Extraction process, products and uses of the extract from the seeds of the Euterpe palm with cytotoxic and antibacterial activity.

Description

1 / 14 Extraction process, products and uses of the extract from the seeds of the Euterpe palm with cytotoxic and antibacterial activity. FIELD OF THE INVENTION

[001] The present invention relates to a process for obtaining an aqueous extract from the seeds of the palm tree of the genus Euterpe. The aqueous extract or powder obtained after drying can be used in the pharmaceutical, cosmetic, phytotherapeutic and food industries due to its bioactive properties. The process minimizes time and energy, and the extract obtained does not require the use of chemical reagents for its production. Therefore, the process and use of the extract can be considered a green technology invention, thus contributing to the bioeconomy and sustainable development. FUNDAMENTALS OF THE INVENTION

[002] The Amazon has a wide variety of fruits that contain bioactive compounds, which have healing, anti-inflammatory, anticarcinogenic, antifungal, and antioxidant properties. Among these fruits is açaí, which comes from the açaí palm, a palm tree (Euterpe oleracea) native to the Amazon region. Phenolic compounds are bioactive compounds and are present in açaí.

[003] The açaí seed is considered a residue after the fruit pulping process, being discarded into the environment, causing environmental impacts. The extract from Euterpe oleracea seeds has aroused the interest of researchers from various areas, due to its peculiarities, such as presenting bioactive characteristics. Açaí seeds may be an important source of compounds with antitumor and immunoprotective properties (MUNIZ FILHO et al. Antitumor effect of açaí (Euterpe oleracea Mart.) Seed extract in LNCaP celle and in the solid ehrlich carcinoma model. Cancers, v. 15, 2544, p. 1-20, April 2023. DOI 10.3390 / cancers15092544). Petition 870250065872, dated 07 / 29 / 2025, page 4 / 37 2 / 14

[004] Tannins are secondary metabolites, being phenolic compounds that are present in the seed of Euterpe oleracea, are water-soluble, anti-inflammatory, and antioxidant, as they act as free radical scavengers. Chemical and spectrometric analyses revealed that the açaí seed extract is predominantly composed of polymeric procyanidins (MONTEIRO et al. An açaí seed extract rich in polyphenols protects against 5-fluorouracil-induced intestinal mucositis in mice via the TLR-4 / MyD88 / PI3K / mTOR / NF-kBp65 signaling pathway. Nutritional Research. V. 125, May, 2024, p. 1-15. https: / / doi.org / 10.1016 / j.nutres.2024.01.017). Studies corroborate that the functional properties of tannin have stimulated research into the development of phytotherapeutic products that can be used in the treatment of diseases, as well as in the pharmaceutical and dye industries, among others.

[005] Considered one of the largest groups of secondary metabolites in the plant kingdom, flavonoids are responsible for protecting plants against pathogens, as well as acting against oxidizing agents. In the early 1990s, with the intense commercial appeal for the consumption of products based on medicinal plants, research on these chemical compounds was intensified (DA SILVA et al. Flavonoides constituição química, ações medicinais e potencial tóxico. Acta toxicol. Argent. Vol.23 no.1 Ciudad Autónoma de Buenos Aires mayo 2015).

[006] Studies have shown that the functional properties of the seed of the palm tree of the genus Euterpe oleracea have stimulated research and investment in studies that prove its functional action, due to its high antioxidant activity. Therefore, it is important to invest in research that seeks to scientifically prove the beneficial action of the seed of Euterpe oleracea and its cultivars, and thus contribute to strengthening phytotherapy in Brazil, since, according to the Ministry of Health, throughout the centuries, products of plant origin have formed the basis for the treatment of different diseases.

[007] According to the National Health Surveillance Agency, for a plant to be considered medicinal it must possess substances that can treat, prevent or cure illnesses, and any medicine produced from a medicinal plant is called Petition 870250065872, dated 07 / 29 / 2025, page 5 / 37 3 / 14 herbal medicine. According to the Agency, knowledge of the correct use of a medicinal plant is important for its proper use and the protection of people's health (Guidelines on the use of herbal medicines and medicinal plants, 2022. National Health Surveillance Agency - ANVISA). BACKGROUND OF THE INVENTION

[008] The following describes the relevant prior art documents relating to the subject matter of the present invention “Extraction Process, Products and Use of the Extract from the Seeds of the Euterpe Palm with Cytotoxic and Antibacterial Activity”.

