BIOLOGICALLY-ACTIVE TOMATO COMPOSITION HAVING REDUCED AMOUNT OF LYCOPENE

Abstract

The present invention provides a composition comprising lycopene, one or both of phytoene and phytofluene, and phytosterols, wherein the concentration of lycopene is in the range of 0.3%-2% (w/w) and wherein the weight ratio of said lycopene to one or both of phytoene and phytofluene is in the range of 1:1 to 1:2.5. The invention also encompasses a method for preparing said composition.

Claims

1. A composition comprising lycopene, one or both of phytoene and phytofluene, and phytosterols, wherein the concentration of lycopene is in the range of 0.3%-2% (w/w) and wherein the weight ratio of said lycopene to one or both of phytoene and phytofluene is in the range of 1:1 to 1:2.5.

2. The composition according to claim 1, wherein the concentration of phytosterols is at least 2% (w/w).

3. The composition according to claim 1, further comprising vitamin E at a concentration of at least 2% (w/w).

4. The composition according to claim 1, further comprising one or more additional components selected from the group consisting of additional carotenoids, vitamin E, polyphenols and soluble tomato solids.

5. The composition according to claim 1, further comprising beta-carotene.

6. The composition according to claim 1, further comprising carnosic acid.

7. The composition according to claim 1, further comprising soluble tomato solids.

8. The composition according to claim 1, wherein said composition is prepared from tomatoes.

9. A process for preparing a composition comprising lycopene, one or both of phytoene and phytofluene, and phytosterols as defined in claim 1, said process comprising the steps of: a) Providing a tomato product comprising 5-20% (w/w) lycopene; b) Extracting carotenoid-rich material from said tomato product using ethanol, isopropanol, ethyl acetate or acetone at a ratio higher than 1:1 (solvent:tomato extract) as the extraction solvent; c) Heating the solvent/tomato extract mixture at a temperature of about 25°-60° C.; d) Separating crystalline material from the solvent by means of filtering or centrifugal separation; and e) Evaporating the supernatant obtained in step (d), thereby obtaining said tomato composition

10. The process according to claim 9, wherein the solvent is ethanol.

11. The process according to claim 9, wherein the tomato product is an oleoresin.

12. The process according to claim 9, wherein the tomato product comprises tomato peels.

13. Use of a composition according to claim 1 in the preparation of a medicament.

14. Use of a composition according to claim 1 in the preparation of a cosmetic or cosmeceutical agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 schematically illustrates a process for preparing a low-lycopene content tomato composition.

[0042] FIG. 2 graphically illustrates the increase in NO levels in vascular endothelial cells, following treatment with the low-lycopene composition of the present invention.

[0043] FIG. 3 graphically illustrates the increase in peNOS induction in vascular endothelial cells, following treatment with the low-lycopene composition of the present invention.

[0044] FIG. 4 graphically depicts the increase in ARE activity in LNCaP prostate cancer cells caused by the low-lycopene composition of the present invention.

[0045] FIG. 5 graphically depicts the increase in ARE activity in T47D mammary cancer cells caused by the low-lycopene composition of the present invention.

[0046] FIG. 6 graphically depicts the inhibition of the NFkB transcription system in T47D mammary cancer cells that is caused by the low-lycopene composition of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0047] As disclosed hereinabove, the present invention provides composition comprising a reduced lycopene tomato product, wherein a general scheme of the process for manufacturing said composition is summarized in FIG. 1 and comprises the steps of: [0048] Step 1: Providing a tomato product comprising 5%-20% Lycopene (for example, an oleoresin, as described in U.S. Pat. No. 5,837,311, or tomato peels). [0049] Step 2: Adding a solvent or solvent mixture which dissolve the lipids and not the lycopene such as ethanol or isopropanol or acetone at a ratio higher than 1:1 (solvent:tomato product), preferably at a ratio of 3:1 or 4:1. [0050] Step 3: Heating the solvent/tomato extract mixture at a temperature of about 25°-60° C. [0051] Step 4: Separating crystalline material from the solvent by means of filtering or centrifugal separation. [0052] Step 5: Optionally incorporating the crystals, which contain approximately 70% lycopene, into colorant formulations. [0053] Step 6: Partially evaporating the solvent (preferably leaving a liquor having less than 50 ppm solvent), which may then be recycled for use in step 2. [0054] Step 7: Standardizing the resultant liquor to the required concentration, by adding the feed composition.

