Compositions comprising heat-treated clear tomato concentrate

Abstract

The present invention provides a therapeutic composition comprising heat-treated clear tomato concentrate (CTC), which has been found to possess both anti-inflammatory and bone-health promoting effects. The present invention is also directed to a composition comprising heat-treated CTC in combination with one or more carotenoids.

Claims

1. A therapeutic composition for treating inflammation comprising an effective amount of a synergistic combination of heat-treated clear tomato concentrate (CTC) and one or more carotenoids, wherein the total free amino acid concentration is less than 2% w/w, and-wherein the free glutamine concentration is less than 0.1% w/w, wherein the heat treated CTC is concentrated tomato serum that is obtained by the steps of: (a) separating tomato material into pulp and serum, (b) discarding the pulp, (c) concentrating said serum to a Brix value between about 40 and 80, preferably above 55° Bx, and (d) heat-treating the concentrated serum at 90° C. for between 1 and 3 hours.

2. The therapeutic composition according to claim 1, wherein the one or more carotenoids is selected from the group consisting of lycopene, phytoene, phytofluene, beta-carotene and lutein, and/or derivatives thereof.

3. The therapeutic composition according to claim 2, wherein the carotenoid is lycopene or a derivative thereof.

4. The therapeutic composition according to claim 1, wherein the one or more carotenoids are provided by a tomato oleoresin.

5. The therapeutic composition according to claim 1, wherein the total concentration of the carotenoids in said composition is at least 0.1% (w/w).

6. The therapeutic composition according to claim 5, wherein the concentration of lycopene is at least 0.1% (w/w).

7. A method for inhibiting or reducing the production of an inflammatory mediator in a subject in need thereof, comprising administering to said subject an effective amount of the therapeutic composition according to claim 1.

8. The method according to claim 7, wherein the inflammatory mediator is selected from the group consisting of NO, TNF-alpha and interleukin 1.

9. The method according to claim 7, wherein the therapeutic composition is administered in a pharmaceutical dosage form.

10. The method according to claim 7, wherein the therapeutic composition is incorporated into a foodstuff or beverage.

11. A method of treating an inflammatory condition, comprising administering to a subject in need thereof an effective amount of the therapeutic composition of claim 1.

12. The method of claim 11 wherein NO, TNF-alpha and/or interleukin 1-beta acts as a modulator or mediator of said condition.

13. A method for improving bone health in a subject in need thereof, comprising administering to said subject an effective amount of the therapeutic composition according to claim 1.

14. The method according to claim 13, wherein the improvement in bone health comprises the inhibition of bone resorption.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 graphically compares the inhibition of NO production by peritoneal macrophages by treatment regular, non-heated CTC (FIG. 1A) with that caused by treatment with heat-treated CTC (FIG. 1B).

(2) FIG. 2 graphically illustrates the effect of different heating times (0-120 minutes) on the ability of heat-treated treated CTC to inhibit NO production by peritoneal macrophages.

(3) FIG. 3 graphically illustrates the concentration of glucose in each of five separate batches of CTC, both before and after heating.

(4) FIG. 4 graphically illustrates the concentration of fructose in each of five separate batches of CTC, both before and after heating.

(5) FIG. 5 shows the inhibitory effect of heat-treated CTC on TNF production by LPS-stimulated macrophages. The first of each group of three bars represents the inhibition obtained with fresh heat-treated CTC, while the second and third bars represent the results obtained with heat-treated CTC that was stored for six months at room temperature or at 40 C, respectively.

(6) FIG. 6 graphically compares the inhibition of NO production by peritoneal macrophages caused by tomato oleoresin (Lyc-O-Mato) alone and heated CTC alone, with a combination of tomato oleoresin and heated CTC.

(7) FIG. 7 graphically represents the inhibition of NO production by peritoneal macrophages caused by lutein alone and heated CTC alone, with a combination of lutein and heated CTC.

(8) FIG. 8 graphically represent the inhibition of NO production by peritoneal macrophages caused by beta-carotene carotene alone and heated CTC alone, with a combination of beta-carotene and heated CTC.

(9) FIG. 9 graphically illustrates the inhibitory effect of a combination of Lyc-O-Mato and heated CTC on the production of interleukin 1-B in a model of carrageen-induced rat paw inflammation.

