Use of tomato extract as antihypertensive agent and process for making water soluble sugar free tomato extract

Abstract

To prepare tomato extract as an anti-hypertensive agent to modulate blood pressure.SOLUTION: The tomato extract comprises water soluble, sugar free tomato extract or its active fraction.SELECTED DRAWING: None

Description

[Technical field]

[0001] Provided herein is the art relating to tomato extracts, particularly, but not limited to, the preparation and use of tomato extracts as antihypertensive agents to regulate blood pressure. [Background technology]

[0002] As cardiovascular disease is a major cause of morbidity and mortality, primary and secondary prevention of cardiovascular disease is a public health priority. Hypertension, hyperlipidemia, and platelet hyperfunction are recognized risk factors for cardiovascular disease. Among these, hypertension is recognized as the major chronic disease. Hypertension is defined as a systolic blood pressure above 140 mmHg and / or a diastolic blood pressure above 90 mmHg. Hypertension affects up to 30% of the adult population in most countries. However, more than 50% of hypertensive individuals are unaware of their condition. It is estimated that 7.6 million premature deaths (about 13.5% of the global total) and 92 million deaths and disability-adjusted life years (DALYS) (6.0% of the overall total) are attributable to hypertension. Untreated hypertension can lead to stroke, aneurysm, coronary heart disease (CHD), heart attack, atherosclerosis, ischemic heart disease, left ventricular hypertrophy, encephalopathy, congestive heart failure, hypertensive retinopathy, gout, tachycardia, kidney dysfunction, disability and death. Hypertension also exacerbates the health problems associated with diabetes. Essential hypertension does not result from a single definable cause, but rather from population factors including genetics, age, weight, environment, and diet. Treatment of moderate to severe hypertension is lifelong and requires drug therapy in combination with lifestyle changes including weight loss if overweight, alcohol restriction, and reduced salt and fat intake. Essential hypertension can be treated with one of several classes of medications including diuretics, beta-adrenergic receptor blockers, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, alpha-adrenergic receptor blockers, vasodilators, and centrally acting agents.

[0003] The renin-angiotensin system is a powerful mechanism for controlling blood pressure. When blood pressure falls, the kidneys undergo several intrinsic reactions to convert prorenin to renin. When renin enters the bloodstream, it hydrolyzes plasma angiotensinogen, releasing a peptide called angiotensin I. When angiotensin I circulates to the small blood vessels of the lungs, it is immediately hydrolyzed by ACE to release an 8-amino acid peptide, angiotensin II. After angiotensin II circulates in the blood, it is inactivated by angiotensinase. Angiotensin II is a very potent vasoconstrictor, increasing blood pressure by highly constricting arteries and increasing peripheral resistance. Angiotensin II also acts on the kidneys, which can retain both salt and water, resulting in an increase in extracellular fluid volume, and therefore an increase in blood pressure. Finally, angiotensin II causes the adrenal glands to release aldosterone, which in turn increases water and salt reabsorption in the kidneys. Thus, one approach to controlling blood pressure is by treatment with ACE inhibitors. ACE inhibitors block the formation of angiotensin II, which normally narrows blood vessels and increases blood pressure. ACE is not specific for converting angiotensin I to angiotensin II. In addition to its action on angiotensin, ACE also cleaves many other peptides, including bradykinin, a nonapeptide. Bradykinin is a potent endothelium-dependent vasodilator that causes natriuresis, resulting in a decrease in blood pressure. ACE inhibitors not only reduce the formation of angiotensin II, but also increase the amount of bradykinin, further decreasing blood pressure. Summary of the Invention [Problem to be solved by the invention]

[0004] In recent years, there has been considerable interest in the possibility of using natural food ingredients as functional foods to treat hypertension, especially for patients with borderline to mild hypertension that does not warrant the prescription of antihypertensive drugs. The mechanism by which some functional foods and dietary supplements of the phenolic type lower blood pressure is mediated by the inhibition and downregulation of the expression of ACE and renin. Thus, technology is provided herein for foods and food-derived dietary supplements that promote cardiovascular health, for example, by acting as antihypertensives. In one aspect, the technology is directed to potent inhibitors of angiotensin-converting enzyme (ACE) activity. The inhibitors are derived from tomatoes, for example, water-soluble, heat-stable, and have a molecular weight of less than 1000 Da. Regulation of blood pressure with tomato extracts is prophylactically and therapeutically effective in preventing and halting pathological processes that lead to cardiovascular disease. Thus, compositions comprising the described tomato extracts are useful in cardiovascular disease prevention, for example, as oral tomato extract therapeutics that can be safely and effectively consumed to protect a person's cardiovascular system. In particular, the sugar-free tomato extract compositions provided herein are effective as cardioprotective agents, especially for people with obesity, insulin resistance, and a sedentary lifestyle. [Means for solving the problem]

