MX2008005921A - Extracts from the bark of corynanthe species, use thereof, and medicaments, dietary products, and pharmaceutical preparations containing said extracts - Google Patents

Abstract

The invention relates to extracts from the bark of corynanthe species, especially corynanthe pachyceras, and the use thereof for treating and preventing diseases of the urinary tract collecting system, sexual disorders, lipid metabolism disorders, cardiovascular diseases, and acute and chronic pain conditions. The invention further relates to medicaments, dietary products, and pharmaceutical preparations containing said extracts.

Description

EXTRACT OF CORYNANTHE CORTEX SPECIES AND THE USE OF THESE AS WELL AS MEDICINES, DIETARY FOOD PRODUCTS AND PHARMACEUTICAL PREPARATIONS CONTAINING SAID EXTRACTS FIELD OF THE INVENTION The present invention relates to bark extracts of corynanthe species, in particular corynanthe pachyceras, as well as their use for therapy and prophylaxis of lower urinary tract diseases, sexual dysfunctions, disorders of lipid metabolism, cardiovascular diseases and conditions. of acute and chronic pain. The present invention also relates to medicaments, dietetic food products and pharmaceutical preparations containing said extracts.

BACKGROUND OF THE INVENTION Benign prosthetic hyperplasia (BPH) and the symptoms of the lower urinary tract that accompany it (LUTS) are, by far, the most important urological diseases in men. The calculations indicate that one third of men over 50 years of age develop LUTS during their lifetime and that a surgical intervention will be necessary for 25% of them. The percentage of men suffering from BPH / LUTS increases to more than 90% until the ninth decade of his life. In the Federal Republic of Germany approximately 4 million patients are treated every year to combat these sets of symptoms. In view of the increasing life expectancy and the increased health awareness, an additional increase in the frequency of illnesses has to be negotiated for the future. (R.B. Moreland et al., J. Pharmacol, Exp. Ther. 2004, 308, 797). Despite the great clinical importance of BPH / LUTS, the etiology and pathogenesis of this disease have not been clarified to any great extent. In addition to advanced age, an endogenous production of androgens is an important precondition for the development of BPH. In general, BPH is considered, in this way, as an endocrinopathy of senile men, which develops as a consequence of the hormonal reorganization with advanced age. From a histological point of view, BPH is a benign growth of the epithelial and stromal part of the prostate. Due to the location of the prostate in the urethra near the exit of the bladder, urinary obstructions, which are accompanied by symptoms such as polyuria and nocturia as well as incomplete and delayed urination, occur due to an enlarged organ. In advanced stage renal failure, it can occur a uremia as a result of urinary stasis. In addition, due to the stasis of urinary retentions and secretions, an abacterial prostatitis, congestions and recurrent infections of the urinary tract develop, which are responsible for irritative urination disorders as well as obstructive diseases. In addition to a static component that is due to an enlargement of the prostate and the mechanical urination disorders caused by this reason, a dynamic component seems to be involved in BPH / LUTS which is manifested by an increased tone of the smooth muscles. The degree of involvement of both mechanisms can vary, to a large extent, from patient to patient. This explains that there is only a marginal correlation between the size of the prostate and the severity of the symptoms (C. G. Roehrborn and D. A. Schwinn, J. Urol. 2004, 171, 1029). Also basically, similar changes in smooth muscle tone are involved in other conditions of the lower urinary tract, also in women, such as stress incontinence, forced incontinence and bladder emptying disorders. In view of the fact that the term BPH is reserved for the histological or macroscopic diagnosis of hyperplasia of the prosthetic gland, the LUTS that accompany it such as forcing to urinate, polyuria and nocturia as well as incomplete and delayed urination prevails in patients. The variety of alternative treatments for the BPH ranges from a wait-and-see attitude ("awaiting alert") to an open prostatectomy. Because of its effectiveness, the transurethral resection of the prostate, to which about 33,500 men are subject per year in Germany, is considered the Gold Standard of surgical methods. However, the considerable risk of mortality and mortality from invasive treatment methods is not acceptable for many patients, particularly patients with a lower severity of BPH, which explains the growing importance of drug therapy approaches. In addition to phytochemicals, which are frequently used in case of less severe disease stages, the variety of drugs includes, mainly, inhibitors to i-antagonists and 5-ar-reductase (E. Koch, Planta Med.67, 489-500 (2001)). The use of "i-antagonists" is based on the view that the dynamic component of BPH / LUTS is caused by an increased tone of the smooth prosthetic muscles, which is mediated by a high release of noradrenaline from sympathetic neurons. At present, it is generally accepted that a? A-adrenoceptors and a? D-adrenoceptors are expressed, predominantly, in the prostate. The results of the clinical studies show that the A-receptor blockers produce a clinically significant improvement in symptoms and maximum urinary flow. A particular advantage of a -receptor blockers is their rapid action attack. However, at present there is no convincing evidence that they prevent further enlargement of the prostate. Side effects of a -receptor blockers include dizziness, headaches, weakness, orthostatic dysregulation, rhinitis and sexual dysfunction (retrograde ejaculations), which are caused in a prevalent manner by the action of the a-receptor blockers in the CNS and the cardiovascular system. The development of subtype-specific a-receptor blockers, which predominantly inhibit "IA-receptors and a? D-receptors (for example, tamsulosin), aims to reduce the frequency of incidents and the severity of side effects. However, alfuzosin to non-selective i-antagonist exhibits, interestingly, a favorable profile of side effects such as tamsulosin, which is usually described as a blocker of the uroselective i-receptor. In addition to pharmacodynamic actions, the pharmacokinetic properties seem to contribute, significantly, to uroselectivity. Therefore, for example, effects on blood pressure can be prevented by a slow dose or sustained release formulations. In addition, a-A receptors are also involved in the control of blood pressure in addition to a1B-receptors (RB Moreland et al., J. Pharmacol. Exp. Ther 308, 797, 2004; CG Roehrborn and DG Schwinn, J. Urol., 171, 1029, 2004) which limit the possibilities of developing blockers of a-uroselective receptors. In general, a BPH develops in the presence of only biologically effective male sex hormones. A BPH has never been observed virtually in men who have had to undergo castration before age 40 or in whom the lack of androgen formation or insufficient androgen formation only occurs in the gonads due to a hypofunction of the hypophysis. In the same way, the normal development of the prostate and the development of a BPH do not occur in the case of a hereditary defect or in the absence of androgenic receptors (for example, testicular feminization). The most important androgen biologically