MXPA00010424A - Antithrombotic agents - Google Patents

Abstract

The invention provides a fruit extract or active fraction thereof for use in the prophylaxis or treatment of a disease state initiated or characterised by platelet aggregation.

Description

ANTI-THROMBOTIC AGENTS FIELD OF THE INVENTION This invention relates to anti-thrombotic agents and more particularly to compositions prepared from fruit extracts.

BACKGROUND OF THE INVENTION It is known that a high consumption of fruits and vegetables is a preventive measure, important by which the risk of cardiovascular diseases and certain cancers that are nutritionally linked can be reduced, including cancer of the stomach, colon, breast and prostate. factor included in the onset and development of cardiovascular diseases and cancers is the occurrence of abnormal oxidative processes that lead to the generation of free radicals or compounds of hydroxy and peroxy. In part, the beneficial effect of eating fruits and vegetables is explained by the antioxidants contained in them, which inhibit oxidative reactions. Specific antioxidants that are known to account for inhibition include vitamin C, vitamin E, and carotenoids including alpha and beta carotenoids, lycopene, lutein, zeanthin, critoxanthin, and xanthophylls. It has become a considerable; effort with respect to the identification of nutritional compounds derived from tomatoes that have a role in the prevention of heart disease and some cancers. These compounds are described in Abushita et al., Food Chemistry, 1997 60 (2), 207-212 where a tomato carotenoid extract was fractionated and the major components identified as lycopene, beta-carotene and luteum a. Studies with respect to tomato have focused on the role of carotenoids, in particular lycopene, in the antioxidant defense against the oxidation of low density lipoprotein (LDL). In Oshima et al. , J. Agricultural and Food Chemistry, 1996, 44 (8), 2306-2309, it is described that LDL supplemented with lycopene accumulates hydroperoxides more slowly than non-supplemented LDL when stimulated by oxygen alone, thereby providing evidence to support the theory that antioxidants have a potential to trap hydroxyl / peroxyl radicals.

Additionally, in fuhman et al., Nutrition Metabolim and Cardiovascular Diseases, 1997, 7 (6), 433-433, it is described that the dietary supplemented lycopene significantly reduced the levels of human LDL oxidation. In Eisburger, Proceedings for the Society for Experimental Biology and Medicine, 1998, 218 (2), 140-143 it is reported that the optimal absorption of carotenoids, being typically lipid soluble chemicals, is improved in the presence of a small amount of oil or dietary fat. Research in the field of nutrition and health has shown that monounsaturated oils such as olive oil are more desirable, since these oils do not increase the risk of atherosclerosis, coronary heart disease or cancer linked nutritionally.

BRIEF DESCRIPTION OF THE INVENTION Applicants have found that extracts from many fruits exhibit a capacity to inhibit platelet aggregation. The results obtained to date suggest that compositions containing extracts of these fruits can therefore be used in the prevention of coronary disease, for example myocardial infarcts and stroke and in the prevention of thromboembolic cases in patients who have suffered a heart attack. to the myocardium, attack or unstable angina. In addition, these compositions may be useful in the prevention of restenosis after angioplasty and bypass procedures. Additionally, compositions comprising fruit extracts may be useful in the treatment of coronary artery disease resulting from thromboembolic disorders such as myocardial infarction in conjunction with thrombolytic therapy. The results obtained to date indicate that the compounds responsible for the anti-platelet aggregation activity are water-soluble compounds that have a structure very different from the water-soluble compounds such as lycopene identified in the articles referred to above. There are many known platelet anti-platelet aggregation agents that act in different stages of the production and action of platelets. Aspirin (acetic acid salt) is the most widely used and studied. Also they have been -. * --- »used dipyridamole and ticlopidine. The anti-platelet activity of aspirin is due to the irreversible inhibition of platelet cyclooxygenase, thus preventing the synthesis of thromboxane A2, a compound that causes platelet aggregation. Indobufen is a reversible inhibitor of cyclooxygenase in platelets. Some compounds with direct inhibitors of thromboxane A2-synthase, for example pirmagrel, or act as antagonists in thromboxane receptors, for example, sulot steal. The results obtained to date suggest that the active components in the substrates of the fruit may affect one or more steps of the routes leading to the production of thromboxane A2 upstream of that of aspirin and the other anti-platelet drugs. available. It is also well known that adverse effects are common occurrences with therapeutic doses of aspirin. The main effect is gastrointestinal disturbances such as nausea, dixpesia and vomiting. Therefore, it is anticipated that the platelet aggregation inhibiting compound (s) isolated in the fruit extracts will find utility as a desirable alternative to aspirin and other anti-platelet drugs in the prevention of thromboembolic cases and coronary disease. Accordingly, in a first aspect, the invention provides a fruit extract, an active fraction thereof, or one or more active components that can be isolated therefrom, for use in the prophylaxis or treatment of a disease state initiated or characterized by the aggregation of platelets. In another aspect, the invention provides a fruit extract or active fraction thereof or one or more active components that can be isolated therefrom for use as an inhibitor of platelet aggregation. In a further aspect, the invention provides a fruit extract or active fraction thereof or one or more active components that can be isolated therefrom for use as an ant i-thrombotic agent. In another aspect, the invention provides the use of a fruit or an extract or an active fraction thereof or one or more active components that can be isolated therefrom as defined hereinabove for the manufacture of a medicament for use in the prophylaxis or treatment of a disease state initiated or characterized by inhibition of platelets; or for use as an inhibitor of platelet aggregation, or for use as an antithrombotic agent. As used herein, the term "" fraction 1 'refers to purified or partially purified extracts. In another aspect, the invention provides a process for the manufacture of a medicament for the use (i) in the prophylaxis or treatment of a disease state initiated, mediated or characterized by the aggregation of platelets, or (ii) as an inhibitor of platelet aggregation; or (iii) as an ant i-thrombotic agent; process that is characterized by the use, as an essential ingredient of the medicament, of a fruit, or an extract or an active fraction thereof or one or more active components that can be isolated therefrom as defined hereinbefore. In a still further aspect, the invention provides a pharmaceutical composition comprising an active component derived from a fruit or an active extract or fraction or one or more active compounds isolatable therefrom as hereinafter defined and a pharmaceutically acceptable carrier. . It is preferred that the fruit extracts used according to the invention are those that are non-toxic to humans and typically the fruits are those that are usually considered as edible fruits. In this way, fruits may contain seeds or bones, or not, but they have an essentially non-oily, edible flesh. Typically, fruits may have a surface, bark or shell surrounding the meat that may optionally be edible. Examples of fruits that can be used according to the present invention are those selected from the families Solnaceae, Rutaceae, Cucurbi taceae, Rosaceae, Musaceae, Anacardiaceae, Bromeliaceae, Vitaceae, Arecaceae, Ericaceae and Lauraceae. Examples of Solnaceae include tomato, for example the English tomato variety.