[009] Document BR1020210042907 relates to the invention of a process for obtaining and producing a precipitating product for chromium metal ions from phenolic compounds present in açaí seeds by pressing. The process for obtaining the precipitating product begins with drying the açaí seeds in an oven to remove moisture. The drying intervals range from 10°C to 100°C for a period of 30 minutes to 2 hours. The açaí seeds are then pressed in a hydraulic press, and the precipitating product is stored for subsequent freeze-drying. The product is used as a precipitating agent for chromium metal ions.

[010] The state of the art teaching cited above does not correspond to the state of the art regarding the “Extraction Process, Products and Use of the Extract from the Seeds of the Euterpe Genus Palm with cytotoxic and antibacterial activity”, because the state of the art teaching describes an extraction process in which it is necessary for the açaí seed to undergo a drying process from 10°C to 100°C for a period of 30 minutes to 2 hours, in order to remove moisture from the açaí seeds.

[011] While the present invention does not require Euterpe seeds to undergo a drying process before pressing, which contributes to green technology and sustainable development, since there is a saving of time and energy when using the extraction process of said invention “Extraction Process, Products and Use of the Extract of Palm Seeds of the Genus Euterpe with cytotoxic and antibacterial activity”, so that the process proposed here has inventive activity of Petition 870250065872, dated 07 / 29 / 2025, page 6 / 37 4 / 14 Green technology compared to the state of the art. Furthermore, there are other technical steps in the process that are fundamental to its differentiation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the harvesting of the fruit of Euterpe oleracea BRS Pará. Figure 2 shows the harvesting of the fruit of Euterpe oleracea BRS Pai D'Égua. Figure 3 shows the harvesting of the fruit of Euterpe oleracea. Figure 4 shows the açaí blending machine and the extracted juice. Figure 5 shows the collection of Euterpe oleracea seeds after depulping. Figure 6 shows the seeds of Euterpe oleracea after washing. Figure 7 shows Euterpe oleracea seeds placed in trays. Figure 8 shows the seeds of Euterpe oleracea in a morphological analysis. Figure 9 shows the seeds of Euterpe oleracea being weighed for pressing. Figure 10 shows the hydraulic press and its parts, used to press the seeds of Euterpe oleracea. Figure 11 shows the aqueous extract being extracted from the seeds of Euterpe oleracea. Figure 12 shows the aqueous extract of Euterpe oleracea seeds after pressing. Figure 13 shows the aqueous extract of Euterpe oleracea BRS Pará seeds after pressing. Figure 14 shows the aqueous extract of Euterpe oleracea BRS Pai D'Égua seeds after pressing. Figure 15 shows the frozen aqueous extract of Euterpe oleracea seed. Figure 16 shows the frozen aqueous extract of Euterpe oleracea BRS Pará seed. Figure 17 shows the frozen aqueous extract of Euterpe oleracea BRS Pai D'Égua seed. Figure 18 shows the freeze-dried extract of Euterpe oleracea seeds. Figure 19 shows the freeze-dried extract of Euterpe oleracea BRS Pará seed. Figure 20 shows the freeze-dried extract of Euterpe oleracea BRS Pai D'Égua seed. Figure 21 presents the results regarding the particle size distribution of the lyophilized extract of Euterpe oleracea seeds. Figure 22 shows the SEM image of the lyophilized extract of Euterpe oleracea. Figure 23 shows the SEM image of the lyophilized extract of Euterpe oleracea BRS Pará. Petition 870250065872, dated 07 / 29 / 2025, p. 7 / 37 5 / 14 Figure 24 shows the SEM image of the lyophilized extract of Euterpe oleracea BRS Pai D'Égua. Figure 25 shows the EDS of the lyophilized extract of Euterpe oleracea. Figure 26 shows the EDS of the lyophilized extract of Euterpe oleracea BRS Pará. Figure 27 shows the EDS of the lyophilized extract of Euterpe oleracea BRS Pai D'Égua. Figure 28 presents the FT-IR results for lyophilized extracts of Euterpe oleracea. Figure 29 presents the XRD results for the lyophilized extract samples of Euterpe oleracea. Figure 30 shows the hydrogen NMR (H1) spectrum of the lyophilized extract of Euterpe oleracea. Figure 31 shows the hydrogen NMR (H1) spectrum of the lyophilized extract of Euterpe oleracea BRS Pará. Figure 32 shows the hydrogen NMR (H1) spectrum of the lyophilized extract of Euterpe oleracea BRS Pai D'Égua. Figure 33 shows the carbon NMR (C13) spectrum of the lyophilized extract of Euterpe oleracea. Figure 34 shows the carbon NMR (C13) spectrum of the lyophilized extract of Euterpe oleracea BRS Pará. Figure 35 shows the carbon NMR (C13) spectrum of the lyophilized extract of Euterpe oleracea BRS Pai D'Égua. Figure 36 presents the results regarding cell viability in different neoplastic cell lines and a non-neoplastic cell line with Euterpe oleracea extract. Figure 37 presents the results regarding cell viability in different neoplastic cell lines and a non-neoplastic cell line with the extract of Euterpe oleracea BRS Pará. Figure 38 shows bacterial growth, which also demonstrated bactericidal effects against strains of S. aureus. DESCRIPTION OF THE INVENTION

