Solution containing Schinus lentiscifolius Marchand extract as an inhibitor of cell proliferation and mechanisms of resistance to multiple drugs.

Description

1 / 14 SOLUTION CONTAINING EXTRACT OF Schinus lentiscifolius Marchand AS AN INHIBITOR OF CELL PROLIFERATION AND MECHANISMS OF RESISTANCE TO MULTIPLE DRUGS Field of invention

[001] The present invention aims to demonstrate and protect the use of the ethanolic extract of Schinus lentiscifolius Marchand resins and extracts of the fractions with hexane and ethyl acetate solvents, as sources of molecules with cytotoxic and / or cytostatic effects, for the treatment of diseases caused by excessive cell proliferation, such as neoplastic, inflammatory or other diseases, and also inhibitory molecules of the ABCB1 membrane protein to prevent resistance to treatment processes in cells exhibiting the MDR phenotype. Fundamentals of the invention

[002] Natural products are increasingly involved in numerous research projects, with the aim of finding bioactive molecules that show promising results, mainly for medicinal purposes. This is because the wide biodiversity of plants allows for new discoveries of bioactive compounds present in different vegetation types.

[003] The Anacardiaceae family includes the genus Schinus, which has 28 recognized species. Of these, 5 species of pepper trees are found in the state of Rio Grande do Sul, among them Schinus lentiscifolius Marchand, popularly known as gray pepper tree, which has grayish foliage. These are found in tropical and subtropical regions, native to Argentina, Paraguay, Uruguay and Brazil, especially in the southern region.

[004] Plant resins are substances produced by resinous plants that contain various chemical compounds in their composition. They develop a characteristic aroma, with a texture that varies from viscous and / or sticky and colors ranging from translucent to brighter and more colorful. These aspects vary according to the species, environment, surfaces, and atmospheric conditions.

[005] In view of this, resin extracts can be sources of biologically active components with great biotechnological and pharmacological potential for different areas of science. Petition 870250075394, dated 08 / 26 / 2025, page 4 / 19 2 / 14

[006] Some studies have shown that extracts obtained from the maceration of gray pepper tree leaves have been identified as containing phenolic compounds and flavonoids, such as quercetin and rutin, gallic acid, mono- and sesquiterpenes. In the same research, the extract made with ethyl acetate solvent showed the presence of moronic acid and triterpenes. Similarly, another study also found phenolic compounds and flavonoids in the aceto-ethyl extract obtained from the bark of this pepper tree. It is understood that extracts produced from other plant parts (stems, roots, fruits, leaves) can present a range of different compounds, and the chosen extraction method can influence the obtaining of these substances.

[007] In the literature there are still no descriptive reports on the spectrum of biological activities of extracts from the Brazilian peppertree Schinus lentiscifolius Marchand. It is understood that other species of Brazilian peppertrees such as Schinus terebinthifolius Raddi may present antimicrobial, anti-inflammatory, antioxidant, anti-adherent, anti-allergic and healing characteristics. Similarly, Schinus molle Linnaeus has antioxidant and antimicrobial activity, and cytotoxic activity has also been observed in some cell lines.

[008] Due to the limited knowledge about the gray pepper tree and its production of resins rich in compounds, the search for new information is essential. We emphasize the importance of the innovation of the present invention for medicinal purposes, in the possible treatment of neoplastic and inflammatory diseases, inhibitory activity of the ABCB1 membrane protein and / or others.

[009] To date, some products developed from the plant Schinus lentiscifolius Marchand have patent registrations, which are related to pharmaceutical compositions and uses, as well as cosmetic formulations (US20130302419A1 / US20170007652A1 / WO2021173972A1). However, there is still no development of products prepared from active ingredients, nor extracts produced from vegetative parts.

[010] In Brazil, no patents have yet been filed using the aroeira Schinus lentiscifolius Marchand. And to date, no patents have been found related to antiproliferative activity and / or chemotherapeutic or cytotoxic action against different neoplastic cell lines when treated with extracts of the gray aroeira or extracts of its resin. Petition 870250075394, dated 08 / 26 / 2025, page 5 / 19 3 / 14

[011] Consequently, these data reinforce the need and importance of further investigations using extracts with the substrate of Schinus lentiscifolius Marchand, especially the resin of this plant in the models mentioned above.

