SOLUTION CONTAINING AMBER PROPOLIS EXTRACT AS AN INHIBITOR OF CELL PROLIFERATION AND INHIBITOR OF THE MULTIPLE DRUG RESISTANCE MECHANISM

Description

1 / 15 Solution containing amber propolis extract as an inhibitor of cell proliferation and inhibitor of the multidrug resistance mechanism. Field of invention

[001] The field of the invention may refer to pharmaceutical and nutraceutical compositions of alcoholic or hydroalcoholic extracts of amber propolis and / or its sequential fractions with organic solvents and / or its isolated chemical molecules. As well as compositions containing bioactive chemical molecules with cytotoxic, cytostatic and MDR phenotype-blocking effects as therapeutic targets against neoplasms and inflammatory diseases for medicinal and veterinary use. Fundamentals of the invention

[002] To date, there are many products on the market developed from active ingredients obtained from natural products. Regarding propolis from other regions of Brazil or the world, numerous patents have been found for a wide variety of purposes. Among the main ones, intended for human health, are listed groups of products for medical, dental and hygienic purposes (WO2013163714A1, CN102961410A, BR1020210059958, WO2013142936A1). Furthermore, products based on propolis or with adjuvants used in the food sector, such as preservatives (BR1020200177486, BR1020200101005). The use of propolis in the cosmetics sector and the preparation of similar products is also noteworthy (EP0109993A1, KR20120020994A). In Brazil, green propolis has 5 patents filed, related to the aqueous extraction process and the use of green propolis in pharmaceutical formulations used in dentistry. Patents also exist for the use of green propolis extract as a food supplement. Regarding red propolis, Petition 870250014664, dated 24 / 02 / 2025, page 9 / 40 2 / 15 There are 43 patents filed which relate to methods / processes for extracting red propolis and its fractions, pharmaceutical compositions (especially for dentistry), production of micro / nanoencapsulated products, use of extracts as preservatives and food enrichers, among others. Patents related to chemotherapeutic or cytotoxic action against neoplasms using propolis extracts are scarce; however, there is one patent application filed in Brazil using a hydroalcoholic extract of red propolis describing the chemopreventive effect against malignant neoplasms (BR1020150175280). Thus, the importance of the present invention for increasing knowledge about the use of extracts of natural products with chemical fractionation of extracts with chemotherapeutic effects of propolis produced in the Pampa Gaúcho Biome, which we have named "amber propolis," is evident.

[003] The patents described above differ from the present invention due to the use of the ethanolic extract and its sequential extracts of amber propolis as promoters of cytotoxic and / or cytostatic effects in neoplastic, hematopoietic cells and cells with MDR (Multiple Drug Resistance) phenotype. The present invention is innovative because the extracts produced in 3 sequential fractions, taking into account the polarity of the chemical molecules present, exhibit specific therapeutic effects and direct new treatment approaches according to the chemical characteristic of the molecule (nonpolar / polar).Furthermore, the production process of the extracts is relatively simple and inexpensive, since, starting from quality and effective beekeeping management for propolis production, the raw material will be used as a source of molecules for the production of the extracts, which are produced with low-cost solvents (ethanol, hexane and ethyl acetate), and the final product exhibits a large availability of chemical molecules that can be used for the described therapeutic purposes. The present invention acts on cells of the SCC9 cell line (squamous cell carcinoma of...). Petition 870250014664, dated 24 / 02 / 2025, page 10 / 40 3 / 15 tongue), K562 (chronic myeloid leukemia cells), FEPS and Lucena (chemotherapy-resistant cell lines selected from the K562 cell line), allowing the study of the behavior of these separate fractions against neoplastic cell lines.

