WO2007026185A2

WO2007026185A2 - A pharmaceutical composition containing an extract of a medicinal herb belonging to the order of violales

Info

Publication number: WO2007026185A2
Application number: PCT/HU2006/000071
Authority: WO
Inventors: Péter LITERÁTI NAGY; Kálmán TORY; Attila Kolonics; Ágnes KÉRI; Lajos LÁSZLÓ; László JASZLITS; Ágnes BAJZA; Sándor BERNÁTH; László VIGH; Tibor BODNÁR; János EGRI
Original assignee: Elsö Magyar Biodrog Kutató És Fejlesztö Kft.
Priority date: 2005-09-01
Filing date: 2006-08-30
Publication date: 2007-03-08
Also published as: HUP0500813A2; WO2007026185A3

Abstract

An extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient is used for enhancing the mitochondrial genesis thereby strengthening the organism of a mammal or for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxid synthase enzyme.

Description

A pharmaceutical composition containing an extract of a medicinal herb belonging to the order of Violales

Field of the invention

The invention refers to a pharmaceutical composition containing an extract of a medicinal herb belonging to the order of Violales or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient. The composition is useful for increasing the mitochondrial genesis as well as for the prevention and/or treatment of diseases due to damages of the mitochondrion or a reduced function of the enzyme constitutive nitric oxide synthase. Background of the invention

Damages of the mitochondrion plays an important role in the formation of several diseases, while other diseases are developped owing to disturbances in the constitutive nitric oxide synthase system.

The mitochondrion is an essential organelle of the cell which occurs in varying number in the cytoplasm of every cell. That is the site of the cell's energy production. 98 % of the oxygen used by the human organism is applied by the mitochondria for energy production. Oxidative phosphorylation taking place in the mitochondrion produces a considerable amount of ATP (adenosine triphosphate) that stores the energy needed by the cell. Thus, the number and state of mitochondria is determinative from the point of view of life. In function of physical requirement, the oxidative capacity of the striated muscle is able to change by an order of magnitude. The myofibrillar protein type of the muscle is changed and the mitochondrion content of the muscle is increased during accomodation to the load. In the regulation of mitochondrial function and formation, the transcription factor PGC-1α of the coactivator PPARγ (peroxisome proliferator-activated receptor γ) has key role. Mitochondrial biogenesis is also influenced by the calcium/calmoduline dependant kinase IV (CaMKIV), calcineurine, AMP-kinase [Zong H et al.: AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation, Proc. Natl. Acad. Sci., 99, 15983 (2002)], MEF2 (myocyte enhancer factor 2), p38 MAPK as well as CREB, however, their effect is produced mainly through PGC-1α [Nisoli E. et al.: Mitochondrial biogenesis as a cellular signaling framework, Biochemical Pharmacology 67, 1 (2004.)]. CAMKIV and calcineurin have an indirect influence on the activity of the promoter of PGC-1α, while p38 MAPK exerts its effect through the phosphorylation of PGC-1 α and delaying the effect of the endogenic inhibiting domain [Fan M. et al., Genes & Development, 18^.278 (2004)]. According to recent observations, the nitric oxide produced by the endothelial nitric oxide synthase enzyme - through the increase of the activity of the guanilate cyclase enzyme and the cGMP level - plays a fundamental part in inducing the expression of PGC-1α and, thus, in the regulation of mitochondrial genesis [Nisoli, E.: Mitochondrial biogenesis in mammals. The role of endogenous nitric oxide. Science, 299, 896 (2003)].

In addition to the energy production, the mitochondrion takes part also in the regulation of other physiological processes, for example, it plays a role in the regulation of the insulin secretion of β-cells, in the oxygen perception of the pulmonary vessels as well as the sinus caroticus. The mitochondrion contains the key enzymes that define the rate of steroid biosynthesis and the carbonic acid anhydrase enzyme that is essential for the secretion of gastric acid. The mitochondrion has a great part in the regulation of calcium signalization through the uptake of cytosolic calcium [Gunter T. E. et al.: Mitochondrial calcium transport: mechanism and functions, Cell Calcium, 28_^285 (2000)]. The heat generation ability of the brown adipose tissue is based on the detachment of oxidative phosphorylation, however, this process is only of secondary importance in man. The mitochondrion is of key importance in the regulation of the programmed cell death (apoptosis) [Martinou J. C, Green D. R.: Breaking the mitochondrial barrier, Nat Rev. MoI. Cell. Biol., 2^63 (2001)].

The damage of mitochondrion is the cause of several diseases. A specific mutation of mitochondrial DNA results in the development of type I or insulin-dependent diabetes mellitus [Maassen J. A. et al.: Mitochondrial diabetes: molecular mechanisms and clinical presentation, Diabetes, 53 Suppl 1, 103 (2004)]. In the type Il or noninsulin-dependent diabetes mellitus, the basic disorder that starts the patomechanism consists in a reduced sensitivity of the tissues against insulin i.e. insulin resistance. According to recent examinations, a reduced oxidative phosphorylation capacity of the mitochondria can be in the background of insulin resistance [Petersen K. F. et al.: Mitochondrial dysfunction in the elderly: possible role in insulin resistance, Science 300, 1140; Petersen K. F. et al.: Impaired mitochondrial activity in the insulin resistant offspring of patients with type Il diabetes, N. Engl J. Med., 350, 665 (2004)]. A genetic relation between the PGC-1α gene playing a key role in the regulation of mitochondrion function and mitochondrion biogenesis on the one hand, and obesity and diabetes on the other hand was shown in Danish and Japanese population [Ek, J. et al., Diabetologia, 44, 2220 (2001); Hara et al., Diabetologia, 45, 740 (2002)]. Furthermore, reduced levels of PGC-1α were detected in patients suffering from type Il diabetes mellitus [Patti, M. et al.: Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1 , PNAS, 100, 8466 (2003)].

Several chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS (amyotrophic lateral sclerosis) are accompanied by damaged mitochondrial function. It is deemed that the damage of mitochondria contributes to the progression of the disease [Scon E. A., Manfredi G., J. Clin. Invest., 111 , 303 (2003)]. In these chronic neurodegenerative diseases, a change of conformation of certain neuronal proteins can be shown, wherein said change is accompanied by the function alteration and aggregation of the proteins. Partly, through the enhanced formation of free radicals, the mitochondrial dysfunction has a causal role in inducing the change of protein conformation, partly, the damaged mitochondrion itself becomes a target of the metabolic cascade induced by the change of protein conformation, thus, contributing to the progression of the disease. The mitochondrial dysfunction results in the desctruction of nerve cells primarily through enhanced free radical formation, reduced energy generation, disorder of calcium homeostasis and endoplasmatic reticulum.

The study of the lifetime of animals revealed that a long lifetime is coupled with a low level of reactive oxygen species (ROS) [Perez-Campo R. et al.: The rate of free radical production as a determinant of the rate of aging: evidence from the comparative approach, J. Comp. Physiol., 168, 149 (1998)]. According to the mostly accepted aging theories, the process of aging is related to the oxidative damage [Hekimi S., Guarente L.: Genetics and specificity of aging process, Science, 299, 1351 (2003)]. The cumulation of mitochondrial defects and the enhanced formation of free radicals are considered as a cause in the development of diseases related to aging [Fridovich, I.: Mitochondria: are they the seat of senescence? Aging Cell, 3, 13 (2004)]. The role of mitochondrion in the aging process is supported by the fact that the point of attack of the genes that enhance lifetime is either the mitochondrion itself or the antioxidant mechanisms closely connected with the mitochondrion.

The functioning of the constitutive nitric oxide synthase system and its role in the patomechanism of diseases is outlined as follows.

Nitric oxide (NO) is a ubiquitous signal transducer molecule having very significant regulatory roles. Nitric oxide has an important role in the vasodilation through the relaxation of the smooth musculation of vessels. The aggregation and activation of blood platelets as well as the proliferation of the smooth muscle cells of vessel are inhibited by nitric oxide that plays a role also in the regulation of heart muscle contraction and relaxation. Nitric oxide is essential in the regulation of the motility of the gastrointestinal tract, primarily through inhibiting the contraction of the sphincters e.g. pylorus (or pyloric sphincter) [Huang P. L., Am. J. Cardiol., 82, 57S (1998); Takahashi T., J. Gastroenterol., 38, 421 (2003)].

Nitric oxide is produced from L-arginine by at least three different enzymes [neuronal nitric oxide synthase (nNOS, NOS1), inducible nitric oxide synthase (iNOS, NOS2) and endothelial nitric oxide synthase (eNOS, NOS3)].