[0005] Thus, the technology provided herein relates to a composition comprising a tomato extract (e.g., a water-soluble, sugar-free tomato extract) or an active fraction thereof. In some embodiments, the tomato extract comprises or consists of a component having a molecular weight of less than 1000 Da. In some embodiments, the composition is essentially free of lycopene, and in some embodiments, the composition is essentially free of nucleosides. In some embodiments, the composition is in the form of a dry powder. In some embodiments, the composition is in the form of a solution that passes through a 0.2 μm filter without loss of solids. According to some embodiments of the technology, the composition is formulated as an oral formulation, e.g., for oral administration. For example, in some embodiments, the composition is formulated in a form selected from the group consisting of a solution, a suspension, a syrup, a tablet, a capsule, a lozenge, a snack bar, a health drink, an insert, and a patch. Further embodiments of the technology provide a method for producing a tomato extract, the method comprising the steps of homogenizing tomatoes to obtain a tomato homogenate; filtering the tomato homogenate through a filter having a molecular weight cut-off of 1000 Da to obtain a filtrate; and collecting the filtrate to obtain an extract. In some embodiments, the method also comprises freeze-drying the homogenate to obtain a freeze-dried homogenate and dissolving the freeze-dried homogenate in water and / or freeze-drying the filtrate to obtain a freeze-dried filtrate and dissolving the freeze-dried filtrate in water. In some embodiments, the method comprises removing water-soluble sugars from the extract. A related embodiment provides a composition comprising a tomato extract, the composition obtainable by a process comprising the steps of homogenizing tomatoes to obtain a tomato homogenate; filtering the tomato homogenate through a filter having a molecular weight cut off of 1000 Da to obtain a filtrate; collecting the filtrate to obtain the extract; and removing water soluble sugars from the extract.

[0006] Embodiments of the provided technology relate to a composition comprising a tomato extract for use as a medicament for treating hypertension and / or for use as a medicament for inhibiting angiotensin converting enzyme (ACE) and / or conditions associated with increased ACE activity or elevated ACE levels. In some embodiments, the composition comprising a tomato extract or an active fraction thereof is selected from the group consisting of juice, paste, sauce, and soup. In some embodiments, the composition is a water-soluble, sugar-free tomato extract. Some embodiments provide a composition for use as a medicament for treating hypertension and / or for use as a medicament for inhibiting angiotensin converting enzyme, wherein the tomato extract is made from cold-break tomato paste. Further, some embodiments provide a tomato extract for use as a medicament for treating hypertension and / or for use as a medicament for inhibiting angiotensin converting enzyme, the composition being obtainable by a process comprising the steps of diluting cold-broken tomato paste of 28-30° Brix and a browning index of less than 0.350 AU with water; removing particulate matter larger than 0.2 μm to obtain a solution; and concentrating the solution to form a syrup of 62-65° Brix (see, e.g., WO 1999 / 0055350, WO 2007 / 0141495, WO 2006 / 085115, the disclosures of which are incorporated herein by reference in their entireties). Some embodiments provide a tomato extract for use as a medicament for treating hypertension and / or for use as a medicament for inhibiting angiotensin converting enzyme, wherein the composition is obtained by a process comprising the steps of homogenizing tomatoes; removing particulates by centrifugation and / or ultrafiltration; removing soluble sugars; and eluting non-sugar components in methanol (see, e.g., WO 1999 / 0055350, WO 2007 / 0141495, WO 2006 / 085115, the disclosures of which are incorporated herein by reference in their entireties).In some embodiments, the composition is prepared by a method further comprising the step of fractionating the components from non-sugar components using high pressure liquid chromatography.

[0007] In some embodiments, the present invention provides a method of treating a subject, comprising: a) identifying a subject having or at risk of developing hypertension or a condition associated with ACE activity; and b) administering to the subject a composition comprising a tomato extract or an active fraction thereof. In some embodiments, the tomato extract inhibits ACE. In some embodiments, the tomato extract is an aqueous extract. In some embodiments, the tomatoes are peeled prior to the homogenizing step. In some embodiments, the tomato extract is in the form of a concentrate or dehydrate. In some embodiments, the tomato extract is in the form of a concentrate that is at least two-fold concentrated. In some embodiments, the tomato extract is dehydrated to obtain a dry extract. In some embodiments, the dry extract is in the form of a solid or semi-solid dosage form. In some embodiments, the extract is contained within a capsule shell. In some embodiments, the extract contains one or more substantially heat stable water-soluble compounds having a molecular weight of less than 1000. In some embodiments, the condition or disease is selected from the group consisting of stroke, aneurysm, coronary heart disease (CHD), heart attack, atherosclerosis, ischemic heart disease, left ventricular hypertrophy, encephalopathy, congestive heart failure, hypertensive retinopathy, gout, tachycardia, renal dysfunction, diabetes, and pathologies associated with ACE activity and / or hypertension.In some embodiments, the extract is in the form of an active fraction of fruit that can pass through an ultrafiltration filter with a molecular weight cutoff of 1000 and contains one or more substantially heat-stable, colorless or pale yellow, water-soluble compounds with a molecular weight of less than 1000.In some embodiments, the composition comprising tomato extract or an active fraction thereof is selected from the group consisting of juice, paste, sauce, and soup.

[0008] In some embodiments, the present invention provides a method of treating a condition associated with hypertension comprising administering to a patient in need of such treatment a therapeutically effective amount of a fruit extract having antihypertensive activity, wherein the fruit extract is obtained from a fruit of a plant of the family Solanaceae by a process comprising homogenizing the pulp of the fruit to form a homogenate and removing solids therefrom. In other related aspects of the technology, embodiments of the technology provide pharmaceutical formulations comprising a tomato extract and excipients suitable for treating hypertension or inhibiting ACE. Further, embodiments provide antihypertensives and / or ACE inhibitors comprising a tomato extract. Embodiments of the technology also provide for a method of treating a subject, the method comprising identifying a subject having or at risk of developing hypertension; and administering to the subject a composition comprising a tomato extract or an active fraction thereof. Additional embodiments will be apparent to one of skill in the relevant art based on the teachings contained herein. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing the effect of sugar-free tomato extract on inhibiting the activity of pure ACE protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Provided herein is technology relating to compositions and uses for antihypertensive drugs in tomatoes. In particular, during the development of embodiments of said technology, it was discovered that sugar-free tomato extracts prepared as described herein exhibit the ability to reduce ACE activity. The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that the various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily appreciate that the specific order in which the method is shown and performed is illustrative, and it is contemplated that the order may be varied and still remain within the spirit and scope of the various embodiments disclosed herein. All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which each embodiment described herein belongs. In the event that the definition of a term in the incorporated reference differs from the definition set forth in the present teachings, the definition set forth in the present teachings shall control.

