Process of obtaining an extract of astronium sp. and use of the same

Description

1 / 26 PROCESS FOR OBTAINING AN EXTRACT OF ASTRONIUM SP. AND ITS USE FIELD OF APPLICATION

[001] The present invention relates to the field of biotechnology and pharmacology, with a special focus on natural products for the development of anti-inflammatory therapies and potential application in the pharmaceutical and cosmetic industry, in the form of a process for obtaining an extract from the plant Astronium sp. and its use. FUNDAMENTALS OF THE INVENTION

[002] Astronium fraxinifolium, popularly known as "Gonçalo Alves", "Gonçaleiro" or "Aroeira do campo", is a tree native to Brazil, found mainly in the Atlantic Forest region. Belonging to the Anacardiaceae family, this plant is known for having compound, alternate, and lanceolate leaves. In addition, its flowers are small and yellowish, gathered in panicle-type inflorescences. Finally, its fruits are oval drupes, edible and yellow when ripe, attracting birds and other animals that contribute to the dispersal of its seeds.

[003] The plant is recognized for its medicinal properties, the beauty and durability of its wood, used in the manufacture of furniture and handicrafts. In folk medicine, various parts of the plant are used to treat different conditions, such as fevers, headaches, inflammations, tuberculosis, hemorrhoids, and digestive problems. Scientific studies have investigated the therapeutic potential of Astronium fraxinifolium, especially due to Petition 870250008869, dated 03 / 02 / 2025, page 10 / 116 2 / 26 due to the presence of compounds with antioxidant and anti-inflammatory properties (Lorenzi et al., 1992). In addition to its medicinal applications, the wood of Astronium fraxinifolium is valued for its resistance and beauty, being used in the manufacture of quality furniture and decorative objects.

[004] Due to its great potential reported in folk medicine, several research groups explored the potential of Gonçalo Alves between the years 2004 and 2021.

[005] In this context, inflammation represents a defense mechanism of our organism, in which there is recognition of a harmful stimulus and the organism responds to it. Several factors can initiate the inflammatory process, among them: a blood clot that induces an ischemic stroke; an immune system disorder; cancer; a neurological condition, such as Alzheimer's disease; and infections by viral, bacterial, fungal, and protozoan pathogens.

[006] To treat these inflammatory disorders, the most recommended medications are nonsteroidal anti-inflammatory drugs and corticosteroids. However, the use of these medications is associated with several adverse effects. For example, long-term use of corticosteroids is associated with a series of adverse effects, requiring constant monitoring of the patient. Since they alter the body's overall metabolism, they have the ability to reduce glucose uptake and utilization, increase gluconeogenesis, promote rebound glycemia, glycosuria, as well as increase catabolism and reduce protein anabolism. The main adverse effects associated with their use are diabetes, susceptibility to infections, Petition 870250008869, dated 03 / 02 / 2025, page 11 / 116 3 / 26 Osteoporosis, cataracts, hypertension, myopathies, neurological disorders, nephrotoxicity, and increased risk for the development of cardiovascular diseases.

[007] However, during the inflammatory process, the excessive production of pro-inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor alpha (TNFα), interleukins, chemokines, adhesion molecules, and eicosanoids, can contribute to tissue damage. Therefore, numerous inflammatory diseases, autoimmune conditions, allergies, and immunodeficiency syndromes can arise when the immune system is activated in an undesirable or exacerbated way.

[008] Therefore, there is a need for new therapeutic alternatives with fewer adverse effects. Natural compounds represent a promising source for prospecting therapeutic agents. The use of medicinal plants for the treatment of various diseases is a recognized practice that has been used for thousands of years. Currently, more than 50% of available drugs are derived from or inspired by natural products.

[009] In this context, a herbal medicine with anti-inflammatory activity will be useful for the treatment of various inflammatory conditions.

[010] In view of the aforementioned problems, the present invention aims to provide a process for obtaining a bioproduct, which is an extract obtained from the plant Astronium sp., and its use.

[011] The Gonçalo-Alves plant has not yet been explored for its anti-inflammatory potential, therefore it represents a new source of bioactive molecules with Petition 870250008869, dated 03 / 02 / 2025, page 12 / 116 4 / 26 anti-inflammatory properties, expanding therapeutic possibilities. This offers a competitive advantage and contributes to the diversification of the arsenal of natural compounds available on the market.