Neuronal type nitric oxide synthase is predominantly expressed in specific neurons of the brain, in non-adrenergic, non-cholinergic autonomic nerve cells, in muscles and in the macula densa region of the renal tubules, however, it is present at lower level in many other tissues as well. In the activation of nNOS enzyme, elevation of intracellular Ca⁺⁺ concentration and protein phosphorylation plays an immediate role. Furthermore, recent observations have revealed that the alteration of the expression level of the enzyme has a significant effect on the regulation of the activity thereof, too [Sasaki, M. et al., Proc. Natl. Acad. Sci. USA, 97, 8617 (2000)].

The examination of nNOS knockout animals revealed a series of disease conditions where impaired nNOS enzyme function had significant role in the pathogenesis [Mashimo, H., Am. J. Physiol., 277, 745 (1999)]. The proper motility of the whole gastrointestonal tract, especially the relaxation of sphincters, depends on the activation of nNOS in non- adrenergic, non-cholinergic neurons [Takahashi, T., J. Gastroenterol., 38, 421 (2003)]. Nitric oxide produced by the nNOS enzyme regulates the muscle tone of the sphincter in the lower esophagus, pylorus, anus and the sphincter of Oddi through the inhibition of contraction. The diminished relaxation of sphincters disturbs the function, in this way e.g. the insufficient relaxation of pylorus (or pyloric sphincter) disturbs the coordinated mechanism of gastric emptying. For example, in nNOS knockout mice, gastric dilatation and stasis develop due to the long evacuation of the stomach. The level and function of nNOS expression is severely damaged in both type I and type Il diabetes with the consequence that a dysfunction of the gastrointestinal system occurs in about 75 % of the patients. Diabetic gastropathy syndrome is characterized by prolonged gastric emptying, flatulence, nausea, vomiting, abdominal pain that deteriorate life quality [Koch K. L., Dig. Dis. Sci., 44, 1061 (1999)]. Insulin treatment that restores nNOS expression and NO level [Watkins C, J. Clin. Invest., 106, 373 (2000)] or supplementing NO through NO donors eliminates the diabetic gastrointestinal motility disturbance, thus, supporting the pathological role of NO in the disease.

A consequence of insufficient nNOS enzyme activity in the sphincter of Oddi is the syndrome of ,,lazy gall bladder". Owing to the lack of nitric oxide due to nNOS, the relaxation of the sphincter of Oddi is not sufficient resulting in an inhibited flow of bile from the gall bladder which leads to digestive troubles due to acholia as well as to cholecystectasia and cholestasis. The consequence of the latter symptoms is an enhanced risk of inflammatory diseases and formation of gallstones. Since bile plays an essential role in the lipid metabolism, the reduced bile secretion results in higher cholesterol level in the blood which can contribute to the development of metabolic syndrome [JAMA, 285, 2486 (2001 )].

Other gastrointestinal motility and function disturbances in the pathomechanism of which the reduced nNOS function may have significant role include achalasia, hypertrophic pylorus stenosis, Hirschprung's disease, functional digestion disorders, ileus and colitis. Significant therapeutical effect can be expected in these diseases by restoring the nNOS function. Also in simple hypermotility disorders, favourable effects can be awaited through the enhancement of the nNOS function and the restoration of the local neuronal reflexes.

Nitric oxide formation mediated by the nNOS enzyme has an essential role also in the regulation of the urinary bladder. The unsufficient expression and activity of the enzyme may be a cause of urinary retention.

In a similar way, the enzyme plays a fundemental role in the erection of penis [Cuevas A.J. et al, Biochem. Biophys. Res. Commun., 312, 1202], therefore, the unsufficient activity of nNOS enzyme, mainly as a consequence of diabetes, is a frequent cause of erectile disfunctions.

The nNOS enzyme activity has an essential role in normal muscle function primarily by attenuating vasoconstriction, thus, allowing adequate blood supply. Recent data indicate that in certain muscle degenerations, for example in Duchene muscular distrophy, also the function of nNOS enzyme is damaged [S. Froehner, Trends in Molecular Medicine, 8, 51 (2002)]. Restoration of the inadequate nNOS function improved the symptoms of the disease in animals.

The unsufficient function of nNOS enzyme can be responsible also for diseases related to aggressive behaviour since animal studies indicate that diminished expression and function of the enzyme result in serotonin dysfunction (descreased serotonin turnover, deficient serotonin receptor function) leading to aggressive behaviour [Chiavegatto, S. et al., Proc. Natl. Acad. Sci. USA, 98, 1277 (2001)]. It is believed that deficient nNOS function has a role in disease patterns related to aggressive behaviour and certain disturbances of sexual attitude.

Nitric oxide may have both pro- and anti-apoptic effect. Based on experimental observations, the suitable activity of the enzyme is essential in different nerve regeneration processes e.g. for recovery in traumatic peripheral nerve lesion [Keilhoff, G. Et al., Cell. MoI. Biol., 49, 885 (2003)]. Nitric oxide donors can be useful in the inhibition or treatment of arteriosclerosis [Herman, A.G. es Moncada, S.: Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis, Eur. Heart J., 2005 May 25].

Recent observations in animals indicate that the reduction of nNOS expression and function significantly contributes to the progression of certain renal diseases (nephrosis, renal damage) [Ni, Z. and Vaziri, N., Biochim. Biophys. Acta, 1638, 129 (2003)].

The inactive eNOS enzyme is attached to the caveolin in the membrane. During activation, eNOS separates from the caveolin and form complexes with other proteins. To achieve perfect activation, phosphorylation by Akt kinase and PKC-A kinase is needed [Nedvetsky et al., PNAS, 99, 16510 (2002)]. Nitric oxide produced by endothelial nitric oxide synthase plays a role in the regulation of blood pressure [Huang et al.: Hypertension in mice lacking the gene for endothelial nitric oxide synthase, Nature, 377, 239 (1995)], and the reduced enzyme function contributes to the development of hypertension [Forte et al.: Basal nitric oxide synthesis in essential hypertension, Lancet, 349, 837 (1997)]. In insulin resistant individuals, activation of eNOS stimulated by insulin is deficient resulting in a reduced glucose uptake of the muscles [Steinberg et al.: Obesity/insulin resistance is associated with endothelial dysfunction, J. Clin. Invest., 97, 2601 (1996)]. In several cardiovascular diseases, a reduced eNOS function or applicability was shown which contributed to the progression of the disease.

The enzymes nNOS and eNOS are jointly called as constitutive nitric oxide synthase (eNOS) enzyme.

Smoking results in a disorder of the system that produces constitutive nitric oxide partly due to the direct inactivation of neuronal and endothelial nitric oxide synthase enzymes [Lowe, E. R. et al.: Time-dependent inactivation and tetrahydro- biopterin depletion of endothelial nitric oxide synthase caused by cigarettes, Drug Metab. Dispos., 33, 131 (2005)]. Stimulation of the activity of eNOS enzyme moderates the damage caused by smoking [Raveendran et al.: Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells, FEBS Lett., 579, 733 (2005)]. As it is known, smoking causes damages, primarily, in the vascular system, accelerates the plaque formation on the vessel wall and enhances the risk of blood clot formation [Lakier, J. B.: Smoking and cardiovascular disease, Am. J. Med., 93(1A), 8S-12S (1992); Price, J. F. et al.: Relationship between smoking and cardio-vascular risk factors in the development of peripheral arterial disease and coronary artery disease, Edinburgh Artery Study, Eur. Heart J., 20(5), 344-353 (1999)].

As a summary, it can be stated that mitochondrial damage as well as the reduced function of constitutive nitric oxide synthase enzyme may develop various diseases. Although some of the diseases can be treated with available synthetic drugs, however a drawback of synthetic drugs resides in the side-effects, often highly disagreeable unwanted effects thereof. In addition, there is no drug presently available that could restore the activity of the nNOS enzyme in different tissues or could achieve mitochondrial genesis.

The aim of the invention is the prevention or treatment of diseases related with mitochondrial damage and/or a reduced function of cNOS enzyme by a pharmaceutical composition based on a medicinal herb extract.

Some of the medicinal herbs belonging to the order of Violales have been widely used in popular medicine. It is known, for example, that the leaf and root of the species Viola odorata have mucolytic, sweltering, blood-cleansing effect, and the flower therof has mucolytic, depressant and blood pressure lowering effect. Based on the expectorant, diuretic, astringent, antiphlogistic and blood-cleansing effect of the species Viola tricolor [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, 6. Band, 1141-1153], teas or extracts prepared from the parts of the plant growing above the earth are often used in case of cold as well as inflammatory, skin and arthritic diseases [Leporatti, M. L., Ivancheva, S.: Preliminary comparative analysis of medicinal plants used in the traditional medicine of Bulgaria and Italy, J. Ethnopharm., 87, 123-142 (2003)]. Certain parts of the species Viola odorata are used, in general, for the treatment of chronic bronchitis, pertussis and asthma bronchiale due to the expectorant and antitussive properties of the plant, however, the relieving action of Viola odorata in case of migraine is also known. The leaves of Viola odorata are considered to be effective in case of hoarseness, sore throat, impairments of sleep and neurosis.