[012] The registration of activities related to access to genetic resources was carried out in the National Genetic Heritage System (SisGen), registration: AC6F51B. The fruits of Euterpe oleracea (BRS Pará) were collected in the municipality of Barcarena-PA, coordinates S1°38'51” W48°34'05”, on January 12, 2022 (Figure 1). Petition 870250065872, dated 07 / 29 / 2025, page 8 / 37 6 / 14 oleracea (BRS Pai D'Égua) were collected in the municipality of Tomé-Açú-PA, coordinates S2°32'41” W 48°21'37”, on June 4, 2022 (Figure 2), the fruits of Euterpe oleracea were collected in the municipality of Barcarena-PA, coordinates S1°35'25” W48°32'07”, on June 22, 2024 (Figure 3).

[013] The açaí fruits were sanitized with a sodium hypochlorite solution at 150 ppm for 15 minutes, after which the fruits were washed in running water to remove excess chlorine odor. The fruits were placed in a tank containing water at a temperature of 80°C to 85°C for 10 seconds, then cold water was added and the temperature dropped to 40°C to 37°C, and left to stand for 20 to 30 minutes.

[014] For pulping, an açaí (Euterpe oleracea) beating machine was used to extract the açaí juice (Figure 4), which was packaged in a plastic bag and frozen. After pulping the fruits, the seeds were collected (Figure 5) to be properly washed in running water. After washing (Figure 6), the seeds were packaged in plastic bags, properly sealed in a sealing machine, and sent to the Chemical Engineering Laboratory-LEQ, located at the Federal University of Pará.

[015] After the seeds arrived at LEQ, they were placed in trays (Figure 7), and a small sample of the seeds was taken to perform the analysis to determine the percentage of moisture and morphological characterization (Figure 8).

[016] The moisture content analysis was performed using an infrared moisture balance (Bel i-thermo G) configured with temperature and drying time parameters of 105°C and automatic mode, respectively. The test was performed on the seeds in triplicate, which presented a moisture content between 11% and 60%.

[017] The Euterpe oleracea seeds, after being weighed (figure 9), were placed inside the cylinder that is part of the hydraulic press (Figure 10).

[018] After pressing (Figure 11) the extracts (Figures 12, 13 and 14) were stored in an amber bottle, weighed, the pH was checked which was in the range of 3.5 to 6.5; and stored in the refrigerator to be sent to the lyophilization process. Petition 870250065872, dated 07 / 29 / 2025, page 9 / 37 7 / 14

[019] The freeze-drying process was carried out in the Supercritical Technology Laboratory (LABTECS) of the PCT Guamá innovation space, at the Federal University of Pará. The extracts were added to stainless steel trays, which were placed in a vertical freezer for freezing at a temperature of -25°C for 24 hours.

[020] After freezing, the trays (Figures 15, 16 and 17) were placed in the freeze dryer (model: LS3000, TERRONI, Brazil) under freeze-drying conditions at a pressure of 100 micro Hg and a condenser temperature of -50°C for 24h.