[012] The developed extract has been validated as a potent anti-inflammatory, with promising results in preclinical tests indicating high efficacy in controlling inflammation, with a lower risk of adverse effects common in synthetic drugs, such as gastric and renal problems.

[013] The plant used can be cultivated sustainably, favoring the local economy and the preservation of biodiversity. This provides an economic and environmental advantage, guaranteeing the production of raw materials with low environmental impact. STATE OF THE ART

[014] When comparing the process for obtaining and applying the present invention for Astronium sp. extract with the documents listed below, significant differences are observed in the proposed applications:

[015] Patent document KR20200117501 refers to natural compounds, such as baicalin, polyzalin, or ebadiamine, which aim to inhibit the activity of the SHIP2 enzyme to prevent the aggregation of tau protein, associated with neurodegenerative diseases. In contrast, the invention presented here focuses on a plant extract with anti-inflammatory properties for use as a component in supplements, functional foods, or herbal remedies, without specific emphasis on neurodegenerative diseases.

[016] Patent document CN109232560 refers to an insecticidal protein and methods for its use in controlling Petition 870250008869, dated 03 / 02 / 2025, page 13 / 116 5 / 26 agricultural pests. The present invention differs from CN109232560 in terms of application, as it is intended for use in humans.

[017] Patent document CA2253440 refers to a new PAF receptor antagonist, the sulfolipid SQDG, as well as the use of SQDG for the prophylaxis or treatment of inflammatory skin diseases, especially psoriasis. The present invention differs from CA2253440, as it is intended for use as a component of supplements and functional foods with anti-inflammatory action, without specificity of the site of action.

[018] Patent document ES2402039 describes a biofertilizer for non-leguminous plants based on rhizobium strains, aiming to promote plant growth. In contrast, the present invention is intended for human use as a component of supplements and functional foods with anti-inflammatory action, differing completely from the teaching of ES2402039.

[019] With regard to document EP3102684, it refers to insecticidal proteins and methods for their use in pest control. The present invention, on the other hand, is intended for human use as a component in supplements, functional foods or herbal medicines with anti-inflammatory action, without relation to pest control.

[020] Patent documents WO2018232072 and WO2019178042 address insecticidal proteins and methods for their use in the control of agricultural pests. In contrast, the invention presented here proposes a component in supplements, functional foods or herbal medicines with general anti-inflammatory action.

[021] Document CN110582508 refers to insecticidal proteins and methods for their use in pest control. Petition 870250008869, dated 03 / 02 / 2025, page 14 / 116 6 / 26 In contrast, the present invention is intended for use as a component in supplements, functional foods, or herbal remedies, with different methods of preparation and application.

[022] Patent document WO2015120276 describes insecticidal proteins and methods for their use in pest control. The present invention focuses on an extract of Astronium sp. with anti-inflammatory action for human use, which differs completely in application and purpose.

[023] In summary, the present invention differs from the documents mentioned by focusing on a specific extract of Astronium sp. with anti-inflammatory action for use as a component in supplements, functional foods or human phytotherapeutic products, while the other patents address different compounds, mechanisms of action and therapeutic or agricultural applications. SUMMARY OF THE INVENTION

[024] Astronium fraxinifolium, popularly known as “Gonçalo Alves”, “Gonçaleiro” or “Aroeira do campo”, is a tree native to Brazil, found mainly in the Atlantic Forest region. The plant is recognized for its medicinal properties, the beauty and durability of its wood, used in the manufacture of furniture and handicrafts. Due to various side effects from the use of traditional medications to treat inflammatory processes, there is a need for new therapeutic alternatives with fewer adverse effects. Thus, natural compounds represent a promising source for prospecting therapeutic agents. Petition 870250008869, dated 03 / 02 / 2025, page 15 / 116 7 / 26

[025] In view of this context, the present invention discloses a process for obtaining an extract from the plant Astronium sp and its use, which has been validated as a potent anti-inflammatory, with a lower risk of adverse effects common in synthetic drugs, such as gastric and renal problems. BRIEF DESCRIPTION OF THE FIGURES

[026] The invention can be better understood through the brief description of the following Figures:

[027] Figure 1 illustrates the steps in the process of obtaining the extract from the Gonçalo-Alves plant.

[028] Figure 2A illustrates the color of the liquid extracts after filtration.

[029] Figure 2B illustrates the color of the extracts liquid after filtration and drying.

[030] Figure 3 illustrates that the AF_MeOH extract does not exhibit cytotoxicity on macrophage cultures stimulated with LPS + IFNγ.