[012] The innovation of the present invention relates to the use of the ethanolic extract of Schinus lentiscifolius Marchand resin and / or the two fractions produced from it, the hexane and ethyl acetate extracts, as suppliers of molecules with antiproliferative, cytotoxic and / or cytostatic activity and inhibitors of ABCB1 protein activity. Brief description of the drawings

[013] Figure 1 illustrates the extracts prepared from the resin of the Brazilian pepper tree Schinus lentiscifolius Marchand.

[014] Figure 2 shows in the table the yield of the production of ethanolic extracts and sequences with hexane and ethyl acetate.

[015] Figure 3 presents the data for the ethanolic and sequential extracts with hexane and ethyl acetate for their preparation used in the in vitro biological assays. The production of the extracts from the Grey Pepper Tree resin used in the biological assays can be better understood through the protocol summarized in values ​​in the table.

[016] Figure 4 shows the yield of the Ethanolic and sequential extracts with Hexane and Ethyl Acetate obtained after extraction of the chemical compounds using the solid-liquid method.

[017] Figure 5 presents the data from the Cellular Metabolism analysis by the MTT colorimetric assay. A) Results obtained from the MTT assay of the Ethanolic Extract (EE-AC); B) Results obtained from the MTT assay of the Hexane Extract (EH-AC); C) Results obtained from the MTT assay of the Aceto-Ethyl Extract (EAE-AC). Cells from the oral squamous cell carcinoma cell line - SCC9 were treated with concentrations of 100μg / ml - 50μg / ml - 25μg / ml over a 24-hour period. The treated groups were statistically compared with the negative control. The data obtained were statistically calculated by two-way ANOVA followed by Tukey's test, where “.” p < 0.1,* p < 0.05, ** p <0.01 and *** p <0.001 (n = 4).

[018] Figure 6 illustrates the Cell Cycle analysis of the SCC-9 cell line. A) Results obtained from the Ethanolic Extract Cell Cycle assay (EE-AC); B) Results obtained from the assay of Petition 870250075394, dated 08 / 26 / 2025, page 6 / 19 4 / 14 Cell cycle of Aceto-Ethyl Extract (EAE-AC); Cells from the oral squamous cell carcinoma cell line - SCC9 were treated with ethanolic and aceto-ethyl extracts at a concentration of 50μg / ml over a 24-hour period and analyzed by flow cytometry. Treated groups were statistically compared with the negative control. The data obtained were statistically analyzed by two-way ANOVA followed by Tukey's test, where “.” p < 0.1, * p < 0.05, ** p < 0.01 and *** p < 0.001 (n = 4).

[019] Figure 7 presents the data obtained from the Cellular Metabolism analysis by the XTT colorimetric assay. A) Results obtained from the XTT assay of the Ethanolic Extract (EE-AC); B) Results obtained from the XTT assay of the Hexane Extract (EH-AC); C) Results obtained from the XTT assay of the Aceto-Ethyl Extract (EAE-AC). K562 cell line cells were treated with concentrations of 100μg / ml - 85μg / ml - 70μg / ml - 55μg / ml - 40μg / ml - 25μg / ml over a 24-hour period. The treated groups were statistically compared with the negative control. The data obtained were statistically analyzed using two-way ANOVA followed by Tukey's test, where “.” p < 0.1, * p < 0.05, ** p <0.01 and *** p <0.001 (n = 4).

[020] Figure 8 illustrates the results obtained through Cellular Metabolism Analysis by the XTT colorimetric assay. A) Results obtained from the XTT assay of the Ethanolic Extract (EE-AC); B) Results obtained from the XTT assay of the Hexane Extract (EH-AC); C) Results obtained from the XTT assay of the Aceto-Ethyl Extract (EAE-AC). Resistant cells of the FEPS cell line were treated with concentrations of 100μg / ml - 85μg / ml, 70μg / ml - 55μg / ml - 40μg / ml - 25μg / ml over a 24-hour period. The treated groups were statistically compared with the negative control. The data obtained were statistically analyzed using two-way ANOVA followed by Tukey's test, where “.” p < 0.1, * p < 0.05, ** p <0.01 and *** p <0.001 (n = 4).