[0011] It will be understood that "about" before temperatures, concentrations, times, etc., set forth in the present teachings means that minor and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of the singular and plural "include", "including", the singular and plural "contain", "containing", the singular and plural "including" and "including" is not meant to be limiting. It is to be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not restrictive of the present teachings. Furthermore, singular terms include pluralities and plural terms include the singular, unless otherwise required by context. In general, the nomenclature used herein in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. For example, standard methods are used for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). The nomenclature used in connection with, and the laboratory procedures and techniques described herein are those well known and commonly used in the art.

[0012] definition To facilitate the understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. Throughout the specification and claims, unless the context clearly dictates otherwise, the following terms shall have the meanings clearly associated therewith. As used herein, the phrase "in one embodiment" does not necessarily mean the same embodiment, but may. As used herein, the phrase "in another embodiment" does not necessarily mean different embodiments, but may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention. Additionally, as used herein, the term "or" is an inclusive modus operandi and is equivalent to the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and contemplates additional unrecited factors unless the context clearly dictates otherwise. Additionally, throughout the specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on." As used herein, the term "water-soluble" as applied to a tomato extract means a tomato extract that is soluble at room temperature, e.g., at about 25° C. However, this definition is not limiting as some compositions that contain extracts of the art are soluble at low temperatures, e.g., as low as about 4° C. As used herein, the term "active" as applied to a composition, extract, substance, mixture, solid, liquid, etc., means having the ability to regulate (e.g., lower) blood pressure and / or regulate (e.g., inhibit) ACE activity.

[0013] A "subject" is an animal, e.g., a vertebrate, preferably a mammal, e.g., a human. It is understood that mammals include, but are not limited to, murines, simians, humans, bovines, deer, equines, porcines, canines, felines, and the like. An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. As used herein, the term "effective amount" refers to an amount of a composition sufficient to effect a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration. The term "co-administration" as used herein means administering at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those skilled in the art will appreciate that the formulations and / or routes of administration of the various agents or therapies used may vary. The dosages appropriate for co-administration can be readily determined by those skilled in the art. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered at a lower dosage than would be appropriate for administration alone. Thus, co-administration is particularly desirable in embodiments where co-administration of agents or therapies reduces the dosage of potentially harmful (e.g., toxic) agents that require. As used herein, the term "pharmaceutical composition" means a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for therapeutic use.

[0014] As used herein, the term "pharmacologically acceptable" or "pharmacologically acceptable" refers to a composition that, when administered to a subject, does not produce a substantial adverse reaction, e.g., a toxic, allergic, or immunological reaction. Pharmaceutically acceptable compositions are known in the art, e.g., those described in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985), the disclosure of which is expressly incorporated herein for all purposes. The term "treatment" as used herein includes reducing or alleviating at least one adverse effect or symptom of a disease or disorder by introducing a therapeutic composition of the present technology into or onto a subject in any manner. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the subject prevents or slows (alleviates) the targeted condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to the disorder or those in whom the disorder is to be prevented. As used herein, a "therapeutically effective dose" refers to an amount of a therapeutic agent sufficient to produce a beneficial or desired clinical effect. The dose may be administered in one or more administrations. However, the exact quantification of what is considered an effective dose may be based on individual factors for each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of disease, route of administration, type or extent of disease, stage of disease, route of administration, type or extent of supplemental treatment used, ongoing disease course, and type of treatment desired (e.g., active vs. conventional treatment).

[0015] Embodiments of the Technology While the disclosure herein refers to certain illustrated embodiments, it should be understood that these embodiments are presented by way of example and not of limitation.

[0016] Tomato Extract Provided herein is a tomato extract having an activity that is beneficial for cardiovascular health. For example, data collected during the development of specific embodiments of the described technology shows that the extract reduced ACE activity in normal serum in a dose-dependent manner. Furthermore, the collected data also shows that the active compounds in the tomato extract are water-soluble and have a structure that is very different from lipid-soluble compounds. The extract can be fractionated to separate one or more active fractions therein, for example, by molecular weight filtration or chromatography on a suitable support. In some embodiments, lipids are removed by fractionation, for example, using a Lipidex-1000 column. In some embodiments, the extract is fractionated by solid phase extraction. Experiments performed with tomato extract during the development of the provided technology showed that the active components of the extract pass through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. Furthermore, the active extract is colorless, water-soluble, and does not lose activity when boiled. In some embodiments, the active compound in the extract is not lycopene. The technique encompasses extracts from tomatoes as well as other members of the Solanaceae family, a family of flowering plants that includes, for example, Datura, Mandragora (mandrake), Atropa belladonna (soybean), Lycium barbarum (wolfberry), Physalis philadelphica (tomatillo), Physalis peruviana (capsicum), Capsicum (chili pepper, bell pepper), Solanum (potato, tomato, eggplant), Nicotiana (tobacco), and Petunia. In some embodiments, the techniques are directed to the cultivation of Solanum species, such as tomato (e.g., S. lycopersicum); potato (e.g., S. tuberosum); eggplant (e.g., S. melongena); Giant Devil's fig (e.g., S. chrysotrichum); Ethiopian eggplant and gilo (e.g., S. aethiopicum); naranjilla or lulo (e.g., S. quitoense); Turkey berry (e.g., S. torvum); pepino (e.g., S. muricatum); Tustralan "bush tomato"; and extracts from other members of the formerly separate genera Lycopersicon (tomato) and Cyphomandra.