[004] Some results related to propolis extracts and their use or the use of isolated compounds related to the biological activities presented here demonstrate the antitumor or anticancer action of ethanolic or hydroalcoholic extracts of propolis or of compounds isolated from propolis extracts that exhibit antiproliferative and cytotoxic effects in tumor cell lines, affirming the therapeutic potential of propolis against cancer. Some of these examples can be consulted through the following patent codes: US20170226042; WO2013012477;GB2620468;JP2015205824A. The difference of the present invention in relation to the inventions cited herein, according to the biological activities presented, is justified by two important factors: 1) the first is that the biological activities tested in the present invention are not based solely on the ethanolic extract, but also on the testing of its sequential fractions carried out on the principle of separation by polarity, that is, concentrating biological molecules with similar chemical characteristics, and thus guiding the research according to the chemical characteristics of the biological molecules present in the tested fractions.2) And the second important point is that, in the present invention, we demonstrate the biological effect of the sequential fractions, in particular the hexane fraction, regarding the reversal of multidrug resistance (MDR) mechanisms through testing on resistant tumor cells (FEPS) by blocking the ABCB1 membrane protein with the hexane fraction of amber propolis. In other words, no other propolis characterized and classified on the Brazilian market, with patents filed, has data filed or published relating to our research. Furthermore, none... Petition 870250014664, dated 24 / 02 / 2025, page 11 / 40 4 / 15 other propolis not classified in other locations outside of Brazil has data similar to the present invention, since we did not find in the patent data platforms research referring to the study of the reversal of multidrug resistance mechanisms in cancer cell lines using propolis / propolis extracts / isolated propolis compounds. Brief description of the drawings

[005] Figure 1 shows a photograph of raw amber propolis and the ethanolic extracts and their sequential fractions: hexane and acetoethyl, respectively.

[006] Figure 2 illustrates the production of amber propolis extracts.

[007] Figure 3 illustrates the percentage of material extracted from the crude ethanolic extract and the sequential fractions from the crude ethanolic extract.

[008] Figure 4 shows the cytotoxic effect of hexane and acetoethyl extracts of amber propolis on the SCC9 cell line using the cell viability assay.

[009] Figure 5 shows the cytostatic effect of ethanolic, hexanic and acetoethyl extracts of amber propolis on the SCC9 cell line using the cell cycle assay.

[010] Figure 6 shows the antiproliferative effect of ethanolic, hexanic and acetoethyl extracts of amber propolis on the SCC9 cell line by the MTT assay.

[011] Figure 7 shows the antiproliferative effect of ethanolic, hexanic and aceto-ethyl extracts of amber propolis on the K562 cell line.

[012] Figure 8 shows the antiproliferative effect of ethanolic, hexanic and aceto-ethyl extracts of amber propolis on the FEPS cell line. Petition 870250014664, dated 24 / 02 / 2025, page 12 / 40 5 / 15

[013] Figure 9 shows the antiproliferative effect of ethanolic, hexanic and aceto-ethyl extracts of amber propolis on the Lucena strain.

[014] Figure 10 shows the cytotoxic effect of the ethanolic extract on the K562 cell line at 6h-16h-24h intervals using the Annexin V-FITC assay.

[015] Figure 11 shows the cytotoxic effect of the hexane extract on the K562 cell line at 6h-16h intervals using the Annexin V-FITC assay.

[016] Figure 12 shows the cytotoxic effect of the acetoethyl extract on the K562 cell line at 6h-16h-24h intervals using the Annexin V-FITC assay.

[017] Figure 13 shows the cytotoxic effect of the ethanolic extract on the FEPS cell line at 6h-16h-24h intervals using the Annexin V-FITC assay.

[018] Figure 14 shows the cytotoxic effect of the hexane extract on the FEPS cell line at 6h-16h intervals using the Annexin V-FITC assay.

[019] Figure 15 shows the cytotoxic effect of the acetoethyl extract on the FEPS cell line at 6h-16h-24h intervals using the Annexin V-FITC assay.

[020] Figure 16 demonstrates the blocking of ABCB1 protein by amber propolis extracts using the Rhodamine 123 dye exclusion assay.