is dihydrotestosterone (DHT), which is formed locally from testosterone under the influence of 5- a -reductase. Because DHT stimulates, predominantly, the epithelial components of BPH, a pause in growth or an atrophy of the glandular component can be achieved by inhibiting 5-reductase. Without However, the stromal component of BPH is not affected in a virtual way in any way. For example, the absorption of finasteride from the 5- a -reductase inhibitor leads to a reduction of the prosthetic DHT concentration up to 85% while the average reduction in prostate size amounts to approximately 20% only and also requires a period up to 12 months. This effect is of clinical relevance essentially only in cases where the volume of the prostate is greater than 40 ml at the start of therapy. Although androgens play a central role in the development and normal function of the prostate as well as in the development of a BPH, male sex hormones alone are not sufficient to produce the growth of prosthetic cells. Numerous experimental investigations show that the effects that stimulate the growth of androgens in vivo are mediated by the local synthesis of the growth factors and that the dysfunctions in the paracrine and autocrine mechanisms of growth control within the epithelial and stromal components of the prostate are involved, in a substantial way, in the development of PBH. A variety of growth factors (e.g., Epidermal Growth Factor [EGF], Transforming Growth Factor-ar [TGF-a], TGF-?, Growth Factor Basic fibroblastic (bFGF), Keratinocyte Growth Factor [KGF], Nerve Growth Factor [NGF], Insulin Type I Growth Factor [IGF-1] etc.) and its receptors have actually been detected in the prostate . Because BPH is usually accompanied by inflammatory reactions that probably play an important role in the pathogenesis, Platelet-Derived Growth Factor (PDGF), which is released for example by fibroblasts, thrombocytes and leukocytes, is possible which is of particular importance for the proliferation of prosthetic cells (CJVíanos et al., J. Cell, Biochem, 52, 404-413 (1993)). Growth factors mediate their biological action by binding to specific receptors on the surface of cells, which have an intrinsic tyrosine kinase activity. After binding to the ligand, phosphorylation of the tyrosine residues occurs in the intracellular receptor domains, which produce a cascade of intracellular reactions. The stimulation of protein synthesis and DNA synthesis, as well as the activation of cell proliferation, are among these reactions. In this way, it is considered that receptor tyrosine kinase inhibitors are promising substances for the development of new drugs for the treatment of diseases which they are accompanied by increased cell proliferation (eg, cancer, atherosclerosis, psoriasis) (A. Levitzki and A. Gazit, Science 267, 1782-1788 (1995)). However, not enough attention has been paid to this mode of action in BPH therapy until now. In recent years, it has been shown in different epidemiological investigations that there is a close correlation between BPH / LUTS and the occurrence of erectile dysfunction (ED). For example, in the MSAM-7 study, it has been shown that the prevalence of PE in men without LUTS at an age between 50 and 80 years is approximately 25%. This rate increases in patients with severe symptoms to more than 80%. In addition, the frequency of occurrence of ED is increased by other concurrent diseases such as hypertension, diabetes, hypercholesterolemia, angina pectoris and depressions (M. Shabbir et al., Curr. Med. Res. Opin. 20, 603, 2004). The relaxation of the penis is maintained, predominantly, by the binding of noradrenaline to a? A-receptors and a? B-receptors in the corpus cavernosum. Thus, it is not surprising that there is an improvement in sexual function in patients with ED by means of therapy with a -receptor blockers (for example, doxazosin and tamsulosin). In contrast, erections are predominantly mediated by the vasodilatory effect of rust nitric (NO). NO is released from nitrile neurons and is also synthesized by endothelial cells in the corpus cavernosum and corpus spongiosum. By stimulating guanylate cyclase and increasing the synthesis of cGMP, NO causes a relaxation of smooth muscle cells. Therefore, a combination of i-antagonistic and or2-antagonistic effects is considered to be particularly favorable for the treatment of ED because the inhibition of a? -receptors produces, directly, a relaxation of the muscle and the inhibition of a2-presynaptic receptors that is accompanied by an increased release of NO from nitrérgicas neurons. (http: // ww. bioportfolio.com / leaddiscovery / mdi002.htm). The ED treatment has been revolutionized by the development of sildenafil, a PDE5 inhibitor which inhibits the degradation of cGMP. However, sildenafil causes a variety of side effects (such as headaches, impaired vision, dyspepsia, hemodynamic effects). In addition, there is evidence that efficacy decreases in an increasing period of treatment (M. Shabbir et al., Curr. Med. Res. Opin. 20, 603, 2004). In addition, sildenafil can not be used by 30-50% of patients due to the severity of the disease and contraindications. There is currently no broad clinical experience with the new PDE5 inhibitors. PGEi is also an active substance for the treatment of ED; however, it must be injected or administered intraurethrally. As an additional treatment option, apomorphine is available, which acts as a dopamine agonist in the central nervous system. It is suitable for even less serious cases of ED. By having a generally lower side effect rate (nausea, cardiovascular effects), sublingual administration, which is required to avoid a first-pass hepatic metabolism, is considered a disadvantage (RB Moreland et al., J. Pharmacol Exp. Ther. 2004, 308, 797). In this way, there is still a great demand for effective medications for the treatment of ED, which produce few side effects. In 1999, a publication surprised the experts because it shows that the proportion of women with sexual dysfunctions (approximately 43%) is significantly higher than those of men (approximately 31%) in the United States (EO Laumann et al. ., JAMA 281, 537, 1999). In general, sexual dysfunctions in women are classified into four categories: lack of sexual desire or sexual aversion, reduced sexual excitability, painful sexual intercourse (vaginismus, dyspareunia) and orgasmic disorders. The portion of these different disorders are 30% (lack of sexual desire), 20% (reduced sexual excitability), 10 to 15% (painful sexual intercourse), and 10 to 15% (orgasmic disorders), where there is a very close relationship between the different types of sexual dysfunctions. Regular sexual function in men and women is controlled by a response cycle which consists of a mental expectation (sexual desire), effective congestion glass (erection in men, clitoral inflammation and vaginal lubrication in women), orgasm and finally resolution. This general course of events depends on a balanced equilibrium between the sympathetic and parasympathetic nervous systems. Therefore, the vessel congestion of the sexual organs is of vital importance. Because there is an analogy between the penis and the clitoris with respect to the anatomical structure and innervation of the corpus cavernosum, it should be anticipated that the same pharmacological mechanisms, which are efficient in the treatment of erectile dysfunction, can also be used. in the treatment of female sexual disorders and, in particular, in reduced sexual irritability.