[0017] The technology includes compositions comprising embodiments of the extracts obtained as described in the Examples and as described in, for example, WO 1999 / 0055350, WO 2007 / 0141495; WO 2006 / 085115, the disclosures of which are incorporated herein in their entirety. Additionally, the technology includes compositions, methods for preparing compositions, pharmaceutical formulations, and other technology disclosed in, for example, U.S. Pat. No. 6,958,164; EP 1334728; AUS 772923; DK 1083912, the disclosures of which are incorporated herein in their entirety. The technology includes modifications of the compositions, methods, and technology described in these references that are obvious to one skilled in the art. For example, due to the presence of anti-ACE and / or antihypertensive drugs in tomatoes, some embodiments of the technology include the use of whole, chopped, pureed, sliced, or other undivided forms of tomatoes (juice, paste, sauce, soup, garnish, etc.) to reduce blood pressure or inhibit ACE. In preferred embodiments, these various forms contain active fractions that reduce blood pressure and / or inhibit ACE activity in target populations (i.e., people who need to reduce blood pressure or people who need to treat diseases associated with increased ACE activity). Thus, while whole tomatoes or chopped or crushed but not divided tomatoes can be used for the present invention, in embodiments, the tomato extract for reducing blood pressure and / or inhibiting ACE activity is an aqueous extract of tomatoes. The extract can be prepared by homogenizing the pulp of tomatoes, with or without their skin, and then filtering the homogenate to remove solids. Preferably, substantially all water-insoluble solids are removed, for example, by centrifugation and / or filtration.

[0018] In either case, the removal of solids has the effect of removing the fraction of skin-containing lycopene. Thus, in some embodiments, the tomato extract encompassed by the technology is a water-soluble extract that is substantially free of lycopene. The aqueous filtrate can be subjected to fractionation to obtain an active fraction containing one or more compounds involved in the biological or therapeutic action described herein. Alternatively, the filtrate can be evaporated to obtain a water-soluble dry extract. Filtration of the tomato homogenate can be accomplished in a single stage or in a series of filtration steps, starting with a relatively coarse filtration or centrifugation step to remove larger particles of tomato skin and / or other water-insoluble fragments of tomato pulp. Some embodiments provide for peeling the tomatoes as a first step, removing large portions of the tomato skin, followed by homogenization. Further filtration steps can then be used to obtain a substantially clear solution, for example, a solution that passes through a 0.2 μm filter without loss of solids. Thus, in an embodiment of the technology, the tomato extract is a water-soluble extract that is substantially free of lycopene and can pass through a 0.2 μm filter without loss of solids.Furthermore, in some embodiments, native sugars are removed from the tomato extract.The advantage of removing sugars is that the extract's activity is concentrated and the extract tends to be less sticky and easier to process in solid form. Alternatively, commercially available tomato paste may be used as the starting material for the preparation of the extract. The tomato paste is typically diluted with water and then the water-insoluble solids are removed, for example, by centrifugation and / or filtration, to obtain a substantially clear solution. When the starting material for the preparation of the extract is tomato paste, it is preferred that the tomato paste is produced by the "cold break" method rather than the "heat break" method. The terms "cold break" and "heat break" are well known in the art of tomato processing, and commercially available tomato paste is typically sold as either heat break paste or cold break paste. Cold break paste can be prepared by a method that includes homogenization of tomatoes followed by a heat treatment step in which the tomatoes are heated to a temperature not exceeding about 60°C, in contrast to heat break paste, in which homogenized tomatoes are heat treated at a temperature of about 95°C (see, for example, Anthon et al., J. Agric. Food Chem. 2002, 50: 6153-6159).

[0019] In some embodiments, an aqueous extract of tomato or tomato paste can be produced by enzymatic digestion of pectin and starch in homogenized pulp or paste, followed by removal of suspended solids from the homogenate and microfiltration or ultrafiltration to remove larger molecular weight proteins and residual polysaccharides. The extract can be purified by removal of monosaccharides, such as glucose, fructose, and sucrose, leaving a concentrated aqueous extract containing a wide variety of low molecular weight (<1000 Da) non-sugar tomato components. Removal of monosaccharides can be achieved using crystallization, such as low-temperature ultrasound-assisted crystallization, or by ethanol precipitation of crystalline glucose and fructose. Alternatively, monosaccharides can be separated from other extract components by chromatographic techniques, such as selective adsorption of bioactive extract components from an aqueous solution onto a polystyrene-based resin material, allowing selective removal of glucose, fructose, and sucrose from the waste stream. The adsorbed non-sugar components are then recovered from the adsorbent resin material by elution with ethanol followed by removal of the ethanol by evaporation. The non-sugar components can be dried by spray drying or drum drying into a water-soluble powder, which can be resuspended in water to produce an aqueous syrup if necessary. The prepared aqueous extract represents an embodiment of the technology. For example, in some embodiments, the extract is a dry powder, a concentrate, a dehydrate, a semi-solid, etc. In some embodiments, the concentrate is at least 2-fold concentrated, at least 4-fold concentrated, at least 8-fold, at least 40-fold, at least 100-fold, at least 200-fold, or at least 1000-fold concentrated.