In spite of the fact that the above-mentioned species of Viola are considered as important medicinal herb, the phytochemical analysis thereof is by far not complete.

Based on scientific proofs, the active agents of Viola tricolor include the following ones: flavonoids such as rutin, quercetin, luteolin, luteolin-7-glycoside, scoparin, saponarin, saponaretin, violantin, orientin and isoorientin, vicenin-2 and vitexin [Hansel R. et al.: publication cited above]. Violarvensin is a flavone di- C-glycoside isolated from the related genus field pansy (Viola arvensis) [Camat, A. P., Camat, A., Fraisse, D., Lamaison J. L.: Violarvensin, a New Flavone Di-C-glycoside from Viola arvensis, J. Nat. Prod., 61, 272-274 (1998)]. The presence of triterpene saponins based on uriolic acid and containing galactose and galacturonic acid as the sugar component were confirmed by some authors (Hansel R. et al.), while other authors considered that a protein designed as violapeptide I was responsible for the haemolytic property [Schopke, Th., Hasan Agha, M. I., Kraft, R., Otto, A., Hitler, K.: Haemolytisch aktive Komponenten aus Viola tricolor L. und viola arvensis Murray, Sci. Pharm., 61, 145-153 (1993)]. According to literature data, carotenoids that can be isolated from the plant include violaxanthin and derivatives thereof [Hansmann, P., Kleinig, H.: Violaxanthin esters from Viola tricolor flowers, Phytochemistry, 21, 238-239 (1982); Molnar, P., Szabolcs, J.: Occurence of 15-cis-violaxanthin in Viola tricolor, Phytochemistry, 19, 623-627 (1980); Molnar, P., Szabolcs, J., Radios, 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); Radios, L., Molnar, P., Szabolcs, J.: ¹³C NMR evidence for the central mono-cis- stereochemistry of naturally occuring violaxanthin isomers, Phytochemistry, 22, 306 (1983)], violeo- xanthin, lutein, luteinepoxide and neoxanthin [Hansel et al.: publication cited above]. Out of the anthocyans, the presence of delphinidine agliconu violanin, platyconin and violanin chloride is characteristic [Saito, N., Timberlake, C. F., Tucknott, O. G., Lewis, I. A. S.: Fast atom bombardment mass spectrometry of the antho-cyanins violanin and platyconin, Phytochemistry, 22, 1007-1009 (1983)]. Phenolcarboxylic acids and the derivatives thereof: trans- and cis-coumaric acid, gentisic acid, p-hydroxy-benzoic acid, 4-hydroxyphenylacetic acid, trans-caffeic acid, protocatechuic acid, vanillic acid, salicylic acid and derivatives thereof are important components [Hansel R. et al.: publication cited above]. As further components, polysaccharides based on glucose, galactose, arabinose, ramnose, xylose and uronic acid, furthermore vitamines E and C, triacylglycerol as well as fatlike substances are to be mentioned [Hansel R. et al.: publication cited above].

Cyclic peptides such as vitri A, vary A and E isolated from the species Viola tricolor were found to be cytotoxic on 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)]. Data referring to the antioxidant properties of the species Viola tricolor were published by Mantle et al. [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)]. Goun et al. described considerable antithrombin activity of Viola tricolor [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 the species Viola surinamensis were described by Lopes et al. [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 (RoI.) Warb. by Waiipi Amazon Indians, J. Ethnopharm., 67, 313-319 (1999)]. The antipyretic effect of the species Viola odorata was published by Khattak et al. [Khattak, S. G., Naeemuddin, G., Ikram, M.: Antipyretic studies on some indigenous Pakistani medicinal plants, J. Ethnopharm., 14, 24-51 (1985)]. This effect can be related to the salicylic acid content of the plant.

Thus, although some of the medicinal herbs belonging to the order of Violales have been used in the form of a herb tea or extract for the treatment of certain diseases, no pharmaceutical composition has been prepared from these medicinal herbs. Summary of the invention

It has been found that the above aim can be achieved by a composition or pharmaceutical composition containing an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient optionally in addition to one or more conventional pharmaceutical carrier(s). The composition or pharmaceutical composition of the invention is suitable for the enhancement of mitochondrial genesis as well as for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxide synthase enzyme. The most important diseases of these type have been discussed above. Description of preferred embodiments

Under the expression ,,a medicinal herb belonging to the order of Violales" the following medicinal plants are meant in terms of the taxonomical description: Order: Violales Families: - Vϊolaceae

Viola genus

Hybanthus genus → H.calycinus

Hybanthus concolor (T.F. Forst) Spreng Hybanthus linearifolius (Vahl) Urban Rinorea genus → Rinorea moagalensis Anchietea genus → Anchietea salutans Corynostylis genus -> Corynostylis hybnanthus

Hymenanthera genus -> Hymenanthera obovata

- Flacourtiaceae

- Lacistemataceae

- Passifloraceae

- Turneraceae

- Malesherbiaceae

- Fouquieriaceae

- Caricaceae

- Bixaceae

- Cochlospermaceae

- Cistaceae

- Tamaricaceae

- Ancistrocladaceae

- Frankeniaceae

- Achariaceae

- Begoniaceae (according to other sources: Salicales)

- Loasaceae

- Datiscaceae

- Samydaceae

- Gucurbitaceae

As for families, there are significant alterations in literature data, therefore, the following species are also considered to belong to the order of Violales:

Viola acuminata acuminate violet

Viola adunca hookedspur violet; western dog violet

Viola adunca var. adunca hookedspur violet

Viola adunca var. kirkii Kirk's violet

Viola adunca var. oxyceras hookedspur violet; sharptail violet

Viola affinis sand violet

Viola appalachiensis Appalachian violet

Viola arvensis field pansy

Viola bakeri Baker's violet

Viola beckwithii Beckwith's violet; Great Basin violet

Viola beckwithii ssp. beckwithii Beckwith's violet

Viola beckwithii ssp. glabrata western pansy Viola bicolor field pansy

Viola biflora

Viola biflora ssp. biflora twoflower violet

Viola biflora ssp. carlottae Carlott's violet

Viola blanda sweet white violet; willdenow violet; etc.

Viola blanda var. blanda sweet white violet

Viola blanda var. palustriformis sweet white violet; marsh violet

Viola brevistipulata short stipule violet

Viola brittoniana northern coastal violet; coast violet

Viola brittoniana var. brittoniana northern coastal violet

Viola brittoniana var. pectinata combed northern coastal violet

Viola calaminaria Yellow calamine violet

Viola californica California violet

Viola canadensis Canadian white violet; Canada violet; etc.

Viola canadensis var. canadensis Canadian white violet

Viola canadensis var. corymbosa Canadian white violet

Viola canadensis var. rugulosa creepingroot violet

Viola canadensis var. scariosa shriveled Canadian white violet

Viola canadensis var. scopulorum Canadian rock white violet

Viola canina heath violet; dog violet

Viola canina ssp. canina heath dog violet

Viola canina ssp. montana heath dog violet

Viola chamissoniana olopu

Viola chamissoniana ssp. chamissoniana olopu

Viola chamissoniana ssp. robusta large olopu

Viola chamissoniana ssp. tracheliifolia olopu

Viola charlestonensis Charleston Mountain violet

Viola chinensis China violet

Viola collina hillside violet

Viola conspersa American dog violet

Viola cornuta horned violet; bedding pansy

Viola cucullata marsh blue violet

Viola cuneata wedgeleaf violet

Viola diffusa spreading violet

Viola douglasii Douglas' golden violet

Viola egglestonii glade violet

Viola epipsila dwarf marsh violet; large marsh violet

Viola epipsila ssp. repens dwarf marsh violet; creeping marsh violet

Viola flettii Olympic violet; rock violet

Viola frank-smithii Frank-Smith's violet

Viola glabella pioneer violet; stream violet Viola grypoceras Viola grypoceras

Viola guadalupensis Guadalupe violet

Viola guestphalica Violet calamine violet

Viola hallii Oregon violet

Viola hastata halberd leaf yellow violet

Viola hederacea Australian violet, trailing violet

Viola helenae Wahiawa stream violet

Viola hirsutula southern woodland violet

Viola hirsuta Hairy violet

Viola hispida Rouen pansy

Viola howellu Howell's violet

Viola incognita large leaved white violet

Viola japonica Japanese violet

Viola kauaensis pohe hiwa

Viola kauaensis var kauaensis pohe hiwa

Viola kauaensis var wahiawaensis pohe hiwa

Viola keiskei Viola keiskei

Viola labradorica alpine violet, Labrador violet

Viola lactea pale dog violet

Viola lanaiensis Hawaii violet

Viola lanceolata bog white violet

Viola lanceolata ssp lanceolata lanceleaf bog white violet

Viola lanceolata ssp occidentalis eastern bog white violet

Viola lanceolata ssp vittata bog white violet

Viola langsdorfii Aleutian violet, Alaska violet

Viola lithion rock violet

Viola lobata moosehorn violet, yellow wood violet

Viola lobata ssp integrifolia moosehorn violet

Viola lobata ssp lobata moosehorn violet

Viola lobata var lobata moosehorn violet

Viola lovelhana Lovell's violet

Viola lutea mountain pansy

Viola macloskeyi small white violet, western sweet white violet

Viola macloskeyi ssp macloskeyi Macloskey's violet, western sweet white violet

Viola macloskeyi ssp pallens smooth white violet

Viola mandschurica Manchuπan violet

Viola maviensis Hawaii bog violet

Viola mirabilis wonder violet

Viola missouriensis Missouri violet, banded violet

Viola nephrophylla var nephrophylla northern bog violet

Viola novae-anghae New England blue violet Viola nuttallii Nuttall's violet; yellow prairie violet

