Composition for inhibiting side effects of anti-cancer treatment, containing cleavers extract or fraction thereof as active ingredient

Abstract

The present invention relates to a composition for always resisting side effects in an anti-cancer treatment, which contains a Galium aparine extract or a graded product thereof as an active ingredient, and which has an excellent effect of preventing, improving or treating side effects in an anti-cancer treatment. The present invention relates to a pharmaceutical product, a food product or a cosmetic product which can be advantageously used for the purpose of inhibiting side effects of an anti-cancer treatment, including: for example, oxidative stress; immune decline or immunomodulatory abnormality; digestive tract injuries such as vomiting, diarrhea, gastrorrhagia or gastric ulcer; skin injuries such as skin redness, edema, blister, dermatitis, xerosis, keratinization or pigmentation, etc.

Description

Technical Field

[0001] This invention is a result of the Industrial Cluster Competitiveness Enhancement Project (Project No. 1415180942) supported by the Korea Industrial Complex and Industrial Development Organization, and relates to a composition containing *Galium affine* extract or its fractions as an active ingredient for inhibiting the side effects of anticancer treatment. More specifically, it relates to a composition containing a hot water extract of *Galium affine* or its high molecular weight fractions with a molecular weight of 3000 or higher as an active ingredient for inhibiting the side effects of anticancer treatment. Background Technology

[0002] There are various anti-cancer therapies used to treat cancer or inhibit the growth of cancer cells. Examples include surgery to remove tumors, chemotherapy using drugs that destroy cancer cells or inhibit their growth, radiation therapy using high-energy radiation to destroy cancer cells, immunotherapy that activates the body's immune system to attack cancer cells, targeted therapy that targets specific genes or proteins of cancer cells, hormonal therapy that inhibits hormones that promote the growth of cancer cells, stem cell transplantation that generates new blood cells from transplanted stem cells, and combination therapy that uses two or more anti-cancer treatments simultaneously or sequentially.

[0003] In cancer treatment, radiation therapy and chemotherapy are highly effective methods for inhibiting the proliferation and survival of tumor cells, but they also cause serious damage to normal cells, leading to a variety of side effects. These side effects include hair loss, fatigue, nausea and vomiting, decreased immunity, anemia, bleeding, diarrhea or constipation, digestive system damage, peripheral nerve damage, kidney and liver damage, decreased memory and attention, decreased reproductive function, skin inflammation, and cardiovascular disease, among many others. These side effects are known to be primarily caused by oxidative stress and inflammatory responses resulting from the excessive generation of reactive oxygen species (ROS). In particular, radiation dermatitis, a common side effect in patients receiving radiation therapy, not only causes skin redness, ulceration, and blisters, but can also lead to skin fibrosis and pigmentation changes in the long term. To address this problem, the use of antioxidant materials to neutralize ROS and protect normal cells has attracted attention as an effective way to reduce the side effects of radiation and anticancer drug treatments.

[0004] Furthermore, radiation therapy and chemotherapy promote cell suicide by inducing DNA damage, ROS generation, and mitochondrial dysfunction. However, in this process, the DNA of normal cells also suffers damage, leading to various cellular repair mechanisms. If repair fails, cells face the risk of suicide or cancerous transformation. Therefore, developing a composition to inhibit these side effects is crucial. Bax and Bcl gene expression analysis serves as an important indicator for understanding cellular responses to DNA damage induced by radiation and anticancer therapies.

[0005] Therefore, the inventors sought to develop a composition for alleviating the effects of radiation and chemotherapy on normal cells by reducing DNA damage and oxidative stress, controlling inflammatory responses, and protecting normal tissues to minimize side effects. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] One object of the present invention is to provide a composition for inhibiting the side effects of anticancer treatment, comprising cleavers extract or its fractions as an active ingredient.

[0008] Another object of the present invention is to provide a method for suppressing the side effects of anticancer treatment in a subject by administering the composition to the subject.

[0009] (II) Technical Solution

[0010] As one embodiment, the present invention provides a composition for inhibiting the side effects of anticancer treatment, comprising cleavers extract or its fractions as active ingredients.