Examples of Rutaceae include the species Citrus such as Citrus paradisi (grapefruit), Citrus s inensi s (orange), Citrus lemon (lemon) and Citrus uranti folia (lime). Examples of Cucurbi taceae include Cucurni s meló (melon), for example sweet melon. Examples of Anacardiaceae include Mangi fera indica (mango). Examples of rosaceae include Pyrus malus or Pyrus sylvestri s (apple), Pyrus communis (pears), amygdalus persica or Prunus pérsica Var, nectarine (nectarine), Prunus ariaca (chabacano), Prunus domestica (plum), Prunusavi um (cherry), Prunus persica (peach), strawberry and blackberry. Examples of Bromel iaceae include Ananas sativus (pineapples). Examples of Lauraceae include Persea grati ssi a or Persea americana (avocado). Examples of vi taceae include Vitis viní fera (grape). The examples of ArecaceaJ. include Phoenix dactylifera (date). The examples of Ericaeae include blueberry. Particular examples of fruits, extracts or active reactions of which have been found to have inhibitory activity of platelet aggregation are tomato, grapefruit, melon, mango, melon, pineapple, nectarine, strawberry, plum, banana, bilberry, grape, pear, apple and avocado. The extracts of the invention can be prepared by homogenizing the flesh of a fruit, preferably peeled, and then removing the solids therefrom, for example by means of centrifugation. In this way, the extract is typically an aqueous extract, which may consist essentially of the juice of the fruit, optionally with the addition of water added during the homogenization step. These aqueous steps can be concentrated, enriched or condensed for example by normal techniques, for example, evaporation under reduced pressure. Examples of concentrates are those that are concentrated at least 2 times, more usually at least 4 times, for example at least 8 times, or at least 40 times, or at least 100 times, or at least 200 times, or at least 1000 times. times. The extracts can be fractionated to isolate one or more reactive fractions therein for example by molecular weight filtration or chromatography on a suitable solid support such as sepharose gel (for size exclusion chromatography) or ion exchange column using HPLC in a suitably treated silica or alumina, for example, silica coated with ODS; or by extraction with solvents. Experiments carried out on tomato extracts have revealed that the active component (s) of the extract passes through an ultrafiltration filter having a molecular weight cutoff of 1000, is colorless or corduroy color, soluble in water and does not lose significant activity when boiled. Accordingly, the invention also provides for the use as an ant agent. i-1 rhombic or for use as an inhibitor of platelet aggregation, or for use in the prophylaxis or treatment of a disease state initiated or characterized by the aggregation of platelets, of an active fraction of a fruit extract (preferably an extract, of tomato), the active fraction containing a water-soluble, straw-colored or colorless compound, substantially color-stable, or compounds having a molecular weight of less than 1000. The extracts of tomato, and in particularly aqueous tomato extracts, represents a preferred aspect of the invention. An active fraction of the tomato extract has been found to contain a mixture of nucleosides including cit idine. Accordingly, in one embodiment, use is provided as a rhizobotic ant i-1 agent, or for use as an inhibitor of platelet aggregation, or for use in the prophylaxis or treatment of a disease state initiated or characterized by platelet aggregation, an active fraction of a tomato extract, the active fraction containing a water-soluble, straw-colored or colorless, substantially heat-stable compound or nucleoside compounds, having a molecular weight of less than 1000. The fraction Active has been found to be mainly associated with, or may be of, the juice, the flesh surrounding the tomato nuggets. In this way, the use of the compositions prepared from an active fraction consisting essentially of a homogenate or an extract thereof derived from the meat and a peeled tomato consisting essentially of the juice and / or the flesh surrounding the nuggets and / or the nuggets, represents a preferred embodiment of the invention. The active component of the tomato extract has been analyzed by mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy and has been found to contain a mixture of nucleosides. In a further aspect, therefore, the invention provides an active fraction per se that can be isolated from tomato and characterized in that: (a) it is substantially heat stable; (b) is colorless or straw colored; (c) is a water soluble compound; (d) consists of components having a molecular weight of less than 1000; (e) it contains one more nucleosides that have platelet aggregation inhibiting activity; and preferably (f) has a mass appearance when subjected to a MALDI-TOF mass spectrometry, as shown in Figure 7 attached hereto; and preferably (g) exhibits a 1H nuclear magnetic resonance spectrum substantially as shown in Figure 6 appended hereto. j ^ a_ Pharmaceutical and Nutritional Formulations The extracts or active fractions thereof can be formulated in a variety of ways. For example, they can be formulated for oral, sublingual, parenteral, transdermal, rectal, inhalation or oral administration, but are preferably formulated for oral or buccal administration. As such, they can be formulated as solutions, suspensions, syrups, tablets, capsules, pills, snack bars, inserts and patches as an example. These formulations can be prepared according to methods well known per se. It is preferred that the formulations be of a low content of lipid materials, or be substantially free of these. For example, extracts or active fractions can be formed in syrups or other solutions for oral administration, for example health drinks, in the presence of one or more excipients selected from sugars, vitamins, flavoring agents, coloring agents, preservatives. and thickeners. Tonicity adjusting agents such as sodium clhtide, or sugars may be added to provide a solution of a particular osmotic concentration, for example an isotonic solution. One or more pH adjustments may also be used, such as buffering agents to adjust the pH to a particular value preferably to maintain it at that value. Examples of buffering agents include sodium citrate / citric acid buffers and phosphate buffers. Alternatively, extracts or active fractions thereof can be dried, for example, by spray drying or freeze drying, and the dried product is formulated in a solid or semisolid dosage form, eg, a tablet, lozenge , capsule, powder, granule or gel. Instead, dry, simple extracts can be prepared without any additional component. Alternatively, the dry extracts can adsorb only a solid support; for example a sugar such as sucrose, lactose, glucose, fluctuous, mannose or a sugar alcohol such as chilol, sorbitol or mannitol; or a cellulose derivative. Other particularly useful adsorbents include starch based adsorbents such as cereal flours for example wheat flour and corn flour. For tabletting, the dry extract is typically mixed with a diluent such as a sugar, for example sucrose and lactose, and taL sugar alcohols such as chilol, sorbitol and mannitol; or modified cellulose or cellulose derivative such as powdered cellulose or microcrystalline cellulose or carboxymethylcellulose. The tablets will also typically contain one or more excipients selected from granulating agents, binders, lubricants and disintegrating agents. Examples of disintegrants include starch and starch derivatives and other swellable polymers, for example crosslinked polymeric disintegrants such as cross-linked carboxymethylcellulose, crosslinked idrirol Ipirrol idone and starch glycollates. Examples of lubricants include stearates such as magnesium stearate and stearic acid. Examples of binders and granulating agents include polyvinylpyrrolidone. Where the diluent is not naturally; very sweet, a sweetener may be activated, for example ammonium glyceride or an artificial sweetener such as aspartame or sodium saccharinate. The dried extracts can also be formulated as powders, semisolid granules for incorporation into capsules. When used in the form of powders, the extracts can be formulated together with any one or more of the excipients defined above in relation to tablets, or they can be presented in an undiluted form. For presentation in the form of a solid, the dry extracts can be dissolved or dispersed in a viscous liquid or semi-solid carrier such as a polyethylene glycol or a liquid carrier such as a glycol., for example propylene glycol 1 or glycerol or a vegetable or fish oil, for example oil selected from olive oil, sunflower oil, safflower oil, evening primrose oil, soybean oil, cod liver oil , herring oil, etc. These extracts can be filled into capsules of either the hard gelatin or soft gelatin type or to produce hard or soft gelatin equivalents, gelatin-equivalent capsules or soft gelatin which are preferred for semisolid or viscous liquid fillings. . - * z _. ..., _. * fifc .- a ..% * -i $ l £ & a.

