Field of the Invention The present invention relates to the use of a product comprising Catuama extract, comprising species of Trichilia sp., particularly Trichilia ca- tigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae).

Background of the Invention Medicinal plants known as catuaba (Trichilia sp.) have recog- nized uses due to their aphrodisiac activities, as a tonic and in the treatment of physical and mental fatigue.

Already known are, e. g., phytotherapeutic formulations prepared from extracts of catuaba plants, which can be used alone or in combination with other medicinal plant extracts, such as guarana. A number of alterna- tive formulations containing extracts of other species of catuaba are already well-know from the state-of-the-art, all of them being related to the tonic and stimulating effect of this group of plants.

There also exists in the art phytotherapeutic products comprising a combination of extracts of plants from the Trichilia sp. species, particularly <BR> <BR> Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopeta- lum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae).

A commercially available product comprising extracts of the above-mentioned plants in combination with suitable carriers is Catuama0.

More particularly, Catuama0 is a phytotherapeutical widely used in Brazil.

Its composition consists of 4 extracts from medicinal plants including : ca- tuaba (Trichilia catigua, A. juss, Meliaceae- (husk)), guarana (Paullinia cu- pana, K., Sapinadaceae- (seed)), muirapuama (Ptychopetalum olacoides, B., Olacaceae- (root)) and ginger (Zingiber officinale, L., Zingiberaceae- (rhizome)).

Summary of the Invention The present invention refers to the use of a product of the extract of Catuama comprising Trichilia sp., particularly Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae) as an antioxidant or cerebral vasodilator agent.

In another aspect, this invention refers to pharmaceutical com- positions comprising said extracts having antioxidant and cerebral vasodila- tor activities.

In yet another aspect, this invention refers to a method for the prophylaxis or treatment of cerebral vascular dysfunctions using said extract of Catuama.

In still another embodiment, the invention refers to the use of a product comprising extract of Trichilia sp., particularly Trichilia catigua (Melia- ceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), for preparing a pharmaceutical composi- tion for the prophylaxis or treatment of cerebral vascular dysfunctions.

Detailed Description of the Invention After extensive studies, the inventors have found that the extract of Catuama, comprising Trichilia sp., particularly Trichilia catigua (Melia- ceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olaca- ceae) and Zingiber officinale (Zingiberaceae), has extraordinary antioxidant and cerebral vasodilator activities.

As used herein"antioxidant activities or activities for prophylaxis

or treatment of cerebral vascular dysfunctions"includes activities related to disorders such as: loss of memory, vascular alterations, ischemia, difficulty in movements, difficulty in learning and concentrating, and for neurodegenera- tive diseases such as Parkinson's disease and Alzheimer's disease, which are pathologies associated, at least part, with prolonged exposure to the im- proper presence or excess of free radicals.

Examples of dysfunctions and uses according to the present in- vention comprise the prophylaxis and treatment of cerebral vascular dysfunc- tions, arteriosclerosis, stroke (AVC), cerebral ischemia; ischemic disturbances in patients with postrupture sub-arachnoid hemorrhage of congenital aneurysms, neurodegenerative diseases such as Parkinson's disease and Alzheimer's dis- ease; use as a facilitator of the arterial, cerebral and peripheral blood flow, and a protector of the structural integrity of cell membranes against free radical attacks; use as an aid in the treatment of signs and symptoms of mental deterioration, specially those related to aging; dizziness, chronic headache, labyrinthitis, poor concentration, disorientation, compromising of memory, lack of initiative, mood depression, unsociability, difficulties with daily activities and personal care, emo- tional lability, reduction in intellectual capacity, behavioral disorders, psychomo- tor retardation, deficits in learning, senile dementia; neurological deficit in mem- ory, concentration and attention; for the prophylaxis and treatment of symptoms of balance disorders (labyrinthine arteriosclerosis, irritability of the labyrinth, Me- niere's syndrome) such as: vertigo, dizziness, buzz, nystagmus, nausea and vomiting, resulting from use as an aid in the treatment of vascular alterations of the inner ear and labyrinth; neurosensorial disorders of vascular origin, in otorhi- nolaryngology and ophthalmology ; and in the treatment of migraine, of subjec- tive symptoms associated with arterial hypertension, for postapoplectic func- tional consequences.

A number of products comprising varying concentrations of ex- tract of the above plants are commercially available. The use thereof, so far recommended in the art is related to the treatment of physical and mental fatigues, neuromuscular asthenia and weariness.

Studies and research now carried out by the present inventors

show new antioxidant and cerebral vasodilator activities related to products based on the above mentioned extracts as confirmed by the data and tests disclosed herein.