[031] Figure 4 illustrates the quantification of nitrite production in the supernatant of activated peritoneal macrophage cultures treated in vitro with the extract.

[032] Figure 5 shows the quantification of TNF-α production in peritoneal macrophage cultures treated in vitro with the extract.

[033] Figure 6 shows the quantification of IL-2 production in splenocyte cultures from BALB / c mice treated in vitro with AF_MeOH.

[034] Figures 7A, 7B, 7C, 7D, 7E and 7F illustrate different results of lymphocyte proliferation inhibition by the AF_MeOH extract. Petition 870250008869, dated 03 / 02 / 2025, page 16 / 116 8 / 26

[035] Figure 8 illustrates the survival curve of mice treated with different extracts of Astronium fraxinifolium and subjected to endotoxic shock.

[036] Figure 9 reveals the survival curve of mice treated with different extracts of Astronium fraxinifolium and subjected to endotoxic shock. DETAILED DESCRIPTION OF THE INVENTION

[037] The invention may be better understood through the following detailed description, in accordance with the attached Figures.

[038] The present invention describes the process for obtaining a bioproduct, in the form of an extract obtained from the plant Astronium sp. and its use as an anti-inflammatory.

[039] The extract comprises: Astronium sp. plant matrix and solvent, both with a purity ranging from 95 to 99%.

[040] It is noteworthy that for the realization of the present invention, species of Astronium sp. selected from the group comprising: Astronium balansae Engl., Astronium concinnum Schott, Astronium fraxinifolium Schott, Astronium glaziovii Mattick, Astronium graveolens Jacq., Astronium lecointei Ducke, Astronium nelson-rosae Santin, Astronium obliquum Griseb., Astronium pumilum JDMitch. & Daly, Astronium ulei Mattick, Astronium urundeuva (M.Allemão) Engl. may preferably be used, with the species Astronium fraxinifolium Schott being preferred.

[041] The solvent used is selected from the group comprising: methanol (99%), grain alcohol, water, absolute alcohol or a mixture thereof, with methanol (99%) being preferred. Petition 870250008869, dated 03 / 02 / 2025, page 17 / 116 9 / 26

[042] The process of obtaining the extract from the Astronium sp. plant, as can be seen in Figure 1, comprises the following steps: (a) Collection of the Astronium sp. sample; (b) Increase in the contact surface of the Astronium sp. sample matrix; (c) Obtaining the extract; (d) Filtration and concentration of the extract; (e) Storage of the extract.

[043] In step (a), the sample is collected, which is selected from the group comprising: leaves, flowers, root, stem and fruits, preferably the stem bark of Astronium sp. The collected fragments are carefully selected, prioritizing undamaged or uncontaminated areas. A pruning tool is used to remove the fragments.

[044] In step (b), the sample is subjected to a mechanical process to increase the sample's contact surface. The sample is ground with a laboratory mill or a knife mill, resulting in a powder with a particle size ranging from 18-20 mesh. This material is stored in capped tubes, made of plastic or glass, and kept at controlled temperatures between -18°C and -22°C until the extract is prepared.

[045] In step (c), the extract is obtained by immersing 4.5 to 5.5 g of the Astronium sp. plant matrix in 22 to 37 mL of solvent, preferably at a ratio of 1:5 ± 0.5 m / v. The mixture obtained previously is agitated in an oven at a temperature between 44 and 46°C and a rotation speed between 200 and 250 RPM for 47 to 49 h. Petition 870250008869, dated 03 / 02 / 2025, page 18 / 116 10 / 26

[046] In step (d), the extract is filtered with 80g / cm3 qualitative paper or filter compatible with the preferred solvent and pore size between 8 and 10 μm and subsequently concentrated in a vacuum centrifuge, with a temperature variation between 34 and 36°C, until the absence of liquid or semi-liquid viscosity of the extract is observed, taking 8 to 12 h to dry 25 mL of extract, for example.

[047] In step (e), the extract obtained is packaged in bottles with lids and stored at -18°C to -22°C.