Viola oahuensis Oahu violet

Viola obliqua marsh blue violet

Viola obtusa blunt violet

Viola ocellata pinto violet; two eyed violet

Viola odorata sweet violet; garden violet; etc.

Viola orbioulata darkwoods violet; western round leaved violet

Viola orientalis Viola orientalis

Viola palmata early blue violet; palmate violet; wild okra

Viola palmata var. palmata early blue violet

Viola palustris marsh violet; alpine marsh violet

Viola palustris var. brevipes marsh violet

Viola palustris var. palustris marsh violet

Viola patrinii stemless violet

Viola pedata birdfoot violet; bird's foot violet; etc.

Viola pedatifida prairie violet; prairie birdfoot violet; etc.

Viola pedunculata California golden violet; grass pansy; yellow pansy

Viola pedunculata ssp. pedunculata California golden violet

Viola pedunculata ssp. tenuifolia threadleaf California golden violet

Viola pinetorum goosefoot yellow violet

Viola pinetorum ssp. grisea goosefoot violet

Viola pinetorum ssp. pinetorum goosefoot violet

Viola pinnata pinnate violet

Viola praemorsa canary violet

Viola praemorsa ssp. flavovirens upland yellow violet

Viola praemorsa ssp. linguifolia upland yellow violet

Viola praemorsa ssp. praemorsa canary violet

Viola primulifolia primrose leaved violet

Viola prionantha Viola prionantha

Viola psychodes butterfly violet

Viola pubescens downy yellow violet

Viola pubescens var. peckii Peck's downy yellow violet

Viola pubescens var. pubescens smooth yellow violet

Viola pubescens var. scabriuscula rough yellow violet

Viola purpurea goosefoot violet

Viola purpurea ssp. aurea goosefoot yellow violet

Viola purpurea ssp. dimorpha two formed goosefoot violet

Viola purpurea ssp. geophyta rock goosefoot violet

Viola purpurea ssp. integrifolia entire leaved goosefoot violet

Viola purpurea ssp. nπesophyta goosefoot violet

Viola purpurea ssp. mohavensis Mojave yellow violet Viola purpurea ssp. purpurea purple goosefoot violet; pine violet

Viola purpurea ssp. quercetorum goosefoot yellow violet

Viola purpurea ssp. venosa goosefoot yellow violet

Viola rafiπesquii field pansy

Viola reichanbachiana slender wood violet

Viola renifolia northern white violet; kidney leaved white violet

Viola renifolia var. renifolia kidneyleaf white violet

Viola rivianawood violet; dog violet

Viola rostrata longspur violet

Viola rotundifolia eastern roundleaf yellow violet; early yellow violet

Viola rupestris Teesdale violet; rock violet

Viola sagittata arrowleaf violet

Viola selkirkii Selkirk's violet

Viola sempervirens evergreen violet; redwood violet

Viola septemloba southern coastal violet

Viola septentrionalis var. septentrionalis northern woodland violet; northern blue violet

Viola sheltonii Shelton's violet

Viola sororaria woolly blue violet

Viola striata striped cream violet; pale violet; striped violet

Viola subsinuata wavy leaf violet

Viola tokubuchiana Viola tokubuchiana

Viola tomentosa feltleaf violet

Viola tricolorJohnnyjump up; heart's-ease; etc.

Viola trinervata Rainier violet; sagebrush violet

Viola tripartita threepart violet

Viola umbraticola Ponderosa violet

Viola umbraticola var. glaberrima Ponderosa violet

Viola umbraticola var. umbraticola Ponderosa violet

Viola utahensis Utah violet

Viola vaginata sheathed violet

Viola vallicola valley violet

Viola vallicola var. major large valley violet

Viola vallicola var. vallicola sagebrush violet

Viola variegata Viola variegata

Viola verecunda hidden violet

Viola viarum twoflower violet; plains violet; wayside violet

Viola villosa Carolina violet; hairy violet; wrinkled violet

Viola violacea Viola violacea

Viola wailenalenae Alakai Swamp violet

Viola walteri prostrate blue violet; prostrate southern violet

Viola x bernardii Bernard's violet Viola x bissellii Bissell's violet

Viola x brauniae Braun's violet

Viola x conjugens violet

Viola x consobrina violet

Viola x consocia violet

Viola x cooperrider Viola x cooperrider

Viola x cordifolia violet

Viola x davisu Davis' violet

Viola x eamesii Eames' violet

Viola x eclipes violet

Viola x filicetorum violet

Viola x hollickii Hollick's violet

Viola x insolita violet

Viola x luciae Lucy's violet

Viola x malteana violet

Viola x mistura violet

Viola x modesta violet

Viola x mollicula Viola x molilalia

Viola x mulfordiae Mulford's violet

Viola x notabihs violet

Viola x peckiana Peck's violet

Viola x porteπaπa Porter's violet

Viola x primulifolia violet

Viola x ravida violet

Viola x redacta violet

Viola x ryoniae Ryon's violet

Viola x slavinii Slavin's violet

Viola wittrockiana, ladies' delight, etc

Viola x wujekn Viola x wujekn

Viola yedoensis Viola yedoensis

The most important species of the Viola genus include Viola tricolor, Viola arvensis and Viola odorata.

Under ,,a part of a plant or herb that has grown above the earth" the leaf and/or stem and/or flowers (inflorescence) of the plant is/are meant.

The extract is prepared in a manner known perse. For this purpose, the part of the herb that has grown above the earth, optionally after drying and size-reducing, is extracted. The extraction is carried out with water or an organic solvent such as an alcohol e.g. ethanol, or an aqueous solution of an organic solvent e.g. aqueous ethanol generally at 0-100 ⁰C, preferably at 20-100 ⁰C. During the extraction, in most cases, mixing is applied, however, ultrasonication can be used, too. The extract is separated from the parts of the plant by known methods using e.g. sedimentation, pressing of the parts of the plant, filtration, centrifugation or the combination of the procedures listed. The extract obtained can be used as it is or it can be converted to a liquid composition or pharmaceutical composition such as an aqueous solution or syrup. However, it is preferred to remove the solvent content of the extract for example by evaporation, spray drying or freeze drying, and the solid residue is used as an active agent for the preparation of a composition or a pharmaceutical composition. (In the description and claims, the expression ,,active agent" is used in this sense and it refers to the solid residue that is dissolved in the extract and can be obtained from the extract of the medicinal herb.) Both the extract and the solid residue obtained from the extract can be characterized by the determination of the flavonoid and polyphenol contents. For example, the flavonoid content of the solid residue amounts to 2.8-3.4 g/100 g, and the total polyphenol content is 9.7-11.0 g/100 g.

Under a pharmaceutical composition" a known formulation or dosage form is meant which is conventionally used for the prevention or treatment of diseases and which is suitable for peroral, parenteral, rectal or transdermal administration or for local treatment. Thus, the pharmaceutical composition of the invention is solid or liquid and contains, in addition to the active substance obtained from the medicinal herb by extraction, one or more pharmaceutical carrier(s). The pharmaceutical composition of the invention contains, in general, 0.1-100 % by mass, preferably 1-50 % by mass, suitably 5-30 % by mass of the active ingredient. It is to be noted that a 100 % content of active ingredient is possible only in certain cases e.g. in capsules where dilution is not absolutely necessary. In most dosage forms, diluents and/or other auxiliary agents are needed for the preparation of the pharmaceutical composition.

