Process for extraction, molecular modification and isolation of pectin, modified pectin and use of the same

Description

1 / 52 PROCESS FOR EXTRACTION, MOLECULAR MODIFICATION AND ISOLATION OF PECTIN, MODIFIED PECTIN AND USE THEREOF FIELD OF THE INVENTION

[001] The present invention falls within the area of ​​food, more precisely in the manufacture of food supplements involving the reuse of agro-industrial waste. The present invention relates to a process for extraction, molecular modification and isolation of pectin, modified pectin and its use as a food supplement. FUNDAMENTALS OF THE INVENTION

[002] Pectins are important macromolecules of plant origin, responsible for the structure and support of plants. They are defined as heteropolysaccharides composed mainly of galactopyranuronic acid (GalpA) units linked by α-1,4 (^·4)-α-D-GalpA-(1^) glycosidic bonds. Frequently, there are alternating stretches of 4-GalpA α-1,2 rhamnose-1 dimers (Rhap), allowing branching primarily of galactose (Galp) and / or arabinose (Araf) linked to O-2 and / or O-3 of rhamnose, termed rhamnogalacturonan type 1 (RG-I).

[003] In the food industry, they are used as a stabilizing or gelling agent and have prebiotic potential. The literature also indicates some positive effects on human health, such as modulation of the intestinal microbiota and modulation of the intestinal immune system.

[004] The mesocarp of passion fruit (albedo) and, consequently, a large part of the commercially available flours of this fruit, are rich in dietary fiber, mainly pectins.

[005] Dietary fiber and whole foods are an essential part of the human diet, their beneficial biological value having been recognized by health professionals for decades. However, different types of fiber have different physicochemical properties, and consequently, different types of fiber. Petition 870250016807, dated 28 / 02 / 2025, page 24 / 115 2 / 52 solubilization in the intestinal environment, resulting in different effects on the body. Therefore, even if the daily amount of fiber is obtained through diet, certain classes, such as pectins and beta-glucans - which exert pronounced beneficial effects - are more difficult to consume naturally.

[006] Pectins naturally have a very high molecular weight in the plant matrix and end up having a character of action more similar to that of insoluble fibers, increasing fecal bulk and aiding intestinal transit. By reducing the molecular size of pectins, it is possible to increase the bioactive effects of these fibers in the human body, improving processes such as fermentation by the microbiota, in addition to enabling direct molecular effects, such as interaction with immune system receptors, which may culminate in a quite relevant anticancer action, an action that is absent in unmodified fiber fractions (high molecular weight pectins).

[007] While some fruits like papayas and apples have good amounts of pectin in their pulp, other fruits need processing to extract these important structures and thus make them consumable, as is the case with passion fruit. The mesocarp of passion fruit, although edible, is generally discarded by the food industry due to its rigid texture and absent or astringent flavor. In the juice industry, this part of the fruit is discarded after pulp extraction, generating a large amount of waste, most of which is disposed of in landfills or destined for animal consumption. Therefore, promoting a technique that uses passion fruit albedo for pectin extraction, which is effective and at the same time low-cost, can help reduce the disposal of this pectin-rich matrix and generate innovative products based on modified pectins.

[008] Conventionally, pectin extraction from fruits, mainly citrus fruits, is carried out using mineral acids. Petition 870250016807, dated 28 / 02 / 2025, page 25 / 115 3 / 52 or organic, which although they have good extraction yields, generate high amounts of waste with environmental impacts.

[009] Several researchers are seeking alternatives to the conventional extraction method that uses mineral or organic acids, with the aim of reducing the environmental impact.

[010] In this context, the present invention proposes an alternative process for extraction, molecular modification and isolation of pectins from passion fruit, by means of a high temperature and pressure method, which is low cost and has a reduced environmental impact. The said process also alters (reduces) the molecular size of the pectins present in this matrix, increasing their bioactive potential.

[011] Thus, the proposed process is simple and has a hydrothermal character. In addition, the process uses ethanol, which is a sustainable solvent, for the precipitation of soluble dietary fibers rich in modified pectins from the obtained matrix.

[012] Compared to commonly used pectin extraction techniques, the production cost is lower due to less solvent use, the absence of other chemicals, and ease of use. Therefore, this process stands out for being economical, sustainable, and adding value to both the raw material and the waste generated in the passion fruit processing industry, since it adds market value and biological value to passion fruit flours on the market, as well as to albedo waste in the passion fruit processing industry. STATE OF THE ART

[013] In the search for references in the scientific and patent literature related to the present invention, the following documents were found:

[014] The documents by Talma et al. (2019) and Silva et al. (2019; 2023a; 2023b) reveal the conventional extraction of pectin from passion fruit mesocarp, which is carried out Petition 870250016807, dated 28 / 02 / 2025, p. 26 / 115 4 / 52 using nitric acid and is well established. The extraction is carried out with a 1:50 w / v ratio, 25 mL of distilled water added to 25 mL of 1 mol L-1 (1 molar) nitric acid previously heated to 80°C in a water bath, left to stir for 40 minutes, and after cooling, 3 volumes of absolute ethanol added to precipitate the high molecular weight pectins, washed with acetone, and dried at 37°C for 5 hours. The use of mineral acids results in an expensive methodology, difficult to apply on an industrial scale, and generates hazardous waste, such as aqueous residues of mineral acids (in the case of passion fruit, nitric acid). The process proposed by the present invention is clearly different from the process usually used in the art for pectin extraction because it does not use mineral acids, and instead uses a hydrothermal process.

[015] The document by Chandel et al. (2016) discloses a method for pectin extraction by autoclaving apple pomace flour at time intervals of 15, 30, 45, and 60 min at 121°C. Although the authors performed the extraction by autoclaving at 121°C for 60 min followed by precipitation with 95% ethanol only of the aqueous phase, the aforementioned method differs from the present invention mainly in the pre-washing and precipitation steps. Specifically, the pre-washing step of the present invention ensures the removal of pigments and free sugars, and the precipitation with ethanol in the total material, instead of precipitation only of the filtered material (aqueous phase), provides the recovery of semi-soluble fractions still present in the solid part of the autoclaved material. These steps help to ensure a pectic structure different from those described in the prior art.

[016] The document Silva et al. (2020) describes that the mesocarp of passion fruit is considered an excellent source of pectin that can be used in the preparation of products Petition 870250016807, dated 28 / 02 / 2025, page 27 / 115 5 / 52 food products, either in the form of pure pectin extracted from the peel, or by the direct application of mesocarp flour as a source of pectin. However, the aforementioned method uses conventional acid extraction for pectin extraction, unlike the present process which uses the hydrothermal method. In SILVA et al. (2020), only what was solubilized in acid, and not the solid remainder, was precipitated with absolute ethanol for pectin isolation. Furthermore, the pectin obtained by this art document is chemically and structurally different from the pectin obtained by the process of the present invention.

[017] The document by CANTERI (2010) presented a study of the autoclaving process in passion fruit flours with the objective of understanding the influence of temperature on the raw material before extraction. In this study, one of the flours evaluated was subjected to heating in an autoclave under pressure conditions of 1 bar, 121 °C for 15 min. However, it is noted that this art document uses the same conventional nitric acid extraction methodology for pectin extraction. The autoclaving performed in this study was used on the raw material; the pretreatment of the initial flour was not isolated, and the objective was to evaluate physicochemical changes compared to other methods, such as maceration with hot ethanol. Furthermore, the precipitation with ethanol was performed only on the final supernatant, and not on the total content, unlike what is done in the present invention.

[018] The document by NASCIMENTO (2020) aimed to establish the relationship between the chemical composition of unmodified and chemically modified passion fruit pectin (with pH alteration) and possible beneficial effects on the culture of epithelial colon cancer cells. The study concluded that modified passion fruit pectins appear to exert antitumor activity in the treatment of colorectal cancer cell lines in vitro. However, the study by NASCIMENTO (2020) and the present Petition 870250016807, dated 28 / 02 / 2025, page 28 / 115 6 / 52 invention differ in the modification method which uses strong acid and base (HCl and NaOH) and in the characteristics of the modified pectins obtained, since the average weight, the monosaccharide composition and the degree of methylation were different.

[019] The study by Pinkaew and colleagues (2024) proposes obtaining pectin from cocoa through a process carried out in an autoclave for a period of 5 to 40 minutes, at a temperature of 105 to 135 °C. However, the aforementioned process, in addition to not proposing the reuse of passion fruit albedo, does not present the pre-washing and ethanol precipitation steps as in the present invention and provides a pectin with different physicochemical characteristics.

[020] Similarly, Jackson and colleagues (2007) perform pectin extraction by autoclaving. However, the authors of the article use commercially available citrus for extraction, and the process described does not include the pre-washing and ethanol precipitation steps. Furthermore, the characteristics of the pectin obtained are not revealed.

[021] Leclere et al. (2015) perform a process similar to that of Jackson et al. (2007) and, likewise, the process described does not have the pre-washing and ethanol precipitation steps, differing from the present invention. SUMMARY OF THE INVENTION

[022] The present invention aims to provide a process for extraction, molecular modification and isolation of pectin, modified pectin and its use as a food supplement.