[021] Figure 17 illustrates the production of ethanolic, hexanic and acetoethyl extracts of amber propolis for in vitro biological assays.

[022] Figure 18 shows the production table in volume and grams of the ethanolic extract of amber propolis and sequential fractionation for the production of the hexane and acetoethyl extract.

[023] Figure 19 shows the table of the final formulation of the ethanolic, hexanic and aceto-ethyl extracts of amber propolis at a concentration of 30% w / v.

[024] Figure 20 presents the formulation table of the extracts for assays. Petition 870250014664, dated 24 / 02 / 2025, page 13 / 40 6 / 15 biological in vitro of ethanolic, hexanic and acetoethyl extracts of amber propolis.

[025] Figure 21 presents the table generated from the analysis with the SIRIUS software of candidate formulas of the ethanolic extract of amber propolis (positive ionization mode) with their respective masses / charge (m / z), annotation of the chemical structure, class of the chemical compound and the respective SIRIUS (for chemical formula) and TANIMOTO (for chemical structure) scores of each possible molecule. Description of the invention

[026] The extracts were produced from samples of Gaucho Amber Propolis, collected from the experimental apiary of the Federal University of Pampa (UNIPAMPA) São Gabriel campus, by the Pampa apiculture research group (APIPAMPA) in the city of São Gabriel, in the state of Rio Grande do Sul. Production of Ethanolic, Hexanic, and Acetoethyl Extracts of Amber Propolis Ethanol Extract

[027] For ethanol extraction, 99.8% Absolute Ethanol was used in a 30% mass / volume ratio. Specifically, 10 g of raw propolis was weighed and diluted in 34 ml of 99.8% Absolute Ethanol. The extract was agitated in a shaker (Marconi MA-420) for 12 hours at room temperature. The crude ethanolic extract was centrifuged at 200 xG for 15 minutes at room temperature. After removing the supernatant (extract), the undissolved material (propolis) was diluted again in 34 ml of 99.8% Absolute Ethanol, and the process of agitation, centrifugation, and removal of the solubilized material was repeated. In total, this procedure was performed four times, totaling [amount] at the end of the extraction. Petition 870250014664, dated 24 / 02 / 2025, page 14 / 40 7 / 15 a volume of approximately 100ml of crude ethanolic extract. At the end of the extraction, the crude ethanolic extract (100ml) was separated into two samples: Ethanolic Extract with wax and Ethanolic Extract without wax. To remove the wax from the Ethanolic Extract, the samples were placed in a freezer at -20°C and 3 successive centrifugations were performed at a speed of 200xG for 10 min and a temperature of 10°C with 20 min freezing intervals to allow the wax to settle in the extract. At the end of this process, the remaining crude ethanolic extract was dried in an oven at 40°C for a period of 48 hours. Hexane Extract

[028] After drying the crude ethanolic extract, the dried material was weighed and diluted in PA Hexane in the same proportion as the ethanolic extract, 30% mass / volume. The dried material obtained from the wax-free ethanolic extract weighed 3.149g and was diluted in 10.5ml of PA Hexane, following the 30% mass / volume ratio. After this, the hexane extract was stirred for 12 hours at room temperature, with the lid sealed with parafilm to prevent evaporation of the hexane. The produced hexane extract was centrifuged at 200 xG for 15 min at room temperature and the supernatant was removed. This process was repeated 3 times, totaling a total volume of hexane extract of 33ml. From this extract, 5 ml of sample corresponding to the hexane extract were taken for testing on cell lines, which were dried in a vacuum concentrator and diluted in DMSO to a final concentration of 100 mg / ml, and the remainder of the material was dried in an oven at 40°C for a period of 48 hours. Aceto-Ethyl Extract

[029] The dry material of the hexane extract was weighed and 2.096g of this material was diluted in Ethyl Acetate PA in a volume of approximately 7ml. The extraction protocol followed the same protocol as the hexane extract, with 3 repetitions, totaling an acetoethyl extract volume of Petition 870250014664, dated 24 / 02 / 2025, page 15 / 40 8 / 15 20ml. After removing the sample of the acetoethyl extract for testing on cell lines following the same procedure as with the hexane extract, the extract was dried to weigh the unextracted material, which totaled 1.891g of remaining unextracted material.