[021] After lyophilization was complete, the lyophilized extracts (Figures 18, 19 and 20) were weighed and vacuum-packed in plastic bags and stored for later analysis.

[022] The particle size analysis of the 3 (three) lyophilized extracts was performed by sieving, in which a series of sieves with known mesh openings were used. The results can be seen in Figure 21.

[023] The quantification of total phenolic polyphenols was performed using a UV 1800 spectrophotometer (Shimadzu®, Kyoto, Japan). A standard curve of gallic acid was constructed at concentrations of 5 to 75 mg / mL. In a 10 mL volumetric flask, 500 μL of Folin-Ciocalteu reagent and 6 mL of distilled water were added. After 2 min, 2 mL of 20% (w / v) sodium carbonate solution were added. The solution was stirred for 30 seconds and the volume was completed with distilled water. The solutions were left to stand for 2 hours, after which readings were taken using a spectrophotometer at a wavelength of 760 nm. To determine the phenolic content in the sample, the same procedure was repeated with the addition of 100 μL of the sample. The results were expressed in milligrams of gallic acid equivalents Sigma (Steinhein, Germany) per gram of sample (mg Eq GA. g -1) (FUNARI and FERRO 2006). The resulting total polyphenol content can vary from 81.67 ± 1.78 g / g.

[024] Total flavonoid quantification was performed using a UV 1800 spectrophotometer (Shimadzu®, Kyoto, Japan). A quercetin standard curve was constructed at concentrations of 5 to 30 mg / mL. In a 10 mL volumetric flask, the different concentrations of the standard were added, followed by 1 mL of 2.5% (w / v) aluminum chloride solution. The solutions were left to stand for 30 min, then... Petition 870250065872, dated 07 / 29 / 2025, page 10 / 37 On August 14th, readings were taken using a spectrophotometer at a wavelength of 425 nm. To determine the flavonoid content in the sample, 800 μL of the sample was used, along with 1 mL of a 2.5% (w / v) aluminum chloride solution and ethanol (q.s.). The results were expressed in milligrams of rutin per gram of sample (Eq QUER. g⁻¹) (FUNARI and FERRO 2006). The resulting total flavonoid content can vary from 22.51 ± 0.54 g / g.

[025] To determine the total anthocyanins present in the extracts, the pH differential spectrophotometric method was used, based on the method described in AOAC (2005) and adapted by Rogez et al. (2012) for products derived from Euterpe oleracea fruits. The result was an anthocyanin content of 95.66 ± 3.02 mg / mL.

[026] The concentration of condensed tannins in the extract was determined by the reaction of 1% vanillin and 8% hydrochloric acid (HCl). A standard curve was constructed at concentrations of 20 to 100 mL using catechin as a standard. In 25 mL test tubes, 5 mL of the 1:1 vanillin-HCl mixture was added (in triplicate). The tubes were preheated in a water bath at 30 °C for 30 min. Then, the different concentrations of catechin were added and vortexed for 30 seconds. The reaction was maintained at 30 °C for 30 minutes. Readings were taken at 500 nm within a maximum of 1 hour. For the determination of tannins in the samples, the same procedure above was repeated with the addition of 1 mL of the sample. The same procedure was repeated for the blank. The results were expressed in micrograms of catechin per gram of extract. The resulting condensed tannin content can vary from 89.81 ± 1.02 g / g.