Dry extracts may also be provided in a powder form for incorporation into snack food bars for example fruit bars, nut bars and cereal bars. For the presentation in the form of snack food bars, the dry extracts may be miwith any one or more ingredients selected from dried fruits such as sun-dried tomatoes, raisins and sultana grapes, ground nuts or cereals such as oats and wheat. Dry extracts may be provided in a powder form for reconstitution as a solution. As such, they can also contain soluble excipients such as sugars, buffering agents such as citrate and phosphate buffers, and effervescent agents formed from carbonates, for example bicarbonates such as sodium or ammonium bicarbonate, and a solid acid eg acid. citric or an acid citrate salt. In a preferred embodiment, the dry extract is optionally provided in powder form together with a solid or preferred excipient (eg, powder) for incorporation into capsules, for example, a hard gelatin capsule.; A solid or semisolid dosage form of the present invention may contain up to about 1000 mg of the dry extract, for example up to about 800 mg. In certain circumstances it may be desirable to present the extracts for administration by injection or infusion. As such, they will be presented in the form of filtered sterile solutions, preferably; in physiological serine solution buffered to approximately pH 7. Alternatively, they can be presented as sterile powders to be prepared in injectable or infusible solutions. The extracts may be presented as food supplements or food additives, or they may be incorporated into foods, for example functional or nutrient foods. The compositions of the invention can be presented in unit dosage forms containing a defined concentration of extract or active fraction thereof. These unit dose forms can be selected to achieve a desired level of biological activity.