[021] Figure 9 presents the analysis of cell death by the Annexin V FITC-Propidium Iodide assay. The K562 and FEPS cell lines were treated with extracts of Aroeira Cinzenta resins and analyzed by discriminating live, dead, and apoptotic cells. For comparisons, untreated cells were designated as negative controls, DMSO was used as a vehicle, and nigericin as a positive control. A) Ethanolic Extract (EE-AC) treated in the K562 cell line at a concentration of 100μg / ml at 6h-16h-24h intervals. B) Hexanic Extract (EH-AC) treated in the K562 cell line at a final concentration of 100μg / ml at 6h-16h intervals. C) Acetoethyl extract (EAE-AC) treated in the K562 strain at a concentration of 100 μg / ml at 6h-16h-24h intervals. D) Ethanolic extract (EE-AC) treated in the strain Petition 870250075394, dated 08 / 26 / 2025, page 7 / 19 5 / 14 E) Hexane extract (EHAC) treated in the FEPS strain at a concentration of 100μg / ml at 6h-16h-24h intervals. F) Acetoethyl extract (EAE-AC) treated in the FEPS strain at a concentration of 100μg / ml at 6h-16h-24h intervals. Data were statistically analyzed by two-way ANOVA followed by Tukey's test, “.” p < 0.1, * p < 0.05, ** p < 0.01 and *** p < 0.001 (n = 4)

[022] Figure 10 shows microscopic images of cells treated with extracts of gray pepper tree labeled with propidium iodide. A) Untreated K562 cell line (negative control) and treated over a 1-hour period with 100 μg / mL of ethanolic (EEAC), hexane (EH-AC), and acetoethyl (EAE-AC) extracts, visualized under bright light, or by fluorescence excited with green light and observed with a red filter. B) Untreated FEPS cell line (negative control) and treated over a 2-hour period with 100 μg / mL of ethanolic (EE-AC), hexane (EH-AC), and acetoethyl (EAE-AC) extracts, visualized under bright light and by fluorescence excited with green light and observed with a red filter.

[023] Figure 11 presents the data obtained from pyroptosis cell death assays using the Caspase-1 inhibitor Ac-YVAD-cmk in K562 and FEPS cell lines treated with extracts of gray pepper trees. A) K562 cell line treated with gray pepper tree extracts, ethanolic (EE-AC), hexane (EH-AC) and acetoethyl (EAE-AC) at concentrations of 100 μg / ml over a 1-hour interval. B) FEPS cell line treated with gray pepper tree extracts, ethanolic (EE-AC), hexane (EH-AC) and acetoethyl (EAE-AC) at concentrations of 100 μg / ml over a 2-hour interval. For statistical comparison, positive controls (cells with extracts) were compared with the negative control, while cells treated with the blocker together with the extracts were compared with the positive controls. The data were statistically analyzed by two-way ANOVA followed by Tukey's test, “.” p < 0.1, * p < 0.05, ** p < 0.01 and *** p < 0.001, 'a' p < 0.001 'b' 0.01 'c' 0.05 '.' 0.1 ' ' 1 (n = 4).

[024] Figure 12 illustrates the analysis of iron involvement in pyroptosis cell death. Cells from the K562 and FEPS cell lines were co-treated with the iron chelator Deferoxamine (DFO) and extracts of gray pepper trees. A) The K562 cell line treated with gray pepper tree extracts, ethanolic (EE-AC), hexanic (EH-AC) and acetoethyl (EAE-AC) at concentrations of 100 μg / ml over a 1-hour period. B) The FEPS cell line treated with gray pepper tree extracts, ethanolic (EE-AC), hexanic (EH-AC) and acetoethyl (EAE-AC) at concentrations of 100 μg / ml over a 2-hour period. For statistical comparison, the positive controls (cells with extracts) were compared with the negative control (cells without extracts). Petition 870250075394, dated 08 / 26 / 2025, page 8 / 19 6 / 14 extracts), while cells treated with the blocker and extracts were compared with positive controls. The data obtained were statistically analyzed by two-way ANOVA followed by Tukey's test, where “.” p < 0.1, * p < 0.05, ** p < 0.01 and *** p < 0.001, 'a' p < 0.001 'b' 0.01 'c' 0.05 '.' 0.1 ' ' 1(n = 4).