The solid pharmaceutical compositions suitable for peroral administration may be powders, capsules, tablets, film-coated tablets, microcapsules etc., and can comprise binding agents such as gelatine, sorbitol, poly(vinylpyrrolidone) etc.; filling agents such as lactose, glucose, starch, calcium phosphate etc.; auxiliary substances for tabletting such as magnesium stearate, talc, poly(ethylene glycol), silica etc.; wetting agents such as sodium laurylsulfate etc. as the carrier.

The liquid pharmaceutical compositions suitable for peroral administration may be solutions, suspensions or emulsions and can comprise e.g. suspending agents such as gelatine, carboxymethylcellulose etc.; emulsifiers such as sorbitane monooleate etc.; solvents such as water, oils, glycerol, propylene glycol, ethanol etc.; preservatives such as methyl or propyl p-hydroxybenzoate etc. as the carrier.

Pharmaceutical compositions suitable for parenteral application contain, in general, a sterile solution of the active agent.

Pharmaceutical compositions suitable for local treatment include solutions, creams, liniments etc.

The dosage forms listed above as well as other dosage forms are known perse, see e.g. the manual Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, USA (1990)

The pharmaceutical composition contains dosage unit, in general. The daily dose can be administered in one or more portions. The actual dosage depends on many factors and is determined by the doctor. In general, a typical dose for adult patients of 70 kg body weight amounts to 0.1 to 10 g, preferably 1 to 5 g of active agent, daily.

In general, the pharmaceutical composition is prepared by admixing the active ingredient to one or more carrier(s) and transforming the mixture obtained into a pharmaceutical composition in a manner known perse. The methods that can be used are known from the literature e.g. the manual Remington's Pharmaceutical Sciences cited above. Of course, as a further possibility, the solid residue obtained from the extract can be directly filled into capsules or the extract itself can be converted to a liquid pharmaceutical composition by the addition of further carriers, if needed.

In cases when the extract or active ingredient of the invention is applied to achieve mitochondrial genesis for e.g. roboration, muscle-building etc. purpose, the composition administered is not necessarily a pharmaceutical composition, however, the contents and preparation thereof corresponds to those of the pharmaceutical compositions described herein. Consequently, the expression ..composition" used in the description and claims without the marking ..pharmaceutical" refers to a dosage form that is applied to induce favourable biological effects related to mitochondrial genesis, however, such treatment is not definitely medication.

The effect of the medicinal herbs belonging to the order of Violales was studied by the following biological tests.

Cell cultures and cultural characteristics employed in the in vitro tests

Primary pig endothelial cell culture

The thoracic aorta of a pig was excised, aseptically, and placed into a PBS solution containing 50 μg/ml of streptomycin for transport (PBS is a physiological saline that contains phosphate buffer). The connective tissue was removed from the aorta which latter was cut to pieces of several cm length, and the rings obtained were opened. A sterile, close-meshed plastic net impregnated with trypsin (0.25 % of trypsin in PBS) was placed onto the surface covered by endothelium and the tissues were kept at 37 ⁰C for 1 or 2 minute(s). The endothelium layer loosened under the action of trypsin was removed by washing, the cells were collected by centrifugation, then removed to culturing dishes coated with collagen and grown in a 1 :1 mixture of DMEM (Dulbecco's modified Eagle's medium) culture medium (GibcoBRL, Eggenstein, Germany) supplemented with 10 % of FCS (fetal calf serum) and F-12. A homogenous culture was obtained in which more than 95 % of the cells showed endothelial morphology. The cells from the first ten passage were used in the tests.

Primary rat glia culture

The cortex of an 8 day's old Wistar rat was removed aseptically, cut into pieces, and the cells were set free by diggestion with trypsin (0.08 % of trypsin in PBS). The larger pieces of tissue were removed, the fraction containing individual cells and lumps consisting of some cells was washed twice with RPMI (Roswell Park Memorial Institute) culture medium containing 10 % of FCS. At last, the cells suspended in RPMI culture medium containing 10 % of FCS were placed into grow dishes (Greiner) without special surface treatment and grown at 37 ⁰C under an atmosphere containing 5 % of carbon dioxide. A mixed cell culture was obtained containing mainly glia (astrocyte, oligodendrocyte and microglia) cells. Cells obtained in the first passage were used in the tests.

HaCaT cell culture

Human immortalized HaCaT skin cells were grown in a DMEM culture medium containing 10 % of FCS in dishes (Greiner) without special surface treatment at 37 ⁰C under an atmosphere containing 5 % of carbon dioxide. The culture medium was supplemented with 25 mM or 50 mM of D-glucose and the cells were grown for at least 1 week in this culture medium.

Quantitative determination and morphological study of mitochondria

Staining with the fluorescent stain MitoTracker

The cells were incubated with 100 nM of the fluorescent stain MitoTracker at 37 ⁰C for 30 minutes. The stain cumulating in the active mitochondria exhibits a fluorescent emission at 516 nm following an excitation at 490 nm. The fluorescence of the mitochondria was determined partly in a microscope, partly using a FACS (fluorescence activated cell sorter) apparatus. For the determination with the microscope, a fluorescence microscope Zeiss-Axioskop was employed. Exposures were prepared with a Nicon Coolpix 995 digital camera using identical exposure time, diaphragm aperture and digital picture size. The exposures were evaluated by densitometry. The intensity of the cell fluorescence is proportional to the amount of mitochondria. In addition, the observation under a microscope allows the study of the morphology of the mitochondrial network.

Determination of a mitochondrion specific protein by means of Western blot

The cells were rinsed and collected in ice-cold PBS (pH=7.4) containing 5 mM of ethylenediaminetetraacetic acid (EDTA), 5 mM of sodium fluoride and 100 μM of Na₃VO₄. Lysis of the cell pellet was carried out on ice under weak shaking for 10 minutes in a buffer solution containing 250 mM of sodium chloride, 50 mM of HEPES [4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid] (pH=7,4), 1 mM of EDTA, 1 mM of EGTA [ethyleneglycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid], 1 ,5 mM of magnesium chloride, 0,1 % of Nonidet P-40 [nonylphenyl-poly(ethylene glycol)], 40 mM of β-glycerol phosphate, 1 mM of Na₃VO₄, 1 mM of phenylmethylsulfonyl fluoride, 10 mM of benzamidine, 20 mM of NaF, 10 mM of sodium pyrophosphate, 10 μM/ml of aprotinin, 10 μg/ml of leupeptin, and 10 μg/ml of antipain. The insoluble cell debris was removed by centrifugation (13000 g, 12 minutes, +4 ⁰C). The clear supernatant was admixed to VT. volume of 2x Laemmli gel loading buffer, the samples were boiled for 3 minutes, then maintained at -20 ⁰C before use. The protein concentration was determined by means of Bio-Rad D₀ Protein Assay reagent (Bio-Rad Laboratories, Hercules, California, USA). Samples containing identical amount of protein were separated by polyacrylamide gel electrophoresis in the presence of 10 % of sodium dodecylsulfate (10 % SDS-PAGE) and blotted on PVDF [poly(vinylidene difluoride)] membrane using a Trans-Blot SD Blotting Kit (Bio-Rad Laboratories). The imune detection was carried out using anti-COX-IV antibody (A21348, Molecular Probes). Anti-mouse IgG HRP (horse radish peroxidase) conjugated antibody (Cell Signalling Technologies) was used as secondary antibody. For the detection, ECL (enhanced chemiluminescence) Plus System (Amersham) was employed.

Quantitative determination of stress proteins and cNOS on Western blot The method is identical with the one described above. The following antibodies were used: anti-HSP72 (Citomarker Research & Development, Hungary), anti-HSP90 alpha (Affinity BioReagents, Golden, USA), anti-eNOS (Transduction Laboratories, USA), anti-HSP25 (Affinity BioReagents, Golden, USA).

The evaluation of the microscope exposures and Western blots was carried out densitometrically by means of a UTHSCA Image Tool Version 3.0 computer program. In case of the microscope exposures, three confluent cell layers selected randomly were examined. From the optical density values obtained, the mean value was determined. Statistical comparison and calculation were carried out by the one-way analysis of the difference using the Posthoc Newman-Keuls test (Pharmacological Calculation System). The statistical significance was p< 0.05.

Quantitative determination of nitric oxide in human lymphocyte in vitro

Human blood was drawn before use and heparin was added to inhibit clotting, then the blood was divided into two portions. One portion was treated at 37 ⁰C for 3 hours with an aqueous solution containing 8 μg/ml of an active substance prepared from Viola tricolor by process A of Example 1. Then the nucleated cells were separated by centrifugation at Ficoll gradient, then DAF-FM [4-amino-5-methylamino-2',7'-difluoro- fluorescein diacetate, Molecular Probes] as fluorescent NO- indicator was added to the cells at a concentration of 2x10^"6 M. Following the reaction with nitric oxide, the fluorescence of DAF-FM (excitation peak at 490 nm, emission peak at 515 nm) becomes 160-times higher, therefore it is suitable for the measurement of the amount of intracellular NO. After incubation for 30 minutes, the DAF specific fluorescence of the cells in the lymphocyte cell fraction was determined by means of a Becton Dickinson FACS Calibur apparatus.

