DRUG DELIVERY SYSTEM BASED ON CALCIUM PHOSPHATE NANOPARTICLES FUNCTIONALIZED WITH BIOACTIVE COMPOUNDS FROM EUPHORBIA EXTRACT AND THE USES THEREOF

Abstract

The present invention relates to a composition comprising the bioactive molecules esculetin and euphorbetin, which present antitumor activity, so it may be used for the treatment and/or prevention of cancer, especially for colorectal, pancreatic and glioblastoma. Furthermore, it relates to a drug delivery system composed of nanoparticles of calcium phosphate functionalized with said bioactive molecules, preferably obtained from a Euphorbia extract. The invention also refers to the obtaining of an ethanolic extract of plant origin from defatted flour of mature seed of Euphorbia.

Claims

1. A composition characterized in that it comprises: a) 0.2 to 30 mg of esculetin per g of total composition and b) 2 to 15 mg of euphorbetin per g of total composition.

2. The composition of claim 1, characterized in that it comprises from 2 to 25 mg of esculetin per g of total composition, preferably from 10 to 20 mg of esculetin per g of total composition.

3. The composition of claim 1, characterized in that it comprises from 5 to 10 mg of euphorbetin per g of total composition.

4. The composition of claim 1, wherein said composition is in the form of an ethanolic extract of a plant from genus Euphorbia.

5. The composition of claim 4, wherein the plant from genus Euphorbia is selected from Euphorbia lathyris, Euphorbia anguiata, Euphorbia cyparissias, Euphorbia dulcis, Euphorbia helioscopia, Euphorbia maculata, Euphorbia peplus, Euphorbia prostrata, Euphorbia valliniana or Euphorbia verrucosa , preferably from Euphorbia lathyris.

6. The composition of claim 4, which further comprises gaultherin, kaempferol-3-rutinoside and carnosol.

7. A drug delivery system comprising a plurality of calcium phosphate nanoparticles (CP-NPs) surface functionalized with the composition of claim 1-6, characterized in that said CP-NPs comprise esculetin and euphorbetin adsorbed to the surface of the nanoparticle.

8. The drug delivery system of claim 7, characterized in that it said CP-NPs comprise: a) 0.1 to 7 mg of esculetin per g of the functionalized CP—NPs, preferably 0.2 to 5 mg of esculetin per g of the functionalized CP—NPs and b) 2 to 10 mg of euphorbetin per g of the functionalized CP—NPs, preferably 3 to 8 mg of euphorbetin per g of the functionalized CP—NPs.

9. The drug delivery system of claim 7, characterized in that the average size of the CP-NPs is between 20 and 50 nm, preferably between 30 to 40 nm.

10. The composition of claim 1 or the drug delivery system of claim 7 te-9-for use as a medicament.

11. The composition of claim 1 or the drug delivery system of claim 7 for use in the treatment of cancer, preferably colorectal, pancreatic or glioblastoma.

12. Pharmaceutical composition comprising the composition of claim 1 or the drug delivery system of claim 7 and at least a pharmaceutically acceptable excipient or carrier, and optionally a further active compound.

13. A process for obtaining an ethanolic plant extract from the flour of mature seeds of Euphorbia according to claim 4, comprising the steps of: a) grinding the seed to obtain flour; b) extracting the flour from step (a) by means of a cold hydroalcoholic extraction solution at acid pH, c) optionally, defatting the mature seed by mechanical cold pressing prior to steps (a) and (b).

14. The process according to claim 13, wherein: a) the seed is ground to flour with a particle size between 100 pm and 150 gm; and b) the flour obtained in step a) is extracted with the extraction solution under the following operating conditions: i. temperature equal to 4° C., ii. under nitrogen atmosphere, iii. the extraction solution is composed of ethanol, double distilled water and hydrochloric acid in the proportions 50:50:0.2 by volume, iv. pH equal to 2, and/or v. the mixture of the flour and the extraction solution is kept stirred for 30 minutes after conditions i to iv. have been reached, and wherein the ethanolic extract is obtained by centrifuging the mixture of extraction solution and flour and collecting the supernatant.

15. The process according to claim 13 , wherein the degreasing step of the mature seed is carried out at a temperature of 40 to 50° C. and an extraction rate of 2 to 3 kg of seed/hour.

16. The process according to claim 14 , wherein: vi. the precipitate resulting from centrifuging the mixture of flour and extraction solution is resuspended in extraction solution, vii. the suspension obtained is again stirred for 30 minutes under the conditions of step (b) as defined in claim 14, viii. the suspension is centrifuged and the supernatant is collected; and ix. the ethanolic extract results from mixing the supernatant obtained in (iii) with the first supernatant obtained.

17. The process according to claim 13, comprising a final additional step wherein the ethanol is partially or completely evaporated from the ethanolic extract obtained.

Description

BRIEF DESCRIPTION OF FIGURES

[0099] FIG. 1. UPLC-ESI(-)-MS total ion current chromatogram of Euphorbia extract (A). 1 = esculetin; 2 = euphorbetin. The chemical structures of the phytochemicals are also shown. UV-vis spectra (B) of Euphorbia extract and a solution containing equal concentrations of standards of esculetin and euphorbetin.

[0100] FIG. 2. XRD diffractograms (A) and FTIR spectra (B) of CP—NP and BC—CP—NP.

[0101] FIG. 3. TEM images of CP—NP (A), BC—CP—NP (B).

[0102] FIG. 4. UPLC-ESI(-)-MS total ion current chromatogram of the solution resulting from the partial dissolution of BC—CP—NP (A). Retention times corresponding to molecular weight of euphorbetin and esculetin are shown in B and C (1 = esculetin; 2 = euphorbetin).

[0103] FIG. 5. BC release profiles from amorphous BC—CP—NP in Potassium Phosphate Buffer pH 7.4 (empty squares) and in Sodium Citrate Buffer pH 5.5 (empty circles) during 9 days. The experimental data were fitted by first order kinetic models to obtain the theoretical release curves represented by dashed lines (release constants, k, are shown in brackets).

[0104] FIG. 6. Antiproliferative activity of BC—CP—NP and CP—NP in colon cells. T84 (A) and CCD18 (B) cells were exposed to nanoparticles at different concentrations (.Math.g nanoparticle/ml) for 72 h to obtain the Ic50 values (C). Data are presented as the mean ± standard deviation of three independent experiments; *P<0.05 vs respective control group. **P<0.01 vs respective control group.