SUMMARY OF THE INVENTION Therefore, it is the fundamental objective of the present invention provide drugs that have a positive effect on both the dynamic and the static component of BPH by inhibiting 1-adrenoceptors and 2-adrenoceptors and by inhibiting the proliferation mediated of the growth factor of epithelial cells and stromal cells and, in this way, it allows the comprehensive treatment of BPH syndrome, LUTS, ED and other sexual dysfunctions in men and women as well as hypercholesterolemia, functional disorders of the bladder, cardiovascular diseases and pain conditions. According to the present invention, this objective is solved by the use of bark extracts of corynanthe species, preferably corynanthe pachyceras for the therapy and prophylaxis of diseases of the lower urinary tract in men and women (for example, benign prosthetic hyperplasia, LUTS, prostate carcinoma, disorders for emptying the bladder, urinary retention, stress incontinence and forced incontinence), sexual disorders in men and women (such as impotence, erectile dysfunction, premature ejaculation, livid disorders, frigidity or anorgasmia) , disorders of lipid metabolism (eg, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia), cardiovascular diseases (eg, endothelial dysfunction, hypertonia, atherosclerosis or restenosis after bypass surgery or vasodilation) and chronic and acute pain conditions such as migraine, neuropathic pain (for example, in the case of diabetes), phantom limb pain, allodynia, pain after tissue injury or inflammation (for example, postherpetic neuralgia). Additional subjects of the present invention are bark extracts of corynanthe species, preferably of the corynanthe pachyceras bark, which has a balanced ratio of effective components, as well as drugs and food products for the therapy and prophylaxis of pathway diseases. lower urinary tract, sexual disorders, lipid metabolism disorders, cardiovascular diseases and chronic and acute pain conditions, which are characterized by an extract content according to the present invention, as well as a pharmaceutical preparation as a form of oral administration, parenteral, topical. The term "food products" used herein, refers in particular to dietetic food products, dietary supplementary products as well as medical foods and dietary supplements.