[025] Figure 13 presents the results of blocking ABCB1 protein activity by extracts of gray pepper trees resin using the Rhodamine 123 dye extrusion assay. A) Controls: K562, K562+Rhodamine, K562+Rhodamine+Verapamil; B) Controls: FEPS+Rhodamine, FEPS+DMSO, FEPS+Rhodamine+Verapamil; C) Treatment with ethanolic extract of gray pepper tree (EE-AC); D) Treatment with hexane fraction of gray pepper tree (EH-AC); E) Treatment with acetoethyl fraction of gray pepper tree (EAE-AC); FEPS cell line cells were treated with extracts or their fractions at a concentration of 100μg / ml for 30 minutes to analyze the blocking of the ABCB1 membrane transporter protein. The experiment was performed in triplicate, along with controls for each treatment (with / without Rhodamine 123 dye and with / without verapamil). Description of the invention

[026] The extracts were produced from resins of the Aroeira Schinus lentiscifolius Marchand, in the municipality of São Gabriel, in the state of Rio Grande do Sul, Brazil.

[027] For the extraction of chemical compounds from the resin, solutions were produced from different solvents with the purpose of separating the molecules present by chemical affinity. Using the method also known as solid-liquid partition, in which highly nonpolar compounds are concentrated in the hexane solvent and less nonpolar compounds in the ethyl acetate solvent. Obtaining the ethanolic extract of Schinus lentiscifolius Marchand resin and the sequential extracts, hexane and ethyl acetate, shown in figure 1.

[028] The ethanolic extract of gray pepper tree resin was produced from 2.28g of this material, in which the resin of Schinus lentiscifolius Marchand (gray pepper tree) was diluted with 99.8% PA ethyl alcohol in a 10% mass / volume ratio and incubated for 24 hours with periodic agitation. After centrifugation, the supernatant (extract) was removed and stored in another container under refrigeration (2°C-8°C). The same process was repeated four more times, as illustrated in Figure 2. Petition 870250075394, dated 08 / 26 / 2025, page 9 / 19 7 / 14

[029] The extract was prepared for use in in vitro biological assays, in which 3 mL corresponding to 100 μg / mL of the ethanolic extracts were dried by vacuum rotary evaporation at 45°C (Eppendorf Concentrator Plus) and subsequently diluted in 99.9% Dimethyl Sulfoxide (DMSO) at a concentration of 10% mass / volume. The material (resin residue) not diluted with ethyl alcohol was dried in an oven at 40°C for a period of 48 hours, methodology shown in Figure 2.

[030] To prepare the hexane fraction, after drying, the material (resin residue) was diluted in hexane at a 10% mass / volume ratio following the same methodology applied to the ethanolic extract (Figure 2); performing three dilutions with the solvent. The resin from the dilutions was again placed in an oven at 40°C for a period of 48 hours to dry it. For biological analyses, before drying, 3 mL (corresponding to 100 μg / mL) of the hexane fraction were taken for drying by rotary evaporation at 45°C, and the dried extract was weighed, diluted in DMSO (10% concentration), and stored under refrigeration at -20°C until use.

[031] Following the same protocol (figure 2), the residue from the hexane extraction underwent two dilutions with 99.9% ethyl acetate PA solvent. The extract was evaporated, weighed, and diluted in DMSO (10% mass / volume), in which it was kept refrigerated at -20°C until use. This extraction produced no residue.

[032] The squamous cell carcinoma of the tongue cell line - SCC-9, used as a biological unit, was maintained in DMEM F12 medium with 1.2 g / L of Sodium Bicarbonate, supplemented with 10% fetal bovine serum and 1% antibiotic and antifungal (Gentamicin Sulfate and Amphotericin B, respectively), maintained in an incubator at 37°C and 5% CO2, for cell exchange and / or replication the culture medium was renewed every 3-4 days, when reaching 80%-100% confluence.

[033] The K562 (non-MDR) and FEPS (MDR) erythroleukemia cell lines are hematopoietic model cell lines of Chronic Myeloid Leukemia (CML), also used as biological units. They were cultured in RPMI 1640 cell medium with 2 g / L of sodium bicarbonate, supplemented with 10% fetal bovine serum and 1% antibiotic and antifungal (gentamicin sulfate and amphotericin B, respectively), maintained in an incubator at 37°C and 5% CO2. The FEPS cell line was treated with the chemotherapeutic agent daunorubicin (466 μM), used for the selection of these cells and maintenance of the MDR phenotype. The exchange and / or Petition 870250075394, dated 08 / 26 / 2025, page 10 / 19 8 / 14 cell replicates of the culture medium were replaced every 2-3 days for the K562 cell line and every 3-4 days for the FEPS cell line.