[0105] FIG. 7. Antiproliferative assays of the Esculetin and Euphorbetin compounds. T84 cells were exposed to Esculetin (A) and Euphorbetin (B) for 72 h in order to determine the Ic50 values (C). Data are presented as the mean ± standard deviation of three independent experiments; *P<0.05 vs respective control group. **P<0.01 vs respective control group.

[0106] FIG. 8. Antiproliferative assay of the combination of Esculetin and Euphorbetin. T84 cells were exposed to different Esculetin and Euphorbetin combinations for 72 h in order to determine the Ic50 values and synergic effect when they are combined. Data are presented as the mean ± standard deviation of three independent experiments.

[0107] FIG. 9. Blood biocompatibility assay of nanoparticles. (A) Representative Images of optical microscopy of erythrocytes after treatment with different concentrations of BC—CP—NP and CP—NP. Scale bar 50 .Math.m. (B) Hemolysis capacity was expressed as the percentage of erythrocytes lysates versus concentration of nanoparticles (.Math.g/ml). (C) Proliferation assay of white blood cells when they were treated with different concentrations of BC—CP—NP and CP—NP during 1 h and 12 h. Data represent the mean value ± SD of triplicate cultures

[0108] FIG. 10. Shows an outline of the methodology for obtaining an ethanolic extract from seed flour.

[0109] FIG. 11. Shows a representative image of the macroscopic difference between ethanolic extracts of undefatted mature Euphorbia lathyris seed flour (a), and previously defatted mature Euphorbia lathyris seed flour (b) based on the different presence of lipid compounds.

[0110] FIG. 12. Shows a plot of the chromatography replicates of the ethanolic extracts from the undefatted mature seed flour of Euphorbia lathyris.

[0111] FIG. 13. Shows a plot of the chromatographic replicates of ethanolic extracts from previously defatted mature seed flour of Euphorbia lathyris.

[0112] FIG. 14. Shows Western Blot membrane reveal for Caspase 3, 8 and 9 expression in colon tumor line T84 cells treated with an IC50 of the ethanolic extract from defatted mature seed flour of Euphorbia lathyris.

[0113] FIG. 15. Shows a representation of the expression level of Caspases 3, 8 and 9 in T84 colon tumor cells treated with an Ic50 of the ethanolic extract from defatted mature seed flour of Euphorbia lathyris.

[0114] FIG. 16. Shows images obtained at different times (0, 8, 24, 24, 48 and 72 hours) of cell migration assays performed with subcytotoxic doses of ethanolic extract of defatted mature seed flour of Euphorbia lathyris on T84 colon tumor cells.

[0115] FIG. 17. Shown, a representation of the percentage of cell migration upon treatment with a subcytotoxic dose of ethanolic extract of defatted mature seed flour of Euphorbia lathyris T84 colon tumor cells at different times.

EXAMPLES

Materials and Methods

Extraction and Characterization of Euphorbia Extract

[0116] Mature seeds of Euphorbia lathyris S3201 were obtained by the seed defatting process of the invention, by separating of the oleaginous part of the mature seed by means of a cold seed oil extraction press without exceeding 40° C. The average working speed of the extraction process was 2-3 kg seed/h, and the average extraction yield ranged 15-25%. As a result of the former processing conditions, a defatted flour with a particle size of 100-150 .Math.m was obtained. Defatted flour samples were stored at -20° C. in absence of light until use. Defatted flour from mature seed of Euphorbia lathyris were used to obtain a polyphenol-rich ethanolic extract. This process was developed mixing 5 g of defatted flour with 15 ml of Ethanol:H2O:HCl(37% w/w) solution (50:50:0.25) at pH 2 and 4° C. in a reducing atmosphere (with nitrogen) for 30 minutes in a magnetic stirrer. After 30 minutes stirring, the extract was centrifuged at 3.000 rpm for 5 minutes. The supernatant was stored, and the pellet recovered to repeat the process. Finally, all the supernatants were mixed and stored at -20° C. After 24 hours, the extracts were centrifuged at 3.000 rpm for 5 minutes and supernatant collected. To determine the ethanolic extract yield and concentration, aliquots (1 mL) were obtained, and ethanol was evaporated using a vacuum evaporator. The evaporated extracts were frozen in liquid nitrogen and lyophilized during 24 hours. Then, the extract dry weight was calculated by difference with the container containing each aliquot which was referred to a volume of 1 mL of initial extract, to the total volume of extract obtained, and finally to the grams of flour.

[0117] Identification of bioactive compounds that are part of the Euphorbia lathyris extract was achieved by means of ultra-high performance liquid chromatography-electrospray ionization tandem mass spectroscopy (UPLC-ESI(-)-MS). The chromatographic method was the same as described for identification of bioactive compounds adsorption on nanoparticles. The identification of major active ingredients from Euphorbia extract was based on their retention times (RT) and mass (MS) fragments. Besides the UPLC-MS analysis, Euphorbia extract was characterized by UV-vis spectroscopy. The UV-vis spectra of the most interesting bioactive compounds, esculetin and euphorbetin, exhibited an absorbance band-centred at 344 nm (ε.sup.344=60.2 mg mL.sup.-1 cm.sup.-1).

[0118] Ultra-high-performance liquid chromatography coupled to a Diode array detection (UPLC-DAD) was employed for quantifying bioactive compounds from Euphorbia extract. Plant extract was filtered through 0.22 .Math.m nylon disk filters and 10 .Math.L of filtered extract was injected into the chromatograph. Analytical separation was carried out in the same conditions as the quantification of bioactive compounds adsorption on nanoparticles.

Synthesis of Biomimetic Nanoparticles

[0119] CP-NP nanoparticles were synthesized following a precipitation method previously reported with modifications (Delgado-López et al., 2012, (WO2016012452A1/en). Two solutions (1:1 v/v, 100 mL total) of (a) 0.12 M K.sub.2HPO.sub.4 + 0.1 M Na.sub.2CO.sub.3 and (b) 0.2 M CaCl.sub.2 + 0.2 M Na.sub.3(cit) were mixed and the resulting aqueous solution became milky. The mixed solution was continuously stirred at around 250 rpm using a stirring hot plate for about 5 min at room temperature. Afterwards, the nanoparticles were repeatedly washed with ultrapure water by centrifugation to remove unreacted salts and dried for further characterizations. Under these conditions the amorphous calcium phosphate nanoparticles were obtained.