Pharmaceutical Uses The present invention also provides a method for the prophylaxis or treatment of a condition or disorder mediated by platelet aggregation, the method comprising administering to a patient (such as a human or other mammal) in need thereof an inhibitory amount of aggregation of platelets, preferably non-toxic and effective of a fruit or an extract or active fraction thereof as defined below in 1 to present. For the treatment of diseases characterized by the aggregation of platelets, the amount of extract or active fraction administered to a patient per day will depend on the concentration of the extract, the particular condition or disease under treatment and its severity, and finally will be at the discretion of the physician . However, the amount administered will typically be a non-toxic amount effective to treat the condition in question. The amount of extract or active fraction administered to a patient will typically vary according to the concentration of the active ingredient or 4. . '.. ingredients in the extract. However, a typical daily dose regimen for a human patient suffering from a platelet aggregation-mediated disease may be from 0.0001 to 0.01 to, for example, 0.001 to 0.05 grams per kilogram of body weight. When an active fraction is isolated and administered, the amount of the solid material administered can be reduced by an amount consistent with the increased purity of the fraction. Typically, administration of at least 100 mg, preferably 200 mg of the active fraction per day to a human suffering from a disease mediated by platelet aggregation, will significantly inhibit platelet aggregation. The compositions can be administered in single or multiple dose units, per day, for example one to four times, preferably one to two times daily. The extracts of the invention can be administered in solid, liquid or semi-solid form. For example, the extracts may be administered in the form of a fruit juice, concentrates of the aqueous extracts or purified active fractions of the extracts in solid, liquid or semi-solid form. When they are administeredIn a non-concentrated state, they can be administered in the form of a juice prepared from 100% fruit. However, the extracts are preferably administered as concentrates and more preferably as concentrates in solid form, for example, the form of tablets, hard gelatin capsules and snack food bars as hereinafter defined. In one embodiment of the invention, at least 300 ml of a 100% fruit juice (for example 600 ml of a 100% fruit juice) can comprise a typical daily dose regimen for a human patient suffering from a disease associated with platelet aggregation, In another embodiment of the invention, at least 300 ml of 100% fruit juice can be administered in multiple doses per day, for example at least twice a day, preferably three times daily. However, the dose regimes mentioned above comprise the consumption of large relative volumes of fluid that may be unacceptable to the patient. Therefore, in an additional modality, the concentrates as defined later herein is _ ^^^^ __ ^^ j ^^^ j ^ can they administer, for example, in multiple doses per day. The extracts of the invention can be administered in conjunction with: therapeutic agents, for example, one or more therapeutic agents selected from cardiac agents or anti-rhombic agents, antiarrhythmics, AC inhibitors, beta-blockers, vasodilators , other inhibitors of platelet aggregation, phosphodiesterase inhibitors, plasminogen activators and ipideaemic hypol, by way of example. the extracts can be formulated separately from the other therapeutic agent or can be formulated together. The compositions of the invention have platelet aggregation inhibition activity. As such, the compositions of the invention are useful in the treatment of conditions and disorders in which the aggregation of blood platelets play a part, or in which the hyperactivity of the platelets is involved. The compositions of the present invention can be used therapeutically in various conditions where the hyperactivity of platelets is a . - i »l *,, -t - -,. »- -, ._. . • -. --_-_. , ^ __: ^^: «- ^ - ¡^ ^, A -? ^ Primary or secondary characteristic such as heart failure, cancers and obesity. Examples of clinical indications in which the compositions of the present invention will be of particular interest include the treatment or management of post-myocardial infarction, coronary thrombosis, coronary artery bypass grafts, replacement of heart valves and peripheral and vascular grafts. The extracts of the invention can be used alone or in combination with other therapeutic agents. In a preferred embodiment, the extracts of the invention are administered in combination with one or more of streptokinase, heparin, insulin, anti-obesity drugs and inhibitors of HMGCoA-reductase.

BRIEF DESCRIPTION OF THE DRAWINGS Now, the invention will be illustrated, but not limited, by the following examples and with reference to the appended figures of which: Figure 1 shows in schematic form a typical procedure for the partial fractionation of tomato extracts; Figure 2 is a gel filtration chromatogram of an ulte raft of the aqueous tomato extract; Figure 3 is an ion exchange chromatogram of high pressure liquid chromatography (HPLC) of an aqueous, gel filtrate, distilled extract; Figure 4 is a graph showing platelet aggregation activity in scruffy fractions, fraction 1 and fraction 2, collected after HPLC ion exchange chromatography; Figure 5 is a XH NMR spectrum of cytidine; Figure 6 is an XH NMR spectrum of an active, scruffy F2 fraction of an aqueous tomato extract; Figure 7 is a mass spectrum of MALDI-TOF of the active fraction F2; Figure 8 is a GC-CIMS chromatogram of the derivatized F2 fraction; and Figure 9 is a graph showing the results of platelet aggregation obtained using extracts from different parts of the tomato.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES EXAMPLE 1 Platelet Aggregation Study Induced by ADP Methods Extracts consisting of 100% fruit juice or diluted fruit juice were freshly prepared on the day of the trial to from the fruits exposed in Table 1 below. To prepare 100% fruit juice, the fruit was peeled and the meat was homogenized. The resulting homogenate was rotated at 3,000 x G for 10 minutes in a centrifuge in Eppendorf 1.5 tubes. ml after which the supernatant (juice) was removed and the pH of the juice was adjusted to pH 7.4 with either 1 M or 0.1 M sodium hydroxide depending on the initial pH of the fruit extract. For relatively fibrous fruits (apple, mango, avocado, A 20% or 50% w / v extract was prepared by homogenizing either 20% or 50% fruit with phosphate buffered saline (PBS) at pH 7.4, the homogenate being processed as described above in relation to the extracts of fruits 100%.

The effect of fruit extracts on the aggregatory properties of human platelets was investigated in volunteers. The venous blood was collected from volunteers who had not taken any medication for at least 14 days before donation Blood was collected (20 ml) using a 19G butterfly needle and the blood samples were prevented from coagulating into the blood samples. acid citrate (135 mM) in the ratio of 9 parts per volume of blood to 1 part per volume of ACD.) Platelet-rich plasma (PRP) was prepared from the samples by centrifuging the blood at 200 g for 15 minutes The fruit juice (50 μl), the pH at which it was adjusted to 7.4 where necessary with either 1 M or 0.1 M sodium hydroxide depending on the initial pH of the fruit extract, was mixed with the PRP (450 μl) and incubated at 37 ° C for 15 minutes, after which the effect of the fruit extract on The aggregation of ADP-induced platelets was monitored with the addition of ADP at a final concentration of 10 μM. controls were run in parallel using PBS at 50 μl, pH 7.4 instead of fruit juice. The aggregation of platelets in PRP is & ^ ^ mm-mm - ^ ~ - - ± M ^ £ ^ L * É ^ * - ^ L * > i - ._. - J - M. I ^ * * .jy monitored using an aggregometer Packs-4 (Helena Labs, USA) at a constant agitation speed of 1000 rpm at 37 ° C. Platelet counts were performed using a Coulter cell counter.