[0020] Sugar-free tomato extracts, such as those described herein, typically contain various compounds with molecular weights of <1000 Da and are presented herein as embodiments for the use of the described technology.Furthermore, it is effective if the tomato extract is free of or has a low content of nucleosides.Thus, in some embodiments, the tomato extracts we find according to the technology contain no nucleosides or less than 10 nM nucleosides therein. The tomato extract encompassed by the technology includes many bioactive components, such as phenolic compounds, amino acids, amino acid complexes, and tomato flavor compounds.In some embodiments, the extract includes phenolic compounds selected from flavonoids and flavonoid derivatives, such as quercetin, kaempferol, or naringenin derivatives; hydroxycinnamic acid and hydroxycinnamic acid derivatives, such as ferulic acid, coumaric acid, and complexes thereof; benzoic acid and benzoic acid derivatives, such as benzoic acid, hydroxybenzoic acid, gallic acid, salicylic acid, and complexes thereof.In some embodiments, the extract includes amino acids selected from tyrosine, hydroxytyrosine, phenylalanine, glutamine, and complexes thereof.In some embodiments, the extract includes flavor compounds, such as hexanal derivatives, dimethyl sulfide, beta-damascenone, 3-methylbutyric acid, eugenol, and / or methional. In some embodiments, the tomato extract is sub-fractionated by HPLC to obtain three sub-fractions based on polarity.

[0021] Pharmaceuticals and Nutraceuticals The extract or its active fraction may be formulated for oral administration. As such, the extract or its active fraction may be formulated, for example, as a liquid, suspension, syrup, tablet, capsule, lozenge, snack bar, insert, and patch. The formulation is prepared according to methods well known in the art. For example, in some embodiments, the extract or active fraction is formed into a syrup or other liquid for oral administration. As an exemplary embodiment of a composition that can be orally administered, the technology contemplates, for example, a drinkable preparation, which may optionally include one or more excipients selected from sugar, vitamins, flavoring agents, coloring agents, preservatives, and thickeners. In some embodiments, an isotonicity agent such as sodium chloride or sugar is added to obtain a solution of a specific osmolarity, for example, an isotonic solution. In some embodiments, one or more pH adjusters, for example, buffers, are used to adjust the pH to a specific value, for example, to maintain the pH at that value. Examples of buffers include sodium citrate / citric acid buffer and phosphate buffer. In some embodiments, the extract or its active fraction is dried, for example by spray drying or freeze drying, and the dried product is formulated as a solid or semi-solid dosage form, for example, tablet, lozenge, capsule, powder, granule, or gel.For example, a simple dried extract is prepared according to the specific embodiment of the above technique without any additional ingredients.Alternatively, some embodiments provide a dried extract prepared by adsorption onto a solid support, for example, sugar such as sucrose, lactose, glucose, fructose, mannose; sugar alcohol such as xylitol, sorbitol, or mannitol; or cellulose derivative.Other particularly useful adsorbents include starch-based adsorbents, such as flour, for example, wheat flour, corn flour.

[0022] For tablet formation, the dried extract is typically mixed with a diluent such as a sugar, e.g., sucrose or lactose; a sugar alcohol, such as xylitol, sorbitol, mannitol; or a modified cellulose or cellulose derivative, such as powdered cellulose, microcrystalline cellulose, or carboxymethylcellulose. Tablets typically also contain one or more excipients selected from granulating agents, binders, lubricants, and disintegrants. Examples of disintegrants include starch and starch derivatives, as well as other swellable polymers, such as cross-linked carboxymethylcellulose, cross-linked polymeric disintegrants, such as cross-linked polyvinylpyrrolidone, and starch glycolate. Examples of lubricants include stearates, such as magnesium stearate, and stearic acid. Examples of binders and granulating agents include polyvinylpyrrolidone. The diluent is not inherently very sweet, and sweeteners, such as ammonium glycyrrhizinate or artificial sweeteners, such as aspartame or sodium saccharinate, can be added. In some embodiments, the dried extract is formulated as a powder, granule, or semi-solid for incorporation into capsules.For example, for the embodiment in the form of a powder, the extract can be formulated with one or more of the excipients mentioned above for tablets, or can be present in undiluted form.For example, for the embodiment in the form of a semi-solid, the dried extract is dissolved or suspended in a viscous liquid or semi-solid vehicle such as polyethylene glycol, or a liquid carrier such as glycerol, for example propylene glycol, or a vegetable or fish oil such as an oil selected from olive oil, sunflower oil, safflower oil, evening primrose oil, soybean oil, cod liver oil, herring oil, etc. The extract can be filled into capsules of hard or soft gelatin type or capsules made of hard or soft gelatin equivalents. In some embodiments, the dried extract is provided in powder form for incorporation into bar-shaped snack foods, such as fruit bars, nut bars, cereal bars, etc. For example, for bar-shaped snack foods, the dried extract is mixed with any one or more ingredients selected from dried fruits, such as dried tomatoes, raisins and sultanas; or peanuts or cereals, such as oats and wheat.