CP-NP Functionalization

[0120] CP-NP nanoparticles (100 mg) were suspended in 5 mL of ultrapure water and sonicated for 30 min. 100 mg of Euphorbia extract was added to the nanoparticles suspension. The mixture was stirred at room temperature for 24 h in the dark to avoid photolytic decomposition of bioactive compounds (BC) in the extract. Subsequently, bioactive compound-loaded CP—NP nanoparticles (BC—CP—NP) were separated from unbound compounds by centrifugation at 10.000 rpm for 5 min. Afterward BC—CP—NP were carefully washed three times with 10 mL of ultrapure water to remove the physically adsorbed bioactive molecules.

Characterization of the Nanoparticles

[0121] Fourier transform infrared (FTIR) spectra were recorded on a FTIR spectrometer using the KBr pellet method. Each pellet was prepared by mixing approximately 3 mg of powdered sample with approx. 200 mg of anhydrous KBr and pressed into 7 mm diameter discs. Pure KBr discs were used as background. FTIR spectra in transmittance mode were registered from 4000 cm.sup.-1 to 400 cm.sup.-1 with a resolution of 4 cm.sup.-1. Powder X-ray diffraction (PXRD) patterns of the samples were collected using a Bruker D8 Advance diffractometer equipped with a Lynx-eye position sensitive detector using Cu Kα radiation (λ = 1.54178 Å) generated at 40 kV and 40 mA. Diffractograms were recorded in the 2θ range from 15 to 70° with a step size (2θ) of 0.02 and a counting time of 1 s. Transmission electron microscopy (TEM) analyses were performed with a Carl Zeiss SMT LIBRA 120 PLUS microscope operating at 120 kV. The powder samples were ultrasonically dispersed in ultrapure water using an Allendale-Ultrasonic cleaner and then few droplets of the slurry were deposited on mesh copper TEM grids covered with thin amorphous carbon films and incubated for several minutes.

Identification and Quantification of Bioactive Compounds Adsorbed on Nanoparticles

[0122] The identification of the bioactive compounds adsorbed on the nanoparticles was carried out by Ultra-high Performance Liquid Chromatography tandem orthogonal acceleration time-of-flight mass spectrometer with an electrospray-ionization technique (UPLC-ESI(-)-MS). BC—CP—NP (1 mg) were partially decomposed in nitric acid (pH=3) by stirring during 24 h. Partial dissolution of BC—CP—NP ensures the release of adsorbed molecules. 10 .Math.L of the final dissolution was filtered through 0.22 .Math.m nylon disk filters and injected into the chromatograph. Analytical separation of bioactive compounds was performed on a C18 column (100 mm x 2.1 mm internal diameter, 1.6 .Math.m) at room temperature. A mobile phase consisting in a gradient program combining deionized water with 0.5% of acetic acid as solvent A and acetonitrile as solvent B was used. The initial conditions were 90% A and 10% B. A linear gradient was then established to reach 100% (v/v) of B at 5 min. Total run time was 8 minutes. Mobile phase flow rate was 0.3 mL min.sup.-1. After chromatographic separation, a high-resolution mass spectrometry analysis was carried out in negative electrospray ionization. The gas used for desolvation (500 L h.sup.-1) and cone (50 L h.sup.-1) was high-purity nitrogen. Spectra were recorded over the mass/charge (m/z) range of 100-1200. All the compounds were identified based on their retention times (RT) and mass (MS) fragments. Based on these data, the compounds were tentatively identified using a specific software.

[0123] The analytical quantification of the adsorbed bioactive compounds was performed by Ultra-Performance Liquid Chromatography coupled to a Diode Array Detection (UPLC-DAD). BC—CP—NP (1 mg) were partially dissolved in an acidic solution (pH=3) by stirring during 24 h to ensure complete release of adsorbed bioactive compounds. After filtering through 0.22 .Math.m nylon disk filters, analytical separation of bioactive compounds was performed as described for identification of bioactive compounds adsorption on nanoparticles. The concentrations of BC were evaluated from peak areas at 344 nm, using calibration curves established with the corresponding standards, esculetin and euphorbetin. Once, the amounts of adsorbed BC were measured, the loading capacity (LC) was calculated as follows:

[00001]$LC=weightofabsorbedBC\left(mg\right)weightofBC-ACP\left(g\right)$

LC represents the mass of bioactive molecules adsorbed per unit mass of BC—CP—NP (mg g-1).

Release of Bioactive Compounds From BC—CP—NP

[0124] The time-dependent release of BC from BC—CP—NP was analyzed at two physiological pH conditions. At a pH of 7.4, the physiological pH of blood and at a pH of 5.5, simulating the pH inside cell lysosomes (pH~5) (Feng et al., 2018). 15 mg of BC—CP—NP were immersed in Potasium Phosphate Buffer (10 mM, 3 mL, pH 7.4) and in Sodium Citrate Buffer (10 mM, 3 mL, pH 5.5), respectively, at room temperature. UV-Vis spectra of the suspensions were recorded every 30 min during 9 days. The UV-vis spectra of the most interesting bioactive compounds, esculetin and euphorbetin, exhibited an absorbance band-centred at 348 nm in Sodium Citrate Buffer (pH 5.5) and at 366 nm in Potasium Phosphate Buffer (pH 7.4). After 9 days, the release of BC from BC—CP—NP was complete.

Example 1 Extraction and Characterization of Euphorbia Extract

[0125] Previously reported analysis of other crude extracts of the genus Euphorbia showed the presence of two molecules of great medicinal interest, named esculetin and euphorbetin. Both compounds (insets FIG. 1A) are structurally-related coumarin derivatives, the esculetin (C.sub.9H.sub.6O.sub.4) is the monomeric coumarin of the euphorbetin (C.sub.18H.sub.10O.sub.8), its dimer. UPLC-ESI(-)-MS total ion current chromatogram (FIG. 1A) showed that Euphorbia extract contained, among others, high concentrations of both phytochemicals. The retention time of 4.174 min, 4.665 and 5.014 min corresponds to the molecular weight of esculetin and euphorbetin, respectively (labelled as 1 and 2 in FIG. 1A). Coumarin derivatives were indeed unambiguously identified in Euphorbia extract by the comparison of their retention times to reference standards (Table 1). Simultaneous quantitative analysis of the two compounds was accomplished by UPLC-DAD. Using external standards of esculetin and euphorbetin, the concentration of the coumarin derivatives in the Euphorbia extract was determined as assessed by their retention times (RT) and absorption band at 344 nm. The contents of esculetin and euphorbetin in the Euphorbia extract were 19.2 mg g.sup.-1 and 7 mg g.sup.-1, respectively.