Resul teds Table 1 shows the antiagregatory properties of the various fruit extracts in human platelets. The results were expressed as% inhibition of the aggregation response to ADP, for several volunteers (n). The table, the extracts marked with an asterisk were boiled for 10 minutes and then centrifuged at 113.00 g for 30 minutes.

TABLE 1 ^ g ^^^^^^ 2 Use Partial Fractionation of Tomato Extract Methods Tomato extracts were fractionated according to the general scheme set forth in Figure 1 and the inhibition activity of platelet aggregation was measured in several stages. In this way, fresh tomato juice, prepared from 100% fruit, boiled for 10 minutes and then centrifuged at 113,000 g for 30 minutes. The inhibition activity of the platelet aggregation of the extract is shown in Table 1 above. After centrifugation, a portion of the supernatant extract was subjected to ultrafiltration to pass through an Amicon YM1 filtration membrane with a molecular weight cut-off of 1000 under nitrogen pressure at 4 ° C. The ullage was collected as it was any remaining fruit juice that remained in the filter (retained), and the last one and the retentate were then tested both for their activities in inhibiting ADP or aggregation of platelets induced by collagen. The anti-platelet activities of the ultrafined and retained were the same indicating that the active component of the extract consists of a compound or compounds having a molecular weight of at least 1000. In order to determine whether the antiplatelet antiplatelet activity was due to water-soluble lipid-soluble components in the tomato ultrifter (molecular weight cutoff of 1000), and late-stage lipid component are extracted with chloroform and methanol according to Bligh and Dyer's method . In this way, 2 ml of the ultralite was mixed with 2.5 ml of methanol followed by 1.25 ml of chloroform to give an individual phase and a ratio of chloroform: methanol: water of 1: 2: 0.8. No precipitate formed. Then chloroform (1.25 ml) and water (1.25 ml) were added to bring the ratio to 2: 2: 1.8 and after a gentle mixture, the mixture was allowed to settle in two layers. The upper layer (methanol / water) was removed and the methanol was completely evaporated under nitrogen at 55CC. The volume was then brought to 2 ml, after adjusting to pH 7.4. The anti- platelet aggregation activity of this aqueous phase was compared with 50 μl of PBS as a control.

The chloroform phase was evaporated under nitrogen and redispersed in ethanol (50 μl). A 10 μl sample of the ethanol phase was then tested for anti- platelet aggregation activity against a 10 μl ethanol control.

Results The ultrafine (molecular weight cutoff of 1000) and the aqueous fraction of the ipydi sada, both to pH 7.4, has similar activity against ADP and platelet aggregation induced poi: collagen. The lipid fraction, on the other hand, does not exhibit primary aggregation, but disaggregation was observed. This is thought to be due to the effects of non-specific lipids in platelets. In conclusion, fractionation experiments suggested that the platelet aggregation inhibition activity is associated with water soluble components of a weight molecular weight of less than 1000. The component (s) is color stable and colorless / straw colored.

EXAMPLE 3 Isolation and Identification of the Anti-platelet Aqreqation Component, Active from the Tomato Extract Methods The tomato extracts were fractionated according to the general scheme set forth in Figure 1 and the platelet aggregation inhibition activity was measured in several stages. In this way, fresh tomato juice, prepared from 100% fruit, was boiled for 10 minutes and then centrifuged at 113.00 g for 30 minutes. After centrifugation, a portion of the supernatant extract was subjected to ultrafiltration by passing through an Amicon YM1 filtration membrane with a molecular weight cut off of 1000, under nitrogen pressure at 4 ° C. The latter, with molecular weight cutoff of 1000, was collected and a sample tested for ADP activity or collagen-induced platelet aggregation. The latter was dried by freezing for further purification. The lyophilized sample was dispersed in 2 ml of water. The anti- i-platelet aggregation activity of this aqueous phase was compared with 50 μl of PBS as a control. Since only the aqueous fraction of the lyophilized sample has the activity of inhibiting platelet aggregation (see Example 2) further purification of the component was carried out using the aqueous fraction. The additional fractionation was carried out on a sepharose column that separates according to the molecular size. In this way, column chromatography on gel filtration of the lyophilized, redispersed sample was carried out using P2 Biogel. A column of P2-Biogel was equilibrated with 0.01 M acetic acid buffer, pH 3.3 containing 0.15 M sodium chloride. The sample was loaded onto the column and boiled with a 0.01 M acetic acid buffer, pH 3.3 , which contains 0.15 M sodium chloride. The aggregation of platelets in each of the fractions collected (designated No. 1 to 8) that corresponded to the peaks of UV spectra shown in the chromatography trace in Figure 2 was evaluated. It was found that the inhibition activity of platelet aggregation is concentrated in which one of the fractions was collected, which corresponded to peak 4. This fraction, referred to as fraction 4, was lyophilized before further purification. The lyophilized sample was redispersed in water to give a 20 mg / ml solution. The desalination of the fraction collected was carried out by loading the sample in a P2 Biogel column and eluting with 0.01 M acetic acid buffer, pH 3.3. The eluate was lyophilized and redispersed in water as before. Further purification was achieved by high pressure liquid chromatography (HPLC), ion exchange chromatography on silica gel Nucleosil. The sample was applied on a 5 μM Nucleosil column with a guard column packed with Persorb A C18. The sample was concentrated in the column while washing the column with solvent A (10 mM sodium acetate adjusted to pH 4 with glacial acetic acid). For the elution, a linear gradient of 100% solvent A at 100% solvent B (10 mM sodium acetate and 1 M sodium chloride, pH 4) over a period of 30 minutes at a flow rate of 1 ml / min. Two fractions were collected: fraction 1 corresponded to the eluate material on peaks 1 to 11 (between 2.3 and 8.1 minutes after the inhibition of the sample) and fraction 2 that corresponded to the material eluted from peak 5. The 5 desal ini The fraction of the collected fractions was carried out by loading the sample in a P2-B? ogel column and eluting with 0.01 M acetic acid buffer, pH 3.3. The eluate was lyophilized and redispersed in water as before. Figure 4 shows the platelet aggregation activity induced by ADP measured in scruffy fractions, fraction 1 (Fl) and fraction 2 (F2). The inhibition activity of platelet aggregation was found to be concentrates in one of the fractions, fraction 2, which corresponded to peak 15 (Figure 3). Fraction 2 was then lyophilized before further analysis. The lyophilized sample is; redispersed in water to give a concentration of; 20 mg / ml retained for structural analysis of the active component (s). The active components present in the active fraction were characterized using nuclear magnetic resonance (NMR), and mass spectroscopy, as described gfe ^^ gy ^ i ^^^^^^^^^ L ^ ^ contuaua tion.