PHARMACEUTICAL COMPOSITIONS

[0055] Although the active agents, lycopene and phytoene/phytofluene, optionally in combinations with other carotenoids, and/or additional agents, can be administered alone, it is contemplated that these compounds will be administered in a pharmaceutical composition containing the active ingredients together with a pharmaceutically acceptable carrier or excipient.

[0056] Pharmaceutical compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The active agents are formulated as pharmaceutical compositions and administered to a mammalian subject, such as a human patient in a variety of forms such as liquid, solid, and semisolid. The pharmaceutical compositions can be administered to a subject by any method known to a person skilled in the art, such as orally, topically, parenterally, transmucosally, transdermal, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially or intratumorally. For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art. The compositions can be formulated in any solid or liquid dosage form known in the art, including but not limited to, tablet, caplet, capsule, microcapsule, pellet, pill, powder, syrup, gel, slurry, granule, suspension, dispersion, emulsion, liquid, solution, dragee, bead and beadlet. The oral compositions can be formulated as immediate release formulations, or as controlled or sustained release formulations allowing for extended release of the active ingredient(s) over a predetermined time period.

[0057] Suitable excipients for solid formulations include but are not limited to fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch based excipients such as maize starch, wheat starch, rice starch, potato starch and the like, gelatin, gum tragacanth, cellulose based excipients as microcrystalline cellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose and the like. Polymers such as polyvinylpyrrolidone (PVP) and cross-lined PVP can also be used. In addition, the compositions may further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), surfactants (e.g. sodium lauryl sulfate), and lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate).

[0058] For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include but are not limited to petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0059] Preferred oral pharmaceutical compositions include capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. In certain preferred embodiments the capsules exclude components of animal origin and are acceptable for vegetarians and vegans.

[0060] Soft gelatin capsules and methods of preparing them are known in the art. Non-limiting examples can be found in U.S. Pat. Nos. 6,217,902; 6,258,380; 5,916,591, and 4,891,229, all of which are incorporated herein by reference.

[0061] Other acceptable excipients and additives known to the person with skill in the art may be included in the compositions of the present invention, for example stabilizers, solubilizers, tonicity enhancing agents, buffer substances, preservatives, thickeners, complexing agents and other excipients, as well as additional therapeutic agents.

[0062] A solubilizer can be for example, tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycols, glycerol ethers or mixtures of those compounds. A specific example of a solubilizer is a polyoxyethylated castor oil for example, the commercial products Cremophor® or Cremophor® RH40. Another example of a solubilizer is tyloxapol. The concentration used depends especially on the concentration of the active ingredient. The amount added is typically sufficient to solubilize the active ingredient. For example, the concentration of the solubilizer is from 0.1 to 5000 times the concentration of the active ingredient

[0063] Examples of buffer substances are acetate, ascorbate, borate, hydrogen carbonate/carbonate, citrate, gluconate, lactate, phosphate, propionate and TRIS (tromethamine) buffers. The amount of buffer substance added is, for example, that necessary to ensure and maintain a physiologically tolerable pH range. The pH range is typically in the range of from 5 to 9, preferably from 5.2 to 8.5.

[0064] The compositions of the present invention may comprise further non-toxic excipients, such as, for example, emulsifiers, wetting agents or fillers, such as, for example, the polyethylene glycols (PEG200, 300, 400 and 600) or Carbowax® (CarbowaxlOOO, 1500, 4000, 6000 and 10000). Other excipients that may be used if desired are listed below but they are not intended to limit in any way the scope of the possible excipients. They can be complexing agents, such as disodium-EDTA or EDTA.sub.5 antioxidants, such as ascorbic acid, acetylcysteine, cysteine, sodium hydrogen sulfite, butyl-hydroxyanisole, butyl-hydroxytoluene; stabilizers, such thiourea, thiosorbitol, sodium dioctyl sulfosuccinate or monothioglycerol; or other excipients, such as, for example, lauric acid sorbitol ester, Methanol amine oleate or palmitic acid ester.

[0065] The amount of a composition to be administered will, of course, depend on many factors including the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. However, the dose employed will generally depend on a number of factors, including the age and sex of the patient, and the severity of the disease being treated.

[0066] Preferably, the preparations are in unit dosage form, intended for oral administration. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active components. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.