[023] Thus, in one embodiment of the present invention, a process for pectin extraction is disclosed comprising the steps of: (i) perform pre-washing of a flour obtained from fruit with solvent in constant magnetic stirring; (ii) filter and dry the material in circulating air Petition 870250016807, dated 28 / 02 / 2025, page 29 / 115 7 / 52 constant; (iii) suspend in an aqueous environment; (iv) place the solution in an autoclave at a temperature of 90 °C to 170 °C for 15 to 90 minutes; (v) precipitate the material in solvent, and leave in a refrigerator at 4 °C overnight; (vi) filter the material through a silkscreen mesh; and (vii) dry it in a vacuum oven.

[024] In a second embodiment, the invention provides modified pectin, which has: a) degree of methyl esterification of 70 to 85%; b) average molecular weight between 70 and 90 kDa; and c) between 40 and 50% galacturonic acid in the total dry mass.

[025] In a third embodiment, the invention provides the use of modified pectin for the preparation of a food supplement for the prevention or adjuvant treatment of cancer. BRIEF DESCRIPTION OF THE FIGURES

[026] For a comprehensive and detailed understanding of the purpose of this invention, the figures mentioned are presented below.

[027] Figure 1 presents a summary of the extraction, modification, isolation and analysis process of the present invention.

[028] Figures 2A and 2B show a comparison of the amount of water-soluble fraction in the extracted samples compared to the unmodified control (A) and between extraction times of 30 and 60 minutes (B).

[029] Figures 3A and 3B show the elution pattern in the size exclusion chromatography system (HPSEC-RID).

[030] Figure 4 shows the FTIR-ATR spectrum of the pectin obtained.

[031] Figure 5 shows the sugar composition by Petition 870250016807, dated 28 / 02 / 2025, page 30 / 115 8 / 52 Methanolysis method with TFA hydrolysis, followed by HPAEC-PAD analysis.

[032] Figure 6 shows the HSQC-NMR spectrum of the LW-MP4 FSA sample.

[033] Figures 7A and 7B show cell viability of human colorectal adenocarcinoma (A) and carcinoma (B) cells after incubation with the different soluble fractions obtained at a dose of 2 mg / mL, at three different exposure times.

[034] Figures 8A to 8F show the cell viability of the MTT assay in both human colorectal adenocarcinoma (HT-29) and carcinoma (HCT-116) cell lines three days after treatment with FSA samples.

[035] Figures 9A to 9H show the IC50 calculated from the dose-dependent values ​​of the cell viability assay.

[036] Figures 10A and 10B present the expression data for proteins related to the cell cycle and apoptosis in the human colorectal adenocarcinoma cell line (HT-29).

[037] Figures 11A and 11B present the expression data for proteins related to the cell cycle and apoptosis in the human colorectal carcinoma cell line (HCT-116).

[038] Figure 12 shows the proliferation rate of colorectal cancer cells (HCT-116) measured by the BrDU colorimetric experiment over 24 h.

[039] Figure 13 shows the flowchart between the different processes tested in the invention.

[040] Figure 14 shows the color difference between the pectins obtained by the different processes tested in the invention.

[041] Figures 15A and 15B show the difference in viscosity and incorporation into food between the modified and unmodified fractions of pectin.

[042] Figures 16A and 16B show the immunoblots of Petition 870250016807, dated 28 / 02 / 2025, page 31 / 115 9 / 52 samples demonstrating positive identification experiments for the presence of specific pectin sequences.

[043] Figures 17A and 17B present viability data for colorectal cancer cells after treatment with unmodified and modified pectin samples.

[044] Figure 18 shows immunofluorescence images of cells hybridized with only primary antibodies (no signal).

[045] Figure 19 shows immunofluorescence images of cells hybridized with only primary and secondary antibodies (no signal).

[046] Figure 20 shows immunofluorescence images of cells hybridized with only secondary antibodies (no signal).

[047] Figure 21 shows immunofluorescence images that detect pectin samples inside cancer cells after treatment with the samples and hybridization with the primary and secondary antibodies. Samples: A) HW-NP; B) LWMP1; C) LW-MP2; D) LW-MP3; and E) LW-MP4. 60X magnification.

[048] Figure 22 shows immunofluorescence images that detect pectin samples inside cancer cells after treatment with the samples and hybridization with the primary and secondary antibodies. Samples: A) HW-NP; B) LWMP1; C) LW-MP2; D) LW-MP3; and E) LW-MP4. 20X magnification. DETAILED DESCRIPTION OF THE INVENTION

[049] The present invention provides a process for the extraction, molecular modification and isolation of pectins from passion fruit, modified pectin and use thereof.

[050] Initially, it should be noted that the following description is based on preferred embodiments of the invention, without being limited to them.

[051] In one modality, the process comprises the following steps: Petition 870250016807, dated 28 / 02 / 2025, page 32 / 115 10 / 52 (i) perform pre-washing with solvent of a flour obtained from fruit in constant magnetic stirring; (ii) filter and dry the material in constant air circulation; (iii) suspend in an aqueous environment; (iv) place the solution in an autoclave at a temperature of 90 °C to 135 °C for 15 to 90 minutes, 1 atm; (v) precipitate the material in solvent, and leave in a refrigerator at 4 °C overnight; (vi) filter the material through a silkscreen mesh, and (vii) dry it in a vacuum oven.

[052] In one embodiment, step (i) of the process described above is carried out using fresh mesocarp, after removing the fruit pulp. In an alternative embodiment, the process uses commercially available passion fruit flours.

[053] In this respect, by transforming a normally discarded by-product into a functional ingredient, the invention contributes to the sustainability of the passion fruit production chain and to the circulation of the economy in the food industry. It is also noteworthy that the process in question is suitable both for use in the fruit processing industry with the by-product while it is still fresh, which would require a prior dehydration and grinding step, and for passion fruit flours already on the market, which have a low production and marketing value.

[054] In one embodiment of the invention, the fruit flour is, but not limited to, selected from the group consisting of citrus, cocoa, apple, papaya, orange, jaboticaba, tomato, carrot, beet, strawberry, grape, eggplant, plum, blackberry, lemon and passion fruit flour.

[055] In a preferred embodiment, the fruit flour is passion fruit flour. Petition 870250016807, dated 28 / 02 / 2025, page 33 / 115 11 / 52

[056] In one embodiment, the process solvent is a green and sustainable solvent.

[057] In a preferred embodiment, the solvent is ethanol.

[058] Specifically, the proposed process involves the use of ethanol to isolate pectins from the matrix, standing out for its simplicity and sustainable nature.

[059] In an alternative embodiment, the process solvent is a polar or nonpolar solvent selected from methanol, butanol, isopropanol, acetonitrile, chloroform, toluene and ethyl acetate.

[060] In a preferred embodiment, the prewashing step of the process is carried out with ethanol in a ratio of 1:2 to 1:20 (m / v) for one hour under constant magnetic stirring.

[061] In an even more preferred embodiment, the pre-washing step of the process is carried out with ethanol in a 1:3 (m / v) ratio for one hour under constant magnetic stirring.

[062] In a preferred embodiment, the suspension stage of the process is carried out with water in a ratio of 1:2 to 1:100 m / v and heating is carried out between 30 and 90 °C, for 15 to 180 minutes.

[063] In an even more preferred embodiment, the suspension stage of the process is carried out with water in a ratio of 1:20 (m / v) and heating is carried out between 50 and 60 °C, for 15 to 30 minutes.

[064] In a preferred embodiment, the autoclaving step is carried out at a temperature of 121 °C for 60 minutes at 1 atm.

[065] In this respect, the present process uses a hydrothermal treatment in an autoclave to achieve the desired extraction and modification. This approach differs significantly from conventional methods of the technique that employ mineral or organic acids, providing a modified molecular profile of pectin with less environmental impact.

[066] In a preferred arrangement, the stage of Petition 870250016807, dated 28 / 02 / 2025, page 34 / 115 12 / 52 Product precipitation is carried out with 99% ethanol solvent in a ratio of 1:2 to 1:4 (v / v).

[067] In an even more preferred embodiment, the product precipitation step is carried out with 99% ethanol solvent in a 1:3(v / v) ratio.

[068] In a preferred embodiment, the filtered material is dried in a vacuum oven (30 °C to 90 °C) for 1 to 8 hours.

[069] In an even more preferred embodiment, the filtered material is dried in a vacuum oven (30 °C to 90 °C) for 4 hours.

[070] In a preferred embodiment, the invention process has the following steps: 1) Starting with a sample of fresh mesocarp (after removing the fruit pulp), the mesocarp was cut, dehydrated (oven at 100 °C and / or oven at 40-100 °C with forced ventilation) and ground to obtain initial flour. When using commercial flours, this step is omitted; 2) Various purification steps were tested, and the one with the best performance will be described. A pre-wash with ethanol was performed at a volume of 1:3 (mass / volume) for one hour under constant magnetic stirring. The material was filtered and dried under constant air circulation; 3) Suspension in water at a ratio of 1:20 m / v, gentle heating between 50 and 60 °C for 15 to 30 minutes; 4) After complete solubilization, the material is transferred to a high-temperature resistant flask, and the solution is placed inside an autoclave. The temperature and pressure are monitored to reach and maintain 121 °C and 1 atm. The ideal time demonstrated by the results was 60 minutes within this temperature and pressure range; 5) The flask was cooled, and the solution was placed in a large beaker, where 99% ethanol was added to the volume. Petition 870250016807, dated 28 / 02 / 2025, page 35 / 115 13 / 52 in a 1:3 ratio (volume / volume), and left in the refrigerator (4 °C) overnight; 6) The material was filtered through a silkscreen mesh, dried overnight in constant air circulation at room temperature, and then dried in a vacuum oven for 4 hours; 7) Chemical analyses and effects on cells were evaluated on the water-soluble fraction of the final flour (extracted by suspension in deionized water at a ratio of 1:20 w / v for one hour), while part of the total flour was incorporated into a suitable diet for mice.