[0011] In this invention, "anti-cancer treatment" refers to radiation therapy, which uses high-energy radiation to destroy cancer cells, and / or chemotherapy, which uses drugs that destroy cancer cells or inhibit their growth, among various anti-cancer treatment methods. For convenience, the chemotherapy is also referred to as an anti-cancer agent.

[0012] In this invention, "side effects of anticancer treatment" refers to the side effects caused by damage to normal cells in addition to the original effect of inhibiting the proliferation and survival of tumor cells during anticancer treatment. Examples of such side effects include: oxidative stress; decreased immunity or abnormal immune regulation; gastrointestinal damage such as vomiting, diarrhea, gastric bleeding or gastric ulcers; and skin damage such as redness, swelling, blisters, dermatitis, xerosis, keratosis and pigmentation changes.

[0013] In this invention, "suppression of side effects" refers to reducing and eliminating the side effects of anticancer treatment. For example, it refers to reducing or eliminating various side effects caused by anticancer treatment through the reduction of oxidative stress caused by excessive generation of reactive oxygen species (ROS), regulation of inflammatory response, prevention of DNA damage, and prevention of skin damage.

[0014] In this invention, "Galium aparine" is an annual vine belonging to the order Gentianales, family Rubiaceae, and genus Galium. The entire plant is approximately 20-90 cm long, with green stems and thorny leaves. Galium aparine is distributed throughout Asia (e.g., Korea, China, and Japan), Europe, North America, North Africa, and Oceania. In traditional Chinese medicine, galium aparine is used to clear heat and detoxify, promote diuresis, and reduce swelling, and is used to treat various diseases such as urinary tract infections, traumatic injuries, and otitis media.

[0015] In this invention, "extract" refers to a product obtained by immersing a desired substance in a variety of solvents, then extracting and fractionating the liquid phase component at room temperature or under heating conditions for a predetermined time, and removing the solvent from the liquid phase component to obtain a solid component, etc.

[0016] As a specific example, the *Galium aparine* extract can be obtained by extracting *Galium aparine* in water, preferably at 70°C to 100°C, more preferably at 70°C to 90°C, more preferably at 75°C to 85°C, more preferably at 78°C to 82°C, more preferably at 79°C to 81°C, and most preferably at 80°C, for 2 to 12 hours, preferably 2 to 10 hours, more preferably 2 to 8 hours, more preferably 3 to 6 hours, more preferably 3 to 5 hours, more preferably 3.5 to 4.5 hours, more preferably 3.8 to 4.2 hours, and most preferably 4 hours.

[0017] When extracting the cleavers extract, some or all of the above-ground or underground parts of the cleavers can be used, preferably the leaves and stems of the above-ground parts of the cleavers, and more preferably the leaves of the cleavers.

[0018] In this invention, "fractionated product" refers to a product obtained by fractionation to separate a specific component or a specific group of components from a mixture containing various components.

[0019] As a specific example, the "gradation material" may depend on the molecular weight, which may be 3000.

[0020] As a specific embodiment, the fractionated form of the *Galium aparine* extract of the present invention is a fractionated form composed of high molecular weight substances with a molecular weight of 3000 or more. The fractionated form composed of high molecular weight substances with a molecular weight of 3000 or more is referred to herein as a high molecular weight fractionated form or GA-H, and the fractionated form composed of low molecular weight substances with a molecular weight of less than 3000 is referred to herein as a low molecular weight fractionated form or GA-L. For the high molecular weight fractionated form, the content of asperulosidic acid is lower than that of the low molecular weight fractionated form, approximately 60% to 70%. Furthermore, although the low molecular weight fractionated form does not contain protein, the high molecular weight fractionated form contains approximately 3.8% to 3.9% of the total high molecular weight fractionated form and contains proteoglycans with a molecular weight of 35 kDa.

[0021] The composition of the present invention for inhibiting the side effects of anticancer treatment contains the extract of Galium aparine or its high molecular weight fraction as an active ingredient.

[0022] The phrase "comprising...as an active ingredient" means that the composition contains a sufficient amount of the extract or its fractions to achieve the desired effect. In this case, the desired effect in this invention may be the suppression of side effects caused by radiation therapy or chemotherapy, and the sufficient amount required to achieve the desired effect can be appropriately adjusted according to the recipient, form of administration, route of administration, etc.