(10) FIG. 10 graphically compares the ability of heated CTC and non-heated CTC to inhibit RANKL-induced osteoclast differentiation, as measured by Tartrate Resistance Acid Phosphatase (TRAP) activity. The results indicate that heated CTC is significantly more potent than non-heated CTC.

(11) FIG. 11 graphically compares the influence of Lycopene alone with CTC alone and with Lycopene and CTC addition on the inhibition of RANKL induced osteoclast differentiation, as measured by TRAP.

(12) FIG. 12 graphically compares the dose dependent activation of the antioxidant response element (ARE) by heated CTC in MC3T3 osteoblastic cells with the lack of such activation that is seen with non-heated CTC.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(13) As disclosed hereinabove, the present invention provides compositions comprising heat-treated CTC alone or combined with one or more carotenoids. The fraction known as CTC may typically (but not exclusively) be prepared by means of the following process: Step 1: Tomatoes are washed and sorted according to their color and quality. Step 2: Clean tomatoes are crushed; at this point the raw crushed tomatoes are sampled for lycopene content and sugar content. Step 3: Raw crushed tomatoes are screened through 4-6 and 12 mm nets. Additional step 3.1: Water is added to the retained solids are further screened on 0.6 and 0.8 mm nets in order to remove peel particles. Step 4: Slurry obtained from step 3 is further screened on 1.5 to 4 mm nets. Step 5: Slurries from step 4 and 3.1 are pooled together and stored in a container; at this point the slurries are sampled and analyzed. Step 6: Slurry is transferred to large storage tanks. Step 7: The slurry is heated to 80-85° C. and sampled. Step 8: Once the temperature has reached 80-85° C., the aqueous phase is separated from the solids in horizontal centrifuges (decanters) Step 9: The solid material (pulp) is packed in laminate bags placed in metal drums which are than sampled and labeled. The pulp contained drums are then frozen and stored in frozen conditions Step 10: Aqueous phase (serum from decanter) is sampled. Step 11: Serum is filtered and de-aerated under vacuum in order to eliminate excess foam Step 12: Serum is stored temporarily in a large container. Step 13: The serum is centrifuged and the sludge is sent back to Step 5. Step 14: the serum is concentrated under vacuum in an evaporator to the desired Bx value.

(14) The above scheme is only one example of a process for producing CTC, and various other processes may also be used, without deviating from the scope of the present invention. However, the key stages of this process may be summarized as follows: Crushed tomatoes are separated into two fractions—serum and pulp—. The tomato serum is concentrated to Brix value between 40 and 80, preferably higher than 55° Bx. This product, which consists of Clear Tomato Concentrate, is commonly referred to as CTC.

(15) Further information concerning the preparation and properties of CTC may be found in co-owned WO 99/60868, which is incorporated herein.

(16) As explained herein above, the CTC of the present invention is subjected to heat treatment. While several different heating regimes may be used to prepare the aforementioned heat-treated CTC, in one preferred embodiment, the CTC (at a concentration corresponding to approximately 60 Bx) is heated at 90 degrees C. for a period of between 1 and 3 hours. In a particularly preferred embodiment, heat-treated CTC is prepared by heating CTC for 1 hour at 90 degrees C.

(17) Preferred daily amounts of the heated CTC present in the compositions that are administered to subjects in need of such treatment are in the range of 100 to 500 mg.

(18) Preferred daily amounts of total carotenoids in the compositions containing both heat-treated CTC and carotenoids, which are administered to subjects in need of such treatment, are in the range of 2 to 20 mg.

(19) Preferably, in the compositions containing both heat-treated CTC and carotenoids, the carotenoids are present at a concentration of at least 0.1%. In one preferred embodiment of the invention, the combination compositions (i.e. those compositions containing both heated-CTC and carotenoids) comprise lycopene at a concentration of at least 0.1%.

(20) The composition of the present invention may be formulated for either systemic or topical use. In the case of systemic administration, the heat-treated CTC may be incorporated into oral dosage forms such as tablets, caplets, capsules, syrups, elixirs, liquids etc.

(21) In other preferred embodiments, the composition of the present invention may be administered topically, for example on the skin or mucous membranes (e.g. as creams, lotions, ointments etc.). Further details of suitable methods of incorporating the heat treated CTC-containing compositions of the present invention into the various different dosage forms may be obtained from any standard reference work known to the skilled artisan, including, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. Easton, Pa., USA (1980).