[071] In another embodiment, the present invention provides modified pectins. In particular, the pectins are structurally and chemically modified in relation to naturally obtained pectins and pectins of the prior art, demonstrating a significant reduction in molecular size as well as exhibiting a higher degree of methylation and a higher proportion of water-soluble fractions compared to the unmodified sample.

[072] Specifically, the modified pectins of the present invention have a molecular weight in the range of 70 to 90 kDa, and exhibit a degree of methylation between 70 and 85%.

[073] In contrast, natural and unmodified pectins have a molecular weight above 750 kDa and a degree of methylation between 50 and 70%.

[074] Structural modification, specifically the decrease in molecular weight, the increase in the degree of methylation and the increase in galacturonic acid levels, provides an increase in the bioactive effects of these fibers in the human body, improving fermentation by the intestinal microbiota and offering potential direct molecular effects, including immunomodulatory and anticancer actions, which are absent in unmodified fiber fractions.

[075] Chemical analyses of modified pectin and its effects on cells were evaluated in the water-soluble fraction of Petition 870250016807, dated 28 / 02 / 2025, page 36 / 115 14 / 52 final flour (extracted by suspension in deionized water at a ratio of 1:20 m / v for one hour), while part of the total flour was incorporated into a suitable diet for mice.

[076] Isolated pectins are structurally characterized and evaluated in various biological models to assess their bioactive potential. Tests in human colon cancer cell lines and in animal models subjected to colorectal cancer induction confirm the efficacy of modified pectin-rich fibers, which demonstrate remarkably positive results compared to control groups.

[077] In another embodiment, the present invention provides the use of modified pectins for the preparation of food additives or food supplements for the prevention and treatment of cancer.

[078] In one embodiment, the cancer of the present invention is selected from among epithelial cancers.

[079] In a preferred embodiment, the cancer is colorectal cancer.

[080] In this context, the present invention offers a viable solution for the food and supplement industry, providing an additive with potentially prebiotic, immunomodulatory and intestinal cancer preventive properties.

[081] In the context of the present invention, a food supplement can be understood as a product that provides nutrients, bioactive substances, enzymes or probiotics in addition to food.

[082] The term food additive can be understood as an ingredient intentionally added to food with the aim of modifying its physical, chemical, biological or sensory characteristics during the manufacture, processing, preparation, treatment, packaging, storage, transport or handling of a food. Petition 870250016807, dated 28 / 02 / 2025, page 37 / 115 15 / 52

[083] The invention may be further described by the following non-limiting examples. The following examples detail the performance of the inventive processes. Those skilled in the art will recognize many variations that are within the scope of the invention and its claims. EXAMPLES OF IMPLEMENTATION

[084] The extracted and isolated modified pectin-rich fibers were structurally analyzed and analyzed in various biological models to assess their bioactive potential. The average molecular weight of the polysaccharides ranges from 70 to 90 kDa (size exclusion chromatography with refractive index detection), their degree of methylation was 70 to 85% (Fourier transform infrared spectrometry), and the main monosaccharide was galacturonic acid (approximately 45% by dry mass). The modified pectins extracted and isolated by combining techniques resulting in a novel procedure were tested in human colon cancer cell lines. Cell viability, proliferation, and expression of key proteins in the control of tumor progression were tested.Fibers rich in modified pectins were also incorporated into the diet of mice that underwent induction of colorectal cancer with increased inflammation, resulting in remarkably positive effects compared to control groups and unmodified pectin groups (high molecular weight).

[085] The tests described above will be detailed by means of the following examples. EXAMPLE 1 - Extraction and characterization of the total cell wall (TCW) of passion fruit

[086] Passion fruits (Passiflora edulis f. flavicarpa) were obtained from a fruit supermarket chain in São Paulo (Oba Hortifruti, Brazil) directly from a producer in Tocantins, Minas Gerais, Brazil (21°09'54.8S 43°04'08.2W). The fruits Petition 870250016807, dated 28 / 02 / 2025, page 38 / 115 16 / 52 ripe (fully yellow) fruits were peeled, cut in half, and the pulp was removed. The remaining white mesocarp (albedo) was cut into smaller pieces and dried in a forced-air oven at 40°C until constant weight. The dried pieces were ground, passed through a 0.48 mesh sieve, and the final flour was stored in desiccators. The main objective of the first extraction was to test different ways of removing small sugars and oligosaccharides, along with other compounds that could interfere in some way with the thermal modification. The extraction flow is summarized in Figure 13. Minor changes to the extraction method were made, resulting in four different modified samples and the unmodified sample.

[087] Unmodified passion fruit polysaccharide-rich flour was washed in three volumes of 80% ethanol to remove contaminants (small sugars, pigments, organic acids), filtered through a polyester membrane (5 μm), and dried to produce higher molecular weight unmodified passion fruit polysaccharides (HW-NP; control fraction). In two of the lower molecular weight modified passion fruit polysaccharide samples (LW-MP), the flour was not washed with ethanol before modification (LW-MP1 and LW-MP2), while two other samples were (LW-MP3 and LW-MP4). All four samples were solubilized in deionized water at a 1:10 (w / v) ratio and autoclaved for 30 or 60 min at 121°C in heat-resistant bottles.The two modified samples, not previously washed with ethanol, were dried in a forced-air oven at 40°C until constant weight, ground, sieved through a 0.48 mesh, and stored in a desiccator, resulting in modified passion fruit polysaccharides of lower molecular weight - LW-MP1 and LW-MP2.

[088] The other two modified samples previously treated with ethanol were precipitated with absolute ethanol in a 1:3 (v / v) ratio, filtered through a polyester membrane (5 μm), and the alcohol-insoluble residue was dried in an oven with Petition 870250016807, dated 28 / 02 / 2025, page 39 / 115 17 / 52 air circulation at 40°C until a constant weight was achieved, producing LW-MP3 and LW-MP4 (Figure 13). 1.1 Extraction of water-soluble polysaccharides from the cell wall

[089] The unmodified and modified samples (500 mg) were extracted three times with 15 mL of deionized water under constant magnetic stirring for one hour. The supernatant fractions were termed the water-soluble fraction (WSF) and were lyophilized and weighed. 1.2 Sample Characterization 1.2.1 Total carbohydrate content

[090] Carbohydrate content was determined by the phenol-sulfuric acid method according to Masuko et al. (Masuko et al., 2005) with adaptations. To each well of a 96-well plate containing 50 μL of glucose standards (0 to 1 mg / mL) or passion fruit pectin samples, 150 μL of concentrated sulfuric acid were added directly on top, along with 30 μL of a 5% phenol-in-water solution. The plate was then incubated at 90°C for 5 min in a temperature-controlled oven, followed by a further 5 min at room temperature to cool. The measurement was performed on a BioTek Epoch 2 plate reader (Agilent, Santa Clara, California, USA) at 490 nm. 1.2.2 Total uronic acid content

[091] Passion fruit samples were pre-hydrolyzed with 72% w / w sulfuric acid at 30 °C for one hour and then hydrolyzed with additional distilled water at 100 °C for three hours. The total uronic acid content was evaluated using the m-hydro-diphenyl assay, using an automated wet chemistry analyzer (Skalar San++® series, Breda, Netherlands). Galacturonic acid was used as a standard for quantification. 1.2.3 Total protein content.

[092] Approximately 10 mg of each sample were weighed. Petition 870250016807, dated 28 / 02 / 2025, page 40 / 115 18 / 52 in aluminum foil containers. They were then folded tightly to prevent nitrogen contamination and / or sample loss. The folded aluminum structures were placed in the FlashSmart™ Elemental Analyzer (ThermoScientific™, Waltham, Massachusetts, USA), and the nitrogen content was obtained. 1.2.4 Total phenolic content

[093] Total bound phenolics were determined according to Attard (Attard, 2013) with minor modifications. Briefly, samples were incubated with 2 M HCl for 1 hour at 100°C to release molecularly bound phenolics. In 96-well plates, 10 μE of these extracts were placed in separate wells, as well as gallic acid standards (ranging from 0 to 250 μg / mL), along with 80 μE of 2 M NaCO2 and 100 μE of diluted Folin-Ciocalteu reagent (1:10 in deionized water). After 20 min of light-protected reaction, the plates were read at 630 nm on a BioTek Epoch 2 plate reader (Agilent, Santa Clara, California, USA). 1.2.5 Total lipid content

[094] Total lipids were measured according to Folch et al. (Folch et al., 1957) with minor modifications. Briefly, a 2:1 chloroform:methanol solution was prepared and 20:1 was added to dried samples in glass tubes. After 30 minutes, the tubes were centrifuged to force the separation of the aqueous, non-extractable phase and the organic phase. Using a pipette, the lower phase (chloroform organic phase) was recovered, transferred to a new, previously weighed tube, and dried under a stream of N2 in a fume hood. After complete drying, the tubes were weighed again and the final mass / initial mass calculations were performed. 1.2.6 Moisture and ash content

[095] The same quantity of samples (500 mg) was placed in pre-dried and weighed ceramic crucibles, and then left inside an oven at 135°C for 2 h (AOAC, 2005, method 930.15). Petition 870250016807, dated 28 / 02 / 2025, page 41 / 115 19 / 52 After this time, it was placed inside a desiccator to cool and weighed again to recover the moisture content. After being weighed, the same crucibles were placed in an oven at 550 °C and kept for 3 hours (AOAC, 2005, method 942.05). The crucibles were then weighed again. The final masses were used to calculate the total ash and moisture content based on the initial weights of the samples, according to formulas (I) and (II). Humidity (%) = (W1-W2) x 100 (I) W1 where W1 is the weight before drying and W2 is the weight after drying. Ash content (%) = (iW - fW) x 100 iW (II) where iW represents the initial weight (after moisture content) and fW represents the final weight (ash only). 1.3 Molecular weight and homogeneity analysis

[096] Samples of each fraction (2 mg / mL) had their molecular weight distribution analyzed by high-performance size exclusion chromatography with refractive index detection (HPSEC-RID) using an Infinity 1250 system (Agilent, Santa Clara, CA, USA) (Pedrosa et al., 2020). The system was equipped with four PL aquagel-OH columns (60, 50, 40, and 30; 300 χ 7.5 mm; Agilent) connected in tandem. The eluent was 0.2 M NaNO3 / 0.02% NaN3 (0.6 mL / min), and the RID temperature was set at 30 °C. Molecular sizes were estimated using Dextran T-series (25, 50, 80, 150, 270, 410, and 670 kDa; Sigma, St. Louis, MO, USA) as molecular size curve standards.