[034] The analyses tested aim to evaluate biological effects, such as cytotoxic, cytostatic and antiproliferative effects, and for this purpose assays of cellular metabolism, cell morphology by microscopy, cell cycle, proliferation and death were performed. The blocking of the activity of the ABCB1 transport protein, present in cells with multidrug resistance, was also tested.

[035] Thus, four repetitions and three triplicates were performed for the biological assays. The SCC9 cell line (oral carcinoma cell line), K562 cell line (erythroleukemic chronic myeloid leukemia cell line), and FEPS cell line (daunorubicin-resistant erythroleukemic cell line derived from the K562 cell line) were used as biological models. The treated cells were compared with the negative control group. For cell cycle and cell proliferation assays, treatments were analyzed after 24 hours. For cell death analyses, treatments were analyzed between 6:16 and 24 hours. The blockade of ABCB1 protein activity was analyzed 30 minutes after treatment. Verapamil was used as a positive control for the evaluation of ABCB1 protein activity.

[036] The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) evaluates cellular metabolism and cytotoxicity. The squamous cell carcinoma of the tongue cell line - SCC9 was analyzed with four replicates and three technical triplicates. The test was performed in 96-well plates, plating 10⁴ cells / well, with 24-hour treatment at the following concentrations: 100 μg / ml - 50 μg / ml - 25 μg / ml of the ethanolic, hexane, and acetoethyl extracts. For each treatment, a blank was prepared consisting of the culture medium plus the treatment, but without cells, as well as a negative control and the vehicle (100 μg / ml DMSO). Metabolically active cells reduce the substrate with NADH-dependent oxidoreductase enzymes, changing its color, which is quantified by absorbance spectrophotometry in the 560nm range.

[037] The cell cycle of the squamous cell carcinoma of the tongue cell line - SCC9 was analyzed with four repetitions and three technical triplicates in a 24-well plate, with 105 cells plated per well. The treatments for cell cycle evaluation were performed at the IC50 concentration of each extract, obtained from MTT analysis. Thus, the ethanolic and Petition 870250075394, dated 08 / 26 / 2025, page 11 / 19 Nine out of fifteen cents of gray pepper tree resin were tested at a concentration of 50 pg / ml and analyzed after a 24-hour interval. Following the treatment period, all cells were treated with lysis buffer for cell cycle assay, which contains the dye Propidium Iodide-PI (5 pg / ml), and evaluated by flow cytometry, analyzing 10,000 events using the FL3-A vs. FL3-H density plot and histograms in FL3-A.

[038] The XTT assay is based on a colorimetric method, which marks metabolically active cells, but which, in comparison to MTT, produces a soluble compound. The K562 erythroleukemic cell line (non-MDR) and the resistant FEPS cell line (with MDR phenotype) used as a biological model were plated at 10⁴ cells / well in 96-well plates, using four biological units and three technical triplicates. To analyze the experiment, different concentrations of the extracts, ethanolic, hexane and acetoethyl, were prepared, 100 pg / ml; 85 pg / ml; 70 pg / ml; 55 pg / ml; 40 pg / ml and 25 pg / ml, and were compared with the control group and vehicle (DMSO). For each treatment, a blank was performed consisting of the culture medium plus the treatment, but without cells. After the 24-hour treatment interval, the XTT reagent was added and the cells were incubated for an additional 4 hours until absorbance readings were obtained in the 450 and 650 nm ranges.

[039] To analyze cell death, samples were treated with the respective ethanolic, hexane, and acetoethyl extracts, incubated with the Annexin V FITC-PI apoptosis detection kit conjugated to fluorescein isothiocyanate (FITC). The K562 and FEPS biological models were plated at 105 cells / well in 24-well plates, and the gray pepper tree resin extracts were tested at a concentration of 100 pg / ml, evaluating at 6-16-24 hour intervals. The experiments were performed in triplicate, with a negative control, nigericin as a positive control, and a vehicle control (DMSO). After treatment, the samples were labeled with the Annexin V / PI kit following the manufacturer's recommendations and analyzed by flow cytometry, collecting 10,000 events per sample in FL1-H vs. FL3-H density plots.