In vivo tests in animals

Study of the brown adipose tissue in mice

NMRI mice having a body mass of 23-25 g were treated, once daily, with a dose of 30 mg/kg of the active substance prepared from Viola tricolor by process A of Example 1 for 4 days. On day 5, the animals were anaesthetized by an injection of pentobarbital [5-ethyl-5-(1-methylbutyl)-2,4,6- (1 H,3H,5H)-pyrimidintrione], and samples were taken from the brown adipose tissue of the animals. Then, the level of proteins in the tissue was determined.

Test of glucose tolerance

Groups of male CFLP mice having a body mass of 25 g were treated, once daily, with a dose of 30 or 100 mg/kg of the active substance prepared from Viola tricolor by process A of Example 1 for 5 days, wherein each group consisted of 5 mice. On the fifth day, 1 hour after the treatment, the animals were anaesthetized by an injection of pentobarbital, blood was drawn from the corner of the eye and the glucose level was determined from the blood using an AccuCheck glucose meter. Then, 2 g of glucose/kg body weight were administered, intraperitoneally, and blood glucose levels were determined in the animals 30, 60 and 90 minutes after the glucose administration. The mice of the control group were treated with an identical amount of the carrier (i.e. distilled water).

The results obtained by the treatment with the active substance prepared from Viola tricolor by process A of Example 1 are given in Table 1 at a later part of the description. The active substances prepared from Viola tricolor by processes B and C have shown rather similar activity, therefore these results are not shown in Table 1. Study of the intestinal motility in rats The test was carried out on rat ileum preparations according to Anjaneyulu [Anjaneyulu, M. and Pamarao, P.: Studies on gastrointestinal tract functional changes in diabetic animals, Methods Find. Exp. Clin. Pharmacol., 24, 71-75 (2002)]. In male Sprague-Dowley rats (Charles River Hungary) of 230-260 g body mass, diabetes was induced by the intravenous administration of 60 mg/kg of streptozocin. The blood sugar level of the animals was determined from the fourth week following the induction of diabetes, weekly, and the animals having stable high blood sugar level (>20 mM/litre) were drawn into the test after the tenth week. The 7 animals of the test group were treated orally, once daily, with a dose of 30 mg/kg of the active substance prepared from Viola tricolor by process A of Example 1 in aqueous solution for 5 days, while the 7 animals of the control group were treated with an identical volume of physiological saline. On the day after the last treatment, the animals were anaesthetized by the administration of pentobarbital, the ileum was removed, cleaned, suspended in a Krebs-Henseleit's solution at 37 ⁰C using 1 g of initial load, contracted with acetylcholine, then the relaxation ability of the ileum was evaluated based on the relaxation response to encreasing doses of isoproterenol [4-[1 - hydroxy-2-[(1-methylethyl)amino]ethyl]-1 ,2-benzenediol]. The measurements were carried out in an lsosys System apparatus (Experimetria, Budapest, Hungary).

Test of aorta relaxation in rat

The test was carried out on rat aorta preparations according to Kobayashi [Kobayashi, T. and Kamata, K.: Effect of insulin treatment on smooth muscle contractility and endothelium- dependent relaxation in rat aorta from established STZ- induced diabetes, Br. J. Pharmacol., ^27, 835-842 (1999)]. In male Sprague-Dowley rats (Charles River Hungary) of 270-290 g body mass, diabetes was induced by the intravenous administration of 60 mg/kg of streptozocin. The tests were started after 9-14 weeks following the induction of diabetes using animals having a stable high blood sugar level of above 20 mM/litre. The 7 animals of the test group were treated orally, once daily, with a dose of 30 mg/kg of the active substance prepared from Viola tricolor by process A of Example 1 in aqueous solution for 5 days, while the 7 animals of the control group were treated with an identical volume of physiological saline. On the day after the last treatment, the animals were anaesthetized by the administration of pentobarbital, the thoracic aorta was removed and placed into ice cold Krebs' solution containing 118,0 mM of sodium chloride, 4,7 mM of potassium chloride, 2,52 mM of calcium chloride, 1 ,64 mM of magnesium sulfate, 24,88 mM of sodium hydrogen carbonate, 1 ,18 mM of potassium dihydrogen phosphate and 11 ,0 mM of D-glucose. A gas mixture consisting of 95 % of oxygen and 5 % of carbon dioxide was led through the solution, constantly. The descending aorta was cut to obtain rings of 5 mm width, the rings were suspended in a Krebs' solution at 37⁰C using 2 g of initial load under constant introduction of the oxygen gas, contracted with phenylephrine [(R)-3-hydroxy-α-[(methylamino)methyl]- benzenemethanol], then the relaxation ability of the rings was determined based on the relaxation response to increasing doses of acetylcholin. The measurements were carried out in an lsosys System apparatus (Experimetria, Budapest, Hungary).

The following results were obtained in the tests: Increase of nitric oxide level in human lymphocyte, in vitro In the freshly isolated human blood lymphocytes, the intracellular nitric oxide level could be well determined using DAF fluorescent stain. The average DAF fluorescence that is in direct proportion to the amount of intracellular NO had a value of 448+105 relative units in the untreated samples (i.e. control), while in case of samples treated with an aqueous solution containing 8 μg/ml of an active substance prepared from Viola tricolor by process A of Example 1 , the average DAF fluorescence had a value of 857+124 relative units. Thus, treatment with the active substance extracted from the medicinal herb Viola tricolor raised the intracellular NO concentration by 91 %.

Increase of the mitochondrial number An increase of the mitochondrial number due to the treatment with an aqueous solution containing 8 μg/ml of an active substance prepared from Viola tricolor by process A of Example 1 for 4 days was observed in primary pig endothelial cell culture. After staining with the fluorescent stain MitoTracker, the results were determined using either a fluorescence activated cell sorter (FACS) apparatus or a fluorescence microscope. In the evaluation of the results obtained, the optical density of the contrail cell culture was taken as unit and the optical densities of the treated cell cultures were compared with that of the control cell culture. Evaluation with the FACS apparatus indicated an increase of the mitochondrial number by 30 %, while the evaluation with the fluorescence microscope indicated an increase of the mitochondrial number by 60 % relative to the control value. The exposures prepared by using microscope and digital camera following the treatment with the active substance extracted from the medicinal herb Viola tricolor exhibited a strengthening of the mitochondrial network which is a sign of the vigorous active mitochondrial system.

In a primary rat glia cell culture, the treatment carried out with a dose of 16 ug/ml of the active substance prepared from Viola tricolor in aqueous solution for 4 days resulted in a 20 % increase of the fluorescence characterizing the amount of mitochondria compared with the control. The strengthening of the mitochondrial network could be observed after the treatment in this case, too.

In addition to the direct determination of the mitochondrial number, an increase of the level of COX-IV protein could be also noticed on Western blot. It is to be noted that the COX-IV protein is specific of mitochondrion and plays a key role in oxidative phosphorylation, thus, the increase of the level thereof indicates the increase of the amount of mitochondria. It was found that the COX-IV protein level increased by 60 % compared with the control in the primary pig endothelial cell culture owing to the treatment for 4 days with a dose of 8 μg/ml of the active substance prepared from the medicinal herb Viola tricolor in aqueous solution. In case of the primary rat glia cell culture, the COX-IV protein level increased by 430 % compared with the control due to the treatment for 4 days with a dose of 16 μg/ml of the active substance prepared from the medicinal herb Viola tricolor in aqueous solution. Increase of the expression of cNOS and HSP Nitric oxide synthetized especially by the endothelial nitric oxide synthase (eNOS) enzyme is rather important in the regulation of mitochondrial function and biogenesis. Nitric oxide enhances the expression of the transcription factor PGC- 1α, the main regulator of mitochondrial biogenesis. Based on the Western blot examination, the amount of cytosolic eNOS was higher by about 60 % in pig endothelial cells due to the treatment for 4 days with a dose of 8 μg/ml of the active substance prepared from the medicinal herb Viola tricolor in aqueous solution. Cytosolic eNOS is considered to be active. The heat shock proteins (chaperons) HSP72 and HSP90 are extremely important in the formation and stabilization of the functionally active eNOS complex. Also according to the tests carried out on Western blot, the amount of HSP90α increased by 10 %, while the level of HSP72 increased by 130 % compared with the untreated control due to the treatment of the invention. It is to be noted that, in addition to the stabilization of the eNOS enzyme system, the HSP72 has a key role also in the import of mitochondrial proteins. This coordinated import of protein is essential for the normal mitochondrial function and biogenesis.