[0126] The Euphorbia extract was also analysed by UV-vis spectroscopy (FIG. 1B). The UV-vis spectrum showed a main absorbance band centred at 344 nm, which perfectly matches with the spectrum of an aqueous ethanolic solution containing both standards, esculetin and euphorbetin, at the same concentrations as in the Euphorbia extract. (FIG. 1B). Thus, the absorbance at 344 nm of the Euphorbia extract was the result of the joint contribution of esculetin and euphorbetin.

TABLE-US-00001 Identification of esculetin and euphorbetin from Euphorbia extract. Retention Time (min) Compound (molecular formula) Molecular mass [M-H].sup.- (g mol.sup.-1) PPM 4.10 Esculetin (C.sub.9H.sub.5O.sub.4) 177.018 -6.2 4.63 Euphorbetin (C.sub.18H.sub.9O.sub.6) 353.03 2.8 PPM: Difference between the observed mass and the calculated mass.

Example 2 Synthesis of Nanoparticles Functionalized With Bioactive Species of the Euphorbia Extract

[0127] The precipitation method was designed to obtain biomimetic CP—NP nanoparticles whose composition -including citrate and carbonate ions- mimics the mineral phase of bones. The XRD pattern of native CP—NP showed two broad humps, indicating the lack of long-range periodicity typical of calcium phosphate amorphous phases (FIG. 2A). During the adsorption, CP—NP phase started to crystallize as indicated by the incipient diffraction peak at 25.8 degrees (FIG. 2A) related to the 002 reflection of nanocrystalline apatite.

[0128] Nanoparticles were also analysed by FTIR spectroscopy. FIG. 2B represents the FTIR spectra of CP-NP and BC—CP—NP. FTIR spectrum of CP—NP displays broad bands characteristic of the amorphous nature. Interestingly, BC—CP—NP FTIR spectrum displays two absorption bands at ca. 1510 cm.sup.-1 and 1640 cm.sup.-1 (marked with *) that are associated respectively, to the functional groups C═C aromatic and C═C alkene of the adsorbed bioactive compounds on nanoparticles surface. The change of the nanoparticles colour from white CP—NP to yellowish BC—CP—NP—yellowish being similar to the plant extract colour- confirms the effective adsorption (data not shown).

[0129] TEM images of CP—NP (FIG. 3A) revealed aggregates of round-shaped amorphous nanoparticles with morphological features previously observed in nanoparticles obtained using the same synthesis route. TEM images of BC—CP—NP (FIG. 3B) showed very similar amorphous nanoparticles as pointed out by the XRD pattern (FIG. 2A). After functionalization, BC—CP—NP morphology remained practically unchanged.

[0130] FIG. 4 shows UPLC-ESI(-)-MS total ion current chromatograms of BC—CP—NP dissolution (A-C). Both bioactive compounds, esculetin and euphorbetin, were unambiguously identified once desorption of BC from the surface of BC—CP—NP occurred (FIG. 4A). In BC—CP—NP dissolution chromatogram, the intensity of the peak of euphorbetin (FIG. 4A, labelled as 2) is higher than esculetin peak (labelled as 1) suggesting higher concentration of euphorbetin after desorption.

[0131] Mass spectrometry qualitative analysis confirmed the specific adsorption of both molecules from Euphorbia extract (esculetin and euphorbetin) on BC—CP—NP surface. Furthermore, we assessed the mayor peak in FIG. 4B at 1.378 min, which corresponds to citrate (labelled as *), since the mass fragment spectrum indicated a molecular weight of 191.124 g mol.sup.-1. Upon particle dissolution, citrate was desorbed as the rest of molecules. The adsorption of citrate (used during the synthesis) on CP—NP is well reported.

[0132] Quantitative analysis of the two coumarins from Euphorbia extract adsorbed on BC—CP—NP was accomplished by UPLC-DAD. Equation 1 was used to find out LC for both coumarin compounds, esculetin and euphorbetin. Results are listed in Table 3. LC were 0.3 mg of esculetin and 3.4 mg of euphorbetin per gram of BC—CP—NP nanoparticles. This is in line with the intensity ratio of the peaks of esculetin and euphorbetin previously depicted in FIG. 4B, which suggested a much higher loading of euphorbetin on BC—CP—NP surface than esculetin.

TABLE-US-00002 BC-CP-NP Loading capacity (LC) of the two coumarin derivatives. Compound LC (mg g.sup.-1) Esculetin 0.3 ± 01.1 Euphorbetin 3.4 ± 0.9

Example 3. Slow and Gradual Release of BC

[0133] The pH effect on BC release kinetic was analyzed to assess the use of BC—CP—NP as a controlled drug delivery system, since pH-dependent dissolution of BC—CP—NP can result in different release profiles depending on the pH of different body fluids. The physiological pH of blood is close to 7.4, whilst the pH of the tumor microenvironment is more acidic (pH~6.5) and the pH inside cell lysosomes is around 5.0. Thus, we evaluated BC release kinetic at a pH of 7.4 (physiological pH of blood) and at a more acidic pH of 5.5 (pH value between the pH of tumor microenvironment and the pH inside cell lysosomes). FIG. 5 displays the time-dependent BC release from BC—CP—NP nanoparticles, during 9 days. The initial burst effect can be due to the desorption of weakly bound BC at the CP—NP surface. For both pH conditions, BC then follows a gradual and slow-release profile, which can be fitted to first order kinetics with release rates in the range 0.02 h.sup.-1 < k < 0.03 h.sup.-1.

[0134] Calcium phosphates exhibit a pH-dependent solubility, i.e., the lower the pH, the higher is the solubility. This behavior can explain the slightly higher release at the more acidic pH 5.5 (k(pH 5.5) = 0.03 h.sup.-1; k(pH 7.4) = 0.02 h.sup.-1). These kinetics profiles are in agreement with the constants of nanoparticles dissolution (Ramírez-Rodríguez, 2020). This means that the adsorbed species are released upon the slow particle dissolution occurring in the aqueous media, slightly faster at the more acidic pH 5.5.

Example 4. In Vitro Biological Assays

[0135] The human colon adenocarcinoma cell line T84was purchased from the American Type Culture Collection (Rockville, MD, USA). The non-tumor colon cell line CCD18 (human colon epithelial cell line) was used as control. All cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and ATB (antibiotic, streptomycin + amphotericin B) at 1% and maintained in an incubator at 37° C. and 5% CO.sub.2 humidified atmosphere.