[040] For the pyroptosis assay, K562 and FEPS cell lines were plated with 105 cells / ml and treated first with the caspase-1 inhibitor Ac-YVAD-cmk (5μM- Sigma) and then treated with the ethanolic extracts or their fractions at a concentration of 100 pg / ml. Untreated cells were used as a negative control, and cells treated with the extracts or fractions, but without the Ac-YVAD-cmk inhibitor, as positive controls. The experiments were incubated at 37°C in a CO2 incubator. The assay was Petition 870250075394, dated 08 / 26 / 2025, page 12 / 19 10 / 14 analyzed at 1-hour intervals for the K562 cell line and 3-hour intervals for the FEPS cell line. After incubation, they were centrifuged and analyzed by PI-labeled flow cytometry, collecting 5,000 events per sample in FL1-H vs. FL3-H density plots.

[041] To evaluate the influence of iron on the pyroptosis process, K562 and FEPS cell lines were plated with 105 cells / ml and treated first with the iron chelator Deferoxamine (50μM) following the manufacturer's recommendations, and then the extracts were treated at concentrations of 100μg / ml. Untreated cells were used as a negative control, and cells treated with the extracts or fractions, but without the chelator, as positive controls. The experiments were incubated at 37°C in a CO2 incubator. The assay was analyzed at 1-hour intervals for the K562 cell line and at 2-hour intervals for the FEPS cell line. After incubation, they were centrifuged and analyzed by PI-labeled flow cytometry, collecting 5,000 events per sample in FL1-H vs. FL3-H density plots.

[042] The ABCB1 transport protein is found in the cell membrane, and one of its characteristics is multidrug resistance (MDR) in hematopoietic and / or neoplastic cells, and for this reason it is widely studied. Rhodamine 123 is one of the most used fluorescent dyes to evaluate the activity of this protein, and the drug Verapamil is used as a blocker of the same. For the ABCB1 protein blocking assay, the K562 cell line was used as a negative control because it did not show ABCB1 protein activity, and its derived cell line FEPS was used as an MDR cell model. For the experiment, cells were plated at 105 cells / ml, treated with a concentration of 100 μg / ml of each extract (ethanolic, hexane, and acetoethyl) of gray pepper resin for an incubation period of 30 minutes, and analyzed by flow cytometry, collecting 10,000 events in FL1-H histogram graphs.As controls, cells labeled and unlabeled with Rhodamine 123, treated or not treated with verapamil, were used.

[043] Using the Solid-liquid methodology, the ethanolic extract of gray pepper tree resin produced 1.55g of material, corresponding to 68% of the resin used initially, as shown in figure 3.

[044] In the sequential extracts (figure 3), the hexane fraction produced 1.07g of material, demonstrating that 69% of the molecules dissolved in the ethanolic extract are highly nonpolar. Petition 870250075394, dated 08 / 26 / 2025, page 13 / 19 11 / 14 In the aceto-ethyl fraction, it was possible to obtain 0.48g of extracted material, corresponding to 31% of the ethanolic extract, demonstrating compounds with less non-polar characteristics.

[045] Thus, extracts were produced with ethanol solvent from gray pepper tree resin, in which, sequentially, extracts with hexane and ethyl acetate solvents were made respectively. It is possible to observe all the data obtained from the production of the ethanolic extracts and the two fractions, hexane and ethyl acetate, in figure 3. Similarly, figure 4 presents the results achieved in the second stage of the extracts, used in the treatment of cell cultures.

[046] MTT colorimetric analysis demonstrated that the SCC9 cell line treated with ethanolic, hexane, and acetoethyl extracts at concentrations of 100μg / ml, 50μg / ml, and 25μg / ml over a 24-hour period showed a decrease in cell culture growth. In the ethanolic extract (EE-AC), illustrated in Figure 5A, it was observed that the dosage of 100μg / ml practically showed a 100% reduction in cell proliferation; similarly, the concentration of 50μg / ml reported only 25% proliferation when compared to the negative control. However, the concentration of 25μg / ml induced an increase in cell proliferation, for which several hypotheses could explain this phenomenon of increased proliferation, but it should be further studied.The hexane extract (EH-AC), shown in Figure 5B, behaved similarly to the ethanolic extract, where dosages of 100 μg / ml and 50 μg / ml showed metabolically active cells with rates of approximately 5% and 25%, respectively. However, cell proliferation at a concentration of 25 μg / ml remained around 75%. The acetoethyl extract in Figure 5C showed a reduction only at the 100 μg / ml dosage, corresponding to 80%. The 50 μg / ml concentration showed no difference compared to the negative control. However, at a concentration of 25 μg / ml, an increase in cell proliferation was observed, a result that should be better understood. The observed data demonstrate that the extracts had a biological effect on the SCC-9 cell line, which may indicate that bioactive molecules interfere with the cellular metabolic process.