[097] The area under the peaks was sectioned for each sample within the standard curve to estimate the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and the polydispersity index (PDI). First, 17 retention time (RT) periods were selected within each sample peak. Petition 870250016807, dated 28 / 02 / 2025, page 42 / 115 20 / 52 The signal obtained for each RT was also recorded side-by-side. Using the standard linear equation determined (y = 5.1578x + 65.998, R² = 0.997), the log values ​​of the molecular weight were obtained for each selected RT. Each signal value was divided and multiplied by its respective molecular weight, and the results were recorded in separate columns (E and F, respectively). The following formulas (III) and (IV) were then applied: Mn = Σ heights of the signal / Σ column E (III) Mw = Σ column F / Σ signal heights (IV) PDI = Mw / Mn 1.4 Sugar composition by methanolysis and TFA (trifluoroacetic acid) hydrolysis

[098] Two milligrams of each polysaccharide sample were dissolved in 1 mL of distilled water. One-tenth of this volume (of the sample and standards) was transferred to glass tubes in replicate. The contents were dried under a stream of N2 at 40 °C and, after being completely dry, were placed in a vacuum oven at 40 °C to be dried again over P2O5 for four hours. Then, 1 mL of 3 M HCl in dry methanol was added to each tube and they were placed for sixteen hours in a heating block at 80 °C inside a fume hood. After cooling, the contents were dried under a stream of N2 once more and hydrolyzed with 0.5 mL of 2 M TFA for one hour at 121 °C, followed by two additions of 1 mL of methanol and evaporation. The final dried samples were dissolved in 1.0 mL of double-distilled water, and 10 μL were injected into a Dionex ICS6000 DC system equipped with a CarboPac PA1 column (ThermoScientific™, Waltham, Massachusetts, USA).A set of neutral monosaccharides and uronic acids were used as standards for quantification (De Ruiter et al., 1992). 1.5 Attenuated Total Reflectance (ATR) Analysis by Fourier Transform Infrared (FTIR)

[099] The degree of esterification and characterization of Petition 870250016807, dated 28 / 02 / 2025, page 43 / 115 The 21 / 52 polysaccharide was evaluated by Fourier transform infrared spectroscopy (FTIR), as described in the literature (do Prado, Shiga, et al., 2019). The Alpha FTIR spectrometer (Bruker Optic, Ettlingen, Germany) was equipped with a deuterated triglycine sulfate detector and a single-step attenuated total reflectance (ATR) accessory (diamond crystal). The established resolution was 4 cm⁻¹ and 64 scans for all samples. A blank was used after each sample set to increase the signal-to-noise ratio. [10 0] The degree of methyl esterification (DM) was calculated as described in the literature (Manrique & Lajolo, 2002), with minor modifications. Briefly, a linear equation of the log-transformed values ​​was made using pectin standards with 28.5, 46, 64, 78 and 92% DM, with an R2 of 0.9964. The formula to obtain the ratios of the area under the peaks (V) was as follows: DM(%) = AP 1740 (V) (AP 1740 + AP 1630) [10 1] The values ​​found for each sample are described in Table 2. 1.6 Immuno-dot blot assay for pectin detection

[102] Two monoclonal anti-pectic primary antibodies from rats were used in a chemiluminescent immuno-dot blot analysis (LM5, LM16). Reinforced nitrocellulose membrane strips were used, and three different amounts (1, 5, and 10 μg) of each sample previously solubilized in deionized water were applied to membranes as dots. After the membranes were dried, they were placed in a blocking buffer solution (5% bovine serum albumin in 0.1% phosphate-buffered saline [PBS]-Tween, pH 7.8) for one hour at room temperature. The primary antibodies were incorporated at a final 1:100 dilution in 5% albumin in PBS, and the membranes were incubated overnight at 4°C under gentle shaking. The membranes were washed three times in PBS. Petition 870250016807, dated 28 / 02 / 2025, page 44 / 115 22 / 52 Tween membranes were then incubated with murine horseradish antiperoxidase secondary antibody (#31460, Thermo Fisher Scientific, Inc.) for one hour at room temperature. The membranes were washed three times in PBS-Tween, revealed with Bio-Rad Clarity Western ECL substrate, and scanned on an Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). 1.7. Analysis of glycosidic linkages by nuclear magnetic resonance (IWN)

[103] Nuclear magnetic resonance analysis was performed as described in the literature (da Silva et al., 2022). In summary, the 1D and 2D1H spectra of the modified lower molecular weight water-soluble passion fruit polysaccharide were recorded using a 500 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) with a triple resonance probe.

[104] Approximately 20 mg of the sample were resuspended in 99.9% deuterium oxide (Cambridge Isotope Laboratory, Cambridge, MA). HSQC (Multiplicity-Edited HSQC)1H / 13C spectra were recorded with 1024 x 512 points and 256 scans using an Echo-Anti-Echo acquisition mode with globally optimized alternating phase rectangular pulses for decoupling at 35°C. The spectra were processed using TopSpin 4.1.4 software (Bruker Biospin). EXAMPLE 2 - In vitro treatment of colorectal cancer cells 2.1. Cell growth and viability assay

[105] Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and 0.1% penicillin / streptomycin and maintained in an incubator at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. The MTT assay was performed to analyze cell viability. Cells were plated in 96 plates Petition 870250016807, dated 28 / 02 / 2025, page 45 / 115 23 / 52 wells (5 x 103 cells / well) were treated with different concentrations of FSA (water-soluble fraction). The final concentrations were 0.25, 0.5, 1, and 2 mg / mL, and the cells were treated for 24, 48, and 72 hours. Cell viability at each incubation time was expressed in relation to untreated cells (positive control for cell growth - 100%) and cells treated with Triton-X (0.2%, positive control for cell death). 2.2. Cellular immunofluorescence assay

[106] Cells were cultured in 24-well plates containing a glass coverslip at the bottom of each well, at a density of 2.5 x 10⁴. A standard curve was made to verify the best arrangement and distance between the cells. After adhering overnight, the cells were treated with polysaccharides (1 mg / mL solution) for 24 h. Each well had its medium removed, was washed three times with 1x PBS, and the cells were fixed with 10% formalin in PBS for 15 min at room temperature. The procedure was followed by three washes with 1x PBS and placement of 0.5% Triton-X in PBS solution for permeabilization. Therefore, the wells were again rinsed three times with 1x PBS and replaced with 3% bovine serum albumin in 1x PBS solution for blocking for one hour at room temperature.Next, the cell coverslips were retrieved with curved forceps and a bent needle, inverted onto a section of parafilm containing a drop of the antipectic polysaccharide LM-19 (approximately 35 µL of a 1:20 antibody-blocking solution) and incubated for 2 hours in a sealed, humidified chamber. After this period, the coverslips were washed in the wells of the plate twice with PBS and again inverted onto parafilm containing a drop of Alexa Fluor-488-conjugated anti-mouse secondary antibody (#A-11006, ThermoFisher Scientific, 1:100 in blocking buffer) in a sealed, humidified dark chamber for 1 h. A final step of... Petition 870250016807, dated 28 / 02 / 2025, page 46 / 115 24 / 52 washing was performed and the cell coverslips were mounted on glass microscope slides containing a drop of mounting medium (Prolong Gold Anti-Fade reagent, P36930, Thermo-Fisher Scientific) with 300 nM DAPI working solution (#D1306, ThermoFisher Scientific). 2.3 Western Blot Analysis

[107] Cells were seeded at a density of 5 x 10⁵ in 6-well plates and treated with the highest dose of three FSM samples (2 mg / mL) or without any treatment (control). After 24 h, cells were scraped and collected in RIPA buffer and stored at -20°C until use. SDS polyacrylamide gel electrophoresis was performed with the denatured samples prepared with Laemmlli buffer. The gel was then transferred to a nitrocellulose membrane using the Trans-Blot Turbo semi-dry system. The membranes were blocked in 5% bovine serum albumin in Tris-buffered saline (TBS 0.1% Tween) for one hour at room temperature, incubated with primary antibodies targeting the cell cycle and apoptosis-related proteins overnight at 4°C, and a secondary antibody coupled to horseradish peroxidase incubation for one hour at room temperature.The membranes were revealed with Bio-Rad's Clarity Western ECL substrate and scanned on an Amersham Imager 680 (GE Healthcare Life Sciences). 2.4 BrdU proliferation assay

[108] The effect of passion fruit polysaccharides on the cell proliferation of cancer cells was quantified by incorporating 5-bromo-2-deoxyuridine (BrdU) during DNA synthesis, followed by colorimetric detection. For this, 5 x 103 HCT-116 cells / well were seeded in complete DMEM medium, in 96-well plates, and incubated in a cell incubator for 24 h. On the second day, the supernatant was removed and unmodified or modified passion fruit polysaccharides, diluted in complete DMEM medium, were added to the Petition 870250016807, dated 28 / 02 / 2025, page 47 / 115 25 / 52 concentration of 2 mg / mL. Cells that received only complete DMEM medium and cells that received doxorubicin at 1 μM served as controls. After 24 h of treatment, the supernatant was removed and the procedures were performed according to the instructions of the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche, sold by Sigma-Aldrich, 11.647.229.001). The plates were read with Synergy H1 Hybrid Reader (BioTek) at 450 and 690 nm. Cell proliferation was expressed as a percentage relative to the untreated control. EXAMPLE 3 - Statistical analysis

[109] Mean and standard deviation values ​​were represented as mean ± SD.