[0136] Cells were seeded in 48-well plates with DMEM (Dulbecco’s modified Eagles Medium) (300 ul) at a density of 4 x 10.sup.3 cells/well in CCD18 and 5 x 10.sup.3 cells/well in T84. After 24 hours, cell cultures were exposed 72 h to increasing concentrations of the nanoparticles dissolved in DMEM. In addition, bioactive components of the extract, Esculetin (range from 0.1 .Math.g/ml to 5 .Math.g/ml) and Euphorbetin (range from 0.02 .Math.g/ml to 0.7 .Math.g/ml), were combined at different concentration in order to determine their antitumor activity and to compare with nanoparticle activity and ethanolic extract activity. After incubation time, cells were fixed with trichloroacetic acid (TCA) at 10% for 20 minutes at 4° C. Once dried, the plates were stained with 0.4% sulforhodamine B (SRB) in 1% of acetic acid (20 min, in agitation). After three washes with 1% of acetic acid, the SRB was solubilized with Trizma® (10 mM, pH 10.5). Finally, the optical density (OD) at 492 nm was measured in a spectrophotometer. Cell survival (%) was calculated according to the following equation:

[00002]$CellSurvival\left(\%\right)=\frac{TreatedcellsOD-blank}{ControlOD-blank}x100$

[0137] A hemolysis assay was carried out following a modified version of the protocol reported by Leiva et al., 2017. Briefly, human blood (25 mL) from a healthy donor transferred by Andalusian Public Health System Biobank was recovered into collection tubes with EDTA and centrifuged (500×g for 5 min). The plasma was discarded, and the erythrocytes were washed with 150 mM NaCl, mixed by inversion, and centrifuged at 500×g for 5 min (twice). The supernatant was then aspirated and replaced with phosphate buffered saline (PBS) at pH 7.4. The erythrocytes were diluted (1:50), and 190 .Math.L of the diluted erythrocytes (pH 7.4) was added to each well of a V-bottomed 96-well plate. BC—CP—NP and CP—NP dissolved in DMEM at different concentrations (dose range from 1 .Math.g/mL to 980 .Math.g/mL) were added in a volume of 10 .Math.L per well. Positive and negative controls were 20% Triton X-100 (10 .Math.L) and phosphate buffer pH 7.4 (10 .Math.L), respectively. The plate was incubated for 1 h at 37° C. under stirring (15 rpm), centrifuged at 500×g for 5 min, and then 100 mL of the supernatant was transferred into a flat-bottomed 96-well plate. The percentage of hemoglobin released from the erythrocytes was determined at a wavelength of 492 nm using a colorimeter. This assay was performed in triplicate and the hemolysis percentage was calculated using the following formula:

[00003]$\begin{array}{l}Hemolysis\left(\%\right)=\\ \frac{abs.ofthesample-abs.ofthenegativecontrol}{abs.ofthepositivecontrol}x100\end{array}$

[0138] Optical microscopy images of the erythrocytes treated with the different formulations were taken at the highest dose used (500 .Math.g/ml) to analyze the morphological modifications.

[0139] In addition, white blood cells (WBCs) were isolated and assayed by nanoparticle toxicity. For its isolation, blood was poured into Ficoll-Paque (v/v) avoiding mixing. It was centrifuged at 400 g for 30 minutes. The upper layer of blood plasma was removed and the middle layer containing the white cells was taken. Then, cells were diluted in 10 ml of PBS and centrifuged again at 1000 g for 10 minutes. Finally, after pulling out the supernatant, the cells were resuspended in RPMI 1640 supplemented with 10% FBS and 1% antibiotic. These cells were cultured in 96-well plates at a density of 2 × 10.sup.4 cells/well in a volume of 90 .Math.L of which 10 .Math.L of BC—CP—NP and CP—NP were added to each well from stock solutions of different concentration to reach a final concentration from 1 to 980 .Math.g/mL. The treatments were incubated for 1 and 12 h at 37° C. and 5% CO.sub.2 in a humidified atmosphere, after which the viability of the WBCs was determined by the Cell Counting Kit-8 (CCK-8). This viability assay consists in adding to each well CCK-8 solution directly to the cells, incubate the plate for 3 hours and measure the absorbance at 450 nm using a microplate reader.

[0140] Nanoparticles were assayed in colon cancer cells (T84) and non-tumor cells (CCD18). As shown in FIG. 5, in both lines no toxicity is observed for the CP—NP nanoparticles. However, BC—CP—NP showed a great antitumor effect in the T84 cell line with an IC.sub.50 value of 71.42 .Math.g/ml. In contrast, the IC.sub.50 value in the non-tumor cell line CCD18was 420.77 .Math.g/ml. Thus, BC—CP—NP exhibited a significant and selective cytotoxicity for colon cancer tumor cells.

[0141] In order to compare antitumor activity of the nanoparticle to esculetin/euphorbetin in the ethanolic extract of Euphorbia lathyris, combinations of the isolated compounds were carried out. First, values of IC50 of each compound (Esculetin and Euphorbetin) were obtained (FIG. 5), being Esculetin the compound with a higher antiproliferative activity (0.27 .Math.g/ml). To carry out the combinations of euphorbetin and esculetin, doses lower than the IC50 of each compound were combined. As shown in FIG. 6, the same euphorbetin/esculetin combination (0.3/0.1 .Math.g/ml) as that found in the extract did not modulate IC50 value. However, when euphorbetin/esculetin were combined using the same nanoparticle concentrations (0.25/0.02 .Math.g/ml) only a very low percentage (10%) of cellular proliferation was inhibited. Therefore, the use of the nanoparticle associated to euphorbetin/esculetin represented a very notable antitumor action advantage in relation to the independent extract.

[0142] In order to evaluate the blood cell toxicity of nanoparticles, a hemolysis test was performed using human erythrocytes. As shown in FIG. 9A and B, none of the nanoparticles caused erythrocytes agglutination or modification of their morphology. A very low level of hemolysis (around 2%) was detected at doses of 122 mg/ml. In addition, white cell toxicity of nanoparticles was also tested. As shown in FIG. 9C, nanoparticles showed a total absence of toxicity in white blood cells (viability around 100%) after 1 and 12 h of exposure at all doses tested.

Example 5 Methodology for Obtaining the Ethanolic Extract and Analysis

[0143] The method of obtaining the ethanolic extract that was developed from the flour of the mature seed of Euphorbia lathyris, both undefatted and defatted, is schematized in FIG. 5 and was as follows:

[0144] 1. Depending on the case:

[0145] (a) For the case of flour without defatting: Grind the ripe seeds obtaining flour, which is stored at -20° C.