The concentration of the extract of each plant of Trichilia sp., particularly Trichilia catigua, Paullinia cupana, Ptychopetalum olacoides and Zingiber officinale, in the product or pharmaceutical composition of the pres- ent invention ("Product comprising extract of Catuama") is as follows : Liquid formulation: Component % (m/v) Generic Preferred Extract of Trichilia sp (specially Trichi-0. 50 to 5.50 0.50 to 5.0 lia catigua) Extract of Paullinia cupana 0.10 to 7.50 0.1 to 5.0 Extract of Ptychopetalum olacoides 0.01 to 5.50 0.01 to 5.0 Extract of Zingiber officinale 0.10 to 2.00 0.1 to 0.40 Suitable excipient 79. 50 to 99.29 84.60 to 99.24 Solid formulation : Component % (m/m) Generic Preferred Extract of Trichilia sp (specially Trichilia 5 to 50 30 to 50 catigua) Extract of Paullinia cupana 2 to 30 10 to 21 Extract of Ptychopetalum olacoides 0.2 to 15.0 5.0 to 12 Extract of Zingiber officinale 0.50 to 3.0 0.5 to 1.50 Suitable excipient 2.0 to 92.30 15.5 to 54.5 In its dry and excipient-free form, extract of Catuama comprises: Formulation Component % (m/m) Generic Preferred Extract of Trichilia sp (specially Trichilia ca-17 to 40.0 22.0 to 34.0 tigua) Extract of Paullinia cupana 24.0 to 57.0 32.0 to 48.0 Extract of Ptychopetalum olacoides 17. 0 to 40.0 22.0 to 34.0 Extract of Zingiber officinale 2.0 to 5.0 2.5 to 4.0

The product may comprise usual excipients for formulation such as preservatives, colorants, carriers, etc. Adequate excipients are well known by those skilled in the art and do not constitute limiting aspects of the invention.

For the purposes of the present invention, all plants of the genus Trichilia were found to be useful, such as, e. g., T. catigua A. Juss., T. claus- seni C. DC., T. casaretti C. DC., T. pallida Swartz. and T. elegans A. Juss.

According to a preferred embodiment of the present invention, it was found that, among the genera comprised of species Trichilia sp., Trichilia catigua is particularly suitable for the intended purposes. Additionally, the materials extracted from Trichilia sp. are preferably fragments of the whole plant, more preferably stalk, which are advantageously used as extract, more preferably they are formulated with pharmaceutical acceptable inert carriers. Formu- lations of Trichilia sp. useful for the present invention can be administered, e. g., orally in the form of tablets, coated tablets, hard and soft gelatin cap- sules, solutions, emulsions and suspensions; or rectally, in the form of sup- positories. Suitable carriers include, but are not limited to, lactose, starch or derivatives thereof, talc, stearic acid or salts thereof in the case of solid for- mulations for oral administration. Suitable carriers for soft gelatin capsules include vegetable oils, waxes, fats, semi-solid and liquid polyols. Solutions may be prepared comprising selected carriers such as water, polyols and carbohydrates. In the case of suppositories, suitable carriers comprise natu- ral or hardened oils, waxes, fats and polyols.

In addition to the carriers, the formulations of the Catuama ex- tract according to the present invention may contain preservative agents, solubilizing agents, stabilizers, wetting agents, emulsifiers, sweeteners, col- oring agents, flavoring agents, tonicity adjustment substances, buffers, coating agents or anti-oxidants.

However, an effective dosage for administration to humans was found to be in the range from 10 mg to 0.5 g Catuama extract.

In the case of pharmaceutical formulations containing Catuama extract, the intended effects can be effectively obtained using from 0.2 to

50% by weight of said extract, based on the total formulation.

The invention will now be described specifically referring to the applicant's product having the name Catuama@, that is already commercial- ized in Brazil for the treatment of several chronic diseases such as physical and mental fatigue, neuromuscular asthenia and weariness. The pharma- ceutical formulations available allow the product to be administered orally.

Another advantage of the product is associated with the lack of any reported undesirable or side effects, even when the product is used for long periods of time.

Together, the results discussed herein show that the Catuama extract referred to in this invention (specially Catauma@) exhibits antioxidant and cerebral vasodilator effects, specially when used regularly and for ex- tended periods of time.