[110] Normal distribution was confirmed using the Kolmogorov-Smirnov test.

[111] All experiments and their respective graphs or images were represented by values ​​at least three times. The data were analyzed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA).A one-way ANOVA, followed by Dunnett's or Tukey's post-hoc test, was used for comparisons between samples and controls or between each group. Significant p-values ​​were selected according to each experimental parameter. EXAMPLE 4 - Structure of the soluble polysaccharide of passion fruit 4.1 Overall composition

[112] The composition of the sample was determined by a group of standardized methods. Through the phenol-sulfuric acid assay, it can be seen that the main component of all of them is carbohydrates (>67%, Table 1). Of this amount, uronic acids represented a significant part of all, and the largest part for three of the modified samples (LW-MP 2 to LW-MP 4). The complete composition can be seen in Table 1. 4.2 The modification increased solubility in water and reduced Petition 870250016807, dated 28 / 02 / 2025, page 48 / 115 26 / 52 molecular weight

[113] Modified pectins have been reported to have greater biological activity due to reduced molecular size (Do Prado et al., 2016; J. Li et al., 2023; Zhu et al., 2022). Furthermore, the solubilization of insoluble pectins is potentially beneficial for the isolation of pectins for biological applications. The FSA yield (Figures 2A and 2B) and molecular distribution (Figures 3A and B) showed that heat and pressure treatment increased the yield of water-soluble polysaccharides, and 60 min significantly decreased the apparent molecular weight, compared to the untreated sample. Soluble dietary fibers are generally considered quite fermentable and may be more involved in direct interactions within cellular receptors and proteins (Prado et al., 2020).The data presented in Figure 2A confirmed the success of the heat treatment, as all modified samples (LW-MP 1-4) showed higher proportions of FSM when compared to HW-NP. The absolute yield of FSM increased relative to HW-NP with p < 0.0001 for all samples, except for LW-MP3 treated for 30 min, which had a slightly smaller increase (p = 0.0002). There were also small differences in FSM yield when comparing 30 and 60 min for each sample (Figure 2B). For LW-MP1, there was a slight reduction in yield (36.3 vs. 32.9%, 30 and 60 min respectively, p = 0.0195). LW-MP4 also showed a difference, but this time a longer exposure to high temperatures and pressures was able to increase the FSM yield (38.6 x 44.1%, 30 and 60 min respectively, p = 0.0042). For LW-MP2 there was no difference in FSM productivity, and for LW-MP3 there was only a tendency towards an increase for 60 minutes.Figures 3A and 3B highlight the reduction in average molecular size and polydispersity of the modified FSM samples compared to HW-NP. The 60-minute treatment was much more effective in this respect, as expected (Figure). Petition 870250016807, dated 28 / 02 / 2025, page 49 / 115 27 / 52 3B). The mean molecular weight (Mw) was lower for LW-MP2 and 4 compared to LW-MP1 and 3 (92.4 x 74.3 kDa and 94.7 x 80.4 kDa, respectively), and all of them were much smaller than the HW-NP sample. Although reduced compared to HW-NP, the samples treated for 30 min could not have their molecular weights predicted because they were located outside the dextran standard curve (Figure 3A).

[114] The highest signal found in the FSM of samples treated for 60 min was interpolated to determine the most abundant molecular size that was isolated. The results of the chromatogram sectioning performed as described in the subsection of “Cell growth and viability assay” are summarized in Table 2.

[115] Although the size of the HW-NP fraction was not precisely determined because it had a higher peak than the first standard of 670 kDa, it consisted of heavy polymer chains with a wide molecular distribution, revealing high polydispersity, visible through the HPSEC-RID chromatogram. (Figures 3A and 3B). 4.3 Degree of esterification and molecular characterization by FTIR-ATR

[116] The region between 1800 and 800 cm⁻¹ in FTIR spectra is often called the fingerprint region, as it is highly variable depending on the type of sample analyzed. For pectic polysaccharides, there are some important vibrations to be evaluated, and many of these points have been explored throughout the literature (Ogutu & Mu, 2017; Pedrosa et al., 2020; Pereira et al., 2016; Szymanska-Chargot et al., 2015). Figure 4 shows the FTIR spectrum of the FSA samples. The axial C=O deformation and the asymmetric COO- angular deformation (near 1740 cm⁻¹ and 1600-1630 cm⁻¹, respectively) are directly correlated to galacturonic acid residues, both with and without the presence of alkyl ester vibration. The ratio Petition 870250016807, dated 28 / 02 / 2025, page 50 / 115 The area under the peak integration of the 1738 and 1608 cm⁻¹ regions (28 / 52) can be interpolated onto a standard curve of known pectic DM samples, resulting in the determination of pectin DM. The 1436 cm⁻¹ region can be described as a symmetrical axial deformation of COO⁻ groups from carboxylate groups. The unmodified sample showed almost no such signal, which can be interpreted as fewer resonating terminal carboxylic groups (the HW-NP chain has a larger molecular size because it did not undergo modification). Both CH and CH₂ angular deformations are described at 1369 cm⁻¹, with all samples showing a subtle signal. Xyloglucans may be one of the sources of this signal, but it could also be due to the presence of smaller, water-soluble β-glucans. The angular deformation of 1228 cm-1 is referred to as OH groups located in the pyranose ring of pectin, where all samples had similarly observed resonances.The 1140 cm⁻¹ region is mainly attributed to ring vibrations coupled to the angular vibration of C-OH. Axial deformation of the pectin rings and chains is represented at 1098 and 1018 cm⁻¹ (C₂-C₃, C₂-O₂, and C₁-O₁). The unmodified sample showed less CO and CC axial deformation resonances (1098 cm⁻¹) than all modified samples. The 965 cm⁻¹ region is attributed to hemicellulose ring vibrations, and the modified samples had slightly higher signals when compared to HW-NP, exhibiting only one shoulder, probably due to solubilization during high temperatures (Table 3).

[117] Regarding the DM estimate, all samples were classified as high methoxylated pectin (HMP), due to DM values ​​above 50%.

[118] Overall, although all samples showed high DM values, both modified samples that underwent conventional alcohol precipitation after modification, without the drying process beforehand (LW-MP2 and LW-MP4), showed significantly lower DM values ​​than the Petition 870250016807, dated 28 / 02 / 2025, page 51 / 115 29 / 52 other samples. 4.4 Sugar composition in FSA samples

[119] Sugar composition is a fundamental pillar to be investigated in polysaccharide samples, since many different biological effects have been proposed to be linked to different compositions and side chains. Galacturonic acid was the primary uronic acid and the general monomer in all FSA samples (Figure 5). However, HW-NP, LW-MP1 and, to a lesser extent, LW-MP3, showed significant amounts of glucose. This may be due to the transport of impurities, such as small sugars. LW-MP2 and LW-MP4 were the richest in GalpA abundance, with a more diverse neutral sugar composition, which gives them a pectic characteristic. The purification steps performed for LW-MP2 and LW-MP4 also appeared precise enough to remove unwanted sugars, as indicated by the decrease in glucose values, which is surprising since this was a non-heavy solvent approach.

[120] Molar ratios can help provide an overview in estimating the structures present in pectic polysaccharides. The modified samples had more indications of prevalence of rhamnogalacturonan residues plus proportion of pectic structures, such as homogalacturonan (HG) and elevated. The RG-I bb parameter means principal rhamnogalacturonan calculated by the molar percentage of Rhap residues times two (Rha*2). The following formula (VI) represents the RG-I bb RG - 1 bb (%) (RGIBB)*100 (VI) HG + RGIBB

[121] Finally, the average chain length is calculated by summing the ratios Ara:Rha and Gal:Rha, which means the total ratio of galactose and arabinose side chains against rhamnose residues (Table 4) Among the modified samples, LW-MP 3 and LW-MP had the highest RG contents. (Reference 870250016807, dated 28 / 02 / 2025, p. 52 / 115) 30 / 52 I, RG-I bb % and RG-I side chains, while LW-MP2 and LW-MP4 had the highest HG content, also followed by LW-MP4.