[0146] (b) In the case of defatted flour: Separation of the oleaginous part of the mature seed from the solid part or pellet is carried out using a seed oil extraction press, KOMET series, which is characterized by its special cold pressing process in which, instead of individual compression screws, screw conveyors are used to squeeze the oil. In this machine the oilseeds are gently pressed without exceeding 50° C. With an average working speed of 2-3 kg of seed per hour and an oil-to-seed conversion yield between 15-25%. As a result, a compact and defatted dry cake is obtained. This cake is then milled at 28,000 rpm to obtain a defatted flour with a particle size of between 100 and 150 microns, which is then stored at -20° C. 2. Weigh 5 g. of defatted flour in a beaker and immediately put the beaker on ice to work at a temperature of 4° C.

[0147] 3. Add 15 ml of extraction solution (50% ethanol (EtOH) = 50 ml EtOH + 50 ml double distilled water + 0.2 ml hydrochloric acid (HCl).

[0148] 4. Shake on a magnetic stirrer at 300 rpm.

[0149] 5. Take to pH 2 (adding HCl 6N).

[0150] 6. Add gaseous nitrogen so that there is no oxidizing environment and the polyphenols are properly preserved.

[0151] 7. Leave 30′ in agitation to 4° C.

[0152] 8. Centrifuge to 7000 rpm during 5′ at 4° C.

[0153] 9. Collect the supernatant and preserve it at -20° C.

[0154] 10. The precipitate is resuspended in 10 ml of extraction solution and a second extraction is performed following the steps indicated above.

[0155] 11. Finally, after performing the pertinent extractions, the supernatants of each extraction are pooled and stored at -20° C. until use.

[0156] 12. The pellet originated in the last centrifuge is discarded.

[0157] Following the described extraction protocol, ethanolic extracts were obtained from the flour of the mature, undefatted and defatted seeds of Euphorbia lathyris. The objective of this double process was to compare the activity of the extracts by removing the high percentage of lipid compounds from the mature seed without defatting (see FIG. 11, where the macroscopic difference between both ethanolic extracts can be appreciated based on the different presence of lipid compounds) and to compare their functional activity against the extracts obtained from previously defatted mature seed flour. Table 3 shows the yield, antioxidant activity (reducing capacity) and total polyphenols of the extract from mature, undefatted and previously defatted Euphorbia lathyris seed flour.

[0158] To determine the yield, the ethanolic extract was divided into 1 mL aliquots for ethanol evaporation and subsequent lyophilization of the remaining water in the extract. To remove ethanol from the ethanolic extracts, ethanol was evaporated under vacuum using a Savant DNA 120 evaporation system (Thermo Scientific) for 60 min. After ethanol evaporation, the aliquots with the remaining extract were frozen in liquid nitrogen and lyophilized using a TELSTAR Cryodos-50 lyophilizer where they were kept for 24 hours. After lyophilization, the dry weight of the extract was calculated by difference with the container containing each aliquot and this dry weight was referred to a volume of 1 mL of initial extract, then to the total volume of extract obtained, and finally to the grams of seed flour used to prepare the extract.

[0159] Total polyphenols were determined by the technique of Dewanto et al. (2002) as described by Kapravelou et al. (2015) using in the determination a gallic acid standard line with concentrations between 0 and 500 .Math.g/mL.

[0160] Finally, the reductive capacity of Fe.sup.3+ to Fe.sup.2+ by the different extracts was determined spectrophotometrically by the technique of Duh et al. (1999) as described by Kapravelou et al. (2015) using in the determination a standard line of gallic acid with concentrations between 0 and 500 .Math.g/mL.

TABLE-US-00003 Euphorbia lathyris Yield (mg/g flour) Total Polyphenols (.Math.g gallic ac.equivalent /mg extract) Reductive capacity (.Math.g gallic ac.equivalent /mg extract) Ripe seed flour without defatting 42.9 ± 1.47 15.85 ± 0.21 9.71 ± 0.28 Defatted ripe seed flour 117.52 ± 9.22 33.52 ± 5.79 22.95 ± 1.92

[0161] As can be seen in Table 3, the yield obtained in relation to both extracts showed a large difference, the yield being significantly higher and more homogeneous with the defatted mature seed flour (117.52 ± 9.22 mg/g flour) versus the yield obtained from the undefatted mature seed flour (42.9 ±1.47 mg/g flour).

[0162] Antioxidant capacity was analyzed including biochemical studies of total polyphenols and reducing capacity. In terms of total polyphenols, extracts from undefatted mature seed flour showed values of 15.85 ± 0.21 .Math.g gallic acid equivalents/mg extract, while extracts from defatted mature seed flour showed higher values (33.52 ± 5.79 .Math.g gallic acid equivalents/mg extract), confirming the purity of working without lipid compounds which, in addition, in their elimination process, do not alter or eliminate phenolic compounds. Reducing capacity tests for the biochemical determination of antioxidant capacity showed values of 9.71 ± 0.28 .Math.g gallic acid equivalents/mg extract in the case of flour from mature, undefatted seeds, and 22.95 ± 1.92 .Math.g gallic acid equivalents/mg extract for defatted mature seed flour (see Table 3). Therefore, the antioxidant capacity of the extracts prepared with defatted mature seed flour was higher on the basis that polyphenols are obtained in higher amount.

[0163] Chromatographic studies were carried out to determine the compounds present in the ethanolic extract of mature Euphorbia lathyris seed flours. The technique used consisted of an Ultra Performance Liquid Chromatography (UPLC) (ACQUITYH CLASSWATERS) coupled to a QTOF mass spectrometer (SYNAP G2. WATERS). FIGS. 12 and 13 show the graphs of the chromatographic replicates of the ethanolic extracts obtained either from the mature seed flour without defatting (FIG. 12) or defatted (FIG. 13). Tables 4 and 5 below show the main bioactive compounds identified in each of the extracts and the chromatographic data for each compound.