[027] The determination of antioxidant activity by scavenging the ABTS free radical was performed using a spectrophotometer. The ABTS radical was prepared by reacting 5 mL of ABTS stock solution (7 mM) with 88 μL of potassium persulfate solution (140 mM) and kept at room temperature in the dark for 16 hours. After the required time, 1 mL of the mixture was diluted in ethyl alcohol until an absorbance of 0.7 nm ± 0.05 nm was obtained at 734 nm using a Shimadzu UV 1800 spectrometer (Shimadzu® 1800, Kyoto, Japan). Subsequently, a trolox standard curve (2,000 μM) was constructed with concentrations ranging from 100 μM to 2,000 μM. In a dark environment, a 30 μL aliquot of each trolox solution (100 μM, 500 μM, 1000 μM, 1500 μM, and 2000 μM) was transferred to test tubes and mixed with 3.0 mL of the ABTS radical solution. Then, Petition 870250065872, dated 07 / 29 / 2025, page 11 / 37 9 / 14 was homogenized in a vortex mixer and after 6 min of mixing, readings were taken at 734 nm. Ethyl alcohol was used as a blank to calibrate the spectrophotometer. To determine the antioxidant activity in the sample, the same procedure above was repeated with the dilution of the oil (RE et al., 1999 adapted by RUFINO et al., 2007). The percentage of inhibition was calculated. The analysis was performed in triplicate and the results were expressed in μmol trolox / g of sample. The resulting antioxidant activity can vary from 48.84 ± 2.22% inhibition by the ABTS method with a concentration of 1290.45 ± 57.03 μmol of Trolox / g.

[028] Antioxidant activity by scavenging the DPPH free radical was performed using a UV 1800 spectrophotometer (Shimadzu ®, Kyoto, Japan) at a wavelength of 515 nm. The DPPH radical solution was prepared from 24 mg of DPPH in 100 mL of ethanol. Then, 10 mL of the solution was removed and transferred to a 100 mL volumetric flask and the volume was completed with ethanol to obtain the working solution. In a dark environment, a 150 μE aliquot of the sample was transferred to test tubes, mixed with 5.085 mL of the DPPH radical from the working solution, and homogenized by vortexing.

[029] After 30 minutes of reaction, the reading was performed using a spectrophotometer. Ethyl alcohol was used as a blank to calibrate the spectrophotometer. The antioxidant activity was calculated based on a Trolox standard curve (50 μM-1000 μM). The percentage of inhibition was calculated and the final concentration expressed in μM Trolox (TE) / g (SILVERA et al., 2018). The antioxidant activity had a result that can vary from 82.94 ± 0.29% in DPPH radical inhibition.

[030] Antioxidant activity by the ferric reduction assay (FRAP) was performed on a UV 1800 spectrophotometer (Shimadzu®, Kyoto, Japan) at a wavelength of 595 nm. The FRAP reagent was obtained from the combination of 100 mL of 0.3 M acetate buffer, 10 mL of a 10 mM TPTZ solution, and 10 mL of a 20 mM aqueous ferric chloride solution, used immediately after preparation. In a dark environment, a 90 μL aliquot of the phenolic compound extraction solution was transferred to test tubes, 270 μL of distilled water and 2.7 mL of the FRAP reagent were added, the mixture was vortexed, and incubated in a water bath at 37 °C for 30 minutes. In Petition 870250065872, dated 07 / 29 / 2025, page 12 / 37 10 / 14 followed by a reading at 595 nm. The FRAP reagent was used as a blank to calibrate the spectrophotometer. Antioxidant activity was calculated based on a Trolox standard curve (160 μM-1600 μM). The final concentration was expressed in μM Trolox (TE) / g (BENZIE and STRAIN, 1996; RUFINO et al., 2006). The resulting antioxidant activity was 982,000 μM Trolox (TE) / g using the FRAP method.

[031] The antioxidant potential by the Phosphomolybdenum complex method (ATT %) was performed with 100 μL of the sample solution (1 mg / mL) diluted in ethanol and mixed with 1000 μL of reagent solution (sulfuric acid 600 mM, sodium phosphate 28 mM and ammonium molybdate 4 mM) in test tubes.

[032] The tubes were wrapped in aluminum foil and placed in a water bath at 90°C for 90 min. After this period, the absorbance was measured using a UV 1800 spectrophotometer (Shimadzu ®, Kyoto, Japan) at a wavelength of 695 nm.

[033] As an analytical blank, 1000 μL of reagent and 100 μL of ethanol were used. The total antioxidant activity was expressed relative to ascorbic acid 1 mg / mL and calculated (PRIETO; PINEDA; AGUILAR, 1999) with adaptations). The result was an antioxidant activity that can vary from 68.94 ± 5.01 mg of ascorbic acid / mL by the phosphomolybdenum complex reduction method.