DETAILED DESCRIPTION OF THE INVENTION The corynanthe pachyceras (rubiaceae) is a tree which has a height of 15-20 meters and a trunk diameter of up to 60 centimeters in the evergreen tropical forest in West Africa (from Sierra Leone to Zaire). Wood is used, in a prevalent manner, for construction purposes, but also for the manufacture of mortars and combs. The dry bark of the trunk is used, to a large extent, in traditional medicine. The bark is chewed to fight colds and used as a decoction in case of leprosy, gastric diseases, diarrhea or heart and kidney diseases. The bark is used in the form of tea as an antipyretic agent in case of malaria and as a stimulant and aphrodisiac agent. The bark of corynanthe pachyceras contains approximately 6% of indolalcaloids, which are assigned to the group of alkaloids of coryantein (for example, dihydrocorinantein, corinantein, corinanteidin) or alkaloids of yumbin (for example, corinantin, to -yumbin). For particular corynanthe alkaloids, the focus resides in their antagonistic to -adrenoceptor activity. In addition, a leismanicidal activity is described in a moderate cytotoxicity for mammary cells and plasmodium falciparum (D. Staerk et al., Planta Med. 2000, 66, 531, 2000).

Corinanthin to -Yumbina Corinanteína Dihidrocorinanteína Corinanteidin A patent specification of 1971 claims the antihypertensive and sedative effects of an aqueous dry extract of the corynanthe pachyceras bark (BE758049, Omnium Chimique SA, 1971). It has been observed, surprisingly, that the alcoholic or ketonic extracts, preferably ethanolic-aqueous extracts of the bark of corynanthe species, in particular the corynanthe pachyceras exhibit a variety of additional biological effects such as properties that inhibit cell proliferation, vasorelaxant properties that depend on the endothelium, properties that decrease cholesterol, analgesic and antioxidant properties, in addition to antagonistic effects to -adrenoceptors and a2-adrenoceptors. These different activities suggest the therapeutic use of these extracts against various disease conditions. BPH, LUTS and sexual dysfunction in men and women, functional disorders of the bladder, hypercholesterolemia, atherosclerosis, endothelial dysfunctions and pain conditions are among these diseases. The effectiveness of the extracts, according to the present invention, against these indications is supported by the following pharmacological investigations. In these investigations it has been essential for the effect of the extracts according to the present invention, that the extracts should contain polyphenols as active ingredients in addition to alkaloids. Therefore, the term "polyphenols" refers to aromatic compounds having at least two hydroxyl groups which may be present in the monomeric, oligomeric or polymeric form.

The extracts containing both groups of compounds are superior, clearly, for the ingredients isolated from corynanthe pachyceras with respect to their full effect. According to the present invention, extracts of the bark of corynanthe species, which have a content of polyphenols and alkaloids, can be obtained according to the following method: (a) extract the dried and powdered bark of corynanthe species with an organic solvent or water or a mixture of one or more organic solvents and / or water at a temperature between 10 ° C and 100 ° C, (b) separating the material of the extracted plant from the extract solution, for example by filtration, (c) ) re-extracting, optionally, the extracted plant material with a solvent according to step (a) and separation according to step (b), (d) combining the extract solutions obtained in the steps ( b) and (c), (e) evaporating and drying the combined solution of step (d) to produce the dry extract. The preferred organic solvents in step (a) are alcohols or ketones, wherein the alcohol is preferably ethanol. Mixtures of ethanol and water are particularly preferred. The maceration and percolation can be considered as the extraction method in step (a) preferably. As a general rule, step (c) is carried out once and the ripple step (c) or a plural performance is also possible. The drying in step (e) can be carried out by methods known per se, such as lyophilization or vacuum drying at room temperature or elevated temperature. The corynanthe pachyceras are used as the preferred corynanthe species. According to the present invention, corynanthe species bark extracts contain both polyphenols and alkaloids in a balanced ratio for the intended use. Therefore, the polyphenol content is preferably at least 15%, particularly preferred at least 24%, and the alkaloid content is preferably at least 8%, particularly preferred at least 12% The compounds of epicatechin, procyanidin B2 and procyanidin Cl have been isolated from corynanthe pachyceras as common polyphenols. However, corynanthe polyphenols are not limited to these three compounds.

Epicatecin Procyanidin B2 A determination of the polyphenol contents was made when determining the total phenol content according to Folin-Ciocalteu. Additionally or alternatively, the contents of epicatechin, procyanidin B2 and procyanidin Cl can also be determined. The contents of alkaloids mentioned are the sum of the contents of corinantin, a-yumin, corinantin, dihydrocorinantin and corinantidin of individual alkaloids.