(22) In other preferred embodiments, the composition of the present invention is prepared as a food additive that is suitable for direct incorporation into a foodstuff or a beverage.

(23) The following examples are provided for illustrative purposes and in order to more particularly explain and describe the present invention. The present invention, however, is not limited to the particular embodiments disclosed in these examples.

Example 1

Inhibition of Production of NO Using Heat-Treated CTC

(24) Methods and Materials:

(25) Macrophage Isolation and Cell Culture—

(26) Peritoneal macrophages were collected from the peritoneal cavity of 6-8 week old male ICR mice (Harlan, Israel) that had been given an intraperitoneal injection of 1.5 ml of thioglycollate broth (4%) 4 days before harvest. Peritoneal macrophages were washed three times with PBS and, if needed, a hypotonic lysis of erythrocytes was performed, yielding 90-95% purity. The macrophages were identified by FACS analysis using FITC-conjugated rate anti-mouse F4/80 (MCA497F) (Serotec, Oxford, England) by flow microfluorimetry on FACS (Becton Dickinson, Mountain View, Calif.). For each sample, 10,000 light scatter-gated viable cells were analyzed. Peritoneal macrophages were cultured in RPMI 1640 medium contained 10% FCS, 2 mM L-glutamine; 100 U/ml penicillin; 100 μg/ml streptomycin (Beit-Haemek, Isreal) in 96-well plates (1×10.sup.6 cells/well) at 37° C. in 5% CO.sub.2 atmosphere. Cells were stimulated with LPS (1 μg/ml) in the presence or absence of heated CTC.

(27) Appropriate volumes of DMSO (0.1-0.2%) were added to the controls and the percent inhibition in each test tube was calculated in relation to its control.

(28) Clear tomato Concentrate (CTC) was prepared as described hereinabove, and since it is water soluble, was added directly to warm culture medium at the desired dilutions.

(29) Unless otherwise stated, the heat-treated CTC used in this study refers to CTC that was heated to 90 degrees C. for period of one hours.

(30) NO Production Assay—

(31) NO levels in supernatants of cell cultures were determined by assaying nitrite levels using Griess reagent and sodium nitrite as a standard as described in Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Whishnok, J. S., and Tannenbaum, S. R. (1982) Anal Biochem. 126: 131-138.

(32) Statistical Analysis—

(33) Data are presented as the mean±SEM. Statistical significance for comparisons between groups was determined using Student's paired two-tailed t test.

Results

A. Dose Dependent Inhibition of NO Production by CTC or Heated CTC

(34) As shown in FIG. 1, addition of CTC or heat-treated CTC in different dilutions to the macrophages 1 before addition of 1 mg/ml LPS for 24 h, caused a dose dependent inhibition, which was significantly much more efficient in the case of heat-treated CTC.

(35) As seen in FIG. 1A, the inhibition by (untreated) CTC was first observed at 1:200 dilution and caused 16±4% inhibition while maximal inhibition of 100% was achieved by 1:25 dilution of CTC.

(36) The inhibition by the heat-treated CTC (FIG. 1B) was much more marked than that caused by non-heated CTC at the same concentration. Even at a dilution of 1:8000 (data not shown) a slight inhibition of 3.5±1.5% was detected. At a 1:2000 dilution the inhibition observed was greater than 20%, a dose-dependent increase in efficacy being seen up until a dilution of 1:200, at which point maximum (100%) inhibition of NO production was observed.

Example 2

Determination of Optimal Heat-Treatment Conditions

(37) In order to investigate the length of heating-time needed to produce a maximum increase in anti-inflammatory efficacy, CTC was heated to 90° C. for different time durations before being added to the macrophages. As shown in FIG. 2, 1 h of incubation is sufficient to achieve maximal inhibition of NO production for all of the different CTC dilutions tested.

Example 3

Characterization of Heat-Treated CTC

(38) The concentrations of certain key constituents of heat-treated CTC were determined and compared with the levels of said constituents in regular CTC that has not been subjected to heat treatment.

(39) A) Amino-Acids

(40) The concentrations of various free amino acids in CTC samples (heated and unheated) were determined using a reverse-phase HPLC method. A Zorbax Eclipse XDB-C8 column was used for the separation of Fmoc derivatizated amino acids, in which an acetate buffer/acetonitrile gradient was used as the mobile phase, at a flow rate of 1.5 ml/min. The eluted components were detected and quantified using a 265 nm UV detector.