[0067] The dosing schedule of the compositions of the present invention can vary according to the particular application and the potency of the active ingredients. Determination of the proper dosage is within the skill of the art. For convenience, a single daily dose is preferred. Alternatively, the total daily dosage may be divided and administered in portions during the day such as twice daily, thrice daily and the like. Biweekly, weekly, bimonthly and monthly administration are also contemplated

[0068] The present invention will be further described in the following Examples, which are brought for illustrative purposes only, and do not limit the scope of the invention in any way.

Preparative Example 1

Process for Producing the Low-Lycopene Concentration Tomato-Derived Composition of the Present Invention

[0069] The low-lycopene concentration composition of the present invention was prepared in accordance with the following process steps: [0070] 1. A tomato oleoresin having the following composition was prepared according to the process disclosed in U.S. Pat. No. 5,837,311: [0071] 10.1% lycopene [0072] 1.7% phytoene and phytofluene [0073] 2.5% phytosterols [0074] 2.5% vitamin E [0075] 82.7% free fatty acids [0076] 0.5% water [0077] (It is to be noted that this preparation may be purchased under the trade name of Lyc-O-Mato®, manufactured by LycoRed Ltd. of Be'er Sheva, Israel.) [0078] 2. 100 ml of the tomato oleoresin prepared according to step 1 was added to a mixing vessel containing 300 ml of 98% ethanol, and mixed at 40° C. for 0.5 hour. [0079] 3. A press filter was used in order to separate the crystalline material from the ethanol solvent. This separation yielded 10.94 g of crystalline material comprising 85% lycopene and 15% fatty acids. [0080] 4. The filtrate from step 3 was then subjected to evaporation, in order to remove most of the ethanol, leaving 89 g of a liquor (which is the low-lycopene product of the present invention) having the following composition: [0081] 2.0% lycopene [0082] 2.2% phytoene and phytofluene [0083] 2.8% phytosterols [0084] 2.8% vitamin E

Experimental Examples

General Methods

Cell Culture and Treatments

[0085] Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords. The cells were harvested by 0.1% collagenase treatment (Worthington, Lakewood, N.J., USA). HUVECs were grown on 0.2% gelatin pre-coated tissue-culture flasks (Corning, Cole-Parmer, Vernon Hills, Ill., USA) in M-199 medium with 20% BCS and other supplements as described for EA.hy926. EA.hy926 cells were used until passage 37, HUVECs at passage 3-8. U937 monocytes were cultured in RPMI 1640 medium with 10% BCS, L-glutamine and antibiotics as specified earlier. For most experiments the endothelial cells at 70-90% confluence were starved in 5% BCS DMEM/M199 medium for 24 hours and pre-incubated with vehicle or carotenoids solution in 5% BCS DMEM/M199 for 18 to 24 hours, and activated with 10 ng/mL TNF-α (Peprotech, Rocky Hill, N.J., USA) for specified times.

Carotenoid Solutions

[0086] Stock solutions (400 uM) of oleoresin (Lycored Natural Products Ltd., Beer-Sheva, Israel), containing either 6% or 7% lycopene, 0.1% β-carotene, 1% vitamin E and polyphenols, and the low-lycopene composition of the present invention (as obtained according to Preparative Example 1, above) were prepared in fresh tetrahydrofuran (THF) containing 0.025% butylated hydrohytoluene (BHT) (both from Sigma, St. Louis, Mo., USA) as an antioxidant, and were added to the culture medium under N.sub.2 stream and reduced lighting.

Experimental Example 1

The Effect of the Low-Lycopene Concentration Tomato-Derived Composition of the Present Invention on the Expression of Nitric Oxide (NO)

[0087] NO is a key signaling molecule in many biological processes and tissues. In the case of vascular tissues, the endothelium uses nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increased blood flow. NO further plays a role in vascular function by inhibiting vascular smooth muscle growth, platelet aggregation, and leukocyte adhesion to the endothelium. In many cases of human disease that are characterized by the presence of cardiovascular lesions (such as atherosclerosis, diabetes, or hypertension), impaired NO pathway function may often be found.

[0088] In the present study, a cultured human coronary endothelial cell model was used to assess the effect of the low-lycopene (2%) concentration composition of the present invention on the induction of NO. This was achieved both by the direct measurement of NO production by the cultured cells, and by assay of the activity of the endothelial cell NO synthase enzyme, peNOS. The results achieved with the composition of the present invention were compared with results obtained with the use of a high-lycopene tomato-derived composition, 7% Lyc-O-Mato (manufactured and supplied by LycoRed Ltd., Be'er Sheva, Israel).