TABLE-US-00004 Bioactive compounds from the ethanolic extract of undefatted mature seed flour of Euphorbia lathyris Compound MF [M-H]- TR PPM %Reliab. 177.0185 2.63 -1.7 Esculetin C.sub.9H.sub.6O.sub.4 90-100 177.0188 2.60 0.0 Euphorbetin 353.0293 3.14 -1.1 C.sub.18H.sub.10O.sub.8 90-100 353.0295 3.17 -0.6 445.1344 1.83 -0.4 Gaultherin C.sub.19H.sub.26O.sub.12 90-100 445.1353 1.82 1.6 329.1744 6.11 -2.7 Carnosol C.sub.20H.sub.26O.sub.4 90-100 329.1758 6.13 1.5 593.1512 3.81 1.0 Kaempferol-3- rutinoside C.sub.27H.sub.30O.sub.15 90-100 593.1495 3.82 -1.9 TR: retention time; MF: molecular formula; PPM: error; MS: mass; %Reliab: reliability percentage

TABLE-US-00005 Bioactive compounds of the ethanolic extract of defatted ripe seed flour of Euphorbia lathyris Compound MF [M-H]- TR PPM % Reliab. Esculetin C.sub.9H.sub.6O.sub.4 177.0181 2.62 -4.0 90-100 177.0185 2.61 -1.7 Euphorbetin C.sub.18H.sub.10O.sub.8 353.0298 3.17 -0.6 90-100 353.0301 3.14 1.1 Gaultherin C.sub.19H.sub.26O.sub.12 445.1349 1.89 0.7 90-100 445.1351 1.81 1.1 Carnosol C.sub.20H.sub.26O.sub.4 329.1744 6.10 -2.7 90-100 329.1750 6.11 -0.9 Kaempferol-3-rutinoside C.sub.27H.sub.30O.sub.15 593.1510 3.86 0.7 90-100 593.1520 3.87 2.4 TR: retention time; MF: molecular formula; PPM: error; MS: mass; %Reliab: reliability percentage.

[0164] Among the compounds present in the extracts studied, both from the mature, undefatted and defatted seed flour, esculetin, euphorbetin, gaultherin, nicotiflorin (Kaempferol-3-rutoside) and carnosol stand out. As previously discussed in the section, these are polyphenols that have been previously related to tumor activity in assays performed with the compounds independently, without having proven their activity as part of Euphorbia lathyris extracts. These polyphenols, independently obtained from commercial houses, have been related in previous studies with antitumor activity, without having been performed so far studies with extracts of Euphorbia lathyris, which are included among the assays presented below.

[0165] Likewise, the presence of these compounds has been corroborated and quantified by means of specific standards for each one: esculetin (Sigma-Aldrich, 68923) gaultherin (Cymitquimica,490-67-5), nicotiflorin (Cymitquimica,17650-84-9) and carnosol (Cymitquimica ,5957-80-2), highlighting the lack of the standard for euphorbetin, which being one of the predominant compounds in the chromatogram, is not currently marketed as an isolated compound. The data obtained are shown below in Tables 6 and 7.

TABLE-US-00006 Quantification (ppb: .Math.g/L) of bioactive compounds of replicate ethanolic extracts of mature, undefatted and defatted Euphorbia lathyris seed flour from known standards. Ppb (.Math.g/L) Esculetin Kaempferol-3-Rutinoside Undefatted ripe seed flour of E. lathyris Replica 1 985.9 59.8 Replica 2 1161.3 59.8 Replica 3 1197.2 61.1 Defatted ripe seed flour of E. lathyris Replica 1 2041.5 188.3 Replica 2 2131.8 327 Replica 3 2325.8 354

TABLE-US-00007 Quantification (mg of bioactive compounds per 100 mg of extract) of the bioactive compounds of the replicates of ethanolic extracts of mature, undefatted and defatted seed flour of Euphorbia lathyris from known standards. Concentration mg/ml Esculetin (mg esculetin per each 100 mg extract) Kaempferol-3-Rutinoside (mg kaempferol per each 100 mg extract) Undefatted ripe seed flour of E. lathyris 29.99 ± 4.46 0.4 ± 0.05 0.02 ± 0.003 Defatted ripe seed flour of E. lathyris 99.59 ± 13.84 0.21 ± 0.023 0.03 ± 0.006

[0166] Of these patterns, two appeared in majority, esculetin and kaempferol-3-ruthinoside (see Table 6). As in the yield, the defatted mature seed flour contained more of these compounds, with 2166.37 ppb (.Math.g/L) of esculetin compared to 1114.8 ppb (.Math.g/L) of the same in the extract of the undefatted mature seed flour. The same is true for kamepferol, 289.78 ppb (.Math.g/L) in the defatted mature seed flour extract versus 60.23 ppb (.Math.g/L) in the undefatted mature seed flour extract (Tables 6 and 7).

Example 6. Determination of the Antitumor Capacity of the Extracts

[0167] To determine the antitumor capacity of the extracts, the cell lines T84 (human colon adenocarcinoma cell line) and HCT15 (human colon adenocarcinoma cell line resistant to chemotherapy) were cultured. As a control, the CCD18 cell line (non-tumorigenic human colon epithelial cell line) was selected.

[0168] The ethanolic extracts were previously evaporated to avoid the toxicity caused by ethanol on the cell lines.

[0169] In addition, once evaporated, a part was lyophilized to know the amount of extract obtained and to quantify its concentration (mg/ml) with which the different concentrations to be tested will be calculated. The cell cultures were exposed to increasing concentrations of the evaporated ethanolic extract of mature, undefatted and defatted seed flour, which made it possible to determine the inhibitory dose 50 (IC50) (concentration of the extract at which it inhibits 50% of the cell population). The results obtained are shown in Table 8.

TABLE-US-00008 Antitumor capacity of extracts from mature, undefatted and defatted Euphorbia lathyris seed flour in vitro at 72 hours in different colon cancer lines. Euphorbia lathyris IC.sub.50 (.Math.g/mL) T84 HCT15 CCD18 Ethanolic extract of mature seed flour without defatting 11.04 ± 1.63 34.26 ± 1.1 388.36 ± 30.14 Ethanolic extract of defatted ripe seed flour 16.29 ± 2.54 72.9 ± 1.27 266.02 ± 18.5 T84: human colon adenocarcinoma cell line; HCT15: human colon adenocarcinoma cell line resistant to chemotherapy; CCD18: human healthy colon cell line. IC50: concentration that inhibits 50% of the cells.

[0170] As can be seen in the table above, for the mature seed extract, the IC50s were as follows: 11.04 ± 1.63 .Math.g/ml in T84, 34.26 ± 1.1 .Math.g/ml in HCT15. In the case of the normal colon line CCD18, the IC50 was much higher (388.36 ± 30.14 .Math.g/ml,) indicating lower activity and thus toxicity in this cell type compared to tumor cells.

[0171] For extracts from defatted mature seed flour, the IC50s were: 16.29 ± 2.54 .Math.g/ml in T84, 72.9 ± 1.27 .Math.g/ml in HCT15 and 266.02 ± 18.5 .Math.g/ml in CCD18.