[034] The lyophilized extracts were analyzed using Scanning Electron Microscopy (SEM-EDS). The images (Figures 22, 23, 24, 25, 26 and 27) were obtained at the Institutional Scanning Electron Microscopy Laboratory of the Museu Paraense Emílio Goeldi, using a Tescan Mira3 electron microscope with a FEG-type electron gun. The samples were mounted on 12 mm diameter aluminum supports using double-sided carbon adhesive tape. Then, in order to become conductive, they were metallized with Au for 2' 30'', which deposits a film with an average thickness of 10 to 15 nm on the sample. The images were generated by secondary electron (SE) detection, using a voltage acceleration of 5 kV and working distances around 15 mm.

[035] Infrared Spectroscopy (FT-IR) was performed at the Vibrational and High Pressure Spectroscopy Laboratory of the Federal University of Pará. using the Petition 870250065872, dated 07 / 29 / 2025, page 13 / 37 11 / 14 BRUKER brand equipment, VERTEX 70v model. The spectra (Figure 28) showed broad peaks, which were observed at approximately 3207-3213 cm⁻¹, corresponding to the axial deformation of the hydroxyl group (OH), typical of polysaccharides, mainly from the intra- and intermolecular hydrogen bonds of cellulose.

[036] The bands at 1517-1519 and 1602 cm-1 present in all diagrams correspond to the vibrations and elongations of the C=C groups in the aromatic ring of lignin. While near 1438 cm-1 and from 1359 to 1371 cm-1 the deformations of the CH bonds of lignin are observed.

[037] The bands at 1278-1280 cm⁻¹ correspond to the acetyl functional groups (COR) of hemicelluloses. The bands that appear at approximately 1055-1056 cm⁻¹ correspond to the axial deformation of the CO and COC bonds.

[038] The absorption region of aromatic nuclei can be observed at 763 cm⁻¹, the strong bands in this region indicate the presence of the out-of-plane angular vibration of CH₂ groups (POPESCU, MC; POPESCU, CM; LISA, G.; SAKATA, Y. Evaluation of morphological and chemical aspects of different wood species by spectroscopy and thermal methods. Journal of molecular structure, v. 988, n. 1-3, p. 65-72, 2011.; NETO, ARS; ARAUJO, MA; SOUZA, FV; MATTOSO, LH; MARCONCINI, JM. Characterization and comparative evaluation of thermal, structural, chemical, mechanical and morphological properties of six pineapple leaf fiber varieties for use in composites. Industrial Crops and Products, v. 43, p. 529-537, 2013.).

[039] XRD analyses (Figure 29) were performed on a PANalytical Empyrean X-ray Diffractometer (XRD), ceramic X-ray tubes with Co anode (Ka1= 1.789010 Å), long fine focus, Fe β filter, PIXCEL3D-Medpix3 1x1 detector, in scanning mode, with a voltage of 40 kV, current of 35 mA. Scanning angle 2° to 75° 2Θ, step size 0.026° in 2Θ and time / step of 31 s. Divergent slit: 1 / 4° and antiscatter: 1 / 2°, mask: 10 mm.

[040] For Nuclear Magnetic Resonance analysis (Figures 30, 31, 32, 33, 34 and 35) the lyophilized extract samples were prepared using 20mg of the extract Petition 870250065872, dated 07 / 29 / 2025, page 14 / 37 12 / 14 solubilized in 600μL of deuterated dimethyl sulfoxide (DMSO-De). The Ή and 13C spectra were obtained on a Bruker Ascend™ 400MHz spectrometer.

[041] TopSpin 3.6.2 software was used for data control and processing, with manual calibration through suppression of the residual H2O signal, and adjustment of the Ή enC spectra by the residual solvent signal, DMSO-H6, respectively, at 2.49 ppm and 39.5 ppm (PAVIA, et al., 2010).

[042] The lyophilized extract of Euterpe oleracea and Euterpe oleracea BRS Pará were evaluated for four tumor cell lines (Figures 36 and 37) AGP-01 (Gastric ascites), SK-MEL 19 (Human melanoma), AHOL (Human secondary glioblastoma) and the non-neoplastic cell line, HEK-293 (Human embryonic kidney). These were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin), maintained in an incubator at 37°C and an atmosphere containing 5% CO2.