Methods and Materials:

[0089] The low-lycopene (2%) composition of the present invention (prepared as described hereinabove and the various control solutions (medium, THF and 7% Lyc-O-Mato) were added to confluent cultured human coronary endothelial cells (plated in 2 ml endothelial cell basal medium per 60 mm dish) for 24 hours, following which the supernatant culture fluid was collected.

Nitrate and Nitrite Analysis

[0090] Nitrite and nitrate (NOx), stable oxidized products of nitric oxide, were measured in the culture medium collected 24 h following the exposure to carotenoids using Greiss reagent, using the method described in Miranda K M, Espey M G and Wink D A, 2001 (Nitric Oxide 5: 62-71; “A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite”).

[0091] Experiments were performed at room temperature or at 37° C. in a warm room, as noted.

peNOS Analysis

[0092] Expression of the endothelial NO synthase enzyme, peNOS was assessed using Western blot analysis, as well known in the art. In brief, the protein content of an endothelial cell lysate was quantified using a BCA protein assay kit (Pierce, Rockford, Ill., USA. Equal quantities of cell proteins were separated by 7.5% SDS-PAGE and blotted to nitrocellulose membrane. After blocking and incubation with the primary peNOS antibody, the relative changes in the proteins content were quantified using densitometry in a reflectance mode.

Results

[0093] As shown in FIG. 2, when used at a concentration of 5.0 μM, the low-lycopene (2%) composition of the present invention caused a significant increase in the level of NO induction, when compared with both the medium and THF controls. Unexpectedly, 7% Lyc-O-Mato, while also causing a measurable increase in NO expression, was far less active in this regard (by a factor of approximately four) than the composition of the present invention.

[0094] FIG. 3 presents the results obtained from the Western blot analysis of peNOS levels in the cultured endothelial cells. Thus, it may be seen from this figure that the low lycopene (2%) composition of the present invention (used at 1.0 μM) caused highly significant induction of peNOS, as compared to both the medium and THF controls. This result differs markedly from that obtained using 7% Lyc-O-Mato (1.0 μM) which did not cause any increase in peNOS expression in comparison with untreated cells.

[0095] It may be concluded from these results that the low-lycopene concentration composition of the present invention is capable of significantly increasing the induction of NO in vascular endothelial cells, and that this effect is at least in part caused by increased expression of NO synthase enzyme in said cells. In view of these results, it may be concluded that the composition of the present system has activity of relevance that may be utilized in the treatment and prevention of various types of cardiovascular disease.

Experimental Example 2

Stimulation of the ARE Transcription System by the Low-Lycopene Concentration Tomato-Derived Composition of the Present Invention

[0096] Antioxidant responsive elements (AREs) are identified in gene promoters and mediate a transcriptional induction of a battery of genes which comprise a chemoprotective response system. Said system is essential for resistance against a broad set of carcinogens. In addition, the activation of cytoprotective ARE-regulated genes can suppress inflammatory responses, whereas decreased expression of these genes results in autoimmune disease and enhanced inflammatory responses to oxidant insults. Therefore this ARE model is used as well in order to determine the anti-inflammatory effects of the tested compositions.

[0097] The aim of the present study was to assess the activity of a reduced-lycopene (0.7% w/w) modified oleoresin composition of the present invention, in the ARE system. The results obtained using this composition were compared with those obtained using LycoMato (containing 6% or 7% lycopene), present in equal molar concentrations of lycopene

Methods and Materials:

[0098] A cell culture model, using both mammary (T47D) and prostate (LnCAP) cancer cells, was used to assess the activity of a low lycopene composition of the present invention, prepared as disclosed hereinabove, on the ARE system. Said low lycopene composition comprised 0.7% lycopene, 1.7% phytoene/phytofluene, 2.4% phytosterols and 2.5% vitamin E. Two commercially-available high-lycopene compositions (LycoMato 6% and LycoMato 7%; manufactured by LycoRed Ltd., Be'er Sheva, Israel) were also tested, for the purpose of comparison.

Cell Culture

[0099] LNCaP, human prostate cancer cells were purchased from American Type Culture Collection (Manassas, Va., USA) and grown in RPMI 1640 medium containing sodium pyruvate (0.11 mg/ml) and DHT (10-9 M). To each medium, penicillin (100 units/nil), streptomycin (0.1 mg/ml), nystatin (12.5 μg/ml), Hepes (10 mM), and 10% FCS were added.