[0172] In view of the results, we observed that both the extract of the mature seed flour, undefatted and defatted, have a high antiproliferative activity with very low IC50s, regardless of whether the mature seed flour is undefatted or defatted, so that the defatting process does not affect the antitumor capacity of the extract obtained. It is noteworthy in both cases, the difference between the IC50 of the tumor lines (T84 and HCT15) and the non-tumor line (CCD18), the latter being much higher than the tumor lines.

[0173] Because both the undefatted and defatted mature seed flour extracts possess similar antitumor activity, but the defatted mature seed flour extracts have higher yields and higher concentrations of polyphenolic compounds, we used the ethanolic extract of defatted mature seed flour to perform the remaining molecular tests detailed below.

[0174] Finally, and based on the previous results, the ethanolic extract of defatted mature seed flour, selected for the rest of the molecular tests, was tested in glioblastoma multiforme and pancreatic adenocarcinoma cell lines, two of the most aggressive types of cancer, with the worst prognosis and for which there are few therapeutic possibilities. For this purpose, cell lines A-172 (human glioblastoma cell line), SF-268 and SK-N-SH (human glioblastoma cell lines resistant to chemotherapy), as well as the Panc-1 cell line (human pancreatic adenocarcinoma cell line) were cultured. Using the same procedure described above, the IC50s of the cell lines under study were determined. The results are shown in Table 9.

TABLE-US-00009 Results of the use of ethanolic extract of defatted mature seed flour tested on glioblastoma multiforme and pancreatic adenocarcinoma cell lines. Euphorbia lathyris IC.sub.50 (ug/ml) SF-268 SK-N-SH A-172 Panc-1 Ethanolic extract of defatted ripe seed flour 39.33 ± 13.2 71.42 ± 13.6 18.58 ± 1.64 185.76 ± 25.8 A-172 (human glioblastoma cell line), SF-268 and SK-N-SH (human chemotherapy-resistant glioblastoma cell lines), as well as the Panc-1 cell line (human pancreatic adenocarcinoma cell line).

[0175] Thus, the IC50 in human glioblastoma cell lines were: 39.33 ± 13.2 .Math.g/ml in SF-268, 71.42 ± 13.6 .Math.g/ml in SK-N-SH and 18.58 ± 1.64 .Math.g/ml in A-172 while in the pancreatic adenocarcinoma derived line Panc-1, the IC50 was 185.76 ± 25.8 .Math.g/ml. These results led to the conclusion that the ethanolic extract of defatted mature seed flour, in addition to exhibiting high antitumor activity against colon cancer, also possesses high antitumor activity against glioblastoma and pancreatic adenocarcinoma cell lines.

Example 7- Molecular Study of Proteins Related to Cell Death

[0176] To elucidate the mechanisms by which the extracts of the present invention act, the cell death pathway (apoptosis) mediated by caspases, mainly caspase 8 (extrinsic pathway), caspase 9 (intrinsic pathway) and caspase 3, was studied by western blot, using β-actin as an endogenous control.

[0177] For this purpose, colon tumor line cells (T84) were cultured with an IC50 of the ethanolic extract obtained from defatted mature seed flour and after 72 hours, the cells were harvested for protein extraction.

[0178] To perform the western blot assay, 40 .Math.g of protein from cells treated with the ethanolic extract, as well as from control cells, were loaded onto an SDS-PAGE electrophoresis polyacrylamide gel in a Mini Protean II cell (Bio-Rad, Hercules, CA). Once the proteins were separated by electrophoresis, they were transferred to a nitrocellulose membrane that was supplied with 20 V at room temperature for 1 h. These membranes were treated with blocking solution (PBS-Tween + 5% milk powder) for 1 h and then, after 2 washes with PBS-Tween, incubated with the primary antibody [rabbit polyclonal IgG anti-caspase-3 (1:500 dilution), anti-caspase-8 (1:1000 dilution) and anti-caspase-9 (1:1000 dilution); Santa Cruz Biotechnology, Santa Cruz, CA). Incubate overnight at 4° C. After incubation time, two washes are performed and incubated for 1 h at room temperature with peroxidase-conjugated secondary antibody. Finally, the proteins are detected by ECL (enhanced chemiluminescence) (Bonnus, Amersham, Little Chalfont, UK) (Ortíz et al. 2009).

[0179] Once the Western Blot was performed (see FIG. 14), the bands obtained in the gels were analyzed using Quantity One Bio-Rad analytical software, confirming that the ethanolic extract of defatted mature seed flour of Euphorbia lathyris produces cell apoptosis mediated by the caspases pathway, being expressed up to four times more than the control (FIG. 15).

[0180] Therefore, we can conclude that the ethanolic extract from defatted seeds produces cell death by apoptosis, both intrinsically and extrinsically.

Example 8 Determination of the Migration Capacity of Tumor Cells

[0181] To determine the migration capacity of tumor cells, and therefore their invasiveness and ability to generate metastasis, an in vitro migration assay was performed. For this purpose, cells of the T84 colon tumor line were seeded in twelve-well plates at 100% confluence. At 24 hours, the gap or “wound” is made manually with a sterile tip. This gap consists of generating, in the well, a cell-free space (gap) in the middle of a cell monolayer and being able to observe and quantify the cell displacement on it (Grada et al, 2017). Once carried out and verified that the gap is free of cells, it is changed to medium without fetal bovine serum so that the cells around the gap stop multiplying and a non-cytotoxic dose of the ethanolic extract from the defatted mature seed flour is added in triplicate. Pictures are taken at different times (0, 8, 24, 24, 48 and 72 hours) to observe cell migration, compared to the control (untreated cells). Images of the results obtained can be seen in FIG. 16.

[0182] To evaluate the effect of the ethanolic extract, the percentage of migration is calculated by measuring the area of the gap still free of tumor cells at the different times at which the images were taken using the Image J software.

[0183] After performing the assay, it can be concluded that, at non-cytotoxic doses of the extract, colon tumor cells migrate significantly less than controls, and therefore, the extract of the present invention, at non-cytotoxic doses, would significantly slow down invasiveness and metastasis formation. In fact, already at 8 hours the slowing down of the migration of tumor cells exposed to non-cytotoxic doses of the extract of the present invention was significantly evident. At 72 hours, with the completion of the experiment, this decrease was greater, decreasing from 79% migration in controls to 60% with the extract, decreasing by about 20% (FIG. 17).

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