[048] The extract obtained can be used as a component in supplements, functional foods or herbal medicines, with emphasis on combating inflammatory diseases. The extract can be used in various forms of presentation, including capsules, tablets, sachets, solutions, suspensions, syrups, gels, creams, ointments, lotions, serums, suppositories, transdermal patches, oral or nasal sprays and inhalers. It is indicated for therapeutic and cosmetic treatments, as an anti-inflammatory. Examples Example 1: Phytochemical analysis

[049] Three adapted methodologies (BARRETO et al., 2022) were applied for the analysis of phytochemicals, detailed below. 1. Quantification of Phenolic Acid

[050] The phenolic acid content was measured based on a reaction with the Folin-Ciocalteu reagent, using a plate spectrophotometer at 765 nm. The result was expressed in milligrams of gallic acid (reference standard and marker of the GONÇALO-ALVES plant) per gram of sample (mgGAE / g). A calibration curve was used for this purpose. Petition 870250008869, dated 03 / 02 / 2025, page 19 / 116 11 / 26 (y=0.004x+0.1243; R2: 0.9886), a reference line obtained from the analysis of different concentrations of gallic acid (7.8 to 250 μg / mL), under the same analysis conditions as the samples. 2. Flavonoid Quantification

[051] The amount of flavonoids was measured using a plate spectrophotometer at 415 nm. For this, a calibration curve made with quercetin (flavonoid standard and marker of the GONÇALO-ALVES plant) was used (y=0.02x-0.0105; R2: 0.9984), with concentrations between 4 and 63 μg / mL. The results were expressed in milligrams of quercetin per gram of sample (mgEQ / g). 3. Antioxidant Capacity (AC)

[052] To evaluate the antioxidant activity of the extract, the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method was used, which measures the ability of extracts to neutralize free radicals, molecules that can cause damage to cells. The antioxidant capacity was measured in a spectrophotometer at 517 nm, using an ethanolic solution of the extract at 0.13 mg / mL. The ability to neutralize radicals was expressed as the percentage of inhibition (reduction) of oxidation through equation 1. Equation 1: % CA = 100 - [ (final absorbance of the sample x 100) / absorbance of the blank]. Example 2: Animal testing

[053] BALB / c mice, aged between 4 and 10 weeks, were supplied by the animal facility of the Gonçalo Moniz Institute and kept in the animal facility in cages containing a maximum of 5 mice. The animals had free access to water and food. Petition 870250008869, dated 03 / 02 / 2025, page 20 / 116 12 / 26 throughout the experiment. The animal experiments were submitted to the ethics committee of FIOCRUZ (L-007 / 2024). Example 3: Substances used

[054] Dexamethasone was purchased from SigmaAldrich (St. Louis, MO). Extracts of Astronium fraxinifolium (GONÇALO-ALVES) were produced according to the methodology described above. In all in vitro assays performed, the Astronium fraxinifolium extracts and dexamethasone were first solubilized in dimethyl sulfoxide (DMSO; PanReac, Barcelona, ​​Spain) and subsequently in culture medium, not exceeding a final concentration of 0.1% DMSO in the tests. For the endotoxic shock model assays, the substances were solubilized in 5% DMSO and subsequently in 1x PBS. Example 4: Cytotoxicity assessment with AlamarBlue

[055] To evaluate the cytotoxicity of the substances under test and determine the CC50 values, the AlamarBlue colorimetric method (Invitrogen, Carlsbad, CA) was used. AlamarBlue (resazurin) is an indicator that produces a colorimetric change and a fluorescent signal in response to metabolic activity. Resazurin is reduced to resorufin by metabolically active cells. The oxidized form is blue (non-fluorescent / non-viable cell) and the reduced form is pink (fluorescent / viable cell). The reduction of resazurin to resorufin reflects cell viability.

[056] In the assay in question, peritoneal macrophages, obtained from peritoneal lavage of BALB / c mice stimulated with 3% thioglycolate (Sigma Aldrich), were distributed in 96-well plates at a predefined density of 2 x 105 cells / well. The methanolic extract and the Petition 870250008869, dated 03 / 02 / 2025, page 21 / 116 13 / 26 dexamethasone was added in a series of 6 concentrations (100 to 3.12 μg / mL) in the presence of lipopolysaccharide (LPS, 500 ng / mL, Escherichia coli serotype O111:B4, Sigma-Aldrich) and IFN-γ (5 ng / mL, Sigma-Aldrich). The negative control consisted of cells that received the same amount of DMSO as the drug-containing wells. The plates were incubated for 24 hours in an incubator at 37°C and 5% CO2. After this period, 20 μL / well of AlamarBlue was added and the plates were incubated for another 4 h. The plates were read using a spectrophotometer (Spectramax 190, Molecular Devices, Sunnyvale, CA) at wavelengths of 570 and 600 nm. All analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA). Example 5: Macrophage culture