The present invention relates to the Catauma extract effects on pathologies requiring the use of antioxidants and prophylaxis or treatment of cerebral vascular dysfunctions. The Catuama extract of the invention (par- ticularly Catuama') is particularly used in the treatment of pathologies in- volving vascular, mainly cerebral, alterations caused by an increase of the oxidative stress and the formation of active species of oxygen. The Ca- tuama extract (specially Catuama@) is comprised of substances acting on the central vascular system, neutralizing free radicals, thus being of great clinical importance. Such effects are a result of the synergistic effect be- tween the diverse substances present in the four extracts making up the product. The synergism is defined as the effect arising from the association of low doses of two or more substances exhibiting the same effect. Such concept is particularly important when taking into account the reduction of undesirable effects of said substances. In the specific case of Catuama ex- tract of the present invention (especially the Catuama@), the four plants comprised therein have pharmacological effects that are potentiated when the extracts are used in combination.

Control of free radicals production is of great interest, since this phenomenon is involved in the development of many diseases. Thus, sub-

stances able to inhibit or delay that situation are greatly useful in clinics, loss of memory, vascular alterations, cerebral ischemia, difficulty in movements, difficulty in learning and concentrating, and for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, which are pathologies associated, at least in part, with extended exposure to an excess of free radicals. Furthermore, it is known that drugs exhibiting the property of neu- tralizing free radicals are important in slowing the aging process and its con- sequences. It can therefore be said that continued use of the phyto- therapeutic product Catuama extract (specially Catuama (D) is highly applica- ble, since it shows a great capacity to neutralize free radicals.

Brief Description of the Drawings Figure 1 shows the concentration-response curve for Catuama extract (10-3000 lig/ml) in porcine basilar artery rings with (E+) and without (E-) endothelium pre-contracted with serotonin (1 p. M). Each point repre- sents the average of 4 to 5 experiments and the vertical bars indicate the E. P. M.

Figure 2 shows the effect of Catuama extract (1-100 lig/ml) on nitrite/nitrate levels in peritoneal macrophages of mice stimulated with LPS (1 pg/ml) and INF-y (10 U/ml). The results are expressed as the average E. P. M. The asterisks indicate the significance when compared to the control group (stimulated), *p < 0.05 **p < 0.01 (n = 3).

Figure 3 shows the effect of Catuama extract (1-100 lig/ml) on cell viability of peritoneal macrophages from mice evaluated by means of the MTT method. The results are expressed as the average E. P. M. (n = 3).

Figure 4 shows the effect of Catuama extract (1-500 pLg/ml) on the formation of free radicals from oxidation of deoxyribose. Each result rep- resents the average of 4 to 5 samples and the vertical bars represent the E. P. M.

Figure 5 shows the effect of Catuama extract (10-70 g/kg) on the formation of superoxide anion radical from the xanthine/xanthine oxidase system. Each group represents the average of 4 to 5 samples and the verti- cal bars represent the E. P. M.

Figure 6 shows the effect of Catuama extract (10-70 g/kg) on the formation of superoxide anion radical from the superoxide dismutase en- zyme assay. XO (xanthine oxidase), SOD (superoxide dismutase). Each result represents the average of 4 to 5 samples and the vertical bars repre- sent the E. P. M.

The illustrative test examples below are given for a better de- scription of the present invention. However, the data and procedures illus- trated therein refer to certain embodiments of the present invention and are not to be construed as limiting the scope thereof.

The following tests were carried out using a composition of ex- tracts in the solid and dry form (herein referred to as Catuama extract) as follows : Formulation Component % (m/m) Extract of Trichilia sp (specially Trichilia catigua) 29.5 Extract of Paullinia cupana 37.6 Extract of Ptychopetalum olacoides 29. 5 Extract of Zingiber officinale 3. 4 Cerebral Vasodilator Effect In order to evaluate the relaxing effect of Catuama extract on cerebral vessels, basilar arteries obtained from male and female swine weighing about 100 kg were used. The skulls of the animals were opened and the brain dissected and put into ice-cooled Krebs Henseleit's solution.

The basilar arteries were removed and, after cleaning and removing adjacent tissues, cut into rings (2 to 3 mm) which were mounted within glass vats containing 5 ml Krebs Henseleit's solution having the following composition (mM): NaCI 118.0, KCI 4.4, MgSO4 1.1, CaC'2 2.5, NaHCO3 25.0, KH2PO4 1.2 and C6H, 206 11.0 (pH 7.2 kept at 37°C and aerated with 95% 02 and 5% C02). The tissues were subjected to 1 g tension and remained in equilibrium for 60 min before the addition of the extracts to the bath (equilibrium period), the nutritive solution being replaced by a fresh one every 20 minutes. Sub- sequent to the equilibrium period, the preparations were pre-contracted with

serotonin (1 IlM) and the endothelium integrity was evaluated by the brady- kinin capacity (BK, 1 I1M), a known vaso-relaxing peptide, to promote relaxa- tion. Preparations were considered to have the intact endothelium when the BK caused a relaxation greater than 40%. Changes in tension (contraction and relaxation) were isometrically measured by means of force transducers (TRI-201) and read with polygraph 6006 from Letica lnstrumentos Cientifi- cos. Sub-maximum serotonin-induced contractile response was considered to be 100% and all subsequent responses were calculated as a percentage of such value. The results were expressed as percentage values of contrac- tion and relaxation relative to the controls.