[0023] In some embodiments, the dried extract is provided in a powder form for reconstitution as a solution. As such, the dried extract can also contain soluble excipients such as sugars, buffers such as citrate buffers and phosphate buffers, and effervescent agents formed from carbonates, such as bicarbonates, such as sodium bicarbonate or ammonium bicarbonate, and solid acids, such as citric acid or acid citrate salts. In some embodiments, the dried extract is provided in a powder form, optionally with a suitable solid (e.g., powdered) excipient for incorporation into a capsule, such as a hard gelatin capsule. In some embodiments, the dried extract is one from which substantially all of the unmodified sugar has been removed. In exemplary embodiments, the solid or semi-solid dosage form of the technology contains up to about 1000 mg, such as up to about 800 mg, of the dried extract. The extract can be indicated as a dietary supplement or food additive in some embodiments or incorporated into a food, such as a functional food or dietary supplement. In some embodiments of the technology, the composition is presented in the form of a unit dosage form containing a defined concentration of the extract or its active fraction. The unit dosage form can be selected to achieve a desired level of biological activity. For example, the unit dosage form can contain an amount of the extract or active fraction up to 1000 mg (dry weight), more typically up to 800 mg, for example 50 mg to 800 mg, for example 100 mg to 500 mg. The specific amount of the extract or active fraction that can be included in the unit dosage form can be selected from 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, and 800 mg. Furthermore, the composition can be placed in a container, pack, or dispenser together with instructions for administration.

[0024] Use of medicines and dietary supplements The amount of extract or active fraction administered to a patient per day for use according to the disclosed technology, for example as an antihypertensive and / or ACE inhibitor, will depend on the strength of the extract and the particular condition or disease being treated and its severity, and will ultimately be at the discretion of the physician. However, the amount administered will typically be a non-toxic amount effective to produce the desired result. For example, a typical daily dosage for a human patient potentially at risk for hypertension may range from 0.0001 to 0.1 grams, e.g., 0.001 to 0.05 grams, per kilogram of body weight. If the active fraction is isolated and administered, the amount of solid material administered may be reduced consistent with increased purity of the active fraction. Typically, at least 100 mg (dry mass or dry mass equivalent), at least 200 mg, and usually at least 500 mg of the extract is administered to a human patient per day. In some embodiments involving administration of a composition, the composition is administered in one or more dosage units per day, e.g., one to four times per day, or one or two times per day. Further embodiments include those in which the extract of the present invention is administered in solid, liquid, or semi-solid form. For example, the extract is administered in the form of tomato juice or a concentrate thereof, alone or mixed with other fruit juices such as orange juice. In some embodiments, provided herein is a method of treatment, comprising: administering a pharma- ceutical effective amount of a tomato extract, a fraction or derivative thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents, to a subject having a condition associated with hypertension. In some embodiments, administration causes a reduction in blood pressure or inhibition of ACE. In some embodiments, administration results in the elimination of one or more symptoms of hypertension or ACE activity, prevention of an increase in the severity of one or more symptoms of the condition, and / or reduction, prevention, or elimination of further diseases or conditions.

[0025] In some embodiments, a subject is tested to assess the presence, absence or level of a disease (e.g., hypertension or a condition associated with hypertension and / or ACE activity), for example by analyzing or measuring biomarkers, metabolites, physical symptoms, indications, etc., to determine the risk or presence of hypertension and / or related diseases, and then the subject is treated with a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents based on the results of the test. In some embodiments, a patient is tested, treated, and then tested again to monitor response to treatment. In some embodiments, the cycle of testing and treatment may occur in a testing and treatment pattern (e.g., test / treatment, test / treatment / test, test / treatment / test / treatment, test / treatment / test / treatment / test, test / treatment / treatment / test / treatment / treatment, etc.), periodicity, or intervals between each testing and treatment phase. For example, in some embodiments, the methods described include a step of a subject being examined for hypertension or a disease or condition related to ACE activity, followed by administering a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents. In some embodiments, the methods described include a step of administering a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents, followed by a step of the subject being examined for hypertension or a disease or condition related to ACE activity. In some embodiments, the methods described include a step of a subject being examined for hypertension or a disease or condition related to ACE activity, followed by administering a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents, followed by a second examination for hypertension or a disease or condition related to ACE activity (e.g., to monitor the effectiveness of treatment). In some embodiments, the method comprises the steps of: a subject being tested for hypertension or a disease or condition related to ACE activity; followed by administering a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents, a second time being tested for hypertension or a disease or condition related to ACE activity; and a second time being administered a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof, alone or in combination with other agents, wherein the second administration has been altered in dosage, duration, frequency, or route of administration in a manner dependent on the results of the previous test.

[0026] In some embodiments, the composition comprising the tomato extract, its active fraction, or its pharmaceutical formulation is co-administered with one or more additional therapeutic agents or medical interventions. In some embodiments, co-administration includes co-formulation of two or more agents together in the same pharmaceutical product. In other embodiments, the agents are administered together in separate formulations, either simultaneously or sequentially (e.g., separated by one or more minutes, hours, or days). In some embodiments, when synergistic or additive benefits are achieved, the co-administered agents can be administered in amounts less than would normally be administered if the agent were used alone to treat the disease or condition. For example, in some embodiments, one or more of the following agents or interventions are co-administered or co-applied with a composition comprising a tomato extract, an active fraction thereof, or a pharmaceutical formulation thereof: a low-fat diet, a low-salt diet, exercise, relaxation techniques (e.g., meditation, biofeedback), ACE inhibitors, smoking cessation, a high-fruit diet, weight loss, reduced alcohol consumption, reduced caffeine consumption, vitamin D, and / or other dietary modifications, such as changing the diet to one rich in nuts, whole grains, fish, poultry, fruits, and vegetables. In some embodiments, a single dose of the composition comprising tomato extract, its active fraction, or its pharmaceutical formulation is administered to the subject.In other embodiments, multiple doses are administered over two or more time points separated by hours, days, weeks, etc. In some embodiments, the composition is administered for an extended period (e.g., chronically), for example, for months or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years or more).In such embodiments, the composition can be taken periodically (e.g., daily, weekly, etc.) for an extended period.