[122] The reactivity of each FSA sample against different pectic polysaccharide epitopes was tested in a dot blot experiment using anti-galactan (LM5) and anti-rhamnogalacturonan type I (LM16) antibodies (Figures 16A and 16B). All samples were positive for galactan and RG-I epitopes, but the HW-NP sample showed weaker signals for both antibodies at all concentrations. This may be due to the lower availability of epitopes relative to a preserved molecular structure for antibody recognition. Furthermore, the signal for galactans was somewhat weak, following the results of the sugar composition. Therefore, it can be concluded that although structures such as galactans and RG-I are present in all samples, they consist mainly of homogalacturonans. 4.5 Indications of the LW-MP4 structure by HSQC-NMR

[123] Due to similarities in the previous structural analysis and the effects observed in vitro (which will be discussed later), LW-MP4 was chosen as a representative sample in HSQC-NMR. This technique gave some indications and confirmations in terms of structure. The most relevant resonances identified are described in Table 5 below, according to literature on pectic molecules (da Silva et al., 2022; do Prado, Santos, et al., 2019; Maxwell et al., 2015, 2016).

[124] Following the two previous results, the HPAEC data showed a majority of GalpA residues, and the FTIR showed subtle vibrations associated with other monosaccharides and the high MS attributed to GalpA. The highest signal identified was related to δ 3.79 / 53.0, which is related to methoxylated residues (MeO). Therefore, the highest signals for most resonances are those associated with the ^4)-«-GalpA-(1) linkages. Petition 870250016807, dated 28 / 02 / 2025, p. 53 / 115 31 / 52 ^ (C1-C5) and -4)- / .-6-MeGalpA-(1 ^ (D1-D5) (Figure 6) .

[125] Due to the fact that most of the identified peaks are related to ^4)-«-GalpA-(1^ or ^4)-«-6-MeGalpA-(1^, and the results of the sugar composition, it can be suggested that the core structure of the identified polysaccharide is mainly linear methylated homogalacturonans (Abboud et al., 2019; Lin et al., 2021). The side chains linked to Rhap residues in the type 1 rhamnogalacturonane structures consist of galactans, and a relevant Araf is linked to O-3 residues related to some assignments, although not relevant to the sugar composition (Shivamathi et al., 2019). Secondary structures, such as α-Glcp-(1^, were also present, coinciding with the sugar composition, which may indicate the possible presence of glucomannan, xyloglucans, or soluble β-glucans (Rong et al., 2021). EXAMPLE 5 - Anticancer activity 5.1 The viability of colorectal cancer cells is reduced by treatment with passion fruit polysaccharides.

[126] Cell viability was tested in human colorectal adenocarcinoma (HT-29) and carcinoma (HCT-116) cells. First, an initial screening was performed on both cell lines, with all FSA samples from both heat treatment groups (30 and 60 min), at a dose of 2 mg / mL. Samples treated for 30 minutes were also tested and are shown in figures 17A and 17B.

[127] All modified samples showed a significant reduction in viability, but only LW-MP2 and LW-MP4 statistically and significantly reduced viability in HT-29, by 40.4% and 64.6%, respectively (Figure 7A). 48 h was marked by a strong reduction in viability in all modified samples, except LW-MP1, which showed higher viability than cells exposed to HW-NP. Finally, each sample, including HW-NP, maintained HT-29 viability close to 50%, but LW-MP1 Petition 870250016807, dated 28 / 02 / 2025, p. 54 / 115 32 / 52 did not appear to influence it in any way, remaining alongside the untreated control at 72 h (Figure 7A).

[128] HCT-116 cells were more prone than HT29 cells to all treatments. For the modified samples at 30 minutes, no statistical difference was detected between them and HW-NPP, except for LW-MP2 at 24 h (Figures 17A and 17B). However, modified samples at 60 minutes appeared to be more effective than HW-NP at 24 and 72 hours, with the most significant reductions being attributed to LW-MP3 and LW-MP4, (55 and 52% by 24 h; 62.2 and 61.4% by 72 h, respectively, Figure 7B).

[129] Due to lower efficacy in initial screening, HPSEC data, and FSA yield results, all modified 30-minute FSA samples (LW-MP1, 2, 3, and 4) and the modified 60-minute LW-MP1 were removed from the subsequent dose-dependency test. Almost all sample treatments, including HW-NP, resulted in a reduction in cell viability, except for LW-MP3 at the lowest dose (0.25 mg / mL) for all three exposure periods, HW-NP at 0.5 and 1 mg / mL up to 48 hours, and LW-MP2 and 4 at the lowest dose (0.25 mg / mL) at 48 and 72 hours, respectively (Figures 8A to 8F). The HT-29 adenocarcinoma cell line was more resistant to pectic treatment, producing reductions in cell viability only within the 72-hour period, to a greater extent in LW-MPs.At 24 h, LWMP4 was also significant in reducing cell viability at all doses, while LW-MP2 showed a major impairment of viability only at the two highest doses, and LW-MP3 was not significant (Figures 8A to 8F). HW-NP was able to reduce cell viability at the highest dose, but was not significant at any of the other doses.

[130] The IC50 potential estimate is shown in Figures 8A to 8F. For the HCT-116 cell line, greater precision was obtained, with IC50 ranging between 1.551 and 1.772 mg / mL for the LW-MP3 and 4 samples, at 48 and 72 h. Neither HW-NP nor LW-MP2 Petition 870250016807, dated 28 / 02 / 2025, page 55 / 115 33 / 52 estimated IC50 values ​​at any time point. However, in the HT-29 adenocarcinoma cell, all four samples were able to achieve an IC50 value at 72 h, while all modified samples had the lowest required doses, with an IC50 of 0.8562 mg / mL for LW-MP2, 0.8474 mg / mL for LW-MP3, and 0.8374 mg / mL for LW-MP4 (Figures 9A to 9H).

[131] By cross-referencing the results between both cell lines, the LW-MP3 and LW-MP4 samples were the most effective in reducing the viability of colorectal cancer cells and, therefore, LW-MP2 was excluded from further evaluations.

[132] Before performing any other biological assays related to the treatment of cancer cells, a cellular immunofluorescence assay was performed to better understand whether the polysaccharide samples could interact with cancer cells and even be taken up in some way. The concentration used (1 mg / mL) was chosen based on two aspects: 1) value lower than the IC50 observed in the HW-NP and LW-MP samples in the MTT assay; 2) Intermediate dose to avoid exaggerated fluorescent signals from antibody recognition. The results shown in Figures 21 and 22 showed that the incorporation of the polysaccharide samples into the cytoplasmic compartment of cancer cells (without experimental artifacts, as visualized in Figures 19 and 20) is a novel result, since no other document has addressed this issue with passion fruit polysaccharides.The result actually showed that pectic polysaccharides could have caused metabolic changes in cancer cells at the intracellular level and could corroborate the results discussed later. 5.2 LW-MP4 positively regulates the expression of p53 protein and cleaved caspase-9.

[133] The investigation continued into two main aspects relating to the potential inhibitory functions of Petition 870250016807, dated 28 / 02 / 2025, page 56 / 115 34 / 52 Cancer cell growth: 1) maintenance of the cell cycle and 2) induction of cell apoptosis. Regarding the first, cells can be maintained in G0 arrest under suboptimal circumstances through the activity of negative regulators such as p21, which is a direct inhibitor of cyclin-dependent kinases (CDKs), and p53, which will lead to cell cycle arrest, cell apoptosis, or DNA repair (Engeland, 2022). In the HT-29 cell, no significant difference in p21 expression was observed between control and polysaccharide-treated cells, both unmodified and modified samples (Figures 10A and 10B). However, a circumstantial statistical difference (p = 0.0396) was found in the upregulation of p53 between the control and LW-MP4 groups, and an even greater difference (p = 0.0249) within the control and LW-MP3 groups. This could mean a subtle change in the enhancement of the cellular machinery for cell cycle regulation by both modified samples.However, the TP53 gene is heavily mutated in HT-29 and may develop attenuated anti-oncogenic functionality. For HCT-116, it was observed that this same protein had approximately 1.8 times higher expression after treatment with LW-MP4 than the control (p = 0.0335, Figures 11A and 11B), corroborating the results of the cell proliferation assay. Although not statistically significant, a high upregulation trend for p21 can be observed in this cell line, which would mean continuity of the canonical cell cycle arrest pathway (average of 1.9 times, compared to the control, Figures 11A and 11B).

[134] In HCT-116 it was possible to differentiate the bands derived from both the whole caspase-9 and its cleaved active form of 37 kDa, due to visual clarity and antibody interaction. Procaspase 9 expression was negatively regulated by both LW-MPs, decreasing 1.6-fold with LWMP3 and 1.49-fold with LW-MP4. This decrease was translated to Petition 870250016807, dated 28 / 02 / 2025, p. 57 / 115 35 / 52 showed a 2.8-fold increase in the expression of cleaved caspase-9 for LW-MP4 (p = 0.0002, Figures 11A and 11B), but not for LW-MP3, which could represent that HCT-116 was potentially more susceptible to the induction of intrinsic / mitochondrial apoptosis in this sample. The expression of cleaved caspase-3 was not significantly affected by any of the FSA samples used; however, other pathways that were not investigated could be involved in a descending cascade, as discussed further below. 5.3 Inhibition of HCT-116 proliferation for LW-MP4 is confirmed by BrdU

[135] To assess whether the reduction in HCT-116 cell viability and protein expression values ​​translated into a functional alteration in the cell proliferation rate generating cell stasis, a BrdU colorimetric assay was performed with HW-NP and LW-MP4, comparing the unmodified and modified samples with the greatest effects. A low dose of doxorubicin control was used as a parameter where a proliferation of 83.52 ± 9.97% was observed in relation to the untreated control, with p = 0.0267.