(41) The CTC samples (heated and unheated) were prepared as follows: One gram of CTC was diluted in a 0.2 molar base solution (prepared by dissolving 16.8 g of sodium bicarbonate in one liter of water. 0.5 ml of the diluted CTC was then transferred to a clean vial, to which a 0.5 ml of a standard amino solution was added (prepared by dissolving 20 to 40 mg of each amino acid standard in the aforementioned base solution, to a final volume of 100 ml). Nine ml of an Fmoc solution (prepared by dissolving 40 mg of Fmoc-OnSU in 100 ml of a 75% acetone/25% water mixture) was added to the vial, which was then agitated gently for 30 minutes at room temperature. In order to prevent racemization and dipeptide formation, the samples prepared as described above were analyzed within two hours following the completion of the Fmoc reaction.

(42) A calibration curve was established using standard solutions of free amino acids treated in the same manner as the CTC samples, and used to derive the concentrations of said amino acids in the CTC samples.

(43) The results obtained from a typical HPLC run are as follows:

(44) TABLE-US-00001 Gln Glu Ala Phe Ile Leu total Free amino acids in Heated CTC 0.05% 0.53% 0.35% 0.12% 0.01% 0.01% 1.07% Free amino acids in unheated CTC 0.89% 0.52% 0.32% 0.13% 0.02% 0.01% 1.88%

(45) These results indicate that there is a reduction in the total amount of the measured free amino acids in CTC upon heating (1.07% in heated CTC as opposed to 1.88% in unheated CTC). Furthermore, most of this reduction is due to the marked decrease in the concentration of free glutamine from a level of 0.89% in unheated CTC to 0.05% in heated CTC.

(46) B) Sugars—Glucose and Fructose

(47) The concentrations of glucose and fructose in CTC samples (heated and unheated) were measured using an HPLC technique.

(48) The results obtained for the glucose and fructose assays in five separate batches of CTC (before and after heating) are shown in FIGS. 3 and 4, respectively. The set of bars at the front of each figure represents the results obtained for heated CTC, while the rear set represents the results obtained for non-heated CTC. It can be seen from these figures that heating a CTC sample results in a decrease of about 35% in the glucose concentration of these samples, and a decrease of about 10% in the fructose concentration.

Example 4

Inhibition of Production of TNFα Using Heat-Treated CTC

(49) A further study was undertaken in order to determine the dose dependent inhibition of TNF-alpha production by heated CTC.

(50) Heated clear tomato Concentrate (CTC) was prepared as described hereinabove. Macrophages were stimulated with LPS in the presence of the heated CTC, and the levels of TNF-alpha production measured (as described below) following treatment with the heated CTC.

(51) TNF-Alpha Production Assay—

(52) Concentrations of TNF-alpha were quantified using ELISA kits (Biolegend Inc., San Diego, Calif.).

(53) TNFα production inhibition was observed with the concentrations of heated-CTC indicated, both when said heated-CTC was freshly-prepared and after storage for six months at either room temperate (25 degrees C.) or at an accelerated storage temperature of 40 degrees C.

(54) The results of this study are summarized in FIG. 5. As shown in this figure, addition of heat-treated CTC at different dilutions to the macrophages causes a dose dependent inhibition. A slight inhibition by the heated CTC was first observed at a 1:2000 dilution and was much more marked at a 1:1000 dilution. A dose-dependent increase in efficacy being seen up until a dilution of 1:200, at which point maximum (80%) inhibition of TNFα production was observed.

(55) Further, it may be seen from the bar graph shown in this figure (first bar in each group of three bars) that at all dilutions of heated-CTC that were tested, no loss of TNFα production inhibition was observed after storage for six months at either room temperature (25 degrees C.; second bar in each group) or at an accelerated storage temperature of 40 degrees C. (third bar in each group).