[0023] Regarding the active ingredient of this invention, namely the cleavers extract or its polymer fraction, it may contain 0.0001% to 50% by weight, preferably 0.001% to 50% by weight, based on the total amount of the total composition. If the content of the cleavers extract or its GA-H is less than 0.0001% by weight, the inhibitory effect on the side effects of anticancer treatment is too weak; if it exceeds 50% by weight, there is a problem that the increase in effect based on the increased content is minimal and the stability of the formulation cannot be ensured.

[0024] The active ingredient of this invention, namely the extract of Galium aparine or its high molecular weight fraction, has the following effects: prevention, improvement and treatment of side effects caused by radiation and anticancer drug treatment, such as oxidative stress; decreased immunity or abnormal immune regulation; gastrointestinal damage such as vomiting, diarrhea, gastric bleeding or gastric ulcers; and skin damage such as redness, swelling, blisters, dermatitis, xerosis, keratosis and pigmentation changes.

[0025] As a specific example, the *Galium aparine* extract and its polymer fractions of the present invention have antioxidant effects in removing reactive oxygen species (ROS). Therefore, the *Galium aparine* extract and its polymer fractions of the present invention have the effect of reducing oxidative stress and skin damage caused by radiation and anticancer drug treatment.

[0026] As another specific example, the *Galium aparine* extract and its high-molecular-weight fraction of the present invention exhibit almost no cytotoxic effects. Furthermore, when macrophages are treated alone, it induces the production of NO and TNF-α without inducing the production of inflammatory cytokines such as IL-10. When macrophages treated with LPS are treated, it has the effect of regulating the production of inflammatory cytokines such as NO, TNF-α, and IL-10. Therefore, the *Galium aparine* extract and its high-molecular-weight fraction of the present invention have the following effects in radiation and anticancer therapy: promoting the removal of cancer cells through an immune response based on the regulation of M1 / M2 macrophage differentiation and the regulation of NF-κB and MAPK signaling pathways, while simultaneously inhibiting excessive inflammatory responses, thereby reducing side effects.

[0027] As another specific example, the *Galium aparine* extract and its high-molecular-weight fractions of the present invention activated cell proliferation in H2O2-treated HaCaT cells and exhibited the effects of inhibiting the expression of the Bax gene, which induces cell suicide, and promoting the expression of the Bcl gene, which induces and inhibits cell suicide. Furthermore, the *Galium aparine* extract and its high-molecular-weight fractions of the present invention regulated cellular energy balance in H2O2-treated HaCaT cells and increased phosphorylation of AMP-activated protein kinase (AMPK), which is related to DNA repair. Therefore, the *Galium aparine* extract and its high-molecular-weight fractions of the present invention have the effects of preventing damage to normal cells and inhibiting cell suicide, while promoting the survival of normal cells in radiation and anticancer therapy.

[0028] As another specific example, the *Galium aparine* extract and its high-molecular-weight fractions of the present invention promote the proliferation of mouse bone marrow cells and promote the production of cytokines such as IL-3, IFN-γ, GM-CSF, and IL-5. Therefore, the *Galium aparine* extract and its high-molecular-weight fractions of the present invention have the effect of promoting bone marrow cell differentiation and immune cell activation during radiation and anticancer therapy, thereby increasing the immune response.

[0029] As another specific example, the *Galium aparine* extract and its polymeric fractions of the present invention reduce IL-8 production in AGS cells (a gastric epithelial cell line treated with TNF-α) and activate the NF-κB (p-p65) and MAPK (p-p38) pathways. Furthermore, the *Galium aparine* extract and its polymeric fractions of the present invention increase the production of trefoil factor 3 (TFF-3), which plays a role in the production of mucin 2 and mucosal regeneration in mucin-producing cells (LS174T cells). Therefore, the *Galium aparine* extract and its polymeric fractions of the present invention reduce gastric inflammation during radiation and anticancer therapy and normalize mucus production in the digestive and respiratory organs, thereby reducing side effects such as vomiting, diarrhea, gastric bleeding, or gastric ulcers.