[057] Peritoneal macrophages (2 x 105 cells / well), obtained from peritoneal lavage of BALB / c mice stimulated with 3% thioglycolate, were incubated in 96-well plates in complete DMEM medium, in triplicate, and stimulated or not with LPS (500 ng / mL) and IFN-γ (5 ng / mL), in addition to being treated or not with the evaluated substances. The cells were kept in an incubator at 37°C and 5% CO2 for 24 hours. After this period, the culture supernatants were collected for nitrite and TNFα assay. Example 6: Lymphoproliferative assay

[058] BALB / c mouse splenocytes (5 x 106 cells / well) were cultured in complete DMEM medium in 24-well plates, in triplicate, with or without concanavalin A (Con A; 5 μg / mL) and with or without the substances under evaluation for 72 hours in an incubator at 37°C and 5% Petition 870250008869, dated 03 / 02 / 2025, page 22 / 116 14 / 26 of CO2. Lymphocyte proliferation was assessed by CFSE staining, as previously described in the literature (MEIRA et al., 2017). In addition, a second set of experiments was performed to measure IL-2 and IFNγ in the supernatant of splenocyte cultures from the different experimental groups after 24 hours of treatment. Example 7: Cytokine and nitric oxide dosage

[059] IL-2, IFNγ and TNF-α levels were measured from cell culture supernatants using the sandwich ELISA technique, employing the protocol, antibodies and recombinant cytokines from the Duoset ELISA Development System kit (R&D Systems, Minneapolis, MN). Nitric oxide production in macrophage supernatant was determined through its oxidative product, nitrite, using the Griess method (GREEN et al., 1982). Example 8: Endotoxic shock model

[060] Male BALB / c mice (14-18g) were pre-treated, orally or intraperitoneally, with different doses of methanolic extract (100, 50 and 25 mg / Kg) or aqueous extract (100 mg / Kg) or ethanolic extract (100 mg / kg) or dexamethasone (2 mg / Kg) or vehicle solution (Saline solution containing 5% DMSO). Ninety minutes later, the animals were challenged with 600 μg of lipopolysaccharide (LPS; Escherichia coli serotype O111:B4, Sigma-Aldrich) in saline solution, intraperitoneally. The survival of the mice was then monitored daily for 4 days. Example 9: Results

[061] After the extraction period, both the liquid extract and the extract after the drying process showed Petition 870250008869, dated 03 / 02 / 2025, page 23 / 116 15 / 26 reddish coloration (with intensity varying between medium red (CIELAB code #d11507) to deep red (CIELAB code #52170b), as observed in Figure 2.

[062] The dry extract presented a viscous and uniform texture, with a characteristic woody aroma. Regarding phytochemical quantification, the (dry) extract of the Gonçalo-Alves matrix showed excellent potential in bioactive compounds, in addition to a high antioxidant capacity. The results obtained are presented in Table 1 below. Table 1: Quantification of gallic acid (phenolic acid), quercetin (flavonoid), and antioxidant capacity of the Gonçalo-Alves plant extract.* Phenolic acid* Flavonoid* Antioxidant capacity* mgEAG / g CV% mgEQ / g CV% % CV% 466.24±37.13 8 148.10±5.38 4 80.52±4.46 6 *Results presented as mean ± standard deviation and coefficient of variation (CV%).

[063] All analyses performed had a coefficient of variation (CV%) below 10%, which confirms the reliability of the results. The gallic acid and quercetin content found shows that the extract is likely to have anti-inflammatory properties, antimicrobial activity, as well as antioxidant capacity (Carrillo-Martinez et al., 2024; Harwansh et al., 2024).

[064] The presence of this last property is reinforced by the DPPH radical antioxidant capacity assay, where 0.13 mg / mL of the study matrix extract was able to inhibit 80.52% of the free radical present in the assay system. It is worth remembering that free radical molecules are produced Petition 870250008869, dated 03 / 02 / 2025, page 24 / 116 16 / 26 naturally occur in the body during metabolic processes, such as cellular respiration, and in small quantities, free radicals play an essential role in bodily functions, such as the immune response, helping to destroy invading microorganisms (Yoshikawa & You, 2024). However, an excess of free radicals promotes an imbalance known as oxidative stress. This stress can damage important cellular components, such as lipids, proteins, and DNA, which contributes to premature aging and increases the risk of developing chronic diseases (Krishnamurthy et al., 2024). Therefore, this extract has the potential to be an important component in supplements, functional foods, or as a phytotherapeutic agent due to its high antioxidant capacity and therapeutic activity.