After the equilibrium period, said preparations were contracted by the addition of serotonin (1 I1M) and after a plateau is reached for the contractile response (approximately 5 minutes), a single cumulative concen- tration-relaxation response curve was obtained for the Catuama extract (10- 3000 pg/ml) The results obtained from the assays for determining the cere- bral vasodilator activity are shown hereinbelow.

The addition of increasing cumulative concentrations of Catuama extract caused concentration-dependent relaxation in the isolated porcine basilar artery having an endothelium showing an EC50 588 (182-1095) sLg/ml and RmaX 100 0% (figure 1). When the endothelium was purposely removed, as confirmed by the absence of a relaxing effect to bradykinin, the relaxation caused by Catuama extract was significantly reduced (73 3% inhibition) (figure 1). Considering that the basilar artery is an important ves- sel for the distribution of arterial blood to the brain, these results explain the use of Catuama extract as a cerebral vasodilator.

A) Effect on the Production of Nitric Oxide The direct effect of Catuama extract on the production of the main vaso-relaxing and nitric oxide free radicals-producing agent was evalu- ated in in vitro macrophages. These cells were obtained from the perito- neum of 3 month-old male mice intraperitoneally injected with 3% thioglyco- late (3 ml). Four days later, the peritoneal cavity was washed with 10 ml of

buffered saline (PBS, composition mmol/l : NaCl 137, KCI 2.7, phosphate buffer 10). The peritoneal wash was centrifuged at 1500 rpm and the pre- cipitate washed with DMEM (nutritive medium). Then, the cells were resus- pended in DMEM at a density of 5 x 106 cells/ml. The cells were cultured in 96-well plates and incubated for 2 hours at 37°C inside a C02 incubator, to allow the adherence of the macrophages. The plate was washed with DMEM (heated at 37°C) for removing non-adhered cells. Subsequently, the medium was replenished and Catuama extract at varying concentrations (1- 100 lig/ml) was added. The cells were stimulated with 1 Ag/ml LPS and 10 U/ml IFN-8 and incubated for 36 hours. The cells incubated only with LPS and tFN-5 were used as the control for the maximum production of nitric ox- ide. The incubation time elapsed, and the supernatant was collected for measuring the concentration of nitrite/nitrate (NOX). First, the conversion of nitrate into nitrite was carried out by incubating the supernatants with bacte- rium Escherichia coli (6 x 10° bacteria/ml) for 3 hours. After centrifugation to separate the bacteria, 100 µl of supernatant was added to 100 pI Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0. 1% naphthylethyl- enediamine in H20). The results were spectrophotometrically determined with a device for ELISA at 550 nm and correlated with a standard curve of sodium nitrate (0-150 pM) which was run in parallel with the assay.

B) Evaluation of Cell Viability The cell viability of the peritoneal macrophages from the mice was measured through mitochondrial activity-dependent reduction of MTT [3- (4,5-dimethyl-thiazole-2-yl)-2, 5-diphenyl-tetrazolium bromide] to MTT- formazan. The macrophages were incubated in 96-well plates with culture medium and Catuama extract (1-100 ug/ml), for 36 hours at 37°C, in a CO2 incubator. After this time, the culture medium was removed and the cells were reincubated with MTT (5 mg/ml) for 4 hours at 37°C. Following the in- cubation period, the MTT-formazan crystals formed were dissolved in iso- propyl alcohol containing HCI (0.04 N). The MTT-formazan complex was spectrophotometrically measured at 550 nm. The untreated cells were con- sidered as having a cell viability of 100%.

The results obtained from the assays for determining the effect on the production of nitric oxide are disclosed hereinbelow. a) Effect on the Production of Nitric Oxide The presence of Catuama extract has caused a significant inhi- bition of the production of NOx at every concentration (1-100 pg/ml) (figure 2). The maximum inhibition produced by the Catuama extract was 93.1 1.03% with a Cl50 of 15.53 (11.81-20.41) pg/ml. b) Evaluation of Cell Viability The results obtained with Catuama extract (figure 3) show that incubation of macrophages with the product did not significantly interfere with the cell viability of peritoneal macrophages at any of the concentrations tested. It can therefore be said that at the same concentrations where Ca- tuama extract caused an inhibition of the formation of NO, it did not interfere with the viability of the macrophages, therefore excluding possible toxic ef- fects of the same on the cells.