[0030] As another specific example, the *Galium aparine* extract and its polymeric fractions of the present invention inhibited MMP-1 expression and increased TGF-β expression in H2O2-treated HaCaT cells. Furthermore, the *Galium aparine* extract and its polymeric fractions of the present invention increased the expression of filaggrin and β-defensin 2 in H2O2-treated HaCaT cells. Therefore, the *Galium aparine* extract and its polymeric fractions of the present invention have a skin-protective effect in preventing or alleviating skin side effects such as redness, edema, blisters, dermatitis, xerosis, keratosis, and pigmentation changes during radiation and anticancer therapy.

[0031] Compositions containing cleavers extract and its high molecular weight fractions as active ingredients, which have the above-mentioned effects, and are used to inhibit the side effects of anticancer treatment, can be used as pharmaceuticals, food or cosmetics for the purpose of inhibiting the side effects of anticancer treatment.

[0032] Therefore, as a specific embodiment, the present invention can provide the anticancer treatment side effect suppression composition as a pharmaceutical composition.

[0033] The pharmaceutical composition refers to a composition prepared for the purpose of disease prevention or treatment, and can be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and can be formulated into parenteral dosage forms using diluents or excipients such as lubricants, wetting agents, flavoring agents, emulsifiers, suspensions, preservatives, and surfactants. Furthermore, it can be formulated into topical preparations, suppositories, topical skin preparations, and sterile injectable solutions. As a specific example, the pharmaceutical composition of the present invention can be used orally in oral dosage forms. As another specific example, the pharmaceutical composition of the present invention can be used by methods such as direct application or dispensing onto damaged skin.

[0034] Furthermore, depending on the dosage form, it may be prepared by including pharmaceutically acceptable carriers, such as buffers, analgesics, solubilizers, isotonic agents, stabilizers, bases, and other carriers known in the art.

[0035] In this invention, the term "pharmaceutically acceptable carrier" can refer to a carrier or diluent that does not irritate the organism and does not hinder the bioactivity and properties of the injected active ingredient. There are no particular limitations on the types of carriers that can be used in this invention; any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of such carriers include saline solutions, sterile water, Ringer's solution, buffered saline solutions, albumin injections, glucose solutions, maltodextrin solutions, glycerol, ethanol, etc. These can be used alone or in combination of two or more. The carrier can be a non-naturally occurring carrier.

[0036] The pharmaceutical compositions of the present invention can be administered in pharmaceutically effective amounts. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment without causing side effects. The effective dose level can be determined based on factors including the patient's health status, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and excretion rate, the duration of treatment, factors of concomitant or simultaneous use of other drugs, and other factors known in the medical field. Specifically, the dosage is typically 0.01 mg to 5000 mg per kg of the subject's body weight, and can be administered once daily or in several divided doses at predetermined intervals, but is not limited thereto, depending on the judgment of a physician or pharmacist.

[0037] The pharmaceutical compositions of the present invention can be used alone or in combination with other pharmaceutically active compounds that exhibit preventive, ameliorative, or inhibitory effects on anticancer treatment side effects, or in appropriate combinations thereof.

[0038] The pharmaceutical compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, they can be administered once or multiple times. Taking all the above factors into consideration, it is important to administer an amount that achieves maximum effect with minimal dosage without causing side effects, and this can be readily determined by those skilled in the art.

[0039] In this invention, the term "administration" means introducing the pharmaceutical composition of the invention to a subject by any suitable method, and for the route of administration of the composition of the invention, it can be administered via a variety of routes, either orally or parenterally, as long as it can reach the target tissue.

[0040] The administration method of the pharmaceutical composition according to the present invention is not particularly limited and can be based on methods commonly used in the art. As a non-limiting example of the administration method, the composition can be administered orally or parenterally.

[0041] The pharmaceutical compositions according to the invention can be prepared in various dosage forms according to the desired route of administration.

[0042] There is no particular limitation on the frequency of administration of the composition of the present invention; it can be administered once a day or the dosage can be divided into multiple administrations.

[0043] As another specific embodiment, the present invention can provide the composition for inhibiting the side effects of anticancer treatment as a food composition.

[0044] The food composition can be various foods, beverages, food additives, etc., but is not limited to these.