[0100] T47D, a human mammary cancer cell line, was kindly provided by Dr. Iafa Keydar (Tel Aviv University, Israel). T47D cells were grown in DMEM containing insulin (0.6 μg/ml or 6 μg/ml).

Transient Transfection and ARE Reporter Gene Assay

[0101] T47D cells were transfected using jet PEI reagent (Polyplus Transfection, Illkrich, France) in 24-well plates (100,000 cells per well). Cells were rinsed once with the appropriate culture medium without serum, followed by the addition of 0.45 ml of medium containing 3% DCC-FCS and 50 μl of a mixture containing DNA and jetPEI reagent at a charge ratio of 1:5. The total amount of DNA was 0.25 μg containing 0.2 μg reporter and 0.05 μg Renilla luciferase. The cells were then incubated for 4-6 h at 37° C. in 95% air/5% CO2. Medium was replaced with one supplemented with 3% DCC-FCS plus the test compounds, and cells were incubated for another 16 h.

[0102] LNCaP cells were transfected using the jetPEI reagent. Cells (70,000) were seeded in 1 ml medium without phenol red containing 3% DCCFCS. On the next day, 500 μl of medium was removed and 50 μl of a mixture containing DNA and jetPEI reagent at a charge ratio of 1:10 was added. The total amount of DNA was 0.2 μg containing 0.16 μg reporter and 0.04 μg Renilla luciferase vectors. Cells were incubated for 4-6 h followed by addition of the test compounds for 24 h.

Luciferase Reporter Assay for ARE Activity Evaluation

[0103] Cell extracts were prepared for luciferase reporter assay (Dual Luciferase Reporter Assay (DLR™) System, Promega) according to the manufacturer's instructions. The Dual-Luciferase Reporter Assay System provides an efficient means of performing dual-reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement.

Results

[0104] As demonstrated in FIG. 4, the low-lycopene composition of the present invention induces significantly greater ARE activity than Lyc-O-Mato in LNCaP prostate cancer cells, when compared at equal molar concentrations of lycopene. The observed effect is dose-dependent. Above the inflection point of the curve (around 10 μM) the differences between the presently-claimed composition and Lyc-O-Mato, with regard to ARE induction are more significant.

[0105] As shown in FIG. 5, similar results are also obtained when the composition of the present invention is added to T47D mammary cancer cells. Thus, as in the case of the results obtained with the prostate cancer cells, the presently-claimed composition caused significantly greater activity in the ARE system than was seen with the Lyc-O-Mato controls.

[0106] It may be concluded from these results that the low-lycopene concentration composition of the present invention is a highly active inducer of ARE activity. Said composition therefore has the potential for influencing important cellular machinery that are of great importance in both the prevention of cancer and the reduction of inflammation.

Experimental Example 3

Inhibition of the NFkB Transcription System by the Low-Lycopene Concentration Tomato-Derived Composition of the Present Invention

[0107] Expression of inflammatory cytokines as well enzyme protein expression can be regulated by the activation of the transcription factor nuclear factor-kappa B (NFκB), which is critically involved in several aspects of the pathogenesis chronic inflammatory diseases. NFκB is activated as a consequence of phosphorylation, ubiquitination, and subsequent proteolytic degradation of the IκB protein through activation of IκB kinase (IKK). The liberated NFκB translocates into nuclei and binds to motifs in the promoters of pro-inflammatory genes such as inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 (COX2) TNF-α, and IL-1β, leading to the induction of their mRNA expression. Many of the anti-inflammatory drugs previously developed have been shown to suppress the expression of these genes by inhibiting the NFκB activation pathway. Thus, an NFκB inhibitor may be useful as a potential therapeutic drug in clinical applications for regulating the inflammation associated human diseases.

[0108] The aim of this study was to investigate whether the low-lycopene composition of the present invention can inhibit the NFkB transcription system in T47D mammary cancer cells.

[0109] The results of this study are shown in FIG. 6. It may be seen from this figure that the low-lycopene composition of the present invention causes significantly greater inhibition of the NFkB transcription system in T47D mammary cancer cells than the 6% Lyc-O-Mato control. These results provide further evidence for the high-level anti-inflammatory activity of the low-lycopene composition for the present invention.