[030] The ethanolic fraction concentrated 35.4% of the total material extracted from the raw amber propolis, in a proportion of 30% mass / volume, validating that the extraction was successfully carried out within the pre-established parameters for the production of commercially produced propolis extracts.

[031] The hexane fraction, made from the wax-free ethanolic extract, concentrated most of the chemical compounds, totaling approximately 60.4% from the ethanolic extract, in accordance with what had been previously reported, where amber propolis presented several chemical groups with high non-polarity. At the same time, the aceto-ethyl fraction concentrated 12.2% of material soluble in this solvent, indicating a low content of polar compounds, corroborating the percentage found in the hexane fraction. Cell line culture

[032] The K562 (non-MDR), FEPS and Lucena (MDR) cell lines were cultured in RPMI 1640 medium with 2g / L of Sodium Bicarbonate, supplemented with 10% fetal bovine serum and 1% antibiotic and antifungal, maintained in an incubator at 37°C and 5% CO2. For the resistant FEPS and Lucena cell lines, 4.66μM of the chemotherapeutic agent Daunorubicin and 60μM of the chemotherapeutic agent Vincristine were used in the culture, respectively, which are necessary to maintain the selection of these cells and maintenance of the MDR phenotype. The SCC9 cell line was cultured in DMEM F12 medium with 1.2 g / L of sodium bicarbonate, supplemented with 10% fetal bovine serum and 1% antibiotic and antifungal, and maintained in an incubator at 37°C and 5% CO2. After Petition 870250014664, dated 24 / 02 / 2025, page 16 / 40 Once 9 / 15 reached ideal confluence, the culture medium was changed every 2 to 3 days. Formulation of ethanolic, hexanic, and acetoethyl extracts of amber propolis.

[033] The ethanolic, hexanic and aceto-ethyl extracts were produced following the commercial standard for propolis extracts in a 30% mass / volume ratio to obtain 10-11% dry material in each extract. Preparation of amber propolis extracts for RPHPLC / LC-MS analysis by a third-party company (CEM-BIO / RJ) and data analysis using SIRIUS software.

[034] Ethanolic and acetoethyl extracts of amber propolis were centrifuged in a Concentrator plus rotary evaporator at 45°C for 40-60 min to obtain between 10-20 mg of dry material and dissolved in 99.8% methanol for analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Prominence 20A chromatograph (Shimadzu Co., Japan). Aliquots (20 μL) were loaded onto a C18 column (NST 18 250 mm x 4.6 mm, 5 μm) in a two-solvent system: solvent A - ultrapure H2O and solvent B - methanol (MeOH). The column was eluted using binary gradient mode at a constant flow rate of 1 mL / min with a gradient of 50 to 100% solvent B (0 min = 50%, 15 min = 60%, 25 min = 90%, 55 min = 100%, 60 min = 50%, 60 min = stop) for 60 min. The elution profile was monitored with a Shimadzu SPD-20A / 20AV detector A at 254 nm and 326 nm simultaneously, and a fluorescence detector B at EM = 380 nm, Ex = 488 nm.Mass spectrometry analyses were performed by the Biomolecule Mass Spectrometry Center of Rio de Janeiro using both positive and negative ionization modes of the samples. Petition 870250014664, dated 24 / 02 / 2025, page 17 / 40 10 / 15