[0045] The content of herbal extracts as active ingredients contained in the food composition of the present invention can be appropriately adjusted according to the food form, the intended use, etc., without limitation. For example, it can be added at 0.01% to 15% by weight of the total weight of the food, and at a ratio of 0.02g to 10g, for example, 0.3g to 1g, based on 100ml of health beverage composition.

[0046] When the food composition of the present invention is a health beverage, there are no particular restrictions on the liquid components, except that it contains cleavers extract or its high molecular weight fraction in a specified proportion as an essential ingredient, and it can contain various flavoring agents or natural carbohydrates as additional ingredients, just like ordinary beverages. Examples of the aforementioned natural carbohydrates are monosaccharides, disaccharides (such as glucose, fructose, etc.), polysaccharides (such as maltose, sucrose, etc.), conventional sugars (such as dextrin, cyclodextrin, etc.), and sugar alcohols (xylitol, sorbitol, and erythritol, etc.). As flavoring agents other than those described above, natural flavoring agents (such as sematriene, stevia extract (such as rebaudioside A, glycyrrhizic acid, etc.) and synthetic flavoring agents (such as saccharin, aspartame, etc.) can be used advantageously. The amount of natural carbohydrates in each 100 ml of the food composition of the present invention is typically about 1 g to 20 g, for example, about 5 g to 12 g.

[0047] The food composition according to the present invention may contain a variety of nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0048] In addition, the compositions of the present invention may contain fruit pulp used in the production of natural fruit juices and fruit juice beverages and vegetable beverages. These ingredients may be used alone or in combination. The proportions of these additives are not critical, but are generally selected in the range of 0 to 20 parts by weight per 100 parts by weight of the compositions of the present invention.

[0049] As another specific embodiment, the present invention can provide the composition for inhibiting the side effects of anticancer treatment as a cosmetic material composition.

[0050] The cosmetic material composition described herein is generally applicable to various skin areas such as the face, scalp, arms, back, chest, and legs where skin side effects such as redness, dermatitis, xerosis, keratosis, and pigmentation changes occur. Furthermore, the cosmetic material composition of this invention can also be applied to hair and scalp.

[0051] In addition to the active ingredient, namely the extract of Galium aparine or its high molecular weight fraction, the cosmetic material composition of the present invention also contains ingredients commonly used in cosmetic material compositions, such as conventional additives and carriers for antioxidants, solubilizers, stabilizers, vitamins, pigments and fragrances.

[0052] The cosmetic material compositions of the present invention can be prepared in any dosage form commonly prepared in the art. For example, they can be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, foundations, emulsion foundations, wax foundations, and sprays, but are not limited thereto. Furthermore, they can be formulated as nourishing creams, astringents, softening lotions, lotions, serums, shampoos, nourishing gels, or massage creams.

[0053] For the cosmetic material composition of the present invention, based on the weight of the total composition, it may contain 0.0001% to 50.0% by weight, preferably 0.001% to 50% by weight of *Galium affine* extract or its GA-H as an active ingredient. If the content of *Galium affine* extract or its polymer fraction is less than 0.0001% by weight, the inhibitory effect on anticancer treatment side effects is too weak; if it exceeds 50.0% by weight, there is a problem that the increase in effect based on the increased content is minimal and the stability of the formulation cannot be ensured.

[0054] As another embodiment, the present invention relates to a method for preparing a cleavers extract or its fractions that has the effect of inhibiting the side effects of anticancer treatment.

[0055] The method for preparing the cleavers extract according to the present invention includes: a drying step, drying cleavers; a chopping step, chopping the dried cleavers; and an extract preparation step, preparing the extract by extracting the chopped cleavers with a solvent.

[0056] The first step is the drying of the cleavers. For drying the cleavers, some or all of the above-ground or underground parts of the cleavers can be used. It is preferable to dry the leaves and stems of the above-ground parts of the cleavers, and more preferably to dry the leaves of the cleavers.

[0057] The drying can be performed using methods available in the art, such as natural drying, hot air drying, cold air drying, freeze drying, and microwave drying, and can be carried out by varying the drying hours and temperature depending on the method used.

[0058] The next step is to chop the dried cleavers. This chopping can be done using methods employed in the art, such as a cutter, grinder, hammer mill, etc.

[