The extracts and fractions of extracts according to the present invention can be administered in the form of droplets, powders, granules, tablets, coated tablets (dragees) or capsules, preferably oral. However, a parenteral application in the form of an injection solution or a topical application in the form of creams, ointments, suppositories, patches or similar preparations is also possible. For the preparation of tablets, the extract is mixed with suitable pharmaceutically acceptable adjuvants such as lactose, cellulose, silicon dioxide, croscarmellose and magnesium stearate and pressed into tablets which can, optionally, be supplied with a suitable coating made from , for example, hydroxymethylpropylcellulose, polyethylene glycol, dyes (such as titanium dioxide, iron oxide) and talc. In accordance with the present invention, the extracts may also be filled into capsules, optionally, under the addition of adjuvants such as stabilizers, fillers and the like. The dose of the extract that can be administered per day is from 5 to 2000 mg, preferably 10 to 1000 mg and, particularly preferred, 50 to 500 mg. The effectiveness of the extracts, according to the present invention, the bark of corynanthe species is supported by the experiments described in the following.

Pharmacological investigations Test for agglutination properties of a-adrenoceptors The corynanthe extracts test and fractions of corynanthe extracts for interactions with α-adrenoceptors was carried out by a receptor agglutination test using rat brain cell membranes. For the preparation of cell membranes, male Sprague-Dawley rats (150-250g) were euthanized in C02 narcosis and the brains were removed (without the cerebellum). After the removal of adherent blood and meninges, the brains were immediately increased ten times their volume with an ice homogenization buffer (50mM Tris-HCl, pH 7.4) and homogenized by using a cooled glass homogenizer. The cell homogenate was centrifuged for 10 minutes at 50,000 g (4 ° C) and the pellet resuspended in an ice homogenization buffer. After further centrifugation (10 minutes at 4 ° C and 50,000 g) the cell membranes were raised ten times their volume with an agglutination buffer (50 mM Tris-HCl, 0.5 mM Na-EDTA, 0.01% ascorbic acid, 10 μM pargyline, pH 7.4) and stored in portions (1 ml) at -80 ° C. In accordance with the present invention, the alkaloid extract or fractions or the polyphenol fractions were dissolved using DMSO in a 150 μl binder buffer and incubated together with 50 μl brain cell membranes (2.5 mg / ml protein) and 50 μl. μl of radioactive ligands in the binder buffer for 45 minutes at room temperature. 3H-prazosin (300 pM, specific activity 80 Ci / mmol) was used as radioligand for the test concerning interactions with α-adrenoceptors. The nonspecific binder was measured in the presence of 2 μM phentolamine. 3H-clonidine (1 μM, specific activity 55.5 Ci / mmol) served as the radioligand for the determination of the a2-adrenoceptor binder. The test for the nonspecific binder for a2-adrenoceptors was carried out in the presence of 10 μM of yumbin. The reaction mixtures were subsequently filtered through glass fiber filters (type GF / B) which have been previously treated with polyethylene imine (0.2% in distilled water) overnight. After washing the filters twice using 3 ml of ice-cold binder buffer each time, the The filters were dried for 24 hours at 60 ° C. The determination of the bound radioactivity was carried out after the transfer of filters in 4 ml of scintillation fluid (filter safety, Zinsser-Analytik) in a beta counter. The percent inhibition of specific binding of 3H-prazosin to a? -adrenoceptors or 3H-clonidine to ar2-adrenoceptors was calculated as compared to the solvent control which has been investigated simultaneously. The determination of half of the maximum inhibition concentration (IC 50 values) was performed by non-linear regression calculations. The results of the investigations are summarized in Table 1. It can be seen that the extract, according to the present invention, of corinanta bark inhibits both the 3 H-prazosin aa -adrenoceptor binding and the 3 H bond. -clonidine to er2-adrenoceptors, this effect is essentially based on the presence of alkaloids. Table 1: The inhibition of 3H-prazosin and 3H-clonidine binding to "i-adrenoceptors and a2-adrenoceptors, respectively.