Nuclear Magnetic Resonance Spectroscopy A portion of the sample of active fraction F2 was subjected to 1U NMR analysis and the resulting NMR spectrum is shown as Figure 6. The spectrum of the active fraction was compared to the spectrum of a pure sample of the compound 4-amino-1 -B-D-ribofurans il-2 - (1H) -pyrimidinone (cytidine), see Figure 5, from which it can be seen that there are considerable similarities but clearly the active fraction does not contain histidine pure The NMR data for sample F2 suggest the presence of ribos. The smaller differences in the NMR data differ a different pH or a different salt.

Mass spectroscopic analysis The fractional active fraction, fraction 2 (F2), was subjected to several analytical techniques of mass spectroscopy. The data obtained from the various maea spectra suggest that sample F2 contains several nucleoside species, of which the main component is cit idine.

EIMS probe A portion of sample F2 (42480) was examined by EIMS probe using a slope temperature of room temperature at about 550 ° C to 50 ° C per minute. A VG AutoSpecE mass spectrometer was used, scanning from 950 to 25 amu to approximately five seconds per scan. The EIMS probe data for F2 showed a potentially diagnostic ion at an m / z of 111 that appeared to correspond to 4-aminopyridinidinone (cytosine) formed by thermal / El-induced fragmentation of a nucleoside, by comparison with a mass spectrum The NIST library of cytosine. There was also clear evidence of the presence of HCl, suggesting a hydrochloride. The sample appeared to be contaminated with branched oligomers of octylphenol ethoxylates, giving ions at m / z of 45, 135, 267, 311, 355, 382, 399, 426, 443, 470 and 487.

MALDI-TOF Portions of sample F2 (42480) and several standards including cytodine were dissolved in water and mixed with a matrix (hydrochloric acid / 50 mM ammonium treatment, 9: 1). A PE Biosystems Voyager-STR mass spectrometer was used. A blank matrix was also analyzed. The spectrum of MALDI-TOF (desorption with laser assisted matrix / ionization-time of flight) of sample F2 (Figure 7) was closely similar to that of citedin and to arabinofuranosi 1 - ina cysts. The three samples clearly showed ions m / z 244 (MH +), m / z 266 (Mna +), m / z 487 (2 MH +) and m / z 509 (2 MNa *) suggesting that the main component F2 is citedin or an isomer of citedin. Cyclotidine has the lowest molecular weight, as expected ions and shown at m / z 266 (MH +), m / z 451 (2MH +) and m / z 473 (2 MNa +).

Derivatization / GC-EIMS Portions of sample F2 (42480) and several standards including cytidine were dissolved in water and mixed with the internal standard (arabitol). The resulting and one blank solutions were filtered, N-acetylated using acetic anhydride / pyridine and trimethylated using Tri-Sil-Z. The resulting products were dissolved in hexane and aliquots (ca 1 μl) were analyzed by GC-EIMS (gas chromatography-mass spectroscopy with electron ionization) on a VG Trio-1 mass spectrometer. The samples were injected via a cold column injector on a capillary GC DB-5 column. The GC-EIMS data from the derivatized F2 sample and a derivatized cytidine control sample suggested that the major component in the F2 sample is closely similar to the derivatized cytidine, but imperceptibly different from the cytokine arabinofuranosi 1 - ci cough Derivatization / GC-CIMS Portions of sample F2 (42480) and the cytidine standard were dissolved in water and lyophilized. They were derived in the same way as above and aliquots (approximately 1 μl) in the resulting hexane solutions were analyzed by GC-CIMS (gas chromatography-mass spectroscopy with chemical ionization) on a PE Turbo Mass mass spectrometer.

The sample was injected via a PPS injector onto a capillary GS column DB-5MS. The GC-CIMS data for derivatized F2 and derivatized cytidine confirmed that one of the peaks in sample F2 is cytidine. Examination of Cl spectra also revealed the presence of ions at m / z 259 and * ¿¿^ £ £ &$ 348, which can be associated with the ribofuranosyl unit.