[035] The data obtained were sent and analyzed by the SIRIUS software with the SIRIUS, ZODIAC, CSI:FingerID and CANOPUS tools to identify and list the molecules present in the analyzed samples according to their chemical formula (SIRIUS score), annotation of the chemical structure (TANIMOTO score) and compound class (CANOPUS). The references for the program used and its tools are below: SIRIUS: Kai Dührkop, Markus Fleischauer, Marcus Ludwig, Alexander A. Aksenov, Alexey V. Melnik, Marvin Meusel, Pieter C. Dorrestein, Juho Rousu and Sebastian Bocker SIRIUS4: a rapid tool for turning tandem mass spectra into metabolite structure information Nat Methods, 16, 2019. https: / / doi.org / 10.1038 / s41592-019-0344-8, CANOPUS: Kai Dührkop, LouisFélix Nothias, Markus Fleischauer, Raphael Reher, Marcus Ludwig, Martin A. Hoffmann, Daniel Petras, William H. Gerwick, Juho Rousu, Pieter C. Dorrestein and Sebastian Bocker Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra Nature Biotechnology, 2020. https: / / doi.org / 10.1038 / s41587-020-0740-8, CANOPUS: Yannick Djoumbou Feunang, Roman Eisner, Craig Knox, Leonid Chepelev, Janna Hastings, Gareth Owen, Eoin Fahy, Christoph Steinbeck, Shankar Subramanian, Evan Bolton, Russell Greiner, David S. Wishart ClassyFire: automated chemical classification with a comprehensive, computable taxonomy J Cheminf, 8, 2016. https: / / doi.org / 10.1186 / s13321-016-0174-y, CANOPUS: Kim, Hyun Woo and Wang, Mingxun and Leber, Christopher A. and Nothias, Louis-Félix and Reher, Raphael and Kang, Kyo Bin and van der Hooft, Justin J. J. and Dorrestein, Pieter C. and Gerwick, William H. and Cottrell, Garrison W. NPClassifier: A Deep Neural Network-Based Structural Classification Tool for Natural Products Journal of Natural Products, 84, 2021. https: / / doi.org / 10.1021 / acs.jnatprod.1c00399, CSI:FingerID: Kai Dührkop, Huibin Shen, Marvin Meusel, Juho Rousu and Sebastian Bocker Searching molecular structure databases with tandem mass spectra using CSI:FingerID Proc Natl Acad Sci U S A, 112, 2015. Petição 870250014664, de 24 / 02 / 2025, pág. 18 / 40 11 / 15 https: / / doi.org / 10.1073 / pnas.1509788112, CSI: FingerlD: Martin A. Hoffmann and Louis-Félix Nothias and Marcus Ludwig and Markus Fleischauer and Emily C. Gentry and Michael Witting and Pieter C. Dorrestein and Kai Dührkop and Sebastian Bocker Assigning confidence to structural annotations from mass spectra with COSMIC bioRxiv, 2021. https: / / doi.org / 10.1101 / 2021.03.18.435634, ZODIAC: Marcus Ludwig, LouisFélix Nothias, Kai Dührkop, Irina Koester, Markus Fleischauer, Martin A. Hoffmann, Daniel Petras, Fernando Vargas, Mustafa Morsy, Lihini Aluwihare, Pieter C. Dorrestein, Sebastian Bocker, ZODIAC: database-independent molecular formula annotation using Gibbs sampling reveals unknown small molecules bioRxiv, 2019. https: / / doi.org / 10.1101 / 842740. Preparation of Amber Propolis Extracts for in vitro biological assays

[036] The amber propolis extracts: ethanolic, hexanic, and acetoethyl were previously prepared for use in in vitro biological assays. In detail, each extract was dried in a vacuum concentrator for 40 min 90 min at 45°C in a volume of 1 ml of the ethanolic extract to obtain a final concentration of approximately 100 mg / ml, as well as the hexanic extract in a volume of 3 ml and the acetoethyl extract in a volume of 5 ml to obtain the same concentration. After drying, the dried material was weighed, and the difference between the weights of the reaction tube without material and with dried material was used to calculate the amount of dried extract. After calculating the obtained material, it was diluted in dimethyl sulfoxide to a final concentration of 10% w / v, to be used in the treatment of cell cultures. The illustration in Figure 15 exemplifies the process of preparing the extracts for use in in vitro biological assays. Methodology of the tests Petition 870250014664, dated 24 / 02 / 2025, page 19 / 40 12 / 15