Test for the inhibition of growth factor-mediated cell proliferation The influence of all extracts or extract fractions on the induced cell proliferation of the growth factor was tested in NIH-3T3 fibroblasts of mice. Cells were cultured in Dulbecco's modified Tagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2mM antibiotic / antifungal solution and glutamine. The culture medium was replaced, usually twice a week. Four days after the last sub-culture, the adherent cells were separated using trypsin / EDTA from the bottom of the cell culture bottle and returned to suspend at a density of 50,000 cells per ml in DMEM supplemented with 0.5% FCS. Subsequently, the cells were transferred in a volume of 200 μl per well in microtiter plates (F-shape) and incubated at 37 ° C for an additional 96 hours. After replacing the medium (DMEM without FCS) and adding the substances, 10 mg / ml of recombinant human platelet-derived growth factor BB (PDGF-BB) was added 60 minutes later. Subsequently, the cells were once again cultured for 24 hours at 37 ° C in an incubator. Six hours before harvesting the cells, 0.5 μCi of methyl-3H-thymidine was added per well. After the expiration of the incubation period, the microtiter plates were centrifuged for 5 minutes at 400 g and the supernatant cells were carefully removed from the pipette. The cells were separated from the bottom using trypsin / EDTA and, subsequently, harvested in fiberglass filters (type G-10, ICH-201) using a cell harvester (Inotech). The determination of the incorporation of 3H-thymidine into newly synthesized DNA was carried out by means of a linear analyzer (LB 2842, Berthold). The determination of the inhibition of cell proliferation in the presence of extracts and fractions of extracts was performed in comparison with solvent controls tested simultaneously in each case. A summary of the results is shown in Table 2. It can be seen from these results that the cell proliferation that inhibits the action of the extracts, according to the present invention, can be attributed mainly to the polyphenol fraction. Table 2: Inhibition of PDGF-mediated cell proliferation of NIH3T3 fibroblasts.

Test for vasorelaxant properties For the test of vasorelaxant effects, the influence of the extracts according to the present invention and fractions of extracts on the contraction of the aorta isolated from male Sprague-Dawley rats was examined (Janvier , Le Genest, France). Immediately after removing the organs, they were transferred in Tyrode solution (in mM, NaCl 118.2, NaHCO3, 24.8, KC1 4.6, CaCl2 2.5, MgSO4 1.2, KH2P04 1.2, glucose 10) and released not to adhere to connective tissue. Then vascular rings having a thickness of about 4 mm were prepared. For some experiments, the endothelium was removed. For this purpose, the aortic rings were mounted in a steel cannula, pressed lightly against the steel cannula and the innermost vascular layer was subsequently removed by rotating and moving it simultaneously in the longitudinal direction. The aorta rings were fixed in an organ bath (20 ml, Hugo Sachs, Hugstetten) filled with Tyrode solution using metal hooks. The culture medium (37 ° C) was gasified, permanently, with carbogen (pH 7.4). For experiments concerning the determination of vascular relaxation after pre-contraction of the organs using phenylephrine (PE), the following substances were added to the Tyrode solution: propanol-HCl (6 μM, RBI), corticosterone-HCl (6 μL) Sigma) and desipramine (0.6 μM, Sigma). In tests concerning relaxation, after stimulation using U-46619, indomethacin (2.8 μM, Sigma) was added. The organ tension was measured in an isometric manner using a force transducer (Statham UC2, Hugo Sachs) under a preload of 1.0 g and recorded using a 4-channel writer (Linearcorder, Graphtec). After a 30-minute equilibrium phase, four contractions were produced with PE (0.15 μg / ml, EK 0.74 μM) at 15 minute intervals to achieve reproducible organ contractions. Following the fourth PE addition, the test substance was added in an increasing concentration after reaching the maximum contraction (cumulative dosing effect). At the end of the experiment, the endothelium dependence of the vasorelaxation was tested by the application of acetylcholine (0.25 μg / ml, EK 1.38 μM). A basically similar test procedure was also observed to test the relaxing effect on aortic rings without endothelium. After equilibration for 30 minutes, three contractions with PE (0.15 μg / 1, EK 0.74 μM) occurred at 15 minute intervals. After reaching the maximum contraction subsequent to the third addition of PE, acetylcholine (0.25 μg / ml, EK 1.38 μM) was applied to confirm the complete removal of the endothelium. After rinsing the acetylcholine, PE contraction (0.15 μg / ml, EK 0.74 μM) occurred after 25 minutes and, subsequently, the test substance was added in increasing concentrations. As described above with respect to PE, tests for a relaxant effect after inducing a contraction with U-46619 (0.022 μg / ml, EK 0.063 μM) or KCl (3 mg / ml, 40 mM EK) was carried out in a way identical The relaxing effect of the extracts on the agonists was determined as a percentage. The IC 50 values were determined by a non-linear regression analysis of concentration effect curves using Prism 3.0 software (GraphPad Software Inc.). The results of the experiments are shown in the following table. It is evident, from the data, that the vasorelaxant effect of the extracts according to the present invention after stimulation with PE, is mediated by the alkaloid that contains the fraction and the free fraction of alkaloid. The relaxing effect of the alkaloid-free fraction is completely dependent on the presence of an intact endothelium. The endothelium-dependent vasorelaxant effects of the alkaloid-free fraction could also be observed after precontraction of the vascular rings with U-46619 or KCl depolarization and, of course, are based on increased endothelial NO release. In contrast, the effect of the alkaloid fraction is based mainly on the presence of ingredients that have a -adrenoceptor that blocks the properties and is also, in this way, determinable after removing the endothelium.