EXAMPLE 4 Essay of the Activity of the Derived Extract of the Tomato in the Inhibition of the Aqreqación of Platelets Induced by Agonists or After Addition of Arachidonic Acid It is known that after the injury, the platelets adhere to the damaged vascular endothelium, thus facilitating that the additional platelets adhere to each other, aggregate, become activated and form a plug of platelets. Platelet aggregation was measured via factors that occur at the site of the injury and react with sectors on the surface of the platelet. Some of these factors for example ADP, serotonin and thromboxane A2 are released by themselves by activated platelets, producing a positive feedback loop. During the process of platelet aggregation and activation, ligands such as ADP or collagen in low doses, bind to specific receptors. This leads to the activation of membrane phospholipases and the release of arachidonic acid from the phosphoLipids of the platelet membrane by the activity of the enzyme phospholipase A2. A proportion of the arachidonic acid is then rapidly metabolized by several cyclic endoperoxidases, the main ones being cyclooxygenase and lipoxygenase in 1 ipoxygenase, to prostaglandins and finally to thromboxane A2 via the enzyme thromboxane synthetase. Thromboxane A2 is active of; a biologically high form and measured an increase in intracellular calcium ions and the release of platelet granules that promotes the additional aggregation of platelets. Thromboxane A2 is chemically unstable and decomposes to thromboxane B2 and therefore the measurement of thromboxane levels was carried out by measuring thromboxane B2. The inhibition activity of platelet aggregation of semi-purified tomato extracts was assessed by measuring the production of thromboxane B2 produced by platelets in the presence of ADP agonists or collagen or when exogenous arachidonic acid is added.

Methods The semi-purified tomato extracts were prepared according to Examples 2 and 3. In this way, 50 μl of the gel filtration fraction corresponding to peak 4 (see Figure 2) by fraction 2 purified by HPLC (see Figure 3) were added to 50 μl of PBS buffer and 400 μl of platelet-rich plasma was incubated for 15 minutes at 37 ° C. After incubation, the agonists were added to the desired concentration. The assay mixture was then centrifuged and thromboxane B2 levels in the supernatant were measured. Alternatively, the centrifuged test samples were rapidly frozen by thromboxane B2 analysis at a later date.

Results Table 2 Table 2 shows the effect of the gel filtration fraction corresponding to peak 4 and HPLC fraction F2, in the production of thromboxane - ^^ and-. Y . í- «,? M¿ * B2 in platelets by ADP collagen, and arachidonic acid. The results were expressed as nonagrams / ml of thromboxane B2 produced in response to ADP, collagen or arachidonic acid in the presence of the semi-purified tomato extract. The gel filtration fraction corresponds to peak 4, fraction 4, and the HPLC fraction, fraction 2, have similar potency against the production of thromboxane B2 induced by ADP. Similarly, fraction 2 inhibited the production of thromboxane B2 induced by collagen when compared to the control sample. Fraction 2, on the other hand, did not inhibit the production of thromboxane B2 in the presence of arachidonic acid.

Conclusion These experiments showed that the active component (s) of the tomato juice extract inhibits the production of thromboxane B2 induced by ADP and collagen, but does not stop the metabolism of arachidonic acid to thromboxane B2. The results suggest that the inhibition activity of platelet aggregation does not block the conversion of arachidonic acid to thromboxane B2 - ,, ». ¿. ? ** M & , .. ° m and thus does not inhibit the activity of the enzyme c locxygenase that catalyzes this conversion. In conclusion, the results of this experiment suggest that the activity of component ant i - platelet aggregation, active, in tomato extracts is different from that of aspirin.

Example 5 Location of Active Component in Tomatoes Four tomatoes were peeled and dissected to obtain preparations containing the following: i) the juice surrounding the seeds; referred to as ii ii) tomato meat only; referred to as T2 iii) whole tomatoes including seeds; referred to as T3. Extracts of the TI to T3 preparations were prepared as described in Example 1 and the platelet aggregation activity induced by ADP was measured in each.

Results and Conclusions Figure 9 shows the antiplatelet activity of platelets of tomato preparations TI to T3 in platelets of humans. The TI and T3 preparations have a similar role against AP-induced platelet aggregation. In addition, platelet aggregation activity measured between TI and T3 were much lower compared to T2 suggesting that the active antiplatelet component of platelets is localized to a greater degree in tomato juice and seeds.

EXAMPLE 6 Bioavailability Studies Preliminary studies of the bioavailability of the component that inhibits active platelet aggregation in tomato extracts was performed on four volunteers. The 300 ml dose of 100% tomato juice prepared as described in Examples 1 and 2 was fed to each of the four volunteers. The activity of platelet aggregation was measured in venous blood samples taken from volunteers immediately before (time 0), and one hour after (time 1) of juice consumption.

Table 3 shows the percentage of reduction in platelet aggregation activity, induced by ADP and induced by collagen in blood samples taken from each of the four individuals one hour after consumption of the tomato juice preparation. The results suggest that the consumption of 300 ml of tomato juice is enough to significantly reduce platelet aggregation.

Table 3 EXAMPLE 7 Investigation of the Cumulative Effect of Tomato Juice Consumption 300 ml of tomato juice prepared according to example 6 were fed to two individuals daily for a period of two weeks. Measurements of platelet aggregation activity revealed that there was approximately 12% inhibition of platelet aggregation compared to Day 0 and no activity was retained, i.e., not accumulated in the body.

FORMULATIONS EXAMPLE 8 Capsules Containing Fruit Extract A capsule formulation is prepared by lyophilizing a fruit extract (e.g., a tomato extract as described in Examples 2 and / or 3) and filling the resulting lyophilized powder. in a hard gelatin capsule to give a capsule content of 800 mg per capsule.

EXAMPLE 9 Capsules Containing Fruit Diluted Extract An extender selected from sucrose, lactose and sorbitol is added to an aqueous solution of the active fraction from Example 2 to Example 3. The solution is then freeze-dried to give a powder that is filled into hard gelatin capsules to give a capsule content of 800 mg per capsule (200 mg of tomato extract and 600 mg of diluent).

EXAMPLE 9 Fruit bar of chives containing dry fruit extract A chive food bar is prepared by combining lyophilized tomato extract powder with oatmeal and mixing it with the other ingredients in a mixer comprising a bar and baking form. Active constituent Grams per bar Dry tomato extract 10 Other Constituents Grape passes 30 Laminated corn flakes 20 Oatmeal 20 Honey 10 Hazel wool 10 Vegetable oil 10 Glucose syrup 10 Sugar 10 Malt extract 5 Corn flour 5 Whey powder 1 Salt 1 The inventions illustrated by reference to particular examples, but it will be readily appreciated that various modifications and alterations may be made without departing from the scope of the appended claims.