Evaluation of the Antioxidant Effect a) Production and Detection of the Hydroxyl Radical (HO-) The procedure used is based on the oxidation of deoxyribose for generating HO-from Fenton's reaction between hydrogen peroxide and Fe (III)-NTA. The chromogen was spectrophotometrically determined at 532 nm. 0.1 mM nitriloacetic acid and 0.025 mM FeCI3 were pre-incubated for 10 minutes. Shortly thereafter, 80 mM KH2PO4, 2.8 mM deoxyribose, 1.4 mM H202, in addition to the Catuama extract (1-500, ug/ml) or carrier, were added. 20 minutes later, 1 ml of 1% thiobarbituric acid and 2.3% trichlor- acetic acid were added, the samples were heated at 100°C (5 minutes), and were then immediately put on ice. The samples were spectrophotometrically (532 nm) analyzed and the results were expressed as a percentage of the oxidation of deoxyribose. For each concentration of the product utilized, a blank was employed for the original color of the sample to be deducted from the result. b) Production and Detection of Superoxide Anion (02-) The superoxide anion radical is generated through the reaction

catalyzed by the xanthine/xanthine oxidase system. Based on this principle, the possibility that Catuama extract might act on the xanthine oxidase activ- ity, impeding the formation of the 02-anion was first verified. Xanthine oxi- dase activity was evaluated by the spetrophotometric (295 nm) measurement of the formation of uric acid derived from xanthine in the presence or ab- sence of Catuama extract. Since no changes in the xanthine oxidase en- zyme action were observed, the next step was to verify the product capacity for sequestering 02-radicals. The formation of this free radical was verified in the presence and absence of Catuama extract (10-70 Rg/ml) or carrier.

The reactions were carried out in a medium containing 100 jJvt xanthine, 0.1 M phosphate buffer, pH 7.8,600 M NBT and 0.04 U/ml xanthine oxidase (incubated at 25°C for 10 minutes). The 02-radical formed was spectropho- tometrically monitored at 560 nm for the reduction of nitrobluetetrazolium (NBT). c) Enzymatic Antioxidant Activity The superoxide dismutase assay (SOD) was carried out in order to verify the Catuama extract activity on glutathione S-transferase enzyme.

This enzyme has the capacity to transform 02-radicals, thus being a naturally occurring antioxidant. The 02-radical is generated by the reaction catalyzed with the xanthine/xanthine oxidase system and its formation was monitored by the reduction of NBT, as previously described. The reactions were car- ried out in a medium containing 100 uM xanthine, 0.1 M phosphate buffer, pH 7.8,600 IlM NBT and 0.04 U/ml xanthine oxidase, and 100 U/ml SOD (incubated at 25°C for 10 minutes), aiming to verify that enzyme capacity for sequestering 02-radicals produced by the reduction of NBT. The SOD activ- ity was evaluated by spectrophotometrically (560 nm) measuring the per- centage reduction of NBT. The enzymatic antioxidant activity was evaluated in the presence and absence of Catuama extract (30-60, ug/ml) or carrier.

The results from the assays for determining the antioxidant ac- tion are disclosed hereinbelow. a) Production and Detection of the Hydroxyl Radical (HO-) Catuama extract was shown to be very effective in impeding the

oxidation of deoxyribose (figure 4). The product caused 61% inhibition with a CI50 of 13.21 lig/ml, it being possible to reach the maximum limit for this assay due to the high speed in which the reaction takes place. b) Production and Detection of Superoxide Anion (°2-) Catuama extract (10-70 pg/ml) caused a significant decrease in the formation of xanthine by xanthine oxidase, indirectly suggesting a de- crease in the production of superoxide anion radical (figure 5). This product inhibited about 63% exhibiting a Cl50 of 2.3 lig/ml. Knowing that Catuama extract does not interfere with the enzyme activity, it can be said that the product acts as a sequesterant of 02-radical. c) Enzymatic Antioxidant Activity Catuama extract did not change the enzyme activity in the assay (figure 6), with just a decrease in the quantity of superoxide anion radical produced by the xanthine/xanthine oxidase system occurring, indicating the antioxidant activity thereof is due only to the sequesteradtion of O2- radicals (figures 6 and 7). These results together suggest an important action of Catuama extract as an antioxidant.