[136] Both samples were able to impact cell proliferation in some way. BrdU measures nuclear DNA synthesis when incorporated as a thymidine analog. However, HW-NP had lower significance, with 84.8 ± 13.2%, which means a reduction close to 15%. Meanwhile, the modified sample reached 73.4 ± 12.6%, almost doubling the reduction (Figure 12, approximately 26%). COMPARATIVE EXAMPLES EXAMPLE 6 - Comparison between the processes tested in the present invention

[137] Further purification of samples prior to high-temperature treatment was evaluated to refine the procedure. For this purpose, the inclusion of a step was evaluated. Petition 870250016807, dated 28 / 02 / 2025, page 58 / 115 36 / 52 prior purification of the flour, as well as the evaluation of the absence of this step. For this purpose, a flowchart was developed to evaluate four slightly different extraction / modification scenarios, and to identify potential differences in the final results (Figure 13).

[138] The two main steps that differ between the samples are: 1) Pre-modification with or without washing with 80% ethanol in dry mass of flour for one hour; 2) Drying of the slurry (post-autoclave solution) or precipitation in ethanol. The first step described was added to evaluate the removal of impurities and greater refinement of the final sample, while the second step would be the difference in the amount of ethanol to be used. Post-autoclave drying reduced the mass of the slurry, which decreased the amount of ethanol needed for precipitation. However, direct precipitation without drying can ensure a greater recovery of some pectin fragments present in the solution, because it inhibits possibly undesirable aggregations and complexations derived from drying.

[139] As an indication that the stratification was a correct point, the samples had noticeable visual differences, in which there was less darkening of the samples that were washed with 80% ethanol before autoclaving. This may be an indication that some compounds present in the albedo, such as flavonoids or other phytoactives, may be heat-sensitive, and may, in the unwashed samples, have been condensed, guaranteeing a different color to the extracts.

[140] The difference in purity and color becomes even more pronounced in the next step, when positioning for drying in an oven or when adding absolute ethanol for precipitation.

[141] Finally, after drying, the samples in Figure 14 were crushed and washed with 80% ethanol. During the drying stage, these two samples became resistant to complete crushing by the available mill, generating residues. Petition 870250016807, dated 28 / 02 / 2025, page 59 / 115 37 / 52 above 0.84 mm. These were separated from the rest, as they do not dissolve as easily, and this would create a lack of standardization in the final sample. EXAMPLE 7 - Comparison between the samples obtained

[142] Molecular size analysis was performed again, now with all samples obtained by the new extraction flowchart. Notably, the tendency of the samples modified for 30 minutes to remain close to the unmodified sample was maintained. Among the samples modified for 60 minutes, the two that were precipitated after modification, and not dried, showed between approximately 10 and 20 kDa less than the dried ones. As briefly mentioned, this may be derived from the condensation of macromolecules both present in the initial matrix and formed by the high temperature. In any case, initially, this difference is small and was not an exclusive criterion of samples.

[143] Due to a combination of factors, such as the effects identified in cells, size within the expected range, and especially visual factors of purification and quality, the LW-MP4 sample modified for 60 minutes was defined as ideal. Both periods and different purification steps were evaluated. EXAMPLE 8 - Comparison of the application of modified flour compared to the water-soluble fraction of unmodified passion fruit flour.

[144] Generally, pectins (fiber present in the water-soluble fraction, WSF) are applied industrially to formulations such as yogurts, creams, and jams, which require a certain viscosity as a texture. For this, both citrus pectin and apple pulp pectin are the most commonly used, forming gels depending on the matrix in which they are inserted.

[145] During some tests, it was identified that in the case of flour extracted from passion fruit albedo, the water-soluble fraction Petition 870250016807, dated 28 / 02 / 2025, page 60 / 115 38 / 52 without thermal modification, it took longer to be incorporated into an aqueous solution without other components, even after heating to accelerate dissolution, as can be seen in the figures (concentration of 20 mg / mL of FSA) immediately after adding water (Figure 15A) and 20 minutes after incubation in an oven at 60 °C (Figure 15B).

[146] The modified passion fruit FSA was completely solubilized immediately after the addition of water (Figure 15A), showing how quickly this raw material can be applied. The difference in viscosity between unmodified and modified FSA is also noticeable. Furthermore, this test confirms the use of modified FSA in the incorporation of liquid products, where it does not alter the rheology of the product.

[147] Enriching beverages with modified passion fruit FSA would not initially imply a physical alteration of the product, but would improve the nutritional profile of such products.

[148] Both isolated FSA and modified whole flour can be used as products with distinct purposes. It is understood that modification has a greater influence by increasing the concentration of FSA in the flour, which improves the flour profile. EXAMPLE 9 - Comparison between the process of the present invention and prior art processes

[149] Starting with the study by Pinkaew et al. (2024) which deals with pectin extraction from cocoa, an extraction between 5 and 40 minutes was tested, between 105 and 135 °C, where the authors identified the extraction at 105 °C and for 5 minutes as the most optimized, generating a pectin yield of 26%. Structurally, the extracted pectin showed low esterification (34%), a weight of 80 kDa and 32% GalA, unlike the pectin obtained by the present invention.

[150] For the extraction of pectin from apple pulp by Chandel et al. (2016), the method consisted of evaluating Petition 870250016807, dated 28 / 02 / 2025, page 61 / 115 39 / 52 both heating at 95 °C for 1 hour at ambient pressure, and between 15 and 60 minutes inside an autoclave at 121 °C. The authors identified that extraction for 1 hour in the autoclave generated the best yield. However, the extraction process differed from that performed by the present invention in some points, such as: 1) water ratio for slurry formation (1:3 while 1:20 was used in the present invention, ensuring lower viscosity and greater contact surface for modification); 2) precipitation only in the filtered extract (supernatant) after heating, while precipitation was performed in the total material of the present invention, in order to recover semi-soluble fractions in water whose chain was reduced by the method employed; 3) they did not perform pre-washing steps with ethanol, responsible for removing soluble sugars and pigments.

[151] Furthermore, modified citrus pectin (MCP) by Jackson et al. (2007) was produced by autoclaving, named FPP (fragmented pectin powder), where the extraction is from native citrus pectin of the Sigma brand. In addition, Jackson et al. (2007) and Leclere et al. (2015) briefly indicate that the method consists of heating a 0.1% citrus pectin solution in an autoclave for 30 or 60 minutes at 121 °C. Interestingly, both articles, although citing each other, differ in the post-heating processing method. While one indicates that the solution was left at 4 °C in the refrigerator to form a gel (without the addition of any solvent) and then the aqueous part was lyophilized, the other mentions that all material was frozen at -80 °C and lyophilized.In both cases, precipitation with ethanol was not identified, perhaps because they started from a material already known as citrus pectins, and ethanol is essential for the precipitation of this macromolecule.

[152] Finally, studies such as the documents by Silva and colleagues (2020) and Canteri (2010) used extractions Petition 870250016807, dated 28 / 02 / 2025, p. 62 / 115 40 / 52 acidic solutions, containing nitric acid (1:50 m / V 1M for D2, between 1 and 92 mM for D3) and heating between 80 and 97 °C. In Silva's document, besides the raw material being the same, nothing is directly comparable. The method consisted of dissolving 1g of material in 50 mL of 1M nitric acid and heating at 80 °C for 40 minutes. Only what was solubilized in acid, and not the remaining solid, was precipitated with absolute ethanol for isolation of the pectins.

[153] Canteri's work used the same pectin extraction methodology with nitric acid. Autoclaving was used on the raw material. In fact, the authors used an autoclave for 15 minutes at 121 °C to verify increased yield / structural change. However, this pretreatment of the initial flour was not isolated and aimed to evaluate physicochemical changes compared to other methods, such as maceration with hot ethanol. The pectin was in fact extracted as usual with nitric acid, and precipitation with ethanol was done only on the final supernatant, and not on the total content, unlike what was done in the present invention.

[154] Table 1 below compares the main differences between the extraction methodologies of the present application in relation to the state of the art. Table 1. Differences in methodology between the present invention and the prior art. Processes Step Present invention ABC Obtaining the initial material Mesocarp cutting, dehydration in an oven with air circulation at 45 °C for After juice removal, apple pulp was dehydrated at 55 °C (without time Cocoa shell was cut and dried at 60 °C for 48 hours in an oven, ground and sieved The raw material (citrus pectin) was purchased in powder and commercially Petition 870250016807, dated 28 / 02 / 2025, page 63 / 115 41 / 52 16 hours, and grinding described) and ground available (Sigma) Pre-washing / removal of undesirable compounds Ethanol ratio 1:3 (flour mass / ethanol volume) for 1 hour under constant magnetic stirring, filtration and drying No process No process No process Modification per se Suspension in distilled water at a ratio of 1:10, gentle heating to 60 °C to solubilize the sample, transfer to autoclave and heating for 1 hour at 121 °C Suspension in distilled water at a ratio of 1:3 and heating in an autoclave at 121 °C (between 15 and 60 minutes) or boiling (95 °C 1 hour) Suspension in distilled water at a ratio of 1:30, heating in an autoclave between 5 and 40 minutes, temperature between 105 and 135 °C Solution of 0.1%, heated for 30 minutes (Jackson et al, 2007) or 60 minutes (Leclere et al, 2015) at 123 °C. Precipitation: Total material (slurry with soluble and insoluble parts) precipitated in absolute ethanol at a 1:3 ratio in a refrigerator. Only filtered material (soluble) precipitated in absolute ethanol at a 1:2 ratio (temperature not described). Only filtered material (soluble) precipitated in ethanol at a 1:1 ratio at room temperature (25 °C). No precipitation in ethanol. Finalization: Filtration through a silkscreen mesh and drying of the material in an air oven. Filtration and drying (methodology not specified). Centrifugation, washing with acetone and drying at 60 °C for 4 to 8 hours. Cooling at 4 °C (Jackson et al 2007); Freezing at -80 °C (Leclere et al.). Petition 870250016807, dated 28 / 02 / 2025, page 64 / 115 42 / 52 Circulating and vacuum oven (1 day and 4 hours, respectively) hot air oven 2015); Freeze-drying in both (A) Chandel et al. (2016); (B) Pinkaew et al. (C) Jackson et al. (2007) and Leclere et al. (2015). EXAMPLE 10 - Comparison between modified pectin obtained by the process of the present invention and pectins of the prior art.