Claims (29)

1. CLAIMS 1. An extract of fruit or active fraction thereof for use in the prophylaxis or treatment of a state of impaired disease or characterized by the aggregation of platelets.
2. 2. A fruit extract or active fraction thereof for use as an inhibitor of platelet aggregation.
3. 3. A fruit extract or active fraction thereof for use as an anti-rombotic agent
4. 4. A fruit extract for use according to any of claims 1 to 3, wherein the fruit is selected from the fruits of 15 plants of the families Solnaceae, Rutaceae, Cucurbi taceae, Rosaceae, Musaceae, Anacardiaceae, Bromel iaceae, Vitaceae, Arecaceae, Ericaceae and Lauraceae.
5. 5. An extract of fruits for use according to any of the preceding claims, wherein the fruit is selected from tomato, grapefruit, melon, mango, pineapple, nectarine, strawberry, plum, banana, cranberry, grape, pear, apple and avocado
6. 6. A fruit extract or active fraction ^^ ¡m ^^^^ M ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ j ^^^^^ ^^^^^^^^ thereof for the use according to claim 5, wherein the fruit is tomato.
7. 7. A fruit extract or active fraction thereof for the use according to claim 5, wherein the fruit is grapefruit.
8. 8. A fruit extract or active fraction thereof for use according to any of claims 1 to 3, wherein the fruit is melon.
9. 9. A fruit extract or active fraction thereof for the use according to any of claims 1 to 3, wherein the fruit is strawberry.
10. 10. An extract according to any of the preceding claims, which is an aqueous extract.
11. 11. An extract according to claim 10, consisting essentially of fruit juice.
12. 12. An extract according to any of the preceding claims, which is derived from the flesh of a peeled fruit.
13. 13. An extract according to claim 9 or claim 10, which has been dehydrated to form a dry extract.
14. 14. A pharmaceutical composition comprising an active component or derivative from a fruit or an active extract or fraction thereof as defined in any of the preceding claims and a pharmaceutically acceptable carrier.
15. 15. A pharmaceutical composition according to claim 14, which is selected from tablets, capsules, powders and granules.
16. 16. The use of a fruit or an active extract or fraction thereof as defined in any of claims 1 to 13, for the manufacture of a medicament for use in the prophylaxis or treatment of a disease condition or condition initiated , mediated or characterized by the aggregation of platelets.
17. 17. The use of a fruit or an active extract or fraction thereof as defined in any of claims 1 to 12, for the manufacture of a medicament for use as an inhibitor in platelet aggregation.
18. 18. The use of a fruit or an active extract or fraction thereof as defined in any of claims 1 to 13, for the manufacture of a medicament for use as an anti- thrombotic agent.
19. 19. The use according to claim 15, wherein the disease state is selected from myocardial infarctions, shock, cardiovascular disease and disease states associated with platelet hyperactivity.
20. 20. A process for the manufacture of a medicament for use in the prophylaxis or treatment of a disease condition or condition initiated, mediated or characterized by aggregation of platelets; the process that is characterized in use, as an essential constituent of the medicament, of a fruit or an extract or an active fraction thereof as defined in any of claims 1 to 13.
21. 21. A process for the manufacture of a medicament for use as an inhibitor of platelet aggregation; the process that is characterized in the use, as an essential constituent of the medicament, of a fruit or an extract or an active fraction thereof as defined in any of claims 1 to 13.
22. 22. A process for the manufacture of a medicament for use as an antithrombotic agent; the process that is characterized in use, as an essential constituent of the medicament, of a fruit or an extract or an active fraction thereof as defined in any of claims 1 to 13.
23. 23. A process for the preparation of an extract as defined in any of the preceding claims; process comprising homogenising the flesh of the fruit (for example a peeled fruit), optionally with the addition of water or a buffered aqueous solution, and then centrifuging to remove solids.
24. 24. A method for the prophylaxis or treatment of a disease condition or condition initiated, mediated or characterized by aggregation of platelets; method comprising administering to a patient (such as a human or other mammal) in need thereof an effective and preferably non-toxic amount of a fruit or an active extract or fraction thereof as defined in any of claims 1 to 13.
25. 25. An active fraction for a fruit extract for use as a rhombic anti- 1 agent or for use as an inhibitor of platelet aggregation, or for use in the prophylaxis or treatment of an initiated disease state or characterized by the aggregation of platelets, as defined in any of the preceding claims, the active fraction containing a water-soluble, colorless, substantially heat-stable compound having a molecular weight of less than 1000.
26. 26. An active fraction of a tomato extract, the active fraction containing a water-soluble, colorless, substantially heat-stable nucleoside compound or compounds having a mole weight cular of less than 1000.
27. 27. An active fraction per se that can be isolated from tomato and which is characterized in that: (a) it is substantially heat stable (b) it is colorless or straw colored; (c) is a water soluble compound; (d) consists of the components having a molecular weight of less than 1000; (e) contains one or more nucleosides having the activity of inhibiting platelet aggregation; and preferably (f) has a mass spectrum that is subjected to the MALDI-TOF mass spectrometry, as shown in Figure 7 attached hereto; and preferably (g) exhibits a 1K nuclear magnetic resonance spectrum substantially as shown in Figure 6 appended hereto.
28. 28. An aqueous extract according to any of claims 1 to 12, wherein at least it is twice concentrated, at least 4 times concentrated, or at least 8 times, or at least 40 times, or at least 100 times, or at least 200 times, or at least 1000 times concentrated.
29. 29. A capsule formulation comprising an extract as defined in any of claims 1 to 13, in a dry form enclosed with a capsule shell.