In general, a culture medium having a higher glucose content (25 mM) than the normal blood sugar level is used for growing human immortalized HaCaT keratinocyte cells. In the culture medium, the cells adapted themseves to the high glucose concentration. Therefore, in order to simulate the hyperglycaemic environment, the concentration of glucose was raised by further 25 mM and the cells were grown in this culture medium. Based on staining with MitoTracker, the mitochondrion content of the cells grown in the culture medium containing 25 mM of glucose was higher by 30 % than that of the cells grown under hyperglycaemic circumstances in a culture medium containing 50 mM of glucose. This observation corresponds to the known fact that a hyperglycaemic environment deteriorates mitochondria. The cell culture pretreated with 50 mM of glucose was treated for 4 days with a dose of 4 μg/ml of the active substance prepared from the medicinal herb Viola tricolor. The treatment raised the amount of mitochondria by 20 % i.e. the treatment could partly combat the hyperglycaemic effect.

In the HaCaT keratinocyte cells grown in a hyperglycaemic environment, the proteins examined by us and having a key role in the mitochondrial genesis were expressed only in the smallest amount. However, in the cells treated for 4 days with a dose of 4 μg/ml of the active substance prepared from the medicinal herb Viola tricolor, the amount of PGC-1α increased by a factor of 8, that of HSP90α and HSP72 increased by a factor of 6, that of HSP60 increased by a factor of 3 and that of COX-IV protein increased by a factor of 3.

The above test results prove unambiguously that the treatment of the invention enhances mitochondrial genesis and inhibits mitochondrial damage, in vitro.

The brown adipose tissue of NMRI mice treated for 4 days with a dose of 30 mg/kg of the active substance prepared from the medicinal herb Viola tricolor contained the heat shock protein HSP90α in a higher amount by 120 % and the heat shock proteins HSP72 and HSP60 in a higher amount by 60 % compared with the control group without treatment. Due to the treatment, the level of heat shock protein HSP25 having an important role in the regulation of the redox processes of cells was also higher by 60 %. This in vivo test result indicates that the active substance extracted from the medicinal herb Viola tricolor is absorbed and exerts its effect also in vivo.

The treatment of the invention improved also the glucose tolerance in mouse. The blood sugar levels expressed in nM and determined before the i.p. administration of 2 g/kg of D- glucose (0 minute), then after 30, 60 and 90 minutes following the administration are given in Table I for the control group and the test groups treated with an i.p. dose of 30 or 100 mg/kg of the active substance extracted from the medicinal herb Viola tricolor.

Table I

From Table I it can be seen that the intraperitoneal administration of glucose in a dose of 2 g/kg resulted in a significant increase of the blood sugar level in the control mice. In the control group, from the starting level of 12 mM (note that the animals had not been starved before the test), the glucose concentration raised to as high as 22 mM 30 minutes after the treatment, and even 90 minutes after the treatment the glucose concentration was higher by 5 mM than the starting value. In case of animals treated with an i.p. dose of 30 mg/kg of the active substance extracted from the medicinal herb Viola tricolor, the increase of blood sugar level was slow, and even the highest blood sugar concentration did not exceed a value of 18 mM. The i.p. administration of 100 mg/kg of the active substance extracted from the medicinal herb Viola tricolor raised the blood glucose level by about 2 mM in 30 minutes, and after 60 minutes, the starting blood sugar level was restored. In summary, it can be stated that, in both dosage levels examined, the active substance extracted from the medicinal herb Viola tricolor reduced, significantly, the increase of blood glucose level induced by the glucose load, thus, improved the glucose tolerance of the animals.

Toxicity test

5 male NMRI white mice having a body mass of 23-25 g were treated, once, with a dose of 200 mg/kg of the active substance, intraperitoneally. The behaviour of the animals was evaluated for a week, neither any change of behaviour, nor weight loss was experienced. Thus, it can be stated that an i.p. dose of 200 mg/kg of the active substance extracted from the medicinal herb Viola tricolor does not result in an acute toxic effect in mice.

Restoration of reduced intestine relaxation The intestine motility test described above which was carried out on the ileum isolated from artificially diabetic rats gave the results summarized in Table II.

Table Il

The data of Table Il show that the contraction developed with acetylcholin is considerably compensated by isoproterenol on the ileum of healthy animals in the control group. Treatment with streptozocin reduced the relaxation significantly, however, treatment with the active substance extracted from Viola tricolor restored the relaxation ability of the ileum. Correction of damaged relaxation of vessel The results shown in Table III were obtained in the test of aorta relaxation of artificially diabetic rats. Table

The data of Table III indicate that in animals treated with streptozocin, the relaxation of vessel was reduced to a third of the value obtained in healthy animals. Treatments with the active substance extracted from Viola tricolor improved the relaxation of vessel significantly.

The above in vitro and in vivo tests prove that an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient enhances the mitochondrial genesis and compensates the reduced function of the constitutive nitric oxide synthase enzyme. The effect on mitochondrial genesis becomes pronounced when the usual mitochondrial content of the cells is reduced by a pathological effect e.g. hyper-glycaemia. In the background of the increase of mitochondrial genesis, an enhanced expression and function of chaperon and cNOS proteins were observed. Nitric oxide that depends on cNOS stimulates mitochondrial biogenesis and increases the expression of transcription factors that regulate biogenesis. The chaperons (HSP70, HSP90, HSP60, HSP27) partly contribute to the formation and stabilization of the functionally active cNOS complex, partly have an important role in the transport of mitochondrial proteins as well as in the compensation of any oxidative load (e.g. hyperglycaemia).

Therefore, it is expected that an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient provides for a curing effect in case of disorders owing to a damage of the mitochondrion or a reduced function of the cNOS enzyme; advantages can be awaited in states or diseases when an increase of mitochondrial genesis is useful; furthermore, diseases connected with a damage of the mitochondrion or the reduced function of the cNOS enyzme can be prevented by applying said extract or active ingredient.

An extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient can be effective especially in the following states: I. Through exerting an influence on the mitochondrial function and promoting the mitochondrial biogenesis a) States and diseases requiring fast mitochondrial regeneration:

- long-lasting immobilization, states following a disease accompanied by weight loss,

- regeneration phase of anorexia. b) States requiring increased mitochondrial demand:

- muscle (especially striated muscle) developing training,

- sudden loading of the muscles (especially striated muscles) and the subsequent period,

- muscular strain

- adaptation to high-altitude. c) States accompanied by increased mitochondrial loss or damage:

- oxidative damage,

- myocardial damages caused by hypoxia-reoxigenation, c-i ) diseases connected with the mitochondrial DNA:

- myoclonus epilepsy and ragged red fibers syndrome,

- leber hereditery optic neuropathy,

- MLAS,

- Leight's disease,

- Kern-Sayre's syndrome,

- chronic progressive external ophtalmoplegia,

- Alper's syndrome,

C₂) neurodegenerative diseases which are not connected with the mitochondrial DNA: - ALS (amyotrophic lateral sclerosis),

- Huntington's disease,

- Alzheimer's disease,

- Parkinson's disease. d) Mitochondrial damage caused by exogenic agents:

- states that follow the damage of skin surface owing to ultraviolet light (such as a sunburn or a precancerous state). e) Myopathies, especially certain forms of cardiomyopathy.

II. Treatment or prevention of metabolic diseases caused by insufficient mitochondrial function:

- diabetes

- insulin resistance,

- metabolic syndrome

- obesity.

III. Slowing of aging that has been accelerated due to mitochondrial damages.

IV. Other application possibilities due to improvement of cNOS function: a) Motility disorders of the gastrointestinal system:

- achalasia,

- infantile hypertrophic pylorus stenosis,

- Hirschprung's disease,

- diabetic gastropathy,

- reflux oesophagitis,

- gastrointestinal function disorder in case of diabetes,

- gastroparesis, - functional dyspepsia,

- intestinal pseudoobstruction and colitis,

- common motility disorders of the gastrointestinal system (e.g. obstipation),

- dysfunction of sphincters (e.g. pyloric sphincter, sphincters of the anus). b) Gall bladder dysfunctions:

- biliary dyskinesia,

- formation of gallstone,

- dyslipidemia,

- types Il and III biliary and pancreatic sorts of sphincter of Oddi dysfunction (SOD),

- post-cholecystectomy syndrome, c) Functional disorders of urinary bladder:

- neurogen urinary retention,

- urinary disorder in diabetes. d) Erectile dysfunctions:

- neurogen erectile dysfunction. e) Impaired fertility. f) Muscle degeneration:

- Duchene muscular dystrophy. g) Traumatic peripheral nerve lesion, h) Nephrosis, traumatic renal demage. i) Disorders of the blood circulation:

- arteriosclerosis,

- vasoconstriction. j) Damages caused by smoking especially vasoconstriction, thrombosis, arteriosclerosis, chronic bronchitis, pulmonary emphysema.