[155] Compared to other prior art pectin modification processes, the average weight, monosaccharide composition, and degree of methylation of the pectin of the present invention were different from those of the art. According to Nascimento (2020), while the extracted FSA without modification had the same average weight pattern above 750 kDa, with a similar chromatographic profile, the modification using 3M NaOH and 3M HCl induced an even greater chain breakage, where both samples, both the modified and then extracted, and the extracted and then modified (ME and EM, respectively), presented between 30 and 40 kDa, a value 2 to 2.5 times lower than those presented in the present invention. Furthermore, although GalpA is still the largest component of these pectins, its representation varied between 65 and 90%, with higher amounts of neutral monosaccharides in the EM and native fractions.To conclude the analysis of differences, both EM and ME fractions had a degree of methylation determined between 0 and 22%, much lower than the native fraction in question (64%), and much lower than those resulting from the present invention.

[156] US patent 7,452,871 describes citrus pectins modified by acidic pH and temperature. In addition to the method involving the use of acids to lower the pH of the solution in which the native citrus pectin is dissolved, significant differences in the final structure are observed, such as molecular size (between 5 Petition 870250016807, dated 28 / 02 / 2025, page 65 / 115 The presence of monomeric GalpA residues (between 5 and 19%) and values ​​of 43 / 52 and 16 kDa are evidence of profound differences compared to the product of the present invention. Furthermore, the patent inventors cite that the degree of esterification is less than 5%, and that the structure contains 10% RG-II, a rarer substructure of some pectins.

[157] The documents by Silva and colleagues (2020) and Canteri (2020) do not present chemical and structural characteristics of pectin, and are not relevant to this aspect of the invention.

[158] The article by Jackson et al. (2007) details structural aspects of the modified citrus pectin obtained (MCP / FPP), but not with much precision. For example, regarding molecular weight, only the chromatographic profile is presented, and it is indicated that the dispersion is between citrus pectin (CP) and pH-modified pectin, suggesting an intermediate weight between 20 and 73 kDa. As for its composition, FPP presented approximately 80% GalpA, followed by Ara and Gal in smaller quantities. Finally, although the article by Jackson et al. (2007) indicates the importance of the degree of esterification for bioactivity (removing esterifications indicates loss of function), concrete numbers for the degree of methylation / esterification are not revealed. But in any case, modified citrus pectins do not present high methylation profiles, as does the potential product resulting from the present invention.

[159] Table 2 below presents a comparison between the pectin obtained by the present invention and the pectins of the prior art. Table 2. Differences in the chemical structures of pectins. Pectins Structural factor Present invention ABC Petition 870250016807, dated 28 / 02 / 2025, page 66 / 115 44 / 52 Molecular size 80 kDa Equivalent weight of 942.04 (not commonly used for this type of material) 80 kDa 23 to 71 kDa Degree of esterification Between 70 and 85% (high esterification) 56% (slightly high esterification) 34% (low esterification) 0% Monosaccharide composition 44% GalA in total dry mass, 65% water-soluble fraction (WSF) 43% GalA (dry mass) 32% GalA (dry mass) Not specified (A) Chandel et al. (2016); (B) Pinkaew et al. (C) Jackson et al. (2007) and Leclere et al. (2015).

[160] As can be seen from the table above, the present invention provides modified pectin with different characteristics compared to pectins obtained in the prior art, exhibiting lower molecular weight, high esterification and a higher fraction of GalA. EXAMPLE 11 - Comparison between biological activities evaluated in the present invention and the prior art. Table 3. Differences in pectin extractions and modifications and the resulting biological effects in relation to the thesis of Dr. Raíssa Sansoni do Nascimento. Petition 870250016807, dated 28 / 02 / 2025, page 67 / 115 45 / 52 Use Study Chemical agents in extraction Applicability in aqueous solutions? Cell viability (cancer cell lines) Cellular effects found Effects evaluated in vivo Present Invention Ethanol Yes, up to 30 mg / mL without viscosity change Reduction in the viability of HCT116 and HT29 Increase in cleavage of caspase-9 (apoptosis) and p53 (cell cycle) protein Decrease in intestinal inflammation, fewer polyps and neoplastic lesions, lower clinical scores of animals, improvement in weight gain curve Nascimento (2020) Chloroform, methanol, NaOH, hydrochloric acid Not tested Reduction in the viability of HCT116 and HT29 No identification of specific proteins Not tested

[161] Thus, the comparative examples presented demonstrate that the present process differs from other processes in the pre-modification steps, such as washing with ethanol and its mass / volume ratios, as well as resuspension in water / formation of the slurry; and in the form and stage in which precipitation with solvent (ethanol) occurs to obtain the polysaccharides. The striking differences, such as combinations of fundamental variables such as the presence of a pre-modification step Petition 870250016807, dated 28 / 02 / 2025, page 68 / 115 46 / 52 washing, time and temperature of this, ethanol ratio, water ratio, time, modification temperature, selection of total material or supernatant for precipitation generates a series of different intermediate structures, which in the case of passion fruit pectins, provide novel medium molecular weight structures with a high degree of methylation and also new and surprising biological effects. BIBLIOGRAPHIC REFERENCES

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Claims

1 / 3 CLAIMS 1. Process for pectin extraction, characterized in that it comprises the steps of: (i) pre-washing a fruit flour with solvent using constant magnetic stirring; (ii) filtering and drying the material in constant air circulation; (iii) suspending in an aqueous environment; (iv) placing the solution in an autoclave at a temperature of 90 °C to 135 °C for 15 to 90 minutes, 1 atm; (v) precipitating the material in solvent, and leaving it in a refrigerator at 4 °C overnight; (vi) filtering the material through a silkscreen mesh; and (vii) drying it in a vacuum oven.

2. Process, according to claim 1, characterized in that the fruit flour is citrus flour, cocoa, apple, papaya, orange, jaboticaba, tomato, carrot, beet, strawberry, grape, eggplant, plum, blackberry, lemon and preferably, passion fruit.

3. Process according to claim 1, characterized in that the solvent is selected from polar and non-polar solvents such as methanol, butanol, isopropanol, acetonitrile, chloroform, toluene, ethyl acetate and ethanol.

4. Process according to claim 3, characterized in that the solvent is ethanol.

5. Process, according to claim 4, characterized in that the pre-washing is carried out with ethanol in a ratio of 1:2 to 1:20 (m / v) for one hour under constant magnetic stirring.

6. Process, according to claim 5, characterized in that the pre-washing is carried out with ethanol in a 1:3 (m / v) ratio for one hour under constant magnetic stirring. Petition 870250016807, dated 28 / 02 / 2025, p. 76 / 115 2 / 3 7. Process, according to claim 1, characterized in that the suspension is made with water in a ratio of 1:2 to 1:100 (m / v) and heating is carried out between 30 and 90 °C, for 15 to 180 minutes.

8. Process, according to claim 7, characterized in that the suspension is made with water in a ratio of 1:20 (m / v) and the heating is carried out between 50 and 60 °C, for 15 to 30 minutes.

9. Process according to claim 1, characterized in that autoclaving is carried out at a temperature of 121 °C for 60 minutes, 1 atm.

10. Process according to claim 1, characterized in that the precipitation of the product is carried out with 99% ethanol solvent in a ratio of 1:2 to 1:4 (v / v).

11. Process according to claim 10, characterized in that the precipitation of the product is carried out with 99% ethanol solvent in a 1:3 (v / v) ratio.

12. Process according to claim 1, characterized in that the filtered material is dried in a vacuum oven for 1 to 8 hours.

13. Process according to claim 12, characterized in that the filtered material is dried in a vacuum oven between 30 and 90°C for 4 hours.

14. Modified pectin obtained by the process as defined in any one of claims 1 to 13, characterized in that it has: a) a degree of methyl esterification of 70 to 85%; b) an average molecular weight between 70 and 90 kDa; and c) between 40 and 50% galacturonic acid in the total dry mass.

15. Modified pectin, according to claim 14, characterized by being a food supplement or food additive. Petition 870250016807, dated 28 / 02 / 2025, page 77 / 115 3 / 3 16. Use of modified pectin as defined in claim 14, characterized by the fact that it is for the preparation of a food supplement, prevention or adjuvant treatment of cancer.

17. Use, according to claim 16, characterized in that the cancer is colorectal cancer. Petition 870250016807, dated 28 / 02 / 2025, p. 78 / 115

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