Table 3: Test for vaso-relaxing effects Testing anti-hypercholesterolemic effects The influence of the extract, according to the present invention, on plasma cholesterol level was examined in male NMRI mice (Janvier, Le Genest, France) with Triton-induced hypercholesterolemia WE1339. The mice were preserved under standardized environmental conditions (21 ° C, 60% relative humidity, 12/12 hours light / dark) and had free access to drinking water and compacted food (Altromina 1324). The Triton R1339 (400 mg / kg Sigma) was dissolved in physiological NaCl solution and injected into the animals by the caudal vein (10 ml / kg). For an oral treatment of the test animals, the extract was initially 0.2% agar suspension and was supplied to the animals 24 hours and 1 hour before the injection of Triton WR1339 as well as 6 hours after the injection of Triton WR1339 by fattening (450 mg / kg in 10 ml / kg). The animals in the control group were treated with the carrier (0.2% agar, 10 ml / kg) only. One hour before the injection of Triton WR 1339 and 6 hours, 24 hours and 48 hours after the injection of Triton WR 1339, a blood sample (32 μl) was taken from the caudal vein of the animals using heparinized capillarity and the Cholesterol concentration was determined immediately afterwards (Reflotron, Boehringer Mannheim). Figure 1 shows the influence of oral treatment with the extract according to the present invention (450 mg / kg) on the cholesterol level in mice with hypercholesterolemia induced by Triton WR 1339 (# means P <0.05 compared to the control ( test t)). The results of the experiments show that the treatment with the extract according to the present invention obtained in Example 1 lead to a significant reduction in the level of increased cholesterol. Test for analgesic effects For the test of analgesic effects, the formalin test was used in mice. Local injection of formalin in the hind leg of the mice leads to an increased sensitivity to pain which occurs in two temporarily separated phases. The first phase is mediated by a direct stimulation of pain receptors due to the release of substance P, bradykinin and excitatory amino acids (eg, glutamine). In the second consecutive phase, an accumulation of histamine, serotonin and prostaglandins occurs in the tissue, which leads to a reaction of local inflammation and functional changes in the central nervous system. For the experiments, male NMRI mice (Janvier, Le Genest, France) having a weight of approximately 22-26 g were used. The animals were treated orally with extracts according to the present invention or extracts fractions. One hour later, 20 μl of a 3.5% formalin solution was injected into the plant of the left leg. Therefore, the animals were placed, individually, in wire cages and the number of pain reactions (paw licks) was recorded in a period of 45 minutes. The effect Pain inhibitor was determined in comparison with a control group tested simultaneously. The animals in this group were treated with a carrier medium only (0.2% agar suspension, 10 ml / kg). The results in Table 4 demonstrate the potent analgesic effects of corynanthe extracts which are substantially mediated by the alkaloids contained therein. Table 4: Test for analgesic effects Test for antioxidant properties The autoxidation of lipids is related to the emission of light. The determination of this extraordinarily weak chemiluminescence can be used both to quantify peroxides and to evaluate the efficacy of antioxidants. Brain tissue of male mice (NMRI, 20-30 g; Center d'Elevage Janvier, Le Genest-Saint Isle, France) serves as a tissue rich in lipids in the present investigation. After its removal, the brain was washed in physiological saline regulated with ice-cold phosphate (PBS, pH 7.4) and released from meninges and remaining blood. The tissue samples were homogenized in four times their volume (v / w) constituted by PBS and centrifuged for 10 minutes in 1000 g at 4 ° C. The supernatants were immediately diluted to three times their volume with the same buffer and stored on ice. 250 μl of the diluted supernatant was transferred to a test tube and incubated for 10 minutes at 37 ° C in a 6-channel luminometer (Multi-Biolumat LB 9505 C, Berthold, Bad Wildbad). After adding 25 μl of compound in PBS with 2.5% DMSO, the incubation was continued for another 10 minutes. Then, the intensity of the chemiluminescence (CL) was determined during a period of 60 minutes. The percent inhibition of autoxidation was calculated in comparison with a simultaneously tested solvent control (PBS with 2.5% DMSO). As can be seen from the results summarized in Table 5, the extract, according to the present invention, has potent antioxidant properties which are mediated, mainly, by the polyphenol fraction.