[0027] Exemplary Procedure for Preparation of Tomato Extract Exemplary Procedure 1 A tomato extract for use according to the described technology is prepared, for example, using as starting material a commercially available cold-break tomato paste with 28-30° Brix (e.g., 28-30% solids (w / w)) and a browning index (the browning index is defined as the absorbance at 420 nm of a solution with 12.5 g / L soluble solids) of less than 0.350 AU. The paste is diluted (e.g., about 1:5) with ultrapure water and large particulate matter is removed by centrifugal filtration at room temperature followed by clarification using a Westfalia MSB-14 separator (centrifugal disc clarifier). Smaller particulate matter is then removed by microfiltration at a temperature of 45°C or less to obtain a clear, pale yellow solution that contains no insoluble (capable of settling) solids and that can pass through a 0.2 μm filter without loss of soluble solids. The solution is concentrated by evaporation to form a syrup of 62-65° Brix, using carefully controlled conditions and a temperature of 80°C or less to inhibit non-enzymatic browning reactions. A flash pasteurization step (e.g., 105° C. for 3 seconds) is incorporated at the beginning of the evaporation procedure. The final product is typically characterized by a browning index of less than 0.600 AU and a total viable count of less than 1000. The concentrated extract, in some embodiments, is added to an orange juice matrix for administration, for example to a patient.

[0028] Example Procedure 2 Another preparation of tomato extract and its subfractions is prepared from tomatoes to obtain an aqueous extract. According to this exemplary procedure, an aqueous extract from ripe tomato fruits is prepared by homogenization of fresh tomatoes (Lycopersicon esculentum, locally sourced), centrifugation, and clarification by ultrafiltration (ultrafiltration membrane, MW cut-off 1000 Da, Millipore (UK) Ltd., Watford, UK) of the resulting pale yellow liquid. A typical analysis shows that the tomato aqueous extract consists mostly of soluble sugars (85-90% of the dry matter). These components are removed using solid phase extraction with styrene divinylbenzene (SDVB) cartridges (JT Baker, Mallinckrodt Baker BV, Deventer, The Netherlands) at pH 2.5. The non-sugar components are retained on the cartridge and eluted in methanol. Typically, the non-sugar material represents about 4% of the aqueous extract dry matter. In some embodiments, preparative HPLC is used to sub-fractionate the components from the non-sugar material into three broad groups. For example, HPLC using a Synergy Polar-RP, 4 μm, 250×10 mm column and a Luna C18(2), 3 μm, 250×10 mm column (Phenomenex, Macclesfield, UK) and an acetonitrile / 0.05% TFA gradient is one method for this step. In some embodiments, the HPLC fractions are reconstituted to known concentrations in phosphate buffered saline (PBS, Sigma-Aldrich, Poole, UK) and may be adjusted to pH 7.4, if necessary, before use, for example for in vitro experiments. EXAMPLES

[0029] Preparation of tomato extract During development of embodiments of the technology described herein, tomato extracts were prepared and tested. In some embodiments, tomatoes were homogenized in a Brown Turbo Mixer at maximum speed for 20-30 seconds. The homogenate was then centrifuged at 9000 x g for 15 minutes at 4°C. The supernatant was then boiled for 30 minutes and centrifuged again. The supernatant was removed and lyophilized overnight. The dried material was dissolved in double distilled water and adjusted to pH 7.4. Samples were then homogenized using a Microsep centrifuge with a molecular weight cutoff of 1000 kDa. TM Ultrafiltration was performed using a Centrifugal Device (Pall Corporation, USA). The ultrafiltrate was collected, lyophilized, and reconstituted in water; the pH was adjusted to 7.4 for further experiments.

[0030] Removal of water-soluble sugars from the extract In some embodiments of the tomato extract composition, the tomato extract contained more than 50% inert water-soluble sugars. Thus, these sugars were removed from the extract. To remove sugars, solid-phase extraction column chromatography was used, for example, using a Bond Elut ENV cartridge (Agilent). The Bond Elut ENV cartridge was conditioned with 2×4 ml 100% methanol, then equilibrated with 2×4 ml distilled water. A volume of 4 ml of the prepared sample (containing about 1 g of tomato extract) was loaded onto the cartridge. The cartridge was washed with 2×3 ml double distilled water, and then the water-soluble components were eluted from the cartridge with water. The cartridge was then completely dried, after which the non-sugar components were eluted. After drying, the non-sugar components were eluted with 3×2 ml 100% methanol using a slow (e.g., dropwise) flow rate, and the eluate was collected in a tube. The eluted sample was evaporated to dryness under N2 at 45° C., then dissolved in 500 μl of milliQ water. Both the water and methanol eluates were then tested for inhibitory activity against ACE. The lyophilized material from each of these steps was used to test for the presence of antiplatelet activity.

[0031] Angiotensin-converting enzyme (ACE) assay Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is a circulating enzyme involved in the body's renin-angiotensin system, which regulates blood pressure. During development of embodiments of the technology provided herein, experiments were performed to test the effect of tomato extracts on serum ACE activity. ACE activity was measured using an angiotensin-converting enzyme assay kit.