[037] The cytotoxic, cytostatic, and antiproliferative capacity of the extracts was evaluated through cell viability, cell cycle, cell proliferation, cell death assays, and morphological analysis by fluorescence microscopy. In addition, the ability to block the ABCB1 transport protein, present in cells with multidrug resistance, was evaluated using amber propolis extracts as potential ABCB1 protein blockers.

[038] Biological assays were performed using four cell models: SCC9 (oral carcinoma cell line), K562 (erythroleukemic chronic myeloid leukemia cell line), FEPS (daunorubicin-resistant erythroleukemic cell line derived from the K562 cell line), and Lucena (vincristine-resistant erythroleukemic cell line derived from the K562 cell line). Assays were performed with four biological units per cell line and technical triplicates, and treated groups were always compared with control groups (no treatment and vehicle) at 24-hour intervals for cell viability, cell cycle, and cell proliferation, and at 6h-16h-24h intervals for the apoptosis assay. Specifically for the analysis of ABCB1 protein blockade, assays were performed at 30-minute intervals. MTT test

[039] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed with the oral squamous cell carcinoma cell line - SCC9, the K562 erythroleukemic cell line (non-MDR) and its parents FEPS and Lucena (MDR) with four repetitions and triplicate techniques, plating 104 cells / well in a 96-well plate. The test measures the metabolic capacity of viable cells to transform the MTT salt into formazan crystals by NADH-dependent oxidoreductase enzymes, which reduce the salt into these insoluble crystals that generate the characteristic coloration. For the test, the following concentrations of Petition 870250014664, dated 24 / 02 / 2025, page 20 / 40 13 / 15 hexane / acetoethyl extract: 100μg / ml - 50μg / ml - 25μg / ml. All concentrations were compared with the vehicle (100μg / ml DMSO) and with the negative control. After 24 hours of treatment, MTT salt was incorporated into the cells and the colorimetric reaction was analyzed by absorbance in the 560nm range using the EnSpire 2300 Multilabel Reader plate reader. Cell Viability Assay

[040] Cell viability analysis was performed using the Propidium Iodide assay (500 ng / ml), which is based on binding to the DNA of cells with degraded / compromised plasma membranes due to cell death. This dye fluoresces upon binding to DNA, allowing dead or dying cells to be distinguished from viable cells. For the test, the squamous cell carcinoma cell line of the tongue - SCC9 was used, with four repetitions and triplicate techniques, plating 10⁴ cells / well in a 96-well plate. The concentrations used were 100 μg / ml - 50 μg / ml - 25 μg / ml, compared with the respective vehicle (0.1 l of DMSO) and with the negative control. After 24 hours of treatment, the dye Propidium Iodide (500 ng / ml) was incorporated into the cells for analysis by optical microscopy in white light and green fluorescent light for distinction and visualization of the cells stained with PI.Photographs were taken of each sample under white light and fluorescent light using an optical microscope for subsequent counting of viable and non-viable cells using ImagePro Plus image analysis software. Cell Cycle Assay

[041] For cell cycle analysis, the squamous cell carcinoma of the tongue cell line - SCC9 was evaluated over a 24-hour period, with four repetitions and technical triplicates in a 24-well plate, with 105 cells / well. Considering the results obtained by the MTT assay, the concentration tested was approximately the IC50 dose of each extract. In which, the Petition 870250014664, dated 24 / 02 / 2025, page 21 / 40 14 / 15 Hexane extract and ethyl acetate extract were tested at concentrations of 50 μg / ml and 100 μg / ml, respectively. After 24 hours of treatment, the cells were lysed with lysis buffer (300 μl in each well) containing PI ^g / ml). Following incubation for at least five minutes, the cells were analyzed using a BD Accuri C6 cytometer, collecting 10,000 events analyzed by the FL3-A vs. FL3-H density plot and histograms in FL3-H. Annexin V FITC - PI Assay