Example 5

Inhibition of Production of NO Using Synergistic Combinations of Heat-Treated CTC and Carotenoids

(56) Methods and Materials:

(57) Macrophage Isolation and Cell Culture—

(58) Peritoneal macrophages were collected from the peritoneal cavity of 6-8 week old male ICR mice (Harlan, Israel) that had been given an intraperitoneal injection of 1.5 ml of thioglycollate broth (4%) 4 days before harvest. Peritoneal macrophages were washed three times with PBS and, if needed, a hypotonic lysis of erythrocytes was performed, yielding 90-95% purity. The macrophages were identified by FACS analysis using FITC-conjugated rate anti-mouse F4/80 (MCA497F) (Serotec, Oxford, England) by flow microfluorimetry on FACS (Becton Dickinson, Mountain View, Calif.). For each sample, 10,000 light scatter-gated viable cells were analyzed. Peritoneal macrophages were cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine; 100 U/ml penicillin; 100 μg/ml streptomycin (Beit-Haemek, Israel) in 96-well plates (1×10.sup.6 cells/well) at 37° C. in 5% CO.sub.2 atmosphere. Cells were stimulated with LPS (1 μg/ml) in the presence or absence of Lycomato or CTC and their combinations.

(59) In some experiments, Lyc-o-Mato was dissolved in DMSO (to a final concentration of 5 mM). The mixture was vortexed and incubated in a water bath at 37° C. (with shaking) for 10 min and then sonicated in a sonicator bath three times for 15 seconds each time. Using this stock solution the desired concentrations were prepared by the addition of appropriate volumes thereof to warm culture medium.

(60) The concentration of lycopene in the solution was determined after extraction as follows: 0.5 ml isopropanol+1.5 hexane/dichloromethane (1:5 V/V) containing 0.025% BHT were added to 1 ml of lycopene solution freshly prepared at a concentration of 200 uM in preheated medium. The solution was vortexed and the phases were separated by centrifugation 3000 rpm for 10 min.

(61) A spectrum analysis is conducted to measure the content of lycopene (absorption peak at 471 nm.)

(62) In other experiments, purified beta-carotene or lutein were used in combination with the heated CTC. Stock solutions and dilutions of each of these carotenoids were prepared as described hereinabove in relation to Lyc-O-Mato.

(63) Appropriate volumes of DMSO (0.1-0.2%) were added to the controls and the percent inhibition in each test tube was calculated in relation to its control.

(64) Clear tomato Concentrate (CTC) was prepared as described hereinabove and, since it is water soluble, was added directly to warm culture medium at the desired dilutions.

(65) Unless otherwise stated, the heat-treated CTC used in this study refers to CTC that was heated to 90 degrees C. for a period of one hour.

(66) NO Production Assay—

(67) NO levels in supernatants of cell cultures were determined by assaying nitrite levels using Griess reagent and sodium nitrite as a standard as described in Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishok, J. S., and Tannenbaum, S. R. (1982) Anal Biochem. 126: 131-138.

(68) Statistical Analysis—

(69) Data are presented as the mean±SEM. Statistical significance for comparisons between groups was determined using Student's paired two-tailed t test.

(70) Results

(71) Synergistic Inhibition of NO Production by Combinations of Lycomato with Heated CTC.

(72) As shown in FIG. 6, there is synergistic inhibition of NO production by LPS treated macrophages by combinations of heated CTC in the range of 1:4000 to 1:1000 dilutions with 0.2 μM Lyc-O-mato. The first bar in each pair of bars in the figure represents the result obtained with heated-CTC alone, while the second bar in each pair represents the result obtained with a combination of the indicated dilution of heated-CTC and 0.2 μM Lycomato. The results shown are the mean values for three separate experiments, each performed in triplicate.

(73) The greatest synergistic effect was obtained by a combination of CTC dilution of 1:400 with 0.2 μM Lycomato that caused 28.1±6% inhibition. Heated CTC (1:4000) alone caused 4.9±2.1% inhibition and 0.2 μM lycomato caused negligible inhibition. Thus the effect of this combination was 5.7 fold higher than the additive effect of each of the ingredient.

(74) Synergistic Inhibition of NO Production by Combinations of Lutein with Heated CTC.

(75) As shown in FIG. 7, combinations of heated CTC in the range of 1:1000 to 1:7000 dilution with both 1 μM and 2 μM lutein cause synergistic inhibition of NO production by LPS treated macrophages. The results shown are the mean values for three separate experiments.

(76) It will be noted that the greatest synergistic effect was obtained by a combination of lutein (at both concentrations tested) together with heated CTC dilutions in the range of 1:2000 to 1:5000.

(77) Synergistic Inhibition of NO Production by Combinations of Beta-Carotene with Heated CTC.