[047] The results obtained, in figures 6A-6B, in the cell cycle demonstrated that the buccal squamous cell carcinoma cell line - SCC9 tested over a 24-hour period with ethanolic and acetoethyl extract at a concentration of 50μg / ml did not show blockage or interruption in the cell cycle in the G1-S-G2 phases. These results indicate that in these fractions and in this type of cell line, the concentrations of compounds present are not sufficient for such an effect. Petition 870250075394, dated 08 / 26 / 2025, page 14 / 19 12 / 14

[048] XTT analysis was performed on K562 (non-MDR) and FEPS (MDR) erythroleukemic cell lines over a 24-hour period, with the following concentrations: 100μg / ml, 85μg / ml, 70μg / ml, 55μg / ml, 40μg / ml and 25μg / ml, of the ethanolic (EE-AC), hexane (EH-AC) and acetoethyl (EAE-AC) extracts. The K562 cell line treated with the ethanolic extract, observed in Figure 7A, showed a reduction in metabolic activity, mainly at the higher dosages; it is observed that as the concentrations decrease, the activity of metabolically active cells increases. The hexane extract, illustrated in Figure 7B at concentrations of 100μg / ml, 85μg / ml, and 70μg / ml, exhibited rates of less than 10% of the metabolic activity of untreated cells; in contrast, the dose of 55μg / ml showed a result close to the IC50.Similar to the ethanolic extract, the acetoethyl fraction, observed in Figure 7C, triggered a progressive increase in active cells with decreasing dosages, with the 55 μg / ml concentration corresponding to approximately 50% of the metabolic decrease. These outcomes indicate that the ethanolic extract and its sequential fractions, hexane and acetoethyl, contain chemical compounds capable of gradually influencing cellular activities.

[049] Similarly, the FEPS cell line analyzed by the XTT assay showed a reduction in the metabolic activity of the cells in both fractions, behaving similarly between them, as observed in Figure 8A-BC. All dosages showed a reduction in metabolic activity, except at the concentrations of 25μg / ml. In the ethanolic extract (Figure 8A), the IC50 remained at the concentration of 70μg / ml, while the hexane extract (Figure 8B) was concentrated between the doses of 55μg / ml and 40μg / ml, and finally the acetoethyl fraction (Figure 8C) between the concentrations of 85μg / ml and 70μg / ml. Throughout the results, it is possible to observe that the extract and its fractions, at different concentrations, show an antiproliferative response in both cell lines, being highly efficient in the resistant FEPS cell line, which presents the MDR phenotype.

[050] The effect of treatments with gray pepper tree extracts was tested on the K562 and FEPS cell lines using the Annexin V / PI technique to identify the mechanisms of cell death. For this purpose, both cell lines were tested at a concentration of 100 μg / ml for both the ethanolic extract and the two sequential fractions, hexane and acetoethyl, of gray pepper tree resin, at intervals of 6-16-24 hours. The ethanolic extract of gray pepper tree resin (Schinus lentiscifolius) and its fractions showed a cytotoxic effect on the K562 cell line at all time intervals (Figure 9 A, B and C). In the first 6 hours, the ethanolic extract (Figure 9A) showed a decrease in live cells (50%) and an increase in cells already in cell death (30%). Over time, the number of live cells decreased and Petition 870250075394, dated 08 / 26 / 2025, page 15 / 19 13 / 14 consequently increased the number of cells undergoing apoptosis, where after 24 hours of the experiment, approximately 60% of the cells were apoptotic and 35% were declared dead. The hexane fraction observed in Figure 9B, after 6 hours, showed 30% apoptotic cells and 50% cell death. After 16 hours of analysis, the rate of apoptotic cells increased from 30% to approximately 70%. By the 24-hour period, all cells were dead, losing their integrity in the microscopic analysis, which did not allow for analysis by flow cytometry. The aceto-ethyl fraction, shown in Figure 9C, showed significant apoptosis / cell death in both the 16-hour and 24-hour treatment evaluations, with an increase over the intervals, however. In view of this, the results obtained demonstrate that both extracts show responses when subjected to analysis, and that as the time interval increases, they show a greater quantity of dead cells.Indicating that the chemical groups present in the extracts are involved in cell death pathways.