Thus, the composition or pharmaceutical composition of the invention containing an extract of a part of a medicinal herb belonging to the order of Violates, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient is suitable for:

- exerting an influence on the mitochondrial function and promoting the mitochondrial biogenesis;

- the treatment or prevention of metabolic diseases caused by insufficient mitochondrial function;

- slowing of aging that has been accelerated due to mitochondrial damages;

- various application possibilities due to improvement of cNOS function, as listed above under items I, II, III and IV.

A preferred embodiment of the invention refers to the use of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, for the preparation of a composition suitable for enhancing the energy supply of the cells through mitochondrial genesis thereby strengthening the organism of a mammal.

A preferred embodiment of the composition of the invention containing the solid residue remaining after the removal of the solvent content of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, as the active ingredient and one or more carrier(s) is suitable for enhancing the energy supply of the cells through mitochondrial genesis thereby strengthening the organism of a mammal.

A preferred composition of the invention is a roborant composition that improves the physical condition of the body after illness or anorexia or in case of muscle development trainings.

Furthermore, a preferred embodiment of the invention refers to the use of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, for the preparation of a pharmaceutical composition suitable for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxide synthase enzyme, especially neurodegenerative diseases comprising ALS, Parkinson's disease, Alzheimer's disease and Atkinson's disease, and/or myopathies comprising cardiomyopathy, and/or metabolic diseases comprising diabetes, insulin resistance, metabolic syndrome and obesity, and/or motility disorders of the gastrointestinal system comprising achalasia, infantile hypertrophic pylorus stenosis, Hirschprung's disease, diabetic gastropathy, reflux oesophagitis, gastrointestinal function disorder in case of diabetes, gastroparesis, functional dyspepsia, intestinal pseudoobstruction, colitis, common motility disorders of the gastrointestinal system and dysfunction of sphincters, and/or gall bladder dysfunctions comprising biliary dyskinesia, formation of gallstone, dyslipidemia and types Il and III biliary and pancreatic sorts of sphincter of Oddi dysfunction (SOD), post-cholecystectomy syndrome, and/or damage of the skin surface owing to ultraviolet light, and/or myocardial damages caused by hypoxia-reoxigenation and/or disorders of the blood circulation comprising arteriosclerosis and vasoconstriction.

A preferred embodiment of the pharmaceutical composition of the invention containing the solid residue remaining after the removal of the solvent content of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, as the active ingredient and one or more pharmaceutical carrier(s) is suitable for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxide synthase enzyme, especially neurodegenerative diseases comprising ALS, Parkinson's disease, Alzheimer's disease and Atkinson's disease, and/or myopathies comprising cardiomyopathy, and/or metabolic diseases comprising diabetes, insulin resistance, metabolic syndrome and obesity, and/or motility disorders of the gastrointestinal system comprising achalasia, infantile hypertrophic pylorus stenosis, Hirschprung's disease, diabetic gastropathy, reflux oesophagitis, gastrointestinal function disorder in case of diabetes, gastroparesis, functional dyspepsia, intestinal pseudoobstruction, colitis, common motility disorders of the gastrointestinal system and dysfunction of sphincters, and/or gall bladder dysfunctions comprising biliary dyskinesia, formation of gallstone, dyslipidemia and types II and III biliary and pancreatic sorts of sphincter of Oddi dysfunction (SOD), postcholecystectomy syndrome, and/or damage of the skin surface owing to ultraviolet light, and/or myocardial damages caused by hypoxia-reoxigenation and/or disorders of the blood circulation comprising arteriosclerosis and vasoconstriction.

A preferred pharmaceutical composition of the invention is suitable for the prevention or treatment of the motility disorders of the gastrointestinal system, thus, a preferred pharmaceutical composition of the invention is a prokinetic agent that induces movement in the gastrointestinal system.

An especially preferred pharmaceutical composition of the invention is suitable for the prevention or treatment of reflux oesophagitis.

The invention includes a method for enhancing the mitochondrial genesis thereby strengthening the organism of a mammal or for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxid synthase enzyme, especially neurodegenerative diseases, myopathies, metabolic diseases, motility disorders of the gastrointestinal system, gall bladder dysfunctions, disorders of the blood circulation or damage of the skin surface owing to ultraviolet light in which the patient being in need thereof is treated with a therapeutically effective amount of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient.

The invention is further elucidated by means of the following Examples.

Example 1 Preparation of an extract

Process A

10O g of the dry, finely powdered parts of Viola tricolor grown over the earth (i.e. leaf, stem, flowers) are extracted with water in a mass ratio of 5:200 at 60 ⁰C under intensive stirring over a water bath. The aqueous extract obtained is filtered, the plant matter is pressed, then the extract is sedimented for 4-8 hours, and filtered again. The dry matter content of the aqueous extract obtained amounts to 5.5-5.9 mg/ml. The water is removed by lyophilization while maintaining the temperature of the tray under -50 ⁰C. The dry residue obtained is stored in darkness at room temperature and protected from moisture. The dry matter (i.e. the active substance) has a flavonoid content of 3.0-3.4 g/100 g, total polyphenol content of 9.7-10.0 g/100 g , and total content of polyphenol in the skin powder of 2.0-2.5 g/100 g.

Process B

100 g of the dry, powdered parts of Viola tricolor grown over the earth (i.e. leaf, stem, flowers) are extracted with water in a mass ratio of 5:150 by boiling at 100 ⁰C. The aqueous extract obtained is worked up as described under process A. The aqueous extract has a dry matter content of 5.7-6.2 mg/ml. The lyophilized product (i.e. active substance) prepared as given under process A has a flavonoid content of 2.8-3.1 g/100 g, total polyphenol content of 10.1-11.0 g/100 g , and total content of polyphenol in the skin powder of 1.9-2.2 g/100 g.

Process C

100 g of the dry, powdered parts of Viola tricolor grown over the earth (i.e. leaf, stem, flowers) are extracted with aqueous ethanol containing 75 % by volume of ethanol in a mass ratio of 5:200 in a cold ultrasonic bath. The extract is filtered and the ethanol is removed by evaporation under reduced pressure. The remaining aqueous phase is dried by lyophilization as described under process A.

Example 2 Preparation of capsules

0.5 g portions of the lyophilized active substance prepared according to Example 1 , process B are filled into hard gelatin capsules, the capsules are closed, placed into a glass container that is sealed airtightly..

Example 3 Preparation of syrup

To 1000 ml of the aqueous extract prepared according to Example 1 , process A (dry matter content: 5.8 mg/ml), 20 ml of glycerol, 100 ml of 70 % aqueous sorbitol solution, 0.1 g of aroma substance and 1 g of methyl paraben are added, the mixture is homogenized and filled into bottles of 50 ml.

Claims

Claims:

1. Use of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, for the preparation of a composition suitable for enhancing the energy supply of the cells through mitochondrial genesis thereby strengthening the organism of a mammal.

2. A composition for enhancing the energy supply of the cells through mitochondrial genesis thereby strengthening the organism of a mammal comprising the solid residue remaining after the removal of the solvent content of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, as the active ingredient and one or more carrier(s).

3. A composition of Claim 2 which is a roborant composition.

4. Use of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, for the preparation of a pharmaceutical composition suitable for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxide synthase enzyme, especially neurodegenerative diseases, myopathies, metabolic diseases, motility disorders of the gastrointestinal system, gall bladder dysfunctions, disorders of the blood circulation or damage of the skin surface owing to ultraviolet light.

5. A pharmaceutical composition for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxide synthase enzyme, especially neurodegenerative diseases, myopathies, metabolic diseases, motility disorders of the gastrointestinal system, gall bladder dysfunctions, disorders of the blood circulation or damage of the skin surface owing to ultraviolet light comprising the solid residue remaining after the removal of the solvent content of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, as the active ingredient and one or more pharmaceutical carrier(s).

6. A pharmaceutical composition of Claim 5 suitable for the prevention or treatment of motility disorders of the gastrointestinal system.

7. A method for enhancing the mitochondrial genesis thereby strengthening the organism of a mammal or for the prevention or treatment of diseases deriving from mitochondrial damage and/or reduced function of the constitutive nitric oxid synthase enzyme, especially neurodegenerative diseases, myopathies, metabolic diseases, motility disorders of the gastrointestinal system, gall bladder dysfunctions, disorders of the blood circulation or damage of the skin surface owing to ultraviolet light in which the patient being in need thereof is treated with a therapeutically effective amount of an extract of a part of a medicinal herb belonging to the order of Violales, wherein said part has grown above the earth, or the solid residue remaining after the removal of the solvent content of the extract as the active ingredient.