[0032] Isolation of antihypertensive components from tomatoes. During the development of the embodiments of the technology provided herein, tomato extracts were identified and tested for activity against ACE. These results are summarized in Tables 1, 2, and 3, which collect data related to the inhibitory effects of both tomato juice and sugar-free tomato extract in a dose-dependent manner and isolation of the sugar-free tomato extract embodiments provided herein. After preparation and testing, the active components of the extracts were found to be water-soluble, heat-stable, and have molecular weights less than 1000 Da. Furthermore, the dried material contained a large amount of soluble sugars (about 50%). Inert sugars were removed by solid phase chromatography, for example, by solid phase extraction column chromatography using a Bond Elut ENV cartridge. The yield of sugar-free antihypertensive factor was 67.86 mg per 100 g of tomatoes.

[0033] Table-1: Preparation of tomato extract TIFF2025010139000001.tif46145

[0034] Table-2: Inhibition of human serum ACE activity by fresh tomato juice (juice was prepared after centrifugation of tomato juice at 9000×g for 15 min at 4°C) TIFF2025010139000002.tif30140

[0035] Table-3: Inhibition of human serum ACE activity by sugar-free tomato extract in a dose-dependent manner TIFF2025010139000003.tif46141

[0036] Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is a circulating enzyme involved in the body's renin-angiotensin system regulating blood pressure. The effect of tomato juice and sugar-free tomato extract on serum ACE activity was measured using the Angiotensin-Converting Enzyme Assay REA Direct Kit (Radioenzymatic Assay) (Buhlmann, Germany). Typically, serum (50 μL) was incubated with different amounts of sugar-free extract or kiwi juice volumes at 37° C. for 15 min. After incubation, serum ACE activity was measured using the ACE kit.

[0037] Table 4: Platelet aggregation inhibitory activity of sugar-free tomato extracts TIFF2025010139000004.tif35164

[0038] The antiplatelet activity of sugar-free tomato extracts was measured in platelet-rich plasma (PRP). The pH of all samples was adjusted to 7.4 before testing the effect on platelet aggregation. Venous blood was collected from volunteers who had not taken any medication for at least 14 days before donation. Blood (20-30 ml) was collected using a 19G butterfly needle and coagulation was prevented by mixing the blood sample with citric acid (135 mM) in a ratio of 9 parts by volume of blood to 1 part by volume of citric acid. PRP was prepared from the samples by centrifuging the blood at 180 × g for 15 min at room temperature. The amount of tomato extract as indicated in the table was incubated with 0.225 ml of PRP for 15 min at 37 °C. Platelet aggregation was monitored by adding ADP to a final concentration of 3 μM after the effect of tomato extract on ADP was induced. A control was performed simultaneously using phosphate buffer, pH 7.4, instead of platelets. Platelet aggregation in PRP was monitored at 37 °C with a constant stirring speed of 1000 rpm using Aggram, Helena, USA. Figure 1 is also a graph showing the effect of sugar-free tomato extract on the inhibition of the activity of pure ACE protein. ACE activity was measured by radioactivity assay as described. ACE enzyme from rabbit lung (Sigma product No A6778) EC 3.4.15.1 CAS No 9015-821-1 was used. Increasing amounts of ACE protein were incubated with or without 30 μL of sugar-free tomato extract (Fruitflow) for 30 min at 37°C and then the residual ACE activity was measured. The tomato extract almost completely inhibited ACE activity compared to the untreated control ACE activity. All publications and patents mentioned in the above specification are incorporated herein by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described with reference to specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are apparent to those skilled in the art of pharmacology, biochemistry, medicine, or related fields are intended to be within the scope of the following claims.

Claims

1. A method for preparing a pharmaceutical composition for treating hypertension, comprising a lycopene-free tomato extract, comprising the steps of: a) homogenizing tomatoes; b) removing particulates by centrifugation and / or ultrafiltration; and c) removing soluble sugars; 20. A method for preparing a pharmaceutical composition comprising:

2. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract is a water-soluble, sugar-free tomato extract.

3. 2. The method for preparing the pharmaceutical composition of claim 1, wherein the tomato extract is made from cold-broken tomato paste.

4. The following steps: a) diluting cold break tomato paste of 28-30° Brix and a browning index of less than 0.350 AU with water; b) removing particulate matter larger than 0.2 μm to obtain a solution; and c) concentrating the solution to form a syrup of 62-65° Brix; 2. A method for preparing the pharmaceutical composition of claim 1, comprising:

5. 10. A method for preparing the pharmaceutical composition of claim 1, further comprising the step of fractionating non-sugar components using high pressure liquid chromatography.

6. The following steps: a) homogenizing tomatoes to obtain a tomato homogenate; b) filtering the tomato homogenate through a filter having a molecular weight cut-off of 1000 Da to obtain a filtrate; c) collecting the filtrate to obtain an extract; and d) removing water-soluble sugars from the extract; 2. A method for preparing the pharmaceutical composition of claim 1, comprising:

7. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomatoes are peeled prior to the homogenizing step.

8. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract is in the form of a concentrate or dehydrate.

9. 9. The method for preparing a pharmaceutical composition according to claim 8, wherein the tomato extract is in the form of a concentrate that is at least 2-fold concentrated.

10. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract is a dry extract.

11. The method for preparing a pharmaceutical composition according to claim 10, wherein the dry extract is in the form of a solid or semi-solid dosage form.

12. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract is contained within a capsule shell.

13. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract contains one or more substantially heat-stable, water-soluble compounds having a molecular weight of less than 1000 Da.

14. 2. The method for preparing a pharmaceutical composition according to claim 1, wherein the tomato extract is in the form of an active fraction of the tomato fruit that is capable of passing through an ultrafiltration filter having a molecular weight cut-off of 1000 Da and contains one or more substantially heat-stable, colourless or pale yellow, water-soluble compounds having a molecular weight of less than 1000 Da.