[043] The MTT assay was performed: in vitro cytotoxicity. Cells were seeded in 96-well plates at a concentration of 3 x 10³ cells / well. The two extracts of the species Euterpe oleracea were dissolved in dimethyl sulfoxide (DMSO) to obtain initial concentrations of 29 mg / mL and 21.9 mg / mL, respectively, and added to the plate. A dose-response assay was then performed with a concentration curve ranging from 300 - 4.68 μg / mL. After a treatment period of 72 hours in a 5% CO₂ incubator, the plates were removed and the supernatant was aspirated, followed by the addition of 100 μL of MTT solution at a concentration of 0.5 mg / mL diluted in DMEM medium and incubated in a 5% CO₂ incubator for 3 hours. Next, the supernatant was aspirated and the precipitate was resuspended in 100 μL of DMSO and stirred for 10 minutes, until complete dissolution of the formazan crystals. The plates were analyzed in a plate spectrophotometer at a wavelength of 570 nm.

[044] The average concentration of the extracts capable of producing 50% of the maximum effect (IC50) and its respective confidence interval (IC95%) for each cell line was obtained from non-linear regression in the GraphPad Prism version 8.0 program. The activity of Euterpe oleracea extracts on cell viability was obtained from the percentages relative to the negative control. Where Absexp and Absctr account for the absorbance at 570 nm for the experimental and control samples, respectively. To verify differences Petition 870250065872, dated 07 / 29 / 2025, page 15 / 37 13 / 14 between the experimental groups, the ANOVA test (two-way) was performed, followed by the Bonferroni post-hoc test, with significance levels p > 0.005 (*), p > 0.001 (**) and p > 0.0001 (***).

[045] The results showed cytotoxic activity, with a decrease in cell viability in all cell lines tested. The lyophilized extract of Euterpe oleracea showed IC50 values ​​around 160 μg / mL on gastric cancer cells (AGP01), glioblastoma (AHOL1) and melanoma (SK-MEL 19). However, in the non-neoplastic kidney cell line (HEK-293) the IC50 was 339 μg / mL, twice as high as in tumor cells, demonstrating a certain selectivity. Of all tumor cell models tested, the Euterpe oleracea extract showed better activity on melanoma (SK-MEL 19), with a significant reduction in viability from 75 μg / mL. While in the other cells, statistical significance was only achieved from a concentration of 150 μg / mL.Thus, through the results we observed that the Euterpe oleracea extract showed concentration-dependent cytotoxic activity on gastric cancer (AGP01), glioblastoma (AHOL) and melanoma (SK-MEL 19) cell lines, but without causing damage to non-tumor cells (HEK-293), in the same concentration range.

[046] The results also showed that the Euterpe oleracea BRS Pará extract, similarly to the Euterpe oleracea extract, caused cytotoxicity on all cell lines tested. Especially in melanoma cell lines (SK-MEL 19) in which it demonstrated greater cytotoxic potential, followed by glioblastoma cell lines (A-HOL) and gastric cancer cell lines (AGP01). It is worth noting that the Euterpe oleracea BRS Pará extract maintained the cytotoxic pattern against the non-neoplastic cell line HEK-293, and did not show damage to the cell line when used at the concentrations that were cytotoxic in the neoplastic cell lines.

[047] The antibacterial activity was evaluated with different concentrations of Euterpe oleracea extracts against strains of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In E. coli strains, only one concentration of the Euterpe oleracea BRS Pará extract was able to inhibit growth by 82%. In the evaluation of P. aeruginosa strains, the two highest concentrations of the three extracts showed inhibition between 90 and 100%. Petition 870250065872, dated 07 / 29 / 2025, p. 16 / 37 14 / 14

[048] Regarding the S. aureus strain, three concentrations of the Euterpe oleracea BRS Pai D'Égua extract inhibited growth between 90 and 100%; two concentrations of the Euterpe oleracea BRS Pará extract showed inhibition between 70 and 100%, and regarding the Euterpe oleracea extract, four concentrations showed inhibition between 80 and 100%. The extracts showed a bacteriostatic effect only against the P. aeruginosa and S. aureus strains. After interaction of the extract for 24 h with the concentrations in which bacterial growth occurred, they also showed a bactericidal effect against S. aureus strains (Figure 38).

