied by an improvement in cNOS function. The function of cNOS is directly related to complex I activity:

a) Disorders of the gastrointestinal tract: - aclasia;

- neonatal hypertrophic pylor stenosis;

- Hirschprung's disease;

- diabetic gastropathy;

- esophageal reflux disease;

- diabetic gastrointestinal dysfunction;

- delayed gastric emptying;

- functional dyspepsia;

- intestinal obstruction and colitis;

- uniform movement disorders of the gastrointestinal tract (eg constipation);

- sphincter dysfunction (eg gastric ring, anus ring).

b) Biliary dysfunctions:

- biliary disorders;

- gallstone formation;

- dyslipidaemia;

- II. and III. types of bile and pancreas Oddi occlusive disorders (SOD);

- bile removal syndrome.

c) Erectile dysfunction:

neurogenic erectile dysfunction.

d) Disorders of conception.

e) Muscle degradation:

- Duchene muscular dystrophy.

f) Accidental peripheral nerve damage.

The present invention therefore relates to a pharmaceutical composition comprising the appropriate fractions of Taraxacum officinale and Viola tricolor, wherein the active compound mixture containing the solid remaining after evaporation of the aforementioned above-ground and / or root extracts is supplemented with one or more conventional pharmaceutical excipients. The pharmaceutical composition of the present invention is useful for: improving mitochondrial function and / or promoting mitochondrial biogenesis;

- for the treatment of metabolic diseases due to inadequate mitochondrial function, or prevention;

- to treat accelerated aging due to mitochondrial damage;

- a number of applications have been listed for the improvement of constitutive nitric oxide synthase (cNOS) function, which are listed in Annexes I, II and III. and IV. have been listed under

Examples to further illustrate the invention include:

Example 1: Preparation of extracts Preparation of fraction A of recipe ‘A’ - Taraxacum officinale:

100 g of dry, powdered root of Taraxacum officinale was extracted with 500 ml of chloroform at 40 ^° C with vigorous stirring. The extract was filtered and allowed to stand for 4-6 hours. The filtration step was then repeated. Thin layer chromatography - using reference materials - identified triterpenes (taraxasterols, α-, β-amirin, faradiol), triterpene esters (taraxasterol acetate) and phytosterol in the extract. The chloroform was removed by vacuum drying at 20 ^° C. The dried residue was stored in the dark at room temperature. The dry matter content obtained was 9.6 ± 0.7 g per 100 g of starting material. The freeze-dried product obtained by this process has a faradiol content of approximately 60.6 g / 100 g.

Recipe ‘B’ - Taraxacum officinale - Preparation of Fraction # 3:

100 g of dry, powdered root of Taraxacum officinale was extracted with 500 ml of chloroform at 40 ^° C with vigorous stirring. The extract was filtered and allowed to stand for 4-6 hours. The filtration step was then repeated. The already pure chloroform extract was significantly enriched for faradiol by column chromatography (8080%). The chloroform was removed by vacuum drying at 20 ^° C. Based on analytical studies, the fraction contained little triterpene as an adjuvant. The dried residue was stored in the dark at room temperature. The dry matter content obtained was 1.6 ± 0.2 g per 100 g of starting material. The faradiol content of the freeze-dried product obtained by the above method is approximately 1,21.2 g / 100 g of starting material.

Recipe ‘C’ - Viola tricolor - Fraction # 2 Preparation 86% Water - 14% Ethanol Extraction:

The above-ground part of Viola tricolor, preferably as fresh as possible, was extracted as a dried plant with a mixture of 86% water - 14% ethanol with ultrasonic dispersion - 1: 100 by volume based on the powdered plant at 60 ^o C with vigorous stirring. The resulting extract was pelleted, centrifuged, and the supernatant was freeze-dried, and the extract powder was used to prepare the formulations. The dry matter content obtained was 1.8 ± 0.3 g per 100 g of starting material. The flavonoid content of the active ingredient reached a starting amount of 0,0.9 g / 100 g.

Recipe ‘D’ - Viola tricolor - Fraction # 3 Preparation 86% Ethanol - 14% Water Extraction:

The above-ground part of Viola tricolor, preferably as fresh as possible, was extracted as a dried plant with a mixture of 86% ethanol - 14% water by ultrasonic dispersion - 1: 100 by volume based on the powdered plant at 60 ^o C with vigorous stirring. The resulting extract was pelleted, centrifuged, and the supernatant was freeze-dried, and the extract powder was used to prepare the formulations. The dry matter content / active ingredient obtained was 3.8 ± 0.6 g per 100 g of starting material. The flavonoid content of the active ingredient reached a starting amount of ~ 1.9 g / 100 g.

Recipe ‘E’ - Preparation of a mixture of Viola tricolor and Taraxacum officinale fractions:

Viola tricolor, resp. The combination of fractions of Taraxacum officinale was prepared from a 1: 1 mixture by weight of the corresponding fractions.

Example 2: Preparation of capsules

0.6 g of lyophilized active ingredient powder mixture prepared according to recipes A, B, C, D and E was filled into hard gelatin capsules. The capsules were sealed and sealed in a glass container.

Example 3: Preparation of syrup

6.2 g of the freeze-dried active ingredient powder mixture prepared according to recipes A, B, C, D and E were slowly mixed without lumps with 100 ml of a 70% aqueous alcohol solution. Glycerol (20 ml) was added and the mixture was stirred thoroughly. An additional 0.1 g of flavor and 1 g of methyl paraben were added. Slowly, with stirring, the solution was dissolved to 1000 ml with water. The mixture was homogenized, filtered and filled into a 50 or 100 ml glass container with an airtight seal.

Claims (9)

Patent claims Pharmaceutical composition, characterized in that it contains extracts of Taraxacum officinale and Viola tricolor. The pharmaceutical composition of claim 1, further comprising a conventional pharmaceutical excipient. Pharmaceutical composition according to Claim 1 or 2, characterized in that the extract is derived from the aboveground parts and / or root parts of Taraxacum officinale and Viola tricolor. Pharmaceutical composition according to any one of Claims 1 to 3, characterized in that the extract is prepared from dried plant parts with an ethanol-water extractant. 5. Use of extracts of Taraxacum officinale and Viola tricolor for the preparation of a pharmaceutical composition. A process for preparing a pharmaceutical composition according to any one of claims 1 to 4, which process comprises the steps of: (a) extraction of the above-ground and / or root parts of Taraxacum officinale and Viola tricolor with water and / or an organic solvent; b) separating the extract obtained in step a) from plant parts; c) formulating the extract obtained in step b) into a pharmaceutical composition. Process according to Claim 6, characterized in that the solvent is partially or completely removed after step b). Process according to Claim 6 or 7, characterized in that the solvent used in step a) is chosen from water, alcohol, preferably ethanol or methanol, chloroform or a mixture thereof. Process according to any one of claims 6 to 8, characterized in that step c) is filled into a capsule or formulated into a syrup.