Table 5: Test for antioxidant properties Examples Determination of the total content of phenols according to Folin-Clocalteu The determination of the total content of phenols is carried out, in photometric form, after the reaction with the reagent molybdate-tungstate in analogy with the method of pharmacopedia for tanning agents (DAB) For this purpose, the extract is dissolved in aqueous ethanol, alkalized with sodium carbonate solution and added with the molybdate-tungstate reagent. After centrifugation, the absorbance of the supernatant solution is measured with respect to water at 720 nm. The calculation is based on epicatecin. Example 1: Dry the extract according to the present invention from the corynanthe pachyceras bark. 500 g of ground bark of corynanthe pachyceras were shaken twice for one hour at 60 ° C, using 3.5 kg of 60% by weight of ethanol each time. After filtration on Seitz Supra 1500, the combined extract solution was evaporated at approximately 50 ° C and reduced pressure and dried at 50 ° C under vacuum: 183.8g (36.8%). The extract contains 14.62% alkaloids (4.75% coryntin, 0.81% a-yumbin, 3.86% corinantein, 1.91% dihydrocorinantein and 3.29% corinanteidin) and had a total phenol content of 26.8% (including 2.66% of epicatechin, 3.05% procyanidin B2 and 1.25% procyanidin Cl). Comparative Example 1: Fraction of polyphenol (free of alkaloids). A solution of 445 g of dry extract according to Example 1 in 4 kg of ethanol (50% by volume) is placed in a column with 3.4 L of strongly acidic ion exchanger (Merck I) and eluted with ethanol (50%). in volume). 9L of eluate were collected, evaporated at 50 ° C under reduced pressure and dried in a drying cabinet at 50 ° C and 12 mbar: 352.4 g (79.2%). The extract does not contain alkaloids (corinanthin, a-yumin, corinantein, dihydrocorinantein and corinanteidin could not be detected) and presented a total phenol content of 28.4% (including 2.57% epicatezin, 2.24% procyanidin B2 and 0.76% procyanidin Cl). Comparative Example 2: Alkaloid Fraction The ion exchange column of Comparative Example 1 was further eluted using a mixture of 50% by volume ethanol and 5% NH3 solution (having a concentration of 25%). 16 L of eluate were collected and evaporated and dried as in Example 2: 46.8 g (10.5%). The extract contains 69.28% alkaloids (24.01% corantin, 2.25% "-yumbin, 19.05% corinantein, 9.56% dihydrocorinantein and 14.41% corinanteidin) and has a total phenol content of 13.0% (epicatechin, procyanidin B2 and procyanidin Cl could not be detected). Example 2: Tablets A dried extract of the corynanthe pachyceras bark (extract according to the present invention obtained in Example 1) is mixed with adjuvants and pressed into tablets (tablet center = article 1-6). The tablets are provided with a coating made of hydroxypropyl methyl cellulose (Articles 7-10).

Claims (12)

1. NOVELTY OF THE INVENTION
2. Having described the present invention, it is considered as a novelty and, therefore, the content of the following is claimed as property:
3. CLAIMS 1. Corynanthe pachyceras bark extract having a content of polyphenols and alkaloids, characterized because it is obtained by the following production process: (a) extract the dried and ground crust of corynanthe pachyceras with an organic solvent or a mixture of various organic solvents or a mixture of one or more organic solvents and water at a temperature between 10 ° C and 100 ° C, wherein the organic solvent is an alcohol or a ketone, (b) separating the material from the extracted plant from the extract solution, for example, by filtration, (c) re-extracting, optionally, the material of the extracted plant with a solvent according to step (a) and separating according to step (b), (d) combining the extract solutions obtained in the steps (b) and (c), (e) evaporating and drying the combined solution of step (d) to obtain the dry extract. 2. The extract according to claim 1, characterized in that the alcohol is ethanol. 3. The extract according to claim 1 or 2, characterized in that the extraction solvent is a mixture of ethanol and water.
4. 4. The extract according to any of claims 1 to 3, characterized by a content of polyphenols of at least 15% and alkaloids of at least 8%.
5. 5. The extract according to claim 4, characterized by a polyphenol content of at least 24%.
6. 6. The extract according to any of claims 4 or 5, characterized by an alkaloid content of at least 12%.
7. 7.-. The use of the extracts according to any of claims 1 to 6 for the manufacture of a medicament or a food product for the therapy and / or prophylaxis of diseases of the lower urinary tract, sexual disorders, disorders of lipid metabolism, cardiovascular diseases and conditions of chronic and acute pain, characterized because sexual disorders are selected from impotence, erectile dysfunction, premature ejaculation, frigidity and anorgasmia, where cardiovascular diseases are selected from endothelial dysfunction, atherosclerosis and restenosis after vaso-dilatation or bypass operation.
8. 8. The use according to claim 7, characterized in that diseases of the lower urinary tract are selected from benign prostatic hyperplasia, LUTS, prostate carcinoma, bladder emptying disorders, urinary retention, stress incontinence and urge incontinence. .
9. 9. The use according to claim 7, characterized in that disorders of lipid metabolism are selected from hypercholesterolemia, hyperlipidemia and hypertriglyceridemia.
10. 10.- The use in accordance with the claim 7, characterized in that the chronic and acute pain conditions are selected from migraine, neuropathic pains, phantom limb pain, allodynia, pains after tissue wounds and in case of inflammations.
11. 11.- The medicine or food product for the therapy and / or prophylaxis of diseases of the lower urinary tract, sexual disorders, disorders of the lipid metabolism, cardiovascular diseases and conditions of chronic and acute pain, characterized by an extract content according to any of claims 1 to 6.
12. 12. The pharmaceutical preparation, which consists of an extract according to any of the claims 1 to 6 and suitable adjuvants as an oral, parenteral or topical form of administration.