[049] The present invention is important because it presents a process that minimizes energy consumption, reduces the time to obtain the extract, eliminates the need for chemical reagents for its production, and has bioactive properties, thus contributing to the production of plant-based products for the treatment of various diseases. It can be considered a green technology invention, which contributes to the bioeconomy and sustainable development. Petition 870250065872, dated 07 / 29 / 2025, page 17 / 37

Claims

1 / 2 CLAIMS 1. PROCESS FOR OBTAINING EXTRACTS WITH ANTIOXIDANT, CYTOTOXIC AND ANTIBACTERIAL ACTIVITIES FROM THE SEEDS OF THE PALM TREE OF THE GENUS EUTERPE characterized by step 1 - sanitizing the açaí fruits with a sodium hypochlorite solution at 150 ppm for 15 minutes; step 2 - washing the açaí fruits sanitized in step 1 with running water to remove excess chlorine odor; step 3 - transferring the açaí fruits washed in step 2 to a tank containing water at a temperature of 80°C to 85°C for 10 seconds, then adding cold water and lowering the temperature to 40°C to 37°C, leaving it to rest for 20 to 30 minutes; Step 4 - Depulping the açaí fruits from step 3 in an açaí blending machine to extract the açaí juice, which is then packaged in a plastic bag and frozen, and obtaining the depulped seeds, which are collected to be properly washed in running water;Step 5 - The seeds collected and washed in Step 4, with a moisture content ranging from 11% to 60%, are pressed in appropriate extraction equipment to obtain the aqueous extract; Step 6 - The aqueous extract obtained in Step 5 can be stored, weighed, have its pH checked, and / or be frozen to be sent to the freeze-drying process; Step 7 - The extract frozen in Step 6 is freeze-dried until a powdered material is obtained with a moisture content ranging from 1% to 10%; Step 8 - The powdered material obtained in Step 7 is vacuum-packed in plastic bags and preserved at room temperature or under refrigeration.

2. AQUEOUS EXTRACT WITH ANTIOXIDANT, CYTOTOXIC AND ANTIBACTERIAL ACTIVITIES, derived from Açaí seeds obtained from step 5 of claim 1, characterized by having a pH in the range of 3.5 to 6.5; the presence of chlorine in its composition; having total solids obtained by lyophilization that have antioxidant activity; having total solids obtained by lyophilization that have cytotoxic activity against gastric cancer cells (AGP01), glioblastoma (AHOL1) and melanoma (SK-MEL 19); having total solids obtained by lyophilization that have antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Petition 870250001227, dated 07 / 01 / 2025, page 22 / 57 2 / 2 3. PRODUCT in powder form derived from the aqueous extract with antioxidant, cytotoxic and antibacterial activities of Açaí seeds, described in claim 2 and obtained from step 7 of claim 1, characterized by having a moisture content that can vary from 1% to 10%; presence of chlorine in its composition; total polyphenol content that can vary from 81.67 ± 1.78 μg / g; total flavonoid content that can vary from 22.51 ± 0.54 μg of Rutin / g; anthocyanin content that can vary from 95.66 ± 3.02 mg / mL; condensed tannin content that can vary from 89.81 ± 1.02 μg / g; antioxidant activity that can vary from 82.94 ± 0.29% in DPPH radical inhibition; Antioxidant activity that can vary from 48.84 ± 2.22% inhibition by the ABTS method with a concentration of 1290.45 ± 57.03 μmol of Trolox / g; antioxidant activity that can vary from 68.94 ± 5.01 mg of ascorbic acid / mL by the phosphomolybdenum complex reduction method;Antioxidant activity of 982,000 μM Trolox (TE) / g by the FRAP method; cytotoxic activity against gastric cancer cells (AGP01), glioblastoma (AHOL1) and melanoma (SK-MEL 19); antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

4. USE of the product in powder form, obtained from step 7 of claim 1 and described in claim 3, characterized by being used as an antioxidant agent; cytotoxic agent against gastric cancer cells (AGP01), glioblastoma (AHOL1) and melanoma (SK-MEL 19); antibacterial agent with bacteriostatic potential against Pseudomonas aeruginosa and Staphylococcus aureus, and bactericidal against Staphylococcus aureus. Petition 870250001227, dated 07 / 01 / 2025, page 23 / 57

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