[042] For cell death analysis, the Annexin V FITC-PI apoptosis detection kit was used, where annexin V conjugated to fluorescein isothiocyanate (FITC) has a high affinity for phosphatidylserines exposed on the extracellular surface and binds to them during the apoptosis process, emitting fluorescence. PI (Propidium Iodide) binds to the DNA of cells with a completely damaged membrane, marking necrotic cells. For the assay, two cell lines were used: K562 (erythroleukemic cell line) and FEPS (resistant erythroleukemic cell line), where the ethanolic, hexane, and acetoethyl extracts were tested at concentrations of 100 μg / ml. For cell death analysis, assays were performed at 6h and 16h intervals for the hexane extract and at 6h, 16h, and 24h intervals for the ethanolic and acetoethyl extracts. All treatments were performed in triplicate along with the negative control and vehicle in 24-well plates, with 10x5 cells / well.After the treatments, the cells were analyzed using a BD Accuri C6 flow cytometer, collecting 10,000 events per sample on FL1-H vs. FL3-H density plots. ABCB1 protein blocking assay

[043] For the ABCB1 transport protein blocking assay, the Rhodamine 123 dye exclusion assay was used. Rhodamine 123 is a fluorescent dye that acts as a “reporter” of P-gp protein function. Petition 870250014664, dated 24 / 02 / 2025, page 22 / 40 15 / 15 used in flow cytometry. To evaluate the functioning of the ABCB1 transport protein, one of the main proteins responsible for conferring the multidrug-resistant (MDR) phenotype in neoplastic cells, we used the Rhodamine 123 dye exclusion assay. As an inhibitor of the ABCB1 protein (positive control), we used Verapamil (10μM), a substance commonly used as a reversal agent for resistance to chemotherapeutic agents in cells with the MDR phenotype. The assays were performed using the FEPS cell line, resistant to the chemotherapeutic agent Daunorubicin, to evaluate the blockade of the ABCB1 protein, present in this cell line and being one of the characteristics responsible for conferring resistance in these cells. In the evaluation of the amber propolis extracts: ethanolic, hexanic, and acetoethyl, a concentration of 100μg / ml was tested during an incubation period of 30 minutes, using 10⁵ cells / ml. Next, the samples were analyzed by flow cytometry, collecting 10.000 events in FL1-H / FL1A histogram graphs. All tests were performed in triplicate, along with controls [with / without Rhodamine 123 (for discrimination between labeled and unlabeled) and with / without verapamil (for discrimination between blocked and unblocked)]. After testing, the data were analyzed using FlowJo software version 10. The K562 cell line was used as a "non-MDR phenotype control" in the experiment to verify the authenticity of the FEPS cell line and confirm the functioning of the ABCB1 membrane transporter protein. Petition 870250014664, dated 24 / 02 / 2025, page 23 / 40

Claims

CLAIMS 1. SOLUTION CONTAINING AMBER PROPOLIS EXTRACT AS AN INHIBITOR OF CELL PROLIFERATION AND INHIBITOR OF THE MULTIPLE DRUG RESISTANCE MECHANISM characterized by its composition containing the elements propolis in natura, ethanol, hexane and ethyl acetate.

2. SOLUTION CONTAINING AMBER PROPOLIS EXTRACT AS AN INHIBITOR OF CELL PROLIFERATION AND INHIBITOR OF THE MULTIPLE DRUG RESISTANCE MECHANISM, according to claim 1, characterized in that the propolis element is present at a concentration of 30% w / v, including beeswax and mechanical masses, to obtain dry propolis material between 10% and 1%, and the elements ethanol, hexane and ethyl acetate are present at a concentration of 70%. Petition 870250014664, dated 24 / 02 / 2025, page 24 / 40

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