[051] The assay performed on the FEPS (MDR) erythroleukemic cell line demonstrated that the ethanolic extract, observed in Figure 9 DE and F, and in the ethanolic extract illustrated in Figure 9D, showed cell death processes at all time intervals, resulting in a decrease in live cells. In the hexane fraction shown in Figure 9E, there was no change in the percentage of apoptotic cells between 6 and 16 hours, both remaining between 70% and 80%. However, in this analysis, the number of cells with characteristics of cell death did not increase, remaining between 10% and 15%. At 24 hours, all cells were dead, losing their integrity in the microscopic analysis, which did not allow for analysis by flow cytometry.The same was observed in the aceto-ethyl extract, shown in Figure 9F, in which the 6-hour treatment analysis showed a small reduction in live cells, resulting in an increase in other conditions. The 16- and 24-hour tests showed similar results, in which cells with apoptotic characteristics remained at approximately 75%. The extracts tested so far are able to act on the pathways responsible for cell death processes; it is noteworthy that the FEPS (MDR) cell line...

[052] To morphologically evaluate cell death processes, cells were treated with extracts and fractions at a concentration of 100 μg / ml and labeled with propidium iodide for visualization of dead cells by microscopy (Figure 10A and B). K562 cells treated with extracts of gray pepper tree (Figure 10A) showed morphological changes, evidencing cell swelling (increase in cytoplasmic content) with formations of Petition 870250075394, dated 08 / 26 / 2025, page 16 / 19 14 / 14 apoptotic bodies. In the FEPS cell line (Figure 10B), cell swelling and apoptotic bodies were present in all fractions.

[053] To assess the possibility that the cytotoxic effect occurs via the pyroptosis pathway, K562 and FEPS cells were pre-treated with the Caspase-1 blocker Ac-YVAD-cmk and subsequently treated with extracts and resin fractions of the gray pepper tree together, showing that the blocker was sufficient to prevent cytotoxicity (Figure 11A and B). In Figure 11A, we observe that all extracts of the gray pepper tree remained alive when the Caspase-1 pathway was blocked. This demonstrates that these fractions trigger cell death by pyroptosis when they are not blocked. The same was observed in the FEPS cell line in Figure 11B. These data suggest that the extracts of gray pepper tree resin in both cell lines tested cause cell death by pyroptosis.

[054] The use of the iron chelator deferoxamine in conjunction with treatments using extracts or fractions showed results similar to those obtained when using the Caspase-1 inhibitor. When cells were treated with extracts of gray pepper tree, in the K562 cell line (Figure 12A and B), in the presence of the iron chelator, the extract was not cytotoxic. However, in the FEPS cell line (Figure 12B) the chelator failed to prevent cell death caused by the ethanolic extract and hexane fraction. The effect of the acetoethyl fraction was inhibited in the presence of the chelator.

[055] The ABCB1 membrane transporter protein activity blocking assay, illustrated in Figure 13, demonstrated the inhibition of this protein's activity when tested with the ethanolic extract and its hexane and acetoethyl fractions at a concentration of 100 μg / ml, over a 30-minute interval in the FEPS cell line. The effect observed in Figure 13 was similar to that observed when the cells were treated with the drug Verapamil, an inhibitor of ABCB1 protein activity. These results confirm that both fractions contain molecules that act on the ABCB1 protein. Petition 870250075394, dated 08 / 26 / 2025, page 17 / 19

Claims

1 / 1 CLAIMS 1. Solution containing extract of Schinus lentiscifolius Marchand as an inhibitor of cell proliferation and multidrug resistance mechanisms, characterized by its composition containing the elements of Schinus lentiscifolius Marchand resin in its natural state, ethanol, hexane, and ethyl acetate.

2. Solution containing an extract of Schinus lentiscifolius Marchand as an inhibitor of cell proliferation and multidrug resistance mechanisms, according to claim 1, characterized in that the resin element is present at a concentration of 10% w / v, and the elements ethanol, hexane and ethyl acetate 99.9% are present at a concentration of 90% each. Petition 870250015579, dated 26 / 02 / 2025, p. 23 / 32

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