[065] Regarding biological validation assays, initially the cytotoxicity of the extract was evaluated in peritoneal macrophage culture stimulated with LPS + IFNγ using the Alamar Blue method. As can be seen in Figure 3, the extract and dexamethasone did not show cytotoxicity at the tested concentrations. Peritoneal macrophages were stimulated with LPS + IFNγ and treated with different concentrations of AF_MeOH (100-12.5 μg / mL) or dexamethasone (12.5 μg / mL) for 24 hours. Cell viability was determined by the addition of Alamar Blue dye. Values ​​represent the mean ± SD of four replicates obtained in 1 of 2 experiments performed.

[066] Next, the anti-inflammatory effect of the extract was initially evaluated by quantifying nitric oxide production, using the Griess method, in cultures Petition 870250008869, dated 03 / 02 / 2025, page 25 / 116 17 / 26 of macrophages activated with LPS and IFNγ. Nitric oxide (NO) is essential in the inflammatory response, promoting vasodilation, modulation of immune cells and antimicrobial activity, in addition to controlling the production of reactive oxygen species. As we can observe in Figure 4, nitrite concentrations were determined in the supernatants of peritoneal macrophage cultures treated or not with extract (100, 50, 25 and 12.5 μg / mL) or dexamethasone (Dexa; 12.5 μg / mL) in the presence of LPS (500 ng / mL) and IFN-γ (5 ng / mL). Values ​​represent the mean ± SD of four determinations obtained in one of two experiments performed. *P < 0.05 compared to untreated cultures and those stimulated with LPS + IFN-γ. Thus, the extract significantly reduced (p < 0.05) nitric oxide production in a concentration-dependent manner, especially at concentrations of 100 and 50 μg / mL, where it inhibited nitrite production by 88% and 66%, respectively.

[067] Aiming at a better characterization of the anti-inflammatory effect of Astronium fraxinfolium extract, we quantified tumor necrosis factor alpha (TNF-α) in macrophage cultures activated with LPS and IFNγ, using the ELISA method. TNF-α is a central cytokine in the inflammatory response, acting in the activation of immune cells and in the regulation of other pro-inflammatory cytokines. Produced mainly by macrophages, it contributes to the defense against infections and facilitates the apoptosis of damaged cells. In excess, TNF-α is associated with chronic inflammatory conditions, such as rheumatoid arthritis and autoimmune diseases, and is a target of biological therapies for the control of dysregulated inflammation. As we can see in Figures Petition 870250008869, dated 03 / 02 / 2025, page 26 / 116 18 / 26 5. TNF-α concentrations were determined in the supernatants of peritoneal macrophage cultures treated or not with the methanolic extract (100, 50, 25, and 12.5 μg / mL) or dexamethasone (Dexa; 12.5 μg / mL) in the presence of LPS (500 ng / mL) and IFN-γ (5 ng / mL) by ELISA. Values ​​represent the mean ± SD of four determinations obtained in one of two experiments performed. *P < 0.05 compared to untreated cultures stimulated with LPS + IFN-γ. Thus, the methanolic extract was able to significantly reduce (p < 0.05) TNF-α production, showing inhibition values ​​between 35.5% and 48% at the evaluated concentrations. Under the same conditions, dexamethasone (12.5 μg / mL), an anti-inflammatory drug already used clinically, induced a 76.4% inhibition of TNF-α production.

[068] To investigate the effect of the extract on lymphocyte function, the effect of the methanolic extract on the production of interleukin-2 (IL-2) was initially evaluated. The cytokine IL-2 is essential for the immune system, promoting the proliferation and activation of T lymphocytes and regulating the immune response. Produced by activated T lymphocytes, IL-2 is crucial for the expansion of T cells during the adaptive immune response and in the maintenance of regulatory T cells, which prevent autoimmunity. As can be seen in Figure 6, IL-2 concentrations were determined in the supernatants of splenocyte cultures from BALB / c mice treated or not with the extract (100, 50 and 25 μg / mL) or dexamethasone (Dexa; 12.5 μg / mL) in the presence of Concanavalin A (Con A; 5 μg / mL) by the ELISA method. Values ​​represent the mean ± SD of four determinations obtained in one of two experiments performed. *P<0.05 Petition 870250008869, dated 03 / 02 / 2025, page 27 / 116 19 / 26 comparison with untreated and Con A-stimulated cultures. Thus, treatment with the extracts reduced the production of this inflammatory mediator in a concentration-dependent manner.

[069] Next, the effect of AF_MeOH on lymphocyte proliferation was evaluated by labeling with Carboxyfluorescein Succinimidyl Ester (CFSE), a fluorescent dye mainly used to track cell proliferation in cell biology and immunology experiments. It penetrates the cell and binds to intracellular proteins, emitting green fluorescence. As the cell divides, CFSE is distributed equally among the daughter cells, resulting in a reduction in fluorescence proportional to the number of divisions. This method is widely used to monitor cell proliferation in immunology studies and lymphocyte function assessment. As we can see in Figures 7A, 7B, 7C, 7D, 7E and 7F, splenocytes activated with Con A were treated or not with different concentrations of the extract or dexamethasone for 72 h. Proliferation rates were evaluated by CFSE labeling using flow cytometry.Figure 7A shows untreated splenocytes; Figure 7B shows splenocytes treated with 12.5 μg / mL of dexamethasone; Figure 7C shows splenocytes treated with 25 μg / mL of AF_MeOH; Figure 7D shows splenocytes treated with 50 μg / mL of AF_MeOH; Figure 7E shows splenocytes treated with 100 μg / mL of AF_MeOH; and Figure 7F shows the values ​​representing means ± SD of three determinations obtained in a two-experiment study. * P < 0.05 compared to stimulated and unstimulated cells. Petition 870250008869, dated 03 / 02 / 2025, page 28 / 116 20 / 26 treated. Thus, treatment with the AF_MeOH extract significantly reduced (p < 0.05) the percentage of dividing lymphocytes, especially at concentrations of 50 and 100 μg / mL. Under the same conditions, dexamethasone (12.5 μg / mL) also significantly reduced lymphoproliferation.

[070] Finally, the anti-inflammatory effect of the methanolic extract was evaluated in an experimental model of endotoxic shock induced by a lethal injection of lipopolysaccharide (LPS). Endotoxic shock, induced by a lethal injection of LPS, is a severe systemic inflammatory response triggered by the presence of LPS, a component of the cell wall of Gram-negative bacteria. When injected, LPS activates macrophages and dendritic cells, leading to the massive release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, resulting in an uncontrolled immune system reaction. This process causes excessive vasodilation, increased vascular permeability, a drop in blood pressure, and eventually multiple organ failure, characterizing septic shock. The LPS injection model is widely used in research to study inflammation mechanisms and evaluate potential anti-inflammatory therapies.In this model, the lethal dose of LPS leads to the death of animals within a maximum period of 4 days. In the first set of experiments, three extracts produced with different solvents were evaluated: methanolic extract (AF_MeOH), aqueous extract (AF_H2O), and extract with 80% grain alcohol (AF_ETOH 80%).

[071] As illustrated in Figure 8, male BALB / c mice (n=5) were pretreated with different extracts of Petition 870250008869, dated 03 / 02 / 2025, page 29 / 116 21 / 26 Astronium fraxinifolium (100 mg / kg), dexamethasone (2 mg / kg), or vehicle (5% DMSO in saline solution) were challenged with 600 μg of LPS (LD90-100 = 42.8 mg / kg) 90 min later, administered intraperitoneally. Survival was monitored for 4 days after the LPS challenge. The results are from two independently conducted experiments. *P < 0.05 compared to the vehicle group. *P < 0.05 compared to the vehicle group. Statistical analysis was performed using Logrank (Mantel-Cox). Thus, the AF_H2O extract did not show a protective effect, with all animals dying on the second day after the lethal dose of LPS challenge. The AF_ETOH 80% extract showed a significant protective effect of 60% (p < 0.05).Under the same experimental conditions, the AF_MeOH extract showed the best result, with a significant protective effect (p < 0.05) of 100% on mortality caused by the lethal dose of LPS, a result similar to that of the reference anti-inflammatory drug, dexamethasone (2 mg / Kg).

[072] In a second set of experiments, a dose-response assay was performed to evaluate different doses of the extract, the most active extract in the preliminary tests. As illustrated in Figure 9, male BALB / c mice (n=5) were pre-treated with different doses of the AF_MeOH extract (100, 50, or 25 mg / kg), dexamethasone (2 mg / kg), or vehicle (5% DMSO in saline solution) and challenged with 600 μg of LPS (LD90-100 = 42.8 mg / kg) 90 min later, administered intraperitoneally. Survival was monitored for 4 days after the LPS challenge. The results are from two experiments performed independently. *P< 0.05 compared to the vehicle group. *P< 0.05 compared Petition 870250008869, dated 03 / 02 / 2025, page 30 / 116 22 / 26 with the vehicle group. Statistical analysis was performed using Logrank (Mantel Cox). Thus, the AF_MeOH extract showed a significant effect only at the highest dose evaluated (100 mg / kg), while doses of 50 mg / kg and 25 mg / kg showed a protective effect of 40% and 20%, respectively. Dexamethasone, under the same experimental conditions, exhibited a 100% protective effect.

[073] Based on the data presented in this report, we can conclude that the methanolic extract obtained from the Gonçalo-alves plant (Astronium fraxinifolium) exhibits a potent and selective immunomodulatory effect on macrophage activation and lymphocyte function, reflected in a 100% protective effect on mortality in the LPS-induced endotoxic shock model. These findings not only reinforce the potential of Gonçalo-alves as a promising source of bioactive compounds, but also open the prospect of developing the methanolic extract as a basis for a future phytotherapeutic with anti-inflammatory potential.

[074] The present invention has been disclosed in this descriptive report in terms of its preferred embodiment. However, other modifications and variations are possible from the present description, still falling within the scope of the invention disclosed herein. BIBLIOGRAPHIC REFERENCES

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Claims

1 / 3 CLAIMS 1. Process for obtaining an extract of Astronium sp., CHARACTERIZED by comprising the steps: (a) Harvesting the Astronium sp. sample; (b) Increasing the contact surface of the Astronium sp. sample matrix; (c) Obtaining the extract; (d) Filtration and concentration of the extract; (e) Storage of the extract.

2. Process, according to claim 1, CHARACTERIZED in that in step (a) the sample is harvested, which is selected from the group comprising: leaves, flowers, root, stem and fruits, preferably the stem bark of Astronium sp.

3. Process, according to any one of claims 1 or 2, CHARACTERIZED by the fact that the species of Astronium sp. are selected from Astronium balansae Engl., Astronium concinnum Schott, Astronium fraxinifolium Schott, Astronium glaziovii Mattick, Astronium graveolens Jacq., Astronium lecointei Ducke, Astronium nelson-rosae Santin, Astronium obliquum Griseb., Astronium pumilum JDMitch. & Daly, Astronium ulei Mattick, Astronium urundeuva (M.Allemão) Engl, with the species Astronium fraxinifolium Schott being preferably used.

4. Process, according to any one of claims 1 to 3, CHARACTERIZED in that in step (b) the sample is ground until a powder with a particle size of 18 to 20 mesh is obtained, followed by storage in tubes with plastic or glass caps, at temperatures between -18°C and -22°C, until the moment of extract preparation. Petition 870250008869, dated 03 / 02 / 2025, page 37 / 116 2 / 3 5. Process, according to any one of claims 1 to 4, CHARACTERIZED in that in step (c), the extract is obtained by immersing 4.5 to 5.5 g of the Astronium sp. plant matrix in 22 to 37 mL of solvent, preferably in a 1:5 ± 0.5 m / v ratio, followed by agitation of the resulting mixture in an oven at a temperature between 44 and 46°C and a rotation speed between 200-250 RPM for 47 to 49 h.

6. Process, according to any one of claims 1 to 5, CHARACTERIZED in that the solvent is selected from the group comprising: methanol (99%), grain alcohol, water, absolute alcohol or a mixture thereof, preferably methanol (99%).

7. Process, according to any one of claims 1 to 6, CHARACTERIZED in that in step (d), the extract is filtered through a filter with a pore size between 8 and 10 μm and concentrated in a vacuum centrifuge, at a temperature between 34 and 36°C, for 8 to 12 h.

8. Process, according to any one of claims 1 to 7, CHARACTERIZED in that in step (e), the extract obtained is packaged in bottles with lids and stored at -18°C to -22°C.

9. Use of Astronium sp. extract, as defined in any of claims 1 to 8, CHARACTERIZED by the fact that it is for the manufacture of supplements, functional foods and herbal remedies.

10. Use, according to claim 9, CHARACTERIZED by being in the form of capsules, tablets, sachets, solutions, suspensions, syrups, gels, creams, ointments, lotions, transdermals, oral sprays, serums, suppositories, nasal patches and inhalers. Petition 870250008869, dated 03 / 02 / 2025, page 38 / 116 3 / 3

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