(78) As shown in FIG. 8, combinations of heated CTC in the range of 1:1000 to 1:7000 dilutions with both 0.5 μM and 1 μM beta-carotene cause synergistic inhibition of NO production by LPS treated macrophages. The results shown are the mean values for three separate experiments.

(79) It will be noted that in the case of 0.5 μM beta-carotene, the greatest synergistic effect was obtained by a combination with heated CTC dilutions in the range of 1:2000 to 1:7000.

(80) These results clearly indicate that there is a synergistic interaction between heated CTC and carotenoids with regard to their ability to inhibit inflammation.

Example 6

Inhibition of Production of NO Interluekin 1-Beta Using Synergistic Combinations of Heat-Treated CTC and Carotenoids

(81) A carrageenan-induced paw inflammation model was used in order to investigate the effects of a combination of heat treated CTC and tomato oleoresin (Lyc-O-Mato; Lycored Ltd., Israel) on the production of the pro-inflammatory cytokine, IL1-beta.

(82) Method:

(83) Lyc-O-Mato and heat-treated CTC (prepared as described hereinabove) were administered (separately and together) by the oral route to laboratory rates, once daily, for 7 days. Diclofenac (administered i.p. two hours prior to challenge) was used as a positive control.

(84) At day 8, a carrageenan solution was injected into the left paw of the rats. Following this injection, inflammation, mediated by the influx of neutrophils which generate ROS and pro-inflammatory cytokines, developed in the treated paw. Secretion of the pro-inflammatory cytokine, interleukin-1 beta into the inflamed tissue was measured using a standard ELISA kit.

(85) Results:

(86) It may be seen from FIG. 9 that a combination of 3 mg/kg Lyc-O-Mato (tomato oleoresin) together with 5 mg/kg heated-CTC caused a marked and statistically significant decrease in the secretion of IL-1beta into the inflamed tissue. This decrease was similar in magnitude to that caused by the diclofenac positive control.

Example 7

The Positive Effects of Heated-CTC and Combinations of Heated-CTC and Carotenoids on Bone Health

(87) Both osteoblasts and osteoclasts are involved in bone remodeling. The present inventors have found that heated-CTC improves bone health by at least two complementary mechanisms: 1. Reduction of osteoclast differentiation. 2. Stimulation of Antioxidant Response Element signaling (ARE/NrF2) in osteoblasts.

(88) The inhibitory effect of CTC on RANKL-mediated osteoclast differentiation and activation was studied in vitro.

(89) Methods:

(90) 1. Osteoclast Differentiation

(91) Cells from a mouse monocyte-macrophage cell line RAW 264.7, an osteoclast-precursors lineage, were incubated for 3 days with RANKL 20 ng/ml with or without CTC (non-heated or heated) at the indicated dilutions and stained fro TRAP activity (a marker for osteoclasts differentiation). The percent inhibition was calculated from the activity obtained in the presence of RANKL alone. LycoMato (equivalent to 1 μM lycopene) was added as indicated in FIG. 9. Data are from a representative out of 3 experiments, each performed in triplicates.

(92) 2. Measurement of Antioxidant Response Element (ARE/NrF2) Signaling in Osteoblasts

(93) Cultures of MC3T3-E1 mouse osteoblastic cells were used for this study. CTC (heated and non-heated) alone, or together with the lycopene (6, 14′) oxidation product was added to the cells and ARE reporter gene transcriptional activity was measured, in accordance with the methods described in co-owned WO 2007/043046.

(94) The results of this study are summarized in FIGS. 10-12.

(95) As shown in FIG. 10, both heated and non-heated CTC causes dose-related inhibition of RANKL-mediated osteoclast differentiation, as measured by TRAP. However, heating the CTC sample results in a significant increase in the potency of CTC in inhibition osteoclast differentiation.

(96) FIG. 11 presents the results showing the inhibition of osteoclast differentiation caused by combinations of heat-treated CTC with lycopene. Comparison of the results of the various treatments shown in the graph indicates synergism between lycopene and heat-treated CTC.

(97) FIG. 12 presents results showing stimulation of dose-related ARE signaling in cultured MC3T3 osteoblastic by means of treatment with heated CTC. It may also be observed that non-heated CTC has no effect in this model.

(98) While specific embodiments of the invention have been described for the purpose of illustration, it will be understood that the invention may be carried out in practice by skilled persons with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims.