Immunomodulatory composition isolated from yerba mate

Abstract

The present invention refers to an immunomodulatory composition comprising compounds, comprising at least one dicaffeoylquinic acid isomer, at least one chlorogenic acid or an isomer thereof, more than 1 % by weight rutin, a caffeoyl- syringoylquinic acid or an isomer thereof, nicotiflorin, and one or more compounds selected from the group consisting of 3-O-Feruloylquinic acid, and Eriojaposide A; wherein the compounds are isolated from yerba mate, a pharmaceutical preparation, food or food additives or cosmetics comprising said extracts as well as its use for treatment or prevention of inflammatory diseases. The invention also refers to a method of making the immunomodulatory extract from yerba mate.

Description

[0001] SMA001P 20260227

[0002] -1- IMMUNOMODULATORY COMPOSITION ISOLATED FROM YERBA MATE

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to an immunomodulatory composition comprising at least one dicaffeoylquinic acid isomer, at least one chlorogenic acid or an isomer thereof, more than 1 % by weight rutin, caffeoyl-syringoylquinic acid or an isomer thereof, nicotiflorin, and one or more compounds selected from the group consisting of 3-O-Feruloylquinic acid, and Eriojaposide A; wherein the compounds are obtained from yerba mate. The invention further refers to a pharmaceutical preparation, food, food additives, or cosmetics comprising said compositions as well as its use for treatment or prevention of inflammatory or proliferative diseases. The invention also refers to a method of making the immunomodulatory composition.

[0005] BACKGROUND OF THE INVENTION

[0006] Yerba mate is a species of the holly genus (Ilex), with the botanical name Ilex paraguariensis. Ilex parguariensis is native of the Republic of Paraguay and its dried material is also used to make the beverage known as “mate” (hot infusion) and “terere” (cold infusion), traditionally consumed in central and southern regions of South America, primarily in Paraguay, as well as in Argentina, Uruguay, southern and central-western Brazil, the Chaco region of Bolivia and southern Chile. Yerba mate can also be found in various energy drinks on the market today. One of the most prominent yerba mate industries is Laboratorio y Herboristeria Santa Margarita S.A., founded by Dr. Oscar Esteban Benitez Rapetti (t) and his wife Dr. Martha Cuenca de Benitez ( ). Currently, the family business is run by the second generation, who carried out the research, including Patricia Benitez, Margarita Benitez, Juan Carlos Benitez ( ), Viviana Benitez and Ernesto Benitez.

[0007] Yerba mate has been claimed to have various salutary effects on human health, specifically stimulating properties, and potential immunomodulatory effects.

[0008] The main ingredients of Mate are caffeine (0.3-2.4 %), theobromine (0.1 -0.5 %), but only traces of theophylline. It is rich in caffeoylquinic acids, accounting for 4-14 % of its composition, with n-chlorogenic acid (CGA), neochlorogenic acid, and dicaffeoylquinic acid isomers being the most prominent. Notably, certain caffeine molecules are bound to these acids. The plant also contains flavonoids, triterpenoid saponins (glycosides derived from ursolic and oleanolic acids (referred to as mateSMA001P 20260227

[0009] -2-saponins) as well as an array of vitamins, minerals and trace amounts of volatile compounds (Bauer etal., 2016).

[0010] Chlorogenic acid (5-O-caffeoylquinic acid) is one of the most abundant, highly functional polyphenolic compounds in the human diet. The evidence of its health benefits obtained from clinical studies, as well as basic research, indicates that a higher consumption of the substance is associated with a lower risk of metabolic syndromes and chronic diseases (Santana-Galvez et al., 2017). It shows further neuroprotective, cardiovascular protective, gastrointestinal protective, renoprotective, hepatoprotective, glucose and lipid metabolism regulatory, and anticarcinogenic effects (Lu etal., 2020).

[0011] Caffeic acid (CA, a phenolic acid compound) and its derivatives have been used for many centuries due to their natural healing and medicinal properties. It possesses various biological and pharmacological activities, including antioxidant, antiinflammatory, anticancer, anti-diabetic and neuroprotective effects. Further, its antitumor activity of has attracted much attention, and this potential has been confirmed both in vitro and in vivo (Alam et al., 2022; Pavlikova, 2022). CA can induce apoptosis in cancer cells via enhancing ROS levels and impairing mitochondrial function. Molecular pathways such as PI3K / Akt and AMPK with role in cancer progression, are affected by CA and its derivatives in cancer therapy. CA is advantageous in reducing aggressive behavior of tumors via suppressing metastasis by inhibiting epithelial-to-mesenchymal transition mechanism (Mirzaei et al., 2021).

[0012] Rutin is also a major compound in yerba mate infusions and is a flavonoid present in numerous fruits and vegetables and is therefore widely consumed. Earlier clinical studies revealed that Rutin has the potential to reduce the increased hypertension associated-capillary fragility to normal and hence, it prevents retinal hemorrhage and cerebral accidents in patients. The compound is a potent antioxidant and widely used as a standard for evaluating the antioxidative activity of a crude plant extract as well as a natural or synthetic chemical entity. Besides, it showed protective effects on the liver and blood vessels including coronary arteries. This molecule was also found to exhibit a protective effect against cisplatin-induced ovarian toxicity in mice by improving the levels of various enzymes that are associated with oxidative stress. On the other hand, its protective effect against ethanol-induced hepatotoxicity was also confirmed in human hepatoma HepG2 cells via reducing hepatic aminotransferase activities and inflammatory response (Lins etal., 2020; Semwal etal., 2021; Yang etal., 2019). FurtherSMA001P 20260227

[0013] -3-on, Rutin could be proposed as promising dietary supplements for prevention of urolithiasis according to Nirumand et al. (2018).

[0014] Long-term yerba mate supplementation, as demonstrated in mouse studies, may offer benefits in addressing obesity, improving insulin resistance and managing dyslipidemia (Choi etal., 2017; Kang etal., 2012).

[0015] De Lima et al. showed that yerba mate extracts preserved normal activity and exploratory behavior before rats were exposed to chronic restraint stress (CRS). Further the authors could show that yerba mate extracts prevented morphological damage in the cortex, hippocampus and striatum, and reduced inflammatory markers (de Lima et al., 2019).

[0016] Santiano et al. showed that yerba mate consumption delayed tumor onset, reduced tumor volume, and lowered proliferative activity in mouse in prostate cancer models. In vitro analyses showed that yerba mate intake inhibited the viability, proliferation, adhesion, and migration of prostate cancer cell lines, with the most significant effects in androgen-sensitive cells (Santiano et al., 2023).

[0017] However, the high content of caffeine in yerba mate, especially when taken in large amounts, can worsen diarrhea, can cause irregular heartbeats and might affect blood sugar. High doses of caffeine might cause seizures or decrease the effects of drugs used to prevent seizures. Yerba mate is possibly unsafe when taken by mouth during pregnancy. Yerba mate seems to increase the risk of getting cancer. It's not known whether that risk is transferred to the fetus. Yerba mate also contains caffeine. Avoid consuming more than 300 mg of caffeine daily (about 6 cups of yerba mate) when pregnant. High doses of caffeine have been linked with miscarriage, premature delivery, and low birth weight.

[0018] US11351214B2 discloses methods for making yerba mate extract composition comprising at least one of caffeic acid, monocaffeoylquinic acids, and dicaffeoylquinic acids, and salts thereof and less than 1% by weight of at least one of caffeine, rutin, and rutin isomers.

[0019] Dos Santos Polidoro et al. outline analytical methods used to characterize phenolic and methylxanthine profiles of yerba mate samples (dos Santos Polidoro etal., 2023).

[0020] Souza et al. describe several extraction methods for obtaining bioactive compounds from yerba mate leaves (Souza etal., 2021).SMA001P 20260227

[0021] -4- Kaltbach et al. describe a high-performance thin-layer chromatography (HPTLC) method for analyzing bioactive compounds in mate tea (Kaltbach etal., 2020).

[0022] A dissertation by Puangpraphant examines the anti-inflammatory and anti-cancer properties of bioactive compounds found in yerba mate tea. (Puangpraphant, 2012).

[0023] Bravo et al. examine the phenolic composition and antioxidant activity of mate infusions and extracts, showing strong antioxidant capacity mainly attributed to caffeoylquinic acid derivatives (Bravo et al., 2007).

[0024] Rocio Soledad etal. investigate the anti-metastatic effects of yerba mate extract in breast cancer and show that it inhibits cell proliferation, adhesion, migration, and invasion, while reducing metastasis and increasing survival in mice (Rocio Soledad et al., 2021).

[0025] Ben Sghaier et al. investigate the anticancer effects of rutin on human lung and colon cancer cell lines and show that rutin significantly inhibits cell proliferation, reduces superoxide anion production, and impairs cell adhesion and migration (ben Sghaier et al., 2016).

[0026] While yerba mate infusions are well known and also salutary effects are described, there is still a high and yet unmet demand for optimized preparations derived from yerba mate, specifically because it is not yet known whether yerba mate infusions made from commercially available yerba mate material may contain further ingredients, besides caffeine, which may cause negative side effects, specifically when taken for a long time. Thus, there is a high demand for optimized compositions comprising compounds from yerba mate, specifically comprising compounds which enhance the salutary effect and said compositions being free of any compounds which may cause negative side effects.

[0027] Specifically, there also is a need for a yerba mate derived compositions that specifically have improved immunomodulatory effects.

[0028] SUMMARY OF THE INVENTION

[0029] It is the objective of the present invention to provide improved compositions comprising compounds having an immunomodulatory effect, wherein the compounds have been obtained from yerba mate infusions.SMA001P 20260227

[0030] -5- The objective is solved by the subject matter of the present invention. The present composition comprises selected compounds which have improved immunomodulatory characteristics such as anti-inflammatory, anti-proliferative, and anti-infective properties.

[0031] The inventors surprisingly found that specific compounds obtained, specifically isolated from yerba mate contribute to the immunomodulatory properties of the compositions by impacting on immune cell activation, mobilization, and migration, which are key processes in inflammatory responses. The identified bioactive compounds interact with immune signaling pathways, thereby influencing immune cell behavior.

[0032] According to the invention there is provided a composition comprising the following compounds:

[0033] at least one dicaffeoylquinic acid isomer,

[0034] at least one chlorogenic acid or an isomer thereof,

[0035] more than 1 % by weight rutin, and

[0036] one or more compounds selected from the group consisting of:

[0037] a caffeoyl-syringoylquinic acid or an isomer thereof,

[0038] nicotiflorin,

[0039] 3-O-Feruloylquinic acid, and

[0040] Eriojaposide A;

[0041] wherein the compounds are obtained from, specifically isolated from yerba mate. According to the invention there is further provided a composition comprising the following compounds:

[0042] - at least one dicaffeoylquinic acid isomer,

[0043] - at least one chlorogenic acid or an isomer thereof,

[0044] - more than 1 % by weight rutin,

[0045] - a caffeoyl-syringoylquinic acid or an isomer thereof,

[0046] - nicotiflorin, and

[0047] one or more compounds selected from the group consisting of:

[0048] - 3-O-Feruloylquinic acid, and

[0049] - Eriojaposide A;

[0050] wherein the compounds are obtained from, specifically isolated from yerba mate. According to a specific embodiment of the invention, the compounds are obtained, specifically isolated by liquid chromatography, specifically by high-performance liquid chromatography (HPLC).SMA001P 20260227

[0051] -6- According to a specific embodiment of the invention, the composition comprises Eriojaposide A.

[0052] According to a specific embodiment of the invention, the composition comprises 3-O-Feruloylquinic acid.

[0053] According to a specific embodiment of the invention, the dicaffeoylquinic acid is present in the percent concentration ratio range of 2 to 50, specifically 2.5 to 45, specifically 4 to 41.1 as calculated from Total Ion Chromatogram (TIC), and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0054] According to a specific embodiment of the invention, chlorogenic acid or isomer thereof is present in the percent concentration ratio range of 1 to 5, specifically 2.3 to 2.5 as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0055] According to a specific embodiment of the invention, caffeoyl-syringoylquinic acid or isomer thereof is present in the percent concentration ratio range of 2 to 3 specifically 2.5 to 2.9, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0056] According to a specific embodiment of the invention, nicotiflorin is present in the percent concentration ratio range of 3 to 5 specifically 4 to 5, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0057] According to a specific embodiment of the invention, 3-O-Feruloylquinic acid is present in the percent concentration ratio range of 20 to 30, specifically 25 to 30, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0058] According to a specific embodiment of the invention, the composition is substantially free of benzo[a]pyrene, oxalic acid, tannins, heavy metals, arsenic, cadmium, or lead.

[0059] According to a specific embodiment of the invention, the dicaffeoylquinic acid isomer is selected from the group consisting of 3,4-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid.SMA001P 20260227

[0060] -7- According to a specific embodiment of the invention, the chlorogenic acid isomer is selected from the group consisting of 3-O-caffeoylquinic acid (3-CQA), 4-CQA, and 5-CQA.

[0061] According to a specific embodiment of the invention, the composition further comprises a compound selected from the group consisting of essential oil, polyphenol, polysaccharide, and combinations thereof.

[0062] According to the invention there is provided a food or food additive comprising the composition described herein.

[0063] According to the invention, there is provided a pharmaceutical preparation comprising the composition described herein and a pharmaceutically acceptable excipient.

[0064] According to the invention, there is provided a cosmetic preparation comprising the composition described herein.

[0065] According to the invention, there is provided a food supplement comprising the composition described herein.

[0066] According to the invention there is provided a method for obtaining the composition described herein comprising the sequential steps of:

[0067] a. preparing a yerba mate infusion, specifically placing loose yerba mate material on a filter and infusing with deionized water at a temperature range of 70-80 °C, specifically 75 °C, and allowing the infusion to steep for 25-35 minutes, specifically 30 minutes;

[0068] b. separating the infusion into fractions using a reversed-phase high-performance liquid chromatography (rp-HPLC) system, specifically running the rp-HPLC for 20 to 50 minutes, specifically about 34 minutes, comprising the sequential steps of:

[0069] i. injecting the infusion into the rp-HPLC system, specifically injecting 10 pL of the infusion with a flow rate of 1.0 mL / min,

[0070] ii. eluting with gradient mobile phases containing acetonitrile and / or HPLC-grade water supplemented with about 0.2% formic acid,

[0071] iii. detecting eluants with a DAD-detector, specifically at wavelengths from 190 nm and 450nm

[0072] iv. collecting fractions, specifically collecting 8 fractions comprising elution peaks, more specifically after 10 minutes into the run with each sample being collected for 0.5 minutes (equivalent to 0.5 mL per sample), andSMA001P 20260227

[0073] -8- c. selecting the fraction(s) comprising the compounds of the composition. According to an embodiment of the invention, the composition is used in the treatment of an inflammatory disease, specifically selected from the group consisting of cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE), asthma, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).

[0074] According to a further embodiment of the invention, the composition is used in the treatment of an cancer, specifically selected from the group consisting brain, squamous cell, bladder, head, neck, liver, ovarian, esophageal, nasopharynx, or thyroid cancers, melanomas, lymphomas, leukemias or multiple myelomas, oral cavity, gastrointestinal tract, colon, colorectal, stomach, pancreas, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.

[0075] FIGURES

[0076] Fig. 1 : Chromatogram of yerba mate brew and the 8 collected fractions.

[0077] Fig. 2: Chromatogram of yerba mate, overlay of four runs.

[0078] Fig. 3: HRMS negative ion mode of yerba mate. Raw data overview.

[0079] Fig. 4: HRMS positive ion mode of yerba mate. Raw data overview.

[0080] Fig. 5: HRMS negative ion mode of (A) fraction 6, (B) fraction 7 and (C) fraction 8 raw data overview.

[0081] Fig. 6: HRMS positive ion mode of (A) fraction 7 and (B) fraction 8; raw data overview.

[0082] Fig. 7: Number of THP1 cells / mL x 106before addition and 2 hours; 4 hours and 24 hours after addition of yerba mate infusion in different dilutions.SMA001P 20260227

[0083] -9- Fig. 8: Viable THP1 cells in % (means), before addition of yerba mate infusion in different dilutions, 2 hours after addition, 4 hours after addition and 24 hours after addition.

[0084] Fig. 9: Chemotactic Index (Cl) of THP1 cells treated with yerba mate extracts (****: P value < 0.0001).

[0085] Fig. 10: Chemotactic Index (Cl) of Jurkat cells treated with yerba mate extracts (**: P value < 0.01).

[0086] Fig. 11: Chemotactic Index (Cl) of neutrophils (Donor 1 and Donor 2) treated with yerba mate extracts. The Cl was normalized to 1.

[0087] Fig. 12: The significant immunoregulatory effect was shown by the chemotactic Index (Cl) of Jurkat cells treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (**: P value < 0.01).

[0088] Fig. 13: The significant immunoregulatory effect was shown by the chemotactic Index (Cl) of neutrophils treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (****: P value < 0.0001).

[0089] Fig. 14: The significant immunoregulatory effect was shown by the chemotactic Index (Cl) of THP-1 cells treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (****: P value < 0.0001).

[0090] DETAILED DESCRIPTION

[0091] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to standard handbooks, such as "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.SMA001P 20260227

[0092] -10- The terms “comprise”, “contain”, “have”, and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0093] The term “obtained from” means derived, produced, recovered, isolated, synthesized, or otherwise acquired from a stated source, either directly or indirectly, and includes materials that have been modified, processed, or further treated after acquisition from the source. Specifically, the source is yerba mate.

[0094] The term “isolated from” means removed or separated from at least one component with which it is naturally or originally associated. The term encompasses partial or substantial purification and does not require absolute purity. An “isolated” compound may remain associated with other compounds, provided it is no longer in its native environment. Specifically, the source is yerba mate.

[0095] In a specific embodiment, “obtained from” includes “isolated from.”

[0096] As used herein, a “fraction” of yerba mate refers to a non-naturally occurring composition obtained from plant material, specifically from herbal infusion, by extraction, separation, partitioning, purification, enrichment, concentration, depletion, or other processing steps, specifically by high-performance liquid chromatography, wherein: (a) at least one component is enriched, depleted, or otherwise present in a proportion different from that found in the intact plant and and specifically identified by high-resolution mass spectrometry; and / or

[0097] (b) the overall compositional profile differs from any naturally occurring plant tissue or exudate. A “fraction” does not encompass the intact plant, untreated plant material, or a composition existing in nature in substantially the same composition.

[0098] The term “about” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0099] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0100] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.SMA001P 20260227

[0101] -11- Yerba mate or yerba-mate, Ilex paraguariensis, is a plant species of the holly genus and is native to Paraguay and South America.

[0102] The term “yerba mate material” generally refers to any and all parts of the yerba mate plant, including the yerba mate plant leaves, stalks, stems, tops, roots, and the like. Specifically, the yerba mate material used herein consists of stems and / or leaves of one single plant, Ilex paraguariensis. The yerba mate material can be in any suitable form including in comminuted form resulting from, e.g., from chopping or grinding the yerba mate material, drying, processing etc.. For example, the yerba mate biomass can be comminuted in a suitable container.

[0103] An exemplary process of harvesting and drying yerba mate material may be summarized by the following steps:

[0104] Step 1 : Harvest

[0105] i. Season: The yerba mate harvest in Argentina and Paraguay is mainly carried out between May and September, when the leaves reach their optimal ripeness.

[0106] ii. Selection: Mature leaves are harvested from the branches, avoiding the youngest leaves and stems, as the latter do not contribute flavor and can affect the quality of the final product.

[0107] iii. Method: The harvest is traditionally done by hand, using scissors or special tools to cut the branches. This method allows careful selection of the leaves and avoids damaging the plant.

[0108] iv. Transport: The harvested leaves are transported quickly to the processing site to prevent deterioration. Suitable containers are used that allow ventilation and prevent crushing of the leaves.

[0109] Step 2: Drying

[0110] Drying is a crucial stage in the processing of yerba mate, as it determines its flavor, aroma and preservation. An exemplary method of drying is as follows:

[0111] * Sapecado: A fast process that consists of exposing the leaves from the harvest to high temperatures (400-500 °C) for a few seconds. It is carried out in a cylindrical rotary oven and aims to stop enzymatic oxidation and reduce the humidity of the leaves.

[0112] * Drying: After the sapecado, the leaves are subjected to a slow and controlled drying to reduce their moisture content to optimal levels for their conservation. This process can last several hours and is carried out in dryers that use hot air.

[0113] Step 3: CanchadaSMA001P 20260227

[0114] -12- Once dry, the leaves are crushed and coarsely ground, obtaining what is known as “yerba mate canchada”. This intermediate product is used for the preparation of yerba mate that is usually consumed.

[0115] Step 4: Maturation

[0116] The yerba mate canchada is stored in burlap bags for a period of time that can vary between 6 and 24 months. This maturation process allows the yerba mate to acquire its characteristic flavor and aroma.

[0117] Step 5: Milling

[0118] Finally, after maturation, the maturated yerba mate is ground until the desired granulometry is obtained for consumption. Different types of grinding can be obtained, from the thickest to the finest, according to the preferences of the consumer.

[0119] The process of harvesting and drying yerba mate may vary depending on the producer and the region. The basic principles may be the same or similar and seek to ensure the quality and characteristic flavor of this infusion popular in Paraguay, Argentina, and other countries in South America.

[0120] As used herein, the terms “infusion”, “tea”, and “extract” are used synonymously. Yerba mate infusion is the liquid solution separated from solid components and is obtained by contacting yerba mate material with water or other solvent, specifically with water in the temperature range of about 60-100 °C, during a time ranging from 0.5 to 60 minutes, preferably 15 to 45 minutes, more preferably 30 minutes.

[0121] The term “immunomodulatory” is multifaceted in the context of this application and it includes at least one of the following aspects: In one aspect it refers to the stimulation of the immune response to foreign and harmful materials such as pathogens. It also refers to the overall enhancement of the health, function and potency of the immune system. It also refers to the training of the immune system to response appropriate and healthy ways for different stimuluses and to avoid unhealthy pathological responses. It also covers immunoregulatory and immunostimulatory aspects to tune the immune response in a healthy way and to avoid unhealthy responses. In one aspect it also refers to the maintaining of the healthy status of immune system and immune response or to slowing down the decline of the immune system and immune response for example in the cases of immune system affecting diseases and aging.SMA001P 20260227

[0122] -13- Specifically, “immunomodulation” refers to the general effect of a compound impacting on the mobilization, activation and migration of immune cells such as neutrophils, T-cells, monocytes / macrophages, eosinophils, dendritic cells.

[0123] An “immunomodulatory composition”, ’’immunomodulatory compound”, an “immunomodulator” in the context of the present invention can be termed as a molecule capable of increasing the body's resistance to disease, stress and other debilitating process. An immunomodulator is said to stimulate immune defects, balance body function, normalize body systems, boost recovery after surgery, protect against radiation, counteract the effect of sugar, optimize energy in times of stress, increase stamina, protect against motion sickness, shield against infection.

[0124] The immunomodulatory effect of the extract of the present invention can be determined by methods known in the art, such as cell-based assays, chemotaxis assays, transwell assays and flow chamber assays, revealing a reduction in the activation, mobilization and migration of various immune cell types. The bioactive fractions can be subsequently analyzed by mass spectrometry to determine their chemical composition.

[0125] Additionally, the yerba mate extracts described herein also specifically show antiinflammatory effects. Such effects can be confirmed in biological assays such as the above mentioned methods.

[0126] Additionally, the yerba mate extracts described herein also specifically show antiproliferative effects.

[0127] Further methods to confirm immunomodulatory and anti-inflammatory effects can be, but are not limited to, in vivo experiments such as air pouch and peritonitis models.

[0128] THP1 , Jurkat cells, T-cells, and neutrophil cells are in vitro models for evaluating inflammatory diseases, specifically cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).SMA001P 20260227

[0129] -14- According to a preferable embodiment, the composition described herein is an immunomodulatory composition.

[0130] According to the definition of the present application, any one or all of dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, caffeoyl-syringoylquinic acid or an isomer thereof, and 3-O-Feruloylquinic acid can be present in the composition as salts thereof.

[0131] Specifically, the composition described herein contains the compounds dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, and Eriojaposide A, wherein the compounds are obtained from, specifically isolated from yerba mate. In a specific embodiment, the ratio of the compounds is as follows:

[0132] Chlorogenic acid : rutin : dicaffeoylquinic acid is 1 : 1 : 1, 1 : 1 : 2, 1 : 1 : 3, 1 : 1 : 4, 1 : 1 5, 1 2 1, 1 2 2, 1 2 3, 1 2 4, 1 2 5, 1 3 1, 1 3 2, 1 3 3, 1 3 4, 1 3 5, 1 4 1, 1 4 2, 1 4 3, 1 4 4, 1 4 5, 1 5 1, 1 5 2, 1 5 3, 1 5 4, 1 5 5, 1 6 1, 1 6 2, 1 6 3, 1 6 4, 1 6 5, 1 7 1, 1 7 2, 1 7 3, 1 7 4, 1 7 5, 1 8 1, 1 8 2, 1 8 3, 1 8 4, 1 8 5, 1 9 1, 1 9 2, 1 9 3, 1 9 4, 1 9 5, 1 : 1( ) : 1, : 10 : 2 1 10 : 3, : 10 : 4 1 : 1 0 : 5, spe jcif cally Chi oroge nic acid rutin : dicaffeoylquinic acid = 1 : 10 : 5.

[0133] According to an alternative embodiment, the composition described herein contains the compounds dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, caffeoyl-syringoylquinic acid or an isomer thereof, and nicotiflorin, wherein the compounds are obtained from, specifically isolated from yerba mate. In a specific embodiment, the ratio of the compounds is as follows:

[0134] Chlorogenic acid : caffeoyl-syringoylquinic acid isomer : rutin : nicotiflorin : dicaffeoylquinic acid isomer is 1 : 1 : 1 : 1 : 1, 1 : 1 : 1 : 1 : 2, 1 : 1 : 1 : 1 : 3, 1 : 1 : 1 : 1 : 4, 1 : 1 : 1 : 1 : 5, 1 : 1 : 1 : 1 : 6, 1 : 1 : 1 : 1 : 7, 1 : 1 : 1 : 1 : 8, 1 : 1 : 1 : 1 : 9, 1 : 1 : 1 : 1 : 10, 1 : 1 : 1 : 1 : 11, 1 : 1 : 1 : 1 : 12, 1 : 1 : 1 : 1 : 13, 1 : 1 : 1 : 1 : 14, 1 : 1 : 1 : 1 : 15, 1 : 1 : 1 : 1 : 16, 1 : 1 : 1 : 1 : 17, 1 : 1 : 1 : 1 : 18, 1 : 1 : 1 : 1 : 19, 1 : 1 : 1 : 1 : 20, 1 : 1 : 1 : 1 : 21 , 1 : 1 : 1 : 1 : 22, 1 : 1 : 1 : 1 : 23, 1 : 1 : 1 : 1 : 24, 1 : 1 : 1 : 1 : 25, 1 : 1 : 1 : 2 : 1 , 1 : 1 : 1 : 2 : 2, 1 : 1 : 1 : 2 : 3, 1 : 1 : 1 : 2 : 4, 1 : 1 : 1 : 2 : 5, 1 : 1 : 1 : 2 : 6, 1 : 1 : 1 : 2 : 7, 1 : 1 : 1 : 2 : 8, 1 : 1 : 1 : 2 : 9, 1 : 1 : 1 : 2 : 10, 1 : 1 : 1 : 2 : 11, 1 : 1 : 1 : 2 : 12, 1 : 1 : 1 : 2 : 13, 1 : 1 : 1 : 2 : 14, 1 : 1 : 1 : 2 : 15, 1 : 1 : 1 : 2 : 16, 1 : 1 : 1 : 2 : 17, 1 : 1 : 1 : 2 : 18, 1 : 1 : 1 : 2 : 19, 1 : 1 : 1 : 2 : 20, 1 : 1 : 1 : 2 : 21, 1 : 1 : 1 : 2 : 22, 1 : 1 : 1 : 2 : 23, 1 : 1 : 1 : 2 : 24, 1 : 1 : 1 : 2 : 25, 2 : 1 : 1 : 1 : 1 , 2 : 1 : 1 : 1 : 2, 2 : 1 : 1 : 1 : 3, 2SMA001 P 20260227

[0135] -15- : 1 : 1 : 1 : 4, 2 : 1 : 1 : 1 : 5, 2 : 1 : 1 : 1 : 6, 2 : 1 : 1 : 1 : 7, 2 : 1 : 1 : 1 : 8, 2 : 1 : 1 : 1 : 9, 2 : 1 : 1 : 1 : 10, 2 : 1 : 1 : 1 : 11 , 2 : 1 : 1 : 1 : 12, 2 : 1 : 1 : 1 : 13, 2 : 1 : 1 : 1 : 14, 2 : 1 : 1 : 1 : 15, 2 : 1 : 1 : 1 : 16, 2 : 1 : 1 : 1 : 17, 2 : 1 : 1 : 1 : 18, 2 : 1 : 1 : 1 : 19, 2 : 1 : 1 : 1 : 20, 2 : 1 : 1 : 1 : 21 , 2 : 1 : 1 : 1 : 22, 2 : 1 : 1 : 1 : 23, 2 : 1 : 1 : 1 : 24, 2 : 1 : 1 : 1 : 25, 2 : 1 : 1 : 2 : 1 , 2 : 1 : 1 : 2 : 2, 2 : 1 : 1 : 2 : 3, 2 : 1 : 1 : 2 : 4, 2 : 1 : 1 : 2 : 5, 2 : 1 : 1 : 2 : 6, 2 : 1 : 1 : 2 : 7, 2 : 1 : 1 : 2 : 8, 2 : 1 : 1 : 2 : 9, 2 : 1 : 1 : 2 : 10, 2 : 1 : 1 : 2 : 11 , 2 : 1 : 1 : 2 : 12, 2 : 1 : 1 : 2 : 13, 2 : 1 : 1 : 2 : 14, 2 : 1 : 1 : 2 : 15, 2 : 1 : 1 : 2 : 16, 2 : 1 : 1 : 2 : 17, 2 : 1 : 1 : 2 : 18, 2 : 1 : 1 : 2 : 19, 2 : 1 : 1 : 2 : 20, 2 : 1 : 1 : 2 : 21 , 2 : 1 : 1 : 2 : 22, 2 : 1 : 1 : 2 : 23, 2 : 1 : 1 : 2 : 24, or 2 : 1 : 1 : 2 : 25, specifically Chlorogenic acid : caffeoyl-syringoylquinic acid isomer : rutin : nicotiflorin : dicaffeoylquinic acid isomer = 2 : 1 : 1 : 2 : 24.

[0136] Alternatively, the composition of the invention contains the compounds dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, and 3-O-Feruloylquinic acid, wherein the compounds are obtained from, specifically isolated from yerba mate. In a specific embodiment, the ratio of the compounds is as follows:

[0137] Chlorogenic acid : 3-O-Feruloylquinic acid : rutin : dicaffeoylquinic acid isomer is 1 : 1 : 1 : 1 , 1 : 1 : 2 : 1 , 1 : 1 : 3 : 1 , 1 : 1 : 4 : 1 , 1 : 2 : 1 : 1 , 1 : 2 : 2 : 1 , 1 : 2 : 3 : 1 , 1 : 2 : 4 : 1 , 1 : 3 : 1 : 1 , 1 : 3 : 2 : 1 , 1 : 3 : 3 : 1 , 1 : 3 : 4 : 1 , 1 : 4 : 1 : 1 , 1 : 4 : 2 : 1 , 1 : 4 : 3 : 1 , 1 : 4 : 4 : 1 , 1 : 5 : 1 : 1 , 1 : 5 : 2 : 1 , 1 : 5 : 3 : 1 , 1 : 5 : 4 : 1 , 1 : 6 : 1 : 1 , 1 : 6 : 2 : 1 , 1 : 6 : 3 : 1 , 1 : 6 : 4 : 1 , 1 : 7 : 1 : 1 , 1 : 7 : 2 : 1 , 1 : 7 : 3 : 1 , 1 : 7 : 4 : 1 , 1 : 8 : 1 : 1 , 1 : 8 : 2 : 1 , 1 : 8 : 3 : 1 , 1 : 8 : 4 : 1 , 1 : 9 : 1 : 1 , 1 : 9 : 2 : 1 , 1 : 9 : 3 : 1 , or 1 : 9 : 4 : 1 , specifically Chlorogenic acid : 3-O-Feruloylquinic acid : rutin : dicaffeoylquinic acid isomer = 1 : 9 : 4 : 1.

[0138] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0139] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, and nicotiflorin.

[0140] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate:SMA001P 20260227

[0141] -16-dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-O-Feruloylquinic acid, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0142] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, Eriojaposid A, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0143] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, nicotiflorin, and 3-O-Feruloylquinic acid.

[0144] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, nicotiflorin, and Eriojaposid A.

[0145] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-O-Feruloylquinic acid, and Eriojaposid A.

[0146] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-O-Feruloylquinic acid, nicotiflorin, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0147] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, Eriojaposid A, nicotiflorin, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0148] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-O-Feruloylquinic acid, Eriojaposid A, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0149] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate:SMA001P 20260227

[0150] -17-dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-0-Feruloylquinic acid, nicotiflorin, and Eriojaposid A.

[0151] According to an alternative embodiment, the composition described herein contains the following compounds obtained from, specifically isolated from yerba mate: dicaffeoylquinic acid isomer, chlorogenic acid or an isomer thereof, rutin, 3-0-Feruloylquinic acid, Eriojaposid A, nicotiflorin, and caffeoyl-syringoylquinic acid or an isomer thereof.

[0152] Dicaffeoylquinic acids (diCQA) are molecules consisting of a quinic acid core, acylated with two caffeoyl groups.

[0153] A dicaffeoylquinic acid (diCQA) isomer can be of the following structure:

[0154]

[0155] (Formula 1 )

[0156] wherein two of R1, R3, R4, and R5 are caffeic acid and the remaining two R groups are H.

[0157] Non-limiting examples of R1, R3, R4, and R5 combinations are shown in the following table:

[0158]

[0159] SMA001P 20260227

[0160] Specifically, 3,5-dicaffeoylquinic acid (3,5-diCQA) has the following structure:

[0161]

[0162] (Formula 2) Specifically, 3,4-dicaffeoylquinic acid (3,4-diCQA) has the following structure:

[0163]

[0164] (Formula 3)

[0165] Specifically, 1,4-dicaffeoylquinic acid (1,4-diCQA) has the following structure:

[0166]

[0167] (Formula 4)SMA001P 20260227

[0168] Specifically, 1,5-dicaffeoylquinic acid (1,5-diCQA) has the following structure:

[0169]

[0170] (Formula 5)

[0171] Specifically, 4,5-dicaffeoylquinic acid (4,5-diCQA) has the following structure:

[0172]

[0173] Quinic acid has the following structure:

[0174]

[0175] (Formula 7)SMA001P 20260227

[0176] -20- Caffeic acid has the following structure:

[0177]

[0178] (Formula 8)

[0179] Because dicaffeoylquinic acids can be considered weak acids, they can each exist in at least one of their conjugate acid form, conjugate base form (e.g., in their salt form), and mixed conjugate acid-conjugate base form, wherein a fraction (e.g., mole fraction) of the compounds exist in the conjugate acid form and another fraction exist in the conjugate base form. Examples of salts of dicaffeoylquinic acids include, but are not limited to, quaternary ammonium, sodium, potassium, lithium, magnesium, and calcium salts dicaffeoylquinic acids, and the like.

[0180] Specific examples of dicaffeoylquinic acid isomers are 3,4-dicaffeoylquinic acid (3,4-diCQA), 1,4-dicaffeoylquinic acid (1,4-diCQA), 1,5-dicaffeoylquinic acid (1,5-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), 4,5-dicaffeoylquinic acid (4,5-diCQA), and 1,3-dicaffeoylquinic acid (1,3-diCQA).

[0181] Specifically, a dicaffeoylquinic acid isomer is present in the percent concentration ratio range of 2 to 50, specifically 2.5 to 45, specifically 4 to 41.1 as calculated from Total Ion Chromatogram (TIC), and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0182] Chlorogenic acid (CGA) is the ester formed between caffeic acid and a hydroxyl group at positions 3, 4, or 5 of L-quinic acid. Isomers of chlorogenic acid include the caffeoyl ester at other hydroxyl sites on the quinic acid ring: 1-caffeoylquinic acid (1-CQA), 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (cryptochlorogenic acid or 4-CQA) and 5-O-caffeoylquinic acid (neochlorogenic acid or 5-CQA). Specifically, 3-CQA is the ester formed between caffeic acid and the 3-hydroxyl of L-quinic acid. Specifically, 4-CQA is the ester formed between caffeic acid and the 4-hydroxyl of L-SMA001P 20260227

[0183] -21-quinic acid. Specifically, 5-CQA is the ester formed between caffeic acid and the 5-hydroxyl of L-quinic acid.

[0184] Specifically, caffeoylquinic acids (CQA) are molecules consisting of a quinic acid core, acylated with one caffeoyl group.

[0185] A caffeoylquinic acid isomer can be of the structure of Formula 1 , wherein one of R1 , R3, R4, and R5 is caffeic acid and the remaining three R groups are H.

[0186] Examples of R1 , R3, R4, and R5 combinations are shown in the following table:

[0187]

[0188] Chlorogenic acid (CGA) can exist as multiple isomers, e.g., 1-CQA, 3-CQA, 4-CQA, or 5-CQA.

[0189] 3-caffeoylquinic acid (chlorogenic acid; 3-CQA) can be of the following structure:

[0190]

[0191] (Formula 9)

[0192] 4-caffeoylquinic acid (4-CQA), specifically (+)-Cryptochlorogenic acidSMA001P 20260227

[0193] -22- or (-)-Cryptochlorogenic acid, can be of the following structure:

[0194]

[0195] (Formula 10)

[0196] 5-caffeoylquinic acid (5-CQA) can be of the following structure:

[0197]

[0198] (Formula 11)

[0199] Specifically, a chlorogenic acid isomer is present in the percent concentration ratio range of 1 to 5, specifically 2.3 to 2.5 as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0200] Rutin can be of the following structure:

[0201]

[0202] (Formula 12)SMA001P 20260227

[0203] Rutin is also known as “rutoside”, “quercetin-3-O-rutinoside”, or “sophorin” and is the glycoside combining the flavonol quercetin and the disaccharide rutinose (a-L-rhamnopyranosyl-(1— >6)-|3-D-glucopyranose).

[0204] Specifically, rutin is present in the percent concentration ratio range of 2 to 15 specifically 4 to 12, specifically 4.1 to 10.6 as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0205] Caffeoyl-syringoylquinic acid isomer can be of following structure:

[0206]

[0207] (Formula 13)

[0208] A specific example of a caffeoyl-syringoylquinic acid isomer is 4-O-Caffeoyl-3-O-syringoylquinic acid (Formula 6).

[0209] Specifically, caffeoyl-syringoylquinic acid isomer is present in the percent concentration ratio range of 2 to 3 specifically 2.5 to 2.9, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).SMA001P 20260227

[0210] -24- Nicotiflorin can be of the following structure:

[0211]

[0212] (Formula 14)

[0213] Nicotiflorin is also known as kaempferol-3-O-rutinoside.

[0214] Specifically, nicotiflorin is present in the percent concentration ratio range of 3 to 5 specifically 4 to 5, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0215] The flavonoid nicotiflorin is known for cardioprotective properties and antioxidant effects. It may reduce oxidative damage and inflammation, contributing to cardiovascular health, further on it is showing a hypoglycemic and anti-Alzheimer effect (Tao et al., 2023).

[0216] 3-O-Feruloylquinic acid can be of the following structure:

[0217]

[0218] (Formula 15)

[0219] 3-O-Feruloylquinic acid is present in the percent concentration ratio range of 20 to 30, specifically 25 to 30, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).SMA001P 20260227

[0220] -25-

[0221] Eriojaposide A can be of the following structure:

[0222]

[0223] (Formula 16)

[0224] In positive ion mode rutin and eriojaposide A can be identified. In a specific embodiment, the ratio of the compounds is as follows: rutin : eriojaposide A = 4 : 1 Yerba mate material and infusions derived therefrom may further contain caffeic acid derivates, e.g. caffeoyl ester derivatives, caffeyltartaric acid, caffeic acid amide derivatives, caffeoyl shikimic acid, caffeoyl quinic acid, caffeoyl danshens, caffeoyl glycoside, caffeic acid, ferulic acid, isoferulic acids, caffeine, chlorogenic acid or isomers thereof, citric acid, dicaffeoylqinic acid isomers, and / or coumarin derivates as shown below.

[0225]

[0226] Formula 18

[0227] caffeine

[0228]

[0229] SMA001P 20260227

[0230] -26-

[0231]

[0232] Formula 19

[0233] citric acid

[0234]

[0235]

[0236] Formula 20

[0237] 3-Hydroxycoumarin

[0238]

[0239] Formula 8

[0240] caffeic acid

[0241]

[0242] SMA001P 20260227

[0243] -27- In a specific embodiment, the extract described herein is substantially free of contaminants such as, but not limited to, benzo[a]pyrene, oxalic acid, tannins, heavy metals, arsenic (As), cadmium (Cd), or lead (Pb).

[0244] In a specific embodiment, the extract described herein is substantially free of aromatic compounds, preferably anthraquinone.

[0245] As used herein the term “heavy metals” refers to metal or metalloid elements, specifically that have toxic effects on the human body. Heavy metals may include the elements chromium, manganese iron, cobalt, nickel, copper, zinc, molybdenum, rhodium, palladium, silver, cadmium, tin, iridium, platinum, gold, mercury, thallium, lead, bismuth, or polonium, preferably heavy metals may include copper, silver, cadmium, tin, mercury, thallium, lead, or bismuth.

[0246] The composition described herein can be in any form, such as solid, powder, gel, paste, foamy material, semi-solid, liquid, or mixtures thereof.

[0247] In some instances, the composition can be an aqueous composition and can be buffered with any suitable buffering system, including, but not limited to, a phosphate, citrate, ascorbate, lactate, acetate, and the like. Buffers can be in the range of 1-1000 mM of the anion. Alternatively, water acidified to pH 5-6 with hydrochloric acid, sulfuric acid, nitric acid or the like can be useful in the aqueous composition, with or without a cosolvent. Alternatively pure water made basic to pH 7-11 with hydroxide, such as with sodium or potassium hydroxide, can be useful in the aqueous composition, with or without a co-solvent. In still other instances, it may be suitable to add a suitable nonionic solute that can help balance the osmotic potential of the aqueous composition.

[0248] In a specific embodiment, said composition comprises an essential oil, a polyphenol, a polysaccharide, or any combinations thereof.

[0249] An essential oil is a concentrated hydrophobic liquid containing volatile (easily evaporated at normal temperatures) chemical compounds from plants. Essential oils are generally extracted by distillation, often by using steam. Other processes include expression, solvent extraction, sfumatura, absolute oil extraction, resin tapping, wax embedding, and cold pressing.

[0250] Polyphenols are a large family of naturally occurring phenols. They are abundant in plants and structurally diverse. Polyphenols include phenolic acids, flavonoids, tannic acid, and ellagitannins.SMA001P 20260227

[0251] -28- In some embodiments, the composition provided herein is formulated in the form of a cosmeceutical, a nutricosmetic, a cosmetic composition, or a body care product.

[0252] The composition comprising the extract described herein can specifically be used as food or food additive. Therefore, it can be incorporated into any ingestible composition, including beverages and food products. The formulation is preferably provided as a durable sales product with a shelf-life of at least 6 months, preferably at least 12 months. Therefore, a process step for sterilizing or preserving, respectively, can be performed, which specifically can comprise physical treatment, such as, but not limited to thermal treatment or filtration. Gentle treatment for preservation may be preferred in order to only insubstantially reduce the active compounds.

[0253] For example, the ingestible composition can be a comestible composition or noncomestible composition. By “comestible composition”, it is meant any composition that can be consumed as food by humans or animals, including solids, gel, paste, foamy material, semi-solids, liquids, or mixtures thereof. By “non-comestible composition”, it is meant any composition that is intended to be consumed or used by humans or animals not as food, including solids, gel, paste, foamy material, semi-solids, liquids, or mixtures thereof. The non-comestible composition includes, but is not limited to medical compositions, which refers to a non-comestible composition intended to be used by humans or animals for therapeutic purposes.

[0254] The term “animal” as used herein, includes any non-human animal, such as, for example, farm animals and pets.

[0255] The composition described herein can be added to a composition or product, such as supplements, nutraceuticals, functional food products (e.g., any fresh or processed food claimed to have a health-promoting and / or disease- preventing properties beyond the basic nutritional function of supplying nutrients), pharmaceutical and over the counter medications, oral care products such as dentifrices and mouthwashes, cosmetic products such as lip balms or creams, and other personal care products.

[0256] The composition described herein can also be a medicinal or pharmaceutical preparation, further comprising one or more pharmaceutically acceptable excipients.

[0257] The medicinal product or pharmaceutical composition described herein is specifically provided as human or veterinary medicinal product or pharmaceutical composition. Medicinal products are understood as substances that are used to treat diseases, to relieve complaints, or to prevent such diseases or complaints in the firstSMA001P 20260227

[0258] -29-place. This definition applies regardless of whether the medicinal product is administered to humans or to animals. The substances can act both within or on the body.

[0259] The medicinal product or pharmaceutical composition described herein contains one or more pharmaceutically acceptable auxiliaries or excipients and is in a pharmaceutical form which allows the active pharmaceutical compound to be administered with high bioavailability. Suitable auxiliaries may be, for example, based on cyclodextrins. Suitable formulations might for example incorporate synthetic polymeric nanoparticles formed of a polymer selected from the group consisting of acrylates, methacrylates, cyanoacrylates, acrylamides, polylactates, polyglycolates, polyanhydrates, polyorthoesters, gelatin, albumin, polystyrenes, polyvinyls, polyacrolein, polyglutaraldehyde and derivatives, copolymers and mixtures thereof.

[0260] Specific medicinal products or pharmaceutical compositions described herein comprise the composition as described herein, and a pharmaceutically acceptable carrier or excipient.

[0261] A “pharmaceutically acceptable excipient” refers to an ingredient in a formulation for medicinal or medical use, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, excipient, stabilizer, or preservative, and in particular saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like.

[0262] Pharmaceutically acceptable excipients generally include any and all suitable solvents, dispersion media, coatings, antiviral, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antiviral small molecule compound or related composition or combination preparation described herein.

[0263] According to a specific aspect, the composition as described herein can be combined with one or more carriers appropriate a desired route of administration. The composition as described herein may be e.g., admixed with any of lactose, sucrose, starch, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatine, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, the composition as described herein may be dispersed or dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutSMA001P 20260227

[0264] -30-oil, cotton seed oil, sesame oil, tragacanth gum, and / or various buffers. Other carriers, adjuvants, and modes of administration are well known in the pharmaceutical arts. A carrier may include a controlled release material or time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well-known in the art.

[0265] The compounds as described herein may be provided in controlled release pharmaceutical ("controlled release formulations") in which the release of the composition comprising the composition described herein is controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0266] Pharmaceutical compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject agent is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, and shellac.

[0267] Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Remington: The Science and Practice of Pharmacy, 23ndrevised edition (Adeboye Adejare, ed., Academic Press, 2021). Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

[0268] The preferred preparation is in a ready-to-use, storage stable form, with a shelflife of at least one or two years.

[0269] The term “formulation” as used herein refers to a preparation ready-to-use in a specific way. Specifically, formulations described herein comprise the composition comprising the yerba mate extract as described herein, and a pharmaceutically acceptable diluent, carrier or excipient.

[0270] According to a specific aspect, formulations are provided comprising pharmaceutically acceptable vehicles for nasal, intrapulmonary, oral, topical, mucosal or parenteral administration. Administration may also be intradermal or transdermal. Also, the present disclosure includes such compounds, which have been lyophilized and which may be reconstituted to form pharmaceutically acceptable formulations for administration.SMA001P 20260227

[0271] -31- To administer the composition as described herein by any route other than parenteral administration, it may be necessary to coat the active agents with, or coadminister the active agent with, a material to prevent their inactivation. For example, an appropriate carrier may be used, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0272] The composition as described herein can be orally administered, for example, with an inert diluent or an assimilable or edible carrier. For example, a preparation may be enclosed in a hard- or soft-shell gelatine capsule, or compressed into tablets. For oral therapeutic administration, the composition comprising the yerba mate extract described herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, teas, suspensions, syrups, wafers, and the like. The percentage of the compound in the compositions and preparations may, of course, be varied.

[0273] Tablets will contain excipients, glidants, fillers, binders, disintegrants, lubricants, flavours and the like. Granules may be produced using isomaltose. It is furthermore preferred to provide for a preparation formulated to act at the site of the mucosa e.g., at mucosal sites (such as nose, mouth, eyes, esophagus, throat, lung), e.g. locally without systemic action. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic.

[0274] The term “mucosal” with respect to administration or application or else mucosal use of a preparation for treating a subject or a respective formulation, refers to administration via the mucosal route, including systemic or local administration, wherein an active ingredient is taken up by contact with mucosal surfaces. This includes nasal, pulmonary, oral, or peroral administration and formulations, e.g. liquid, syrup, lozenge, chewing gum, tablet, spray, powder, instant powder, granules, capsules, cream, gel, drops, suspension, or emulsion.

[0275] Peroral formulations may include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically acceptable vehicles suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, tragacanth, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typicalSMA001P 20260227

[0276] -32-preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[0277] Other compositions useful for attaining systemic delivery of the composition as described herein or respective preparations include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose, or glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents.

[0278] The composition described herein or respective preparations therefrom can also be administered topically to a subject, e.g., by the direct laying on or spreading of a composition containing same on the epidermal or epithelial tissue of the subject, or transdermally via a “patch”. Such compositions include, for example, lotions, creams, solutions, gels and solids. Suitable carriers for topical administration typically remain in place on the skin as a continuous film, and resist being removed by perspiration or immersion in water. Generally, the carrier is organic in nature and capable of having dispersed or dissolved therein the therapeutic agent. The carrier may include pharmaceutically acceptable emollients, emulsifiers, thickening agents, solvents and the like.

[0279] According to some embodiments, the composition of this invention is formulated as a cosmetics, such as a body lotion, soap, body wash, moisturizer, self-tanner, hand cream, body scrub, sunscreen, bath product, toothpaste, soap, shampoo, mouth wash, deodorant, antiperspirant, or shaving soap.

[0280] According to some embodiments, the composition of this invention is a cosmetic or cosmeceutical composition selected from an anti-wrinkle composition, a moisturizing cream, a face mask, a makeup, and a lipstick.

[0281] According to some other embodiments, the composition of this invention is a body care product, selected from a body lotion, a soap, a body wash, a moisturizer, a selftanner, a hand cream, a body scrub, a sunscreen, a bath product, a toothpaste, a shampoo, a mouth wash, a dental floss, a deodorant, an antiperspirant, and a shaving product.

[0282] As used herein, the term “cosmeceutical” refers to a cosmetic product with bioactive ingredients purported to have medical or drug-like benefits. TheSMA001P 20260227

[0283] -33- “cosmeceutical” label applies only to products applied topically, such as creams, lotions and ointments. Products which are similar in perceived benefits but ingested orally are termed herein “nutricosmetics”.

[0284] For example, the composition described herein may be used in the manufacture of anti-acne cream or paste.

[0285] Cosmetics and cosmeceuticals, also sometimes collectively referred to herein as “beauty products”, are formulated in a form suitable, e.g., for topical application on an applied area, and may be used as anti-wrinkles, moisturizing creams, face masks, makeup, lipsticks and the like. A beauty product in accordance with some embodiments, may be in the form of a cream, an ointment, a paste, a gel, a lotion, a milk, an oil, a suspension, a solution, an aerosol, a spray, a foam, or a mousse.

[0286] As used herein, “treatment”, “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0287] The composition described herein may be used for the treatment of inflammatory diseases such as, but not limited to cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE - including lupus nephritis), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) in a subject, including a human, comprising the step of administering to said subject in need thereof a therapeutically effective amount of a yerba mate extract or composition or pharmaceutical preparation comprising said extract described herein.SMA001P 20260227

[0288] Specifically, the inflammatory diseases such as, but not limited to cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE - including lupus nephritis), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), are treated by inhibiting the activation, mobilization, or migration of T-cells, monocytes, or neutrophils.

[0289] According to a specific embodiment, the composition described herein may be used to inhibit the activation of T-cells, monocytes, or neutrophils.

[0290] According to a specific embodiment, the composition described herein may be used to inhibit the mobilization of T-cells, monocytes, or neutrophils.

[0291] According to a specific embodiment, the composition described herein may be used to inhibit the migration of T-cells, monocytes, or neutrophils.

[0292] According to a specific embodiment, the composition described herein may be used for the treatment of hyperproliferative diseases and disorders such as cancerous diseases or cancer, including but not limited to brain, squamous cell, bladder, head, neck, liver, ovarian, esophageal, nasopharynx, or thyroid cancers, melanomas, lymphomas, leukemias or multiple myelomas, oral cavity, gastrointestinal tract, colon, colorectal, stomach, pancreas, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.

[0293] The compounds of the composition described herein are specifically obtained from the yerba mate by applying chromatographic separation to the yerba mate infusion, such as, but not limited to electrophoretic methods or liquid chromatography, specifically by HPLC, more specifically by reverse phase HPLC.

[0294] Herein provided is also a method for producing a composition comprising compounds obtained from yerba mate infusions. Said method can comprise the sequential steps of:

[0295] a) preparing a yerba mate infusion with deionized water.SMA001P 20260227

[0296] -35- Specifically, loose yerba mate material is placed on a filter and infused with hot deionized water. The filter can be any filter material appropriate for such purpose, e.g. filter paper, nylon filter.

[0297] The temperature can be in the range of 70-80 °C, specifically 75 °C. The infusion can be allowed to steep for about 20-40 minutes, specifically 25-35 minutes, specifically 30 minutes.

[0298] b) separating the infusion into fractions.

[0299] Specifically, reversed-phase high-performance liquid chromatography (rp-HPLC) system is used, specifically the rp-HPLC is run for 20 to 50 minutes, specifically 30-40 minutes, specifically about 34 minutes. The following sequential steps can be applied:

[0300] i. injection of the yerba mate infusion into the rp-HPLC system, specifically injecting 10 pL of the infusion with a flow rate of 1.0 mL / min,

[0301] ii. elution of fractions with a gradient mobile phase. The mobile phase A can consist of HPLC grade water supplemented with about 0.2 % formic acid. The mobile phase B can consist of HPLC grade acetonitrile supplemented with about 0.2% formic acid. The method employs a gradient approach.

[0302] iii. detection of eluants, specifically with a DAD-detector, specifically operating at wavelengths from 190 nm to 450 nm.,

[0303] iv. collection of the fractions, specifically starting the collection of 8 fractions comprising elution peaks, more specifically after 10 minutes into the run with each sample being collected for 0.5 minutes (equivalent to 0.5 mL per sample). Samples from each fraction were pooled as required.

[0304] c) selecting the fraction(s) comprising the compounds of the composition.

[0305] The present invention also encompasses the following embodiments:

[0306] 1. A composition comprising the following compounds:

[0307] at least one dicaffeoylquinic acid isomer,

[0308] at least one chlorogenic acid or an isomer thereof,

[0309] more than 1 % by weight rutin, and

[0310] one or more compounds selected from the group consisting of:

[0311] a caffeoyl-syringoylquinic acid or an isomer thereof,

[0312] nicotiflorin,

[0313] 3-O-Feruloylquinic acid, andSMA001P 20260227

[0314] -36- Eriojaposide A;

[0315] wherein the compounds are obtained, specifically isolated from yerba mate. 2. The composition of embodiment 1, wherein the compounds are obtained, specifically isolated by liquid chromatography, specifically by HPLC.

[0316] 3. The composition of embodiments 1 or 2, comprising Eriojaposide A.

[0317] 4. The composition of any one of embodiments 1 to 3, comprising caffeoyl-syringoylquinic acid or an isomer thereof and nicotiflorin.

[0318] 5. The composition of any one of embodiments 1 to 4, comprising 3-O-Feruloylquinic acid.

[0319] 6. The composition of any one of embodiments 1 to 5, wherein dicaffeoylquinic acid or isomer thereof is present in the percent concentration ratio range of 2 to 50, specifically 2.5 to 45, specifically 4 to 41.1 as calculated from Total Ion Chromatogram (TIC), and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0320] 7. The composition of any one of embodiments 1 to 6, wherein chlorogenic acid or isomer thereof is present in the percent concentration ratio range of 1 to 5, specifically 2.3 to 2.5 as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0321] 8. The composition of any one of embodiments 1 to 7, wherein caffeoyl-syringoylquinic acid or isomer thereof is present in the percent concentration ratio range of 2 to 3 specifically 2.5 to 2.9, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0322] 9. The composition of any one of embodiments 1 to 8, wherein nicotiflorin is present in the percent concentration ratio range of 3 to 5 specifically 4 to 5, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0323] 10. The composition of any one of embodiments 1 to 9, wherein 3-O-Feruloylquinic acid is present in the percent concentration ratio range of 20 to 30, specifically 25 to 30, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

[0324] 11. The composition of any one of embodiments 1 to 10, which is substantially free of benzo[a]pyrene, oxalic acid, tannins, heavy metals, arsenic, cadmium, or lead.SMA001P 20260227

[0325] -37- 12. The composition of any one of embodiments 1 to 11, wherein in the dicaffeoylquinic acid isomer is selected from the group consisting of 3,4-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid.

[0326] 13. The composition of any one of embodiments 1 to 12, wherein in the chlorogenic acid isomer is selected from the group consisting of 3-O-caffeoylquinic acid (3-CQA), 4-CQA, and 5-CQA.

[0327] 14. The composition of any one of embodiments 1 to 13, further comprising a compound selected from the group consisting of essential oil, polyphenol, polysaccharide, and combinations thereof.

[0328] 15. Food or food additive comprising the composition of any one of embodiments 1 to 14.

[0329] 16. A pharmaceutical preparation comprising the composition of any one of embodiments 1 to 14 and a pharmaceutically acceptable excipient.

[0330] 17. A cosmetic preparation, comprising the composition of any one of embodiments 1 to 14.

[0331] 18. Food supplement comprising the composition of any one of embodiments 1 to 14.

[0332] 19. A method for isolating the composition of any one of embodiments 1 to 14 comprising the sequential steps of:

[0333] a. preparing a yerba mate infusion, specifically placing loose yerba mate material on a filter and infusing with deionized water at a temperature range of 70-80 °C, specifically 75 °C, and allowing the infusion to steep for 25-35 minutes, specifically 30 minutes;

[0334] b. separating the infusion into fractions using a reversed-phase high-performance liquid chromatography (rp-HPLC) system, specifically running the rp-HPLC for 20 to 50 minutes, specifically about 34 minutes, comprising the sequential steps of:

[0335] i. injecting the infusion into the rp-HPLC system, specifically injecting 10 pL of the infusion with a flow rate of 1.0 mL / min,

[0336] ii. eluting with gradient mobile phases consisting of acetonitrile and / or HPLC-grade water supplemented with about 0.2% formic acid,

[0337] iii. detecting eluants with a DAD-detector, specifically at wavelengths from 190 nm and 450nmSMA001P 20260227

[0338] -38- iv. collecting fractions, specifically collecting 8 fractions comprising elution peaks, more specifically after 10 minutes into the run with each sample being collected for 0.5 minutes (equivalent to 0.5 mL per sample), and

[0339] c. selecting the fraction(s) comprising the compounds of the composition. 20. The composition of any one of embodiments 1 to 14 for use in the treatment of an inflammatory disease, specifically selected from the group consisting of cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE), asthma, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).

[0340] 21. The composition of any one of embodiments 1 to 14 for use in the treatment of an cancer, specifically selected from the group consisting brain, squamous cell, bladder, head, neck, liver, ovarian, esophageal, nasopharynx, or thyroid cancers, melanomas, lymphomas, leukemias or multiple myelomas, oral cavity, gastrointestinal tract, colon, colorectal, stomach, pancreas, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.

[0341] EXAMPLES

[0342] The examples described herein are illustrative of the present inventions. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention. Accordingly, it is understood that the examples are illustrative only and are not limiting the scope of the invention.SMA001P 20260227

[0343] -39- Example 1

[0344] Materials & Methods

[0345] Sample preparation

[0346] Yerba mate infusion

[0347] The yerba mate material for preparing infusions were provided by Laboratorio y Herboristeria Santa Margarita S.A., San Juan Bautista, Misiones, Paraguay. The yerba mate material for infusion consists of stems and leaves of Ilex paraguariensis (Aquifoliaceae) plants which had been dried and processed. Yerba mate infusion was brewed following traditional practices: 50 g of loose yerba mate material were placed in a filter and infused with 500 mL of deionized water (dehLO) at a temperature of 75 °C. The infusion was allowed to steep for 30 minutes.

[0348] Reversed-phase High-performance liquid chromatography (rpHPLC) LaChrom Elite

[0349] The high-performance liquid chromatography (HPLC) system utilized in these experiments was sourced from Hitachi, LaChrom Elite. The chromatographic separation column was a C18 column (Avantor™; Avantor® ACE® Equivalence; dimensions: 250 x 4.6 mm; 5 pM). The mobile phase A employed for elution consisted of a composition of HPLC-grade water (HPLC grade; VWR chemicals BDH) supplemented with 0.2 % formic acid (Sigma-Merck). The mobile phase B consisted of a composition of acetonitrile (HPLC grade; VWR chemicals BDH) supplemented with 0.2 % formic acid (Sigma-Merck). This mobile phase composition was meticulously crafted to optimize the separation of compounds within the sample matrix, ensuring robust and reproducible chromatographic performance. The method employed a gradient approach, meaning the concentration of acetonitrile changed throughout the chromatographic run (Table 1).

[0350] Table 1: Yerba mate HPLC LaChrom Elite analysis — method gradient

[0351]

[0352] SMA001P 20260227

[0353] -40- The running time for collecting fractions was 34 minutes. 10 pl of the yerba mate infusion were injected with a flow rate of 1 mL / min. Detection of eluting compounds was achieved using a DAD-detector (VWR, Hitachi, L-2450) operating at wavelengths from 190-450 nm.

[0354] Fractionation of the tea samples was accomplished utilizing an automated sample collection device (Model 2110, Biorad), which ensured precise and efficient collection of eluting peaks. The collection of fractions started at 10 minutes into the HPLC run, with each sample being collected over 0.5 minutes (equivalent to 0.5 mL per sample). Samples from each fraction were pooled as required, and the pooled fractions from 15 runs were combined and subsequently the fractions underwent desiccation under vacuum conditions using the SpeedVac (Concentrator 5301, Eppendorf) set to 60 °C. For the following chemotaxis experiments the fractions were resolubilized in 500 pL deH2O. For further analyzing with high resolution mass spectrometry the fractions were resolubilized in 20 pL of deH2O. HPLC data was evaluated using EZChrom Elite version 3.2.1 (EZChrom Elite Chromatography Data System by Agilent Technologies).

[0355] High Resolution Mass spectrometry

[0356] The experiments aimed at identifying the active ingredients in the fractions were carried out using an ultra-high-performance liquid chromatography (uHPLC) system combined with mass spectrometry. Specifically, a Dionex Ultimate 3000 HPLC system coupled to a QExactive Hybrid Quadrupole Orbitrap MS was employed. The chromatographic separation column was an Acquity™ Premier BEH C18 column (Waters; dimensions: 2.1 x 100 mm; 1.7 pm particle size; PN 186009453), paired with an Acquity™ UPLC BEH C18 VanGuard™ Pre-column (2.1 x 5 mm; 1.7 pm particle size; PN 186003975). The column temperature was maintained at 40 °C to ensure optimal separation conditions.

[0357] The mobile phase A employed for elution consisted of HPLC-grade water (HPLC grade; VWR chemicals BDH) supplemented with 0.2 % formic acid (Sigma-Merck). The mobile phase B consisted of acetonitrile (HPLC grade; VWR chemicals BDH) also supplemented with 0.2 % formic acid (Sigma-Merck). The elution process followed a gradient approach, enabling dynamic changes in the concentration of acetonitrile throughout the chromatographic run to optimize compound separation (Table 2).SMA001P 20260227

[0358] -41- Table 2: Method gradient for YM HRMS analysis

[0359]

[0360] Detection was carried out using a diode array detector (Thermo Scientific Dionex UltiMate 3000-Serie Diodenarray-Detector DAD-3000) with a wavelength range of 190-450 nm. The injection volume for each run was 3 pL of the resolved fractions collected through rpHPLC LaChrom Elite.

[0361] The mass spectrometry (MS) system was operated with the following parameter settings: a spray voltage of 3.5 kV in positive mode and 2.7 kV in negative mode, a capillary temperature of 330 °C, and a probe heater temperature of 350 °C. These parameters were selected to achieve robust ionization and reliable detection of analytes within the sample matrix. The mass spectrometry (MS) system parameters were carefully configured to ensure optimal performance. The sheath gas flow rate was set to 50 arbitrary units, while the auxiliary gas flow rate was maintained at 10 arbitrary units. The S-lens RF level was adjusted to 60 to enhance ion transmission efficiency. The resolution was configured to 70000 (FWHM) for full MS scans and 17500 (FWHM) for data-dependent MS / MS (ddMS2) analysis.

[0362] The system operated in HESI (heated electrospray ionization) mode, with polarity switching enabled to accommodate both positive and negative ion modes during the analysis. Data acquisition employed a “Top 5” MS / MS method, focusing on the five most intense precursor ions for fragmentation, thereby maximizing the detection and identification of analytes within the sample. The raw MS data obtained from UHPLC-HRMS analysis were first converted to the mzML format using MSConvert (version 3.0.24272-bc60c9, ProteoWizard) to ensure compatibility with downstream processing tools. The converted mzML files were then processed using MZmine (version 4.3.0) for data preprocessing, applying parameters such as retention time, intensity threshold, and m / z tolerances. Data was then exported in MGF format and imported into SIRIUS (version 6.0.7) for molecular formula prediction and compound annotation. Furthermore, data was exported from MZmine as a CSV file for the calculation of relative abundancesSMA001P 20260227

[0363] -42-of compounds, considering peak areas of the selected m / z values. Relative quantitation, and the resulting compound ratios, were based solely on identified compounds, with the total identified compound pool set as 100% for each fraction. The relative abundance of each compound was expressed as a percentage of this subset, and compound ratios were determined accordingly. Calculations were based on three replicates for each fraction.

[0364] Cultivation, isolation and stimulation of cells

[0365] Involvement of THP1, Jurkat cells, T-cells, and neutrophil cells is known in inflammatory diseases, specifically cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Thus, the above listed inflammatory diseases may be treated by inhibiting the mobilization, and migration of THP1, Jurkat cells, T-cells, or neutrophils. It follows that THP1, Jurkat cells, T-cells, and neutrophil cells are excellent in vitro models for evaluating inflammatory processes and diseases.

[0366] Inhibiting monocyte (i.e., THP1 cells) mobilization / migration inhibits metastasis formation in a mouse model.

[0367] THP1 cells

[0368] THP1 cells (Monocyte isolated from peripheral blood from an acute monocytic leukemia patient, ATCC TIB-202, a cell line appropriate as model for inflammatory processes) were bought from ATCC (The American Type Culture Collection). THP1 cells were cultured in an appropriate medium consisting of RPMI medium 1640 (1 *, Gibco), 10 % FBS (fetal bovine serum; Gibco), 1 % L-glutamine (Gibco) and 1 % Penstrep (Consisting of 0.5 % penicillin and 0.5 % % streptomycin; Gibco). Cultivation of THP1 cells involves several key steps to ensure their proper growth, maintenance, andSMA001P 20260227

[0369] -43-experimental utility. Twice a week, THP1 cells were harvested through centrifugation (5 minutes, 500 rpm) and cultivated with fresh medium. They were passaged once a week, to maintain healthy cultures and proper growth and to avoid overcrowding, which can lead to nutrient depletion and cell stress. The cells were incubated at 37 °C and 5 % (v / v) CO2.

[0370] Jurkat cells

[0371] Jurkat cells (Clone E6-1 (ATCC TIB-152, lymphoblast cells, a cell line which can serve as model for treatment of blood cancer) were bought from ATCC. Jurkat cells are a stable T-cell line and were grown in a suitable medium, which consisted of RPMI medium 1640 (1x, Gibco), 10 % FBS (fetal bovine serum; Gibco), 1 % L-glutamine (Gibco) and 1 % Penstrep (Mixture of 0.5 % penicillin and 0.5 % % streptomycin; Gibco). Twice a week, Jurkat cells were harvested through centrifugation (5 minutes, 500rpm) and cultivated with fresh medium. They were passaged twice a week to maintain healthy cultures and prevent overgrowing. The cells were incubated at 37 °C and 5 % (v / v) CO2.

[0372] T-cells

[0373] T-cells were isolated from whole blood collected from healthy human donors. T-cells are widely acknowledged to serve as models for studying a wide range of indications, particularly those involving the adaptive immune system, immunotherapy, and chronic inflammation. Isolation was carried out using a Ficoll Paque™ PLUS (Cytiva) density gradient. The buffy coat (containing the T-cells) was collected and followed by washing steps using HBSS - / - (Hanks' Balanced Salt solution without Magnesium and Calcium, Gibco) and centrifugation (10 minutes, 500 rpm). The collected T-cells were transferred into an appropriate medium (RPMI medium 1640, 1 x; Gibco) with 10 % FBS (fetal bovine serum; Gibco), 1 % L-glutamine (Gibco) and 1 % Penstrep (Consisting of 0.5 % penicillin and 0.5 % streptomycin; Gibco). The cells were incubated at 37 °C and 5 % (v / v) CO2. T-cells require a 2-day stimulation period with 2 pl / mL PHA-L (phytohemagglutinin-L, Sigma-Merck) followed by an at least 12-day stimulation with 100 U / mL IL-2 (lnterleukin-2; Sino Biological Inc.). During this process, IL-2 was replenished every 3 days to optimize their functionality and reactivity, which is essential for their effective application in immunological studies.SMA001P 20260227

[0374] -44- Neutrophil cells

[0375] Neutrophil cells were isolated from whole blood collected from healthy human donors. Neutrophil cells can serve as models to study a wide range of indications, such as acute inflammation, bacterial / fungal infections, and cancer-related inflammation. Isolation was carried out using a Ficoll Paque™ PLUS (Cytiva) density gradient followed by a Dextran (Dextran sulphate sodium salt, Sigma-Merck) gradient. The RMNC (polymorphonuclear cells)-rich fraction was diluted with HBSS - / - (Gibco) and centrifuged at 500 g for 10 minutes. After centrifugation ice-cold lysis was performed twice (ice-cold deH2O, 10xHBSS - / -) The ice-cold lysis was followed by 2 washing steps (HBSS - / -, centrifugation: 10 minutes, 500 rpm). Finally, the cell pellet was resuspended in HBSS + / + (final assay buffer) and adjusted to the desired cell concentration for chemotaxis assays

[0376] Cell kill curve

[0377] To provide background context, cell kill curve experiments were performed to confirm that the effects observed in chemotaxis assays are due to the inhibition of migration rather than cytotoxicity caused by the teas. For chemotaxis experiments the cells need to stay viable over a period of 30 minutes (Neutrophils) up to 2 hours (THP1 , Jurkat, T-cells).

[0378] The cells were harvested through centrifugation (5 minutes; 500 rpm) and counted using a Neubauer counting chamber and an automated cell counter (RWD Life Science Co.; C100, software: V01.00.00.27933). 1.5 mL cell suspension was transferred to each well of a 12-well plate resulting in a final cell number of 0.1 x 106cells / well. The cells were incubated at 37 °C, 5 % CO2 (v / v). After 48 hours incubation, the tea was added to the cells in different dilutions (1:10; 1:100; 1:1000). Each concentration was tested in triplicates. The cell viability was determined before the addition of the tea and 2, 4, and 24 hours after addition by mixing the cell suspension with trypan blue (Sigma-Merck). Trypan blue stains dead cells dark blue whereas viable cells appear white. The number of viable cells and dead cells was counted (Neubauer counting chamber and an automated cell counter).SMA001P 20260227

[0379] -45- Chemotaxis assays

[0380] The objective of the chemotaxis assays was to determine whether the teas influence the migration of various immune cells and stable cell lines in response to chemokines. Chemokines are signaling proteins that guide movement of various cell types. Besides attracting effector cells to infection sites, they also facilitate the passage of immune cells through different membranes to reach the inflammation. It was aimed at assessing whether the teas enhance or impede cellular migration.

[0381] The chemotaxis assays were performed with yerba mate infusion in different dilutions, as well as with the through rpHPLC collected fractions from yerba mate infusion (La Chrom Elite) with the above-mentioned cell-types. It was tested if the migration of different cell types is affected. Immediately before performing the chemotaxis assays, cells were washed and resuspended using HBSS + / + (Hanks' Balanced Salt solution with calcium and magnesium, Gibco) to the assigned final concentration. The tea, as well as the chemokines, were diluted with HBSS + / +. A 48-well Boyden chamber system (Neuroprobe) with a polycarbonate, PVP-free cell culture membrane (5pM pore size for THP1 cells, Neutrophils, and T-cells; 8 pM pore size for Jurkat cells; Neuroprobe) was used. Dilution series of the teas reached from undiluted to 1:10000. Various concentrations of chemokines were evaluated to determine the optimal concentration for the experimental protocols described herein. For the experiments, a concentration of 0.15 pM was utilized for each distinct chemokine. For THP1 cell chemotaxis assays as chemokine MCP1 (CCL2) was used. The SDF1 (CXCL12) was utilized for Jurkat cell Boyden chamber assays. For Neutrophil testing IL8 (CXCL8) was used. For T-cell Boyden chamber assays IP10 (CXCL10) was used. All chemokines were produced, purified and characterized in house.

[0382] 29 pl of the chemokine was placed in the lower part of the chamber. As background HBSS + / + was used. 50pl of cell suspension (THP1 cells: 4 x 106cells / mL; Jurkat cells: 4 x 106cells / mL; T-cells: 3.5 x 106cells / mL & 4 x 106cells / mL; Neutrophil cells: 3.5 x 106cells / mL & 4 x 106cells / mL) mixed with the tea dilutions or tea fractions were added in the upper wells of the chamber. After 30 minutes (Neutrophil) - 2 hours (Jurkat cells, THP1 cells, T-cells) of incubation time (migration period) at 37 °C and 5 % CO2 (v / v), the membrane was removed from the chamber and the side of the membrane with the non-migrated cells was rinsed twice with 1 x PBS (phosphate-buffered saline; pH 7.4; Gibco) and then air dried. The migrated cells were fixed on the membrane andSMA001P 20260227

[0383] -46-stained. For Jurkat cells, the Hematoxylin staining solution (Merck) and Eosin staining solution (Sigma-Merck) were used. The Hemacolor Kit (Merck) was employed for staining Neutrophils, T-cells, and THP1 cells. 5 Pictures of each well were taken (NIKON Eclipse E200 Light microscope connected to a PC via Nikon digital sight and operated by NIS-Elements, Version: BR 3.22.00)) and the migrated cells were counted (NIS-Elements Version: BR 3.22.00, Adobe Photoshop Version 25.9.1). The chemotactic index (Cl) was calculated by dividing the number of migrated cells by the migrated cells in the background (HBSS + / + only).

[0384] Results

[0385] rpHPLC fractions

[0386] A reversed-phase high-performance liquid chromatography was used as a reliable analytical technique to separate the components of yerba mate.

[0387] Eight fractions were collected in each run, as illustrated in Fig. 1. A total of fifteen runs for fraction collection of yerba mate were conducted. As shown in Fig. 2, the individual runs were found to be replicable.

[0388] Fig. 1 shows a chromatogram of yerba mate brew, the 8 collected fractions and their respective retention times.

[0389] Fig. 2 shows a chromatogram of yerba mate, overlay of four runs.

[0390] The retention times of the respective fractions under the above rpHPLC conditions are shown in Table 3.

[0391] Table 3: Retention times of rpHPLC fractions.

[0392]

[0393] High Resolution Mass spectrometry

[0394] Mass spectrometry, a highly precise and sensitive analytical technique, was employed to characterize the active compounds of yerba mate and identify keySMA001P 20260227

[0395] -47-molecules responsible for biological activity. By profiling molecular structures and assessing the composition of active fractions, it was aimed to establish a robust method.

[0396] High Resolution Mass spectrometry yerba mate brew

[0397] Fig. 3 shows high resolution mass spectrometry (HRMS) negative ion mode of yerba mate. A raw data overview is shown.

[0398] Fig. 4 shows HRMS positive ion mode of yerba mate. A raw data overview is shown.

[0399] Table 4 summarizes the compounds identified in Fig. 3 and Fig. 4 and their respective chemical details.

[0400] Table 4: compounds found in yerba mate infusion (HRMS)

[0401]

[0402] High Resolution Mass spectrometry verba mate Fraction 6, Fraction 7 and Fraction 8

[0403] Fig. 5 shows HRMS negative ion mode of fraction 6 (A), fraction 7 (B) and fraction 8 (C). A raw data overview is shown.SMA001P 20260227

[0404] -48- Fig. 6 shows HRMS positive ion mode of fraction 7 (A) and fraction 8 (B). A raw data overview is shown.

[0405] The chemical structures in the following Tables have been retrieved from https: / / pubchem.ncbi.nlm.nih.gov / compound / .

[0406] HRMS negative ion mode Fraction 6

[0407] Table 5 summarizes the compounds identified in fraction 6 in Fig. 5 and their respective chemical details.

[0408] Table 5: Compounds identified in Fraction 6, negative ion mode

[0409]

[0410] HRMS negative ion mode Fraction 7

[0411] Table 6 summarizes the compounds identified in Fraction 7 in Fig. 5 and their respective chemical details.

[0412] Table 6: Compounds identified in Fraction 7, negative ion mode

[0413]

[0414] HRMS negative ion mode Fraction 8

[0415] Table 7 summarizes the compounds identified in fraction 8 in Fig. 5 and their respective chemical details.SMA001P 20260227

[0416] -49- Table 7: Compounds identified in Fraction 8, negative ion mode

[0417]

[0418] HRMS positive ion mode Fraction 7

[0419] Table 8 summarizes the compounds identified in fraction 7 in Fig. 6 and their respective chemical details.

[0420] Table 8: Compounds identified in Fraction 7, positive ion mode

[0421]

[0422] HRMS Positive ion mode Fraction 8

[0423] Table 9 summarizes the compounds identified in fraction 8 in Fig. 6 and their respective chemical details.

[0424] Table 9: Compounds identified in Fraction 8, positive ion mode

[0425]

[0426] Summary of compounds identified in Fractions 6-8

[0427] Table 10 summarizes the compounds that have been identified in fractions 6-8 by HRMS and combines positive and negative ion mode.SMA001P 20260227

[0428] -50- Table 10: Compounds identified in Fractions 6-8

[0429]

[0430] Due to its later appearance in the reversed-phase chromatogram, the more hydrophobic character of the compounds contained in fraction 8 is revealed, which refers to a high cell membrane-targeting bio-activity impacting leukocyte activation and mobilization.

[0431] Cell kill curve

[0432] The number of THP1 cells per mL as shown in Fig. 7 suggests that yerba mate infusion, in various dilutions, is non-toxic to the cells. However, it is observed that THP1 cells treated with yerba mate infusion do not proliferate as fast as the untreated cells. The percentage of viable THP1 cells as illustrated in Fig. 8 indicates that yerba mate infusion does not cause cell death when added. Consequently, no cell death due to yerba mate infusion is expected during the 2-hour migration period in the chemotaxis assays.

[0433] Chemotaxis assays

[0434] Charts were visualized using GraphPad Prism 10 and Origin 8.0. The P values were calculated for the extracts / fractions with the highest influence in migration of cells.

[0435] Negative control assay with THP1 cells (MCP1 as a chemokine) and water As a negative control a Boyden-chamber experiment with water (instead of yerba mate tea or fraction) was performed. Water has less to no effect on the migration of THP1 cells.SMA001P 20260227

[0436] -51-

[0437] THP1 cells (MCP1 as a chemokine) treated with yerba mate infusion in different dilutions

[0438] Yerba mate infusion seems to have a concentration (dilution) dependent effect in decreasing the migration of THP1 cells. The 1:10 dilution of yerba mate shows the most influence in migration. Higher dilutions show less influence (Fig. 9).

[0439] Fig. 9 shows the Chemotactic Index (Cl) of THP1 cells treated with yerba mate extracts (****: P value < 0.0001).

[0440] The x axis labels of Fig. 9 are:

[0441] MCP1: Chemokine;

[0442] YM1: Yerba mate infusion Dilution (1:10);

[0443] YM2: Yerba mate infusion Dilution (1:100);

[0444] YM3: Yerba mate infusion Dilution (1:1000);

[0445] YM4: Yerba mate infusion Dilution (1:10000)

[0446] Jurkat cells (SDF1 as a chemokine) treated with yerba mate infusion in different dilutions

[0447] Yerba mate infusion seems to have a concentration (dilution) dependent effect in decreasing the migration of Jurkat cells. The 1:10 dilution of yerba mate shows the most influence in migration, as shown in Fig. 10.

[0448] Fig. 10 shows the Chemotactic Index (Cl) of Jurkat cells treated with yerba mate extracts (**: P value < 0.01).

[0449] The x axis labels of Fig. 10 are:

[0450] SDF 1: chemokine;

[0451] YM1: Yerba mate infusion Dilution (1:10);

[0452] YM2: Yerba mate infusion Dilution (1:100);

[0453] YM3: Yerba mate infusion Dilution (1:1000);

[0454] YM4: Yerba mate infusion Dilution (1:10000)

[0455] As the infusion and its dilutions showed promising effects in decreasing the cell migration of the used cell lines, the focus of experimentation shifted towards immuneSMA001P 20260227

[0456] -52-cells, specifically neutrophils and T-cells, which were isolated from whole blood obtained from healthy human donors.

[0457] Neutrophil cells (IL8 as a chemokine) treated with yerba mate infusion in different dilutions

[0458] In neutrophils, both the 1:10 and 1 : 100 dilutions of yerba mate infusion exhibit the greatest impact on migration, whereas higher dilutions (1:1000 and 1:10000) demonstrate less influence (Fig. 11)

[0459] Fig. 11 shows the Chemotactic Index (Cl) of neutrophils (Donor 1 & Donor 2) treated with yerba mate extracts. The Cl was normalized to 1.

[0460] IL8 = chemokine.

[0461] Jurkat cells (SDF1 as a chemokine) treated with yerba mate fractions (collected through rpHPLC LaChromElite)

[0462] Fractions obtained from reversed-phase-High-performance liquid chromatography (HPLC LaChromElite) were evaluated using chemotaxis assay to determine their effect. Fractions 6, 7, and 8 are particularly influential.

[0463] Fig. 12 shows the significant immunoregulatory effect was shown by the chemotactic Index (Cl) of Jurkat cells treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (**: P value < 0.01)..

[0464] Neutrophils (IL-8 as a chemokine) treated with yerba mate fractions (collected through rpHPLC LaChromElite)

[0465] Fractions 6, 7, and 8 are particularly influential in decreasing the migration of neutrophils as shown in Fig. 13.

[0466] Fig. 13 shows the significant immunoregulatory effect was shown by the chemotactic Index (Cl) of neutrophils treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, andSMA001P 20260227

[0467] -53- Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (****: P value < 0.0001).

[0468] THP-1 cells (MCP-1 as a chemokine) treated with yerba mate fractions (collected through rpHPLC LaChromElite)

[0469] Fractions 6, 7, and 8 are particularly decreasing the migration of THP-1 cells as shown in Fig. 14.

[0470] Fig. 14 shows the significant immunoregulatory effect was shown by the chemotactic Index (Cl) of THP-1 cells treated with fraction 6 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and 3-O-Feruloylquinic acid; fraction 7 comprising a dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, and Eriojaposide A.; and fraction 8 comprising dicaffeoylquinic acid isomer, a chlorogenic acid isomer, rutin, caffeoyl-syringoylquinic acid isomer and nicotiflorin (****: P value < 0.0001).

[0471] Summary of the chemotaxis assays

[0472] With all tested cell types and chemokines, the 1:10 dilution of yerba mate infusion had the biggest effect on cell migration. Interestingly, in neutrophil cells (IL8 as a chemokine), the 1:100 dilution of yerba mate infusion also showed a strong effect on cell migration.

[0473] Furthermore the through rpHPLC collected fractions, specifically fractions 6, 7, and 8 have been shown to reduce cell migration across various cell types, including established cell lines and freshly isolated cells from healthy human donor blood.

[0474] Summary of the relation between the present results and the immunomodulatory effect of yerba mate and the tested fractions

[0475] To identify and isolate bioactive compounds, yerba mate infusion was fractionated using rpHPLC. The immunomodulatory activity of the resulting fractions was assessed using various cell-based assays, revealing a significant reduction in the migration of different immune cell types. The bioactive fractions were subsequently analyzed by mass spectrometry to determine their chemical composition.SMA001P 20260227

[0476] -54- The findings confirmed that specific compounds within yerba mate have immunomodulatory properties by modulating immune cell migration, a key process in inflammatory responses. The identified bioactive constituents may interact with immune signaling pathways, thereby influencing immune cell behavior. These results provide valuable insights into the molecular mechanisms underlying the immunoregulatory potential of these compounds and their therapeutic applications.

[0477] The specific compounds obtained from yerba mate are combined into a composition as described herein to harvest their immunomodulatory effects. Specifically, one, two, three, or more fractions, collected as described herein, are combined into a composition as described herein.SMA001P 20260227

[0478] -55- REFERENCES

[0479] Alam et al. (2022). Therapeutic Implications of Caffeic Acid in Cancer and Neurological Diseases. Frontiers in Oncology, 12.

[0480] Bauer et al. (2016). Teedrogen und Phytopharmaka. (Wolfgang Blaschek, Ed.) (Vol. 6). Wissenschaftliche Verlagsgesellschaft mbH.

[0481] ben Sghaier et al. (2016). Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells. Biomedicine & Pharmacotherapy, 84: 1972-1978.

[0482] Bravo et al. (2007). LC / MS characterization of phenolic constituents of mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to commonly consumed beverages. Food Research International, 40(3): 393-405.

[0483] Choi et al. (2017). Long-Term Dietary Supplementation with Yerba Mate Ameliorates Diet-Induced Obesity and Metabolic Disorders in Mice by Regulating Energy Expenditure and Lipid Metabolism. Journal of Medicinal Food, 20(12): 1168-1175.

[0484] de Lima et al. (2019). Comparing the Effects of Chlorogenic Acid and Ilex paraguariensis Extracts on Different Markers of Brain Alterations in Rats Subjected to Chronic Restraint Stress. Neurotoxicity Research, 35(2): 373- 386.

[0485] dos Santos Polidoro et al. (2023). Analytical and chemometric strategies for elucidation of yerba mate composition. Food Chemistry, 429: 136918.

[0486] Kaltbach et al. (2020). New HPTLC methods for analysis of major bioactive compounds in mate (Ilex paraguariensis) tea. Journal of Food Composition and Analysis, 92: 103568.

[0487] Kang et al. (2012). Anti-obesity and anti-diabetic effects of Yerba Mate (Ilex paraguariensis) in C57BL / 6J mice fed a high-fat diet. Laboratory Animal Research, 28(1): 23-9.

[0488] Lins etal. (2020). Rutin prevents cisplatin-induced ovarian damage via antioxidant activity and regulation of PTEN and FOXO3a phosphorylation in mouse model. Reproductive Toxicology, 98: 209-217.

[0489] Lu etal. (2020). Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action,SMA001P 20260227

[0490] -56- and future directions. Comprehensive Reviews in Food Science and Food Safety, 19(6): 3130-3158.

[0491] Mirzaei et al. (2021). Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways: New hope in the fight against cancer. Pharmacological Research, 171: 105759.

[0492] Nirumand et al. (2018). Dietary Plants for the Prevention and Management of Kidney Stones: Preclinical and Clinical Evidence and Molecular Mechanisms. International Journal of Molecular Sciences, 19(3): 765.

[0493] Pavlikova. (2022). Caffeic Acid and Diseases — Mechanisms of Action. International Journal of Molecular Sciences, 24(1): 588.

[0494] Puangpraphant. (2012, February 1). Anti-inflammatory and anti-colon cancer potential of yerba mate (Ilex paraguariensis St. Hilaire) bioactive constituents (Ph.D. Dissertation). University of Illinois, Urbana, Illinois.

[0495] Rocio Soledad et al. (2021). Yerba Mate Modulates Tumor Cells Functions Involved in Metastasis in Breast Cancer Models. Frontiers in Pharmacology, 12.

[0496] Santana-Galvez et al. (2017). Chlorogenic Acid: Recent Advances on Its Dual Role as a Food Additive and a Nutraceutical against Metabolic Syndrome. Molecules, 22(3): 358.

[0497] Santiano et al. (2023). Protective effects of Yerba mate (Ilex paraguariensis) on prostate cancer development. Nutrition, 108: 111957.

[0498] Semwal etal. (2021). Health benefits and limitations of rutin - A natural flavonoid with high nutraceutical value. Phytochemistry Letters, 46: 119-128.

[0499] Souza etal. (2021). Simultaneous extraction and separation of compounds from mate (Ilex paraguariensis) leaves by pressurized liquid extraction coupled with solid-phase extraction and in-line UV detection. Food Chemistry: Molecular Sciences, 2: 100008.

[0500] Tao etal. (2023). Ethnopharmacology of Rubus idaeus Linnaeus: A critical review on ethnobotany, processing methods, phytochemicals, pharmacology and quality control. Journal of Ethnopharmacology, 302: 115870.

[0501] Yang etal. (2019). Conversion of Rutin to Quercetin by Acid Treatment in Relation to Biological Activities. Preventive Nutrition and Food Science, 24(3): 313-320.

Claims

1. SMA001P 20260227-57-CLAIMS1. A composition comprising the following compounds:- at least one dicaffeoylquinic acid isomer,- at least one chlorogenic acid or an isomer thereof,- more than 1 % by weight rutin,- a caffeoyl-syringoylquinic acid or an isomer thereof,- nicotiflorin, andone or more compounds selected from the group consisting of:- 3-O-Feruloylquinic acid, and- Eriojaposide A;wherein the compounds are isolated from yerba mate.

2. The composition of claim 1 , wherein the compounds are isolated by liquid chromatography, specifically by high-performance liquid chromatography (HPLC).

3. The composition of claims 1 or 2, comprising Eriojaposide A.

4. The composition of any one of claims 1 to 3, comprising 3-O-Feruloylquinic acid.

5. The composition of any one of claims 1 to 4, wherein the dicaffeoylquinic acid isomer is present in the percent concentration ratio range of 2 to 50, specifically 2.5 to 45, specifically 4 to 41.1 as calculated from Total Ion Chromatogram (TIC), and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

6. The composition of any one of claims 1 to 5, wherein chlorogenic acid or isomer thereof is present in the percent concentration ratio range of 1 to 5, specifically 2.3 to 2.5 as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

7. The composition of any one of claims 1 to 6, wherein the caffeoyl-syringoylquinic acid or isomer thereof is present in the percent concentration ratio range of 2 to 3 specifically 2.5 to 2.9, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

8. The composition of any one of claims 1 to 7, wherein nicotiflorin is present in the percent concentration ratio range of 3 to 5 specifically 4 to 5, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).SMA001P 20260227-58- 9. The composition of any one of claims 1 to 8, wherein 3-O-Feruloylquinic acid is present in the percent concentration ratio range of 20 to 30, specifically 25 to 30, as calculated from TIC, and wherein the percent concentration ratio is based on concentrations in weight per volume (g / L).

10. The composition of any one of claims 1 to 9, which is substantially free of benzo[a]pyrene, oxalic acid, tannins, heavy metals, arsenic, cadmium, or lead.

11. The composition of any one of claims 1 to 10, wherein the dicaffeoylquinic acid isomer is selected from the group consisting of 3,4-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid.

12. The composition of any one of claims 1 to 11, wherein in the chlorogenic acid isomer is selected from the group consisting of 3-O-caffeoylquinic acid (3-CQA), 4-CQA, and 5-CQA.

13. The composition of any one of claims 1 to 12, further comprising a compound selected from the group consisting of essential oil, polyphenol, polysaccharide, and combinations thereof.

14. Food or food additive comprising the composition of any one of claims 1 to 13.

15. A food supplement comprising the composition of any one of claims 1 to 13.

16. A cosmetic preparation comprising the composition of any one of claims 1 to 13.

17. A pharmaceutical preparation comprising the composition of any one of claims 1 to 13 and a pharmaceutically acceptable excipient.

18. A method for isolating the composition of any one of claims 1 to 13 comprising the sequential steps of:a. preparing a yerba mate infusion, specifically placing loose yerba mate material on a filter and infusing with deionized water at a temperature range of 70-80 °C, specifically 75 °C, and allowing the infusion to steep for 25-35 minutes, specifically 30 minutes;b. separating the infusion into fractions using a reversed-phase high-performance liquid chromatography (rp-HPLC) system, specifically running the rp-HPLC for 20 to 50 minutes, specifically about 34 minutes, comprising the sequential steps of:SMA001P 20260227-59- i. injecting the infusion into the rp-HPLC system, specifically injecting 10 pL of the infusion with a flow rate of 1.0 mL / min,ii. eluting with gradient mobile phases consisting of acetonitrile and / or HPLC-grade water supplemented with about 0.2% formic acid,iii. detecting eluants with a DAD-detector, specifically at wavelengths from 190 nm and 450 nmiv. collecting fractions, specifically collecting 8 fractions comprising elution peaks, more specifically after 10 minutes into the run with each sample being collected for 0.5 minutes, andc. selecting the fraction(s) comprising the compounds of the composition.

19. The composition of any one of claims 1 to 13 for use in the treatment of an inflammatory disease, specifically selected from the group consisting of cytokine release syndrome, neuroinflammation, vasculitis, arthritis, sepsis, septic shock, endotoxic shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, Malaria, NASH (nonalcoholic steatosis hepatis), multiple sclerosis, acute and chronic pancreatitis, Type 1 diabetes, IgA nephropathy, interstitial cystitis, post COVID syndrome, psoriasis, glomerulonephritis, inflammatory bowel disease, nephritis and peritonitis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).

20. The composition of any one of claims 1 to 13 for use in the treatment of a cancer, specifically selected from the group consisting brain, squamous cell, bladder, head, neck, liver, ovarian, esophageal, nasopharynx, or thyroid cancers, melanomas, lymphomas, leukemias or multiple myelomas, oral cavity, gastrointestinal tract, colon, colorectal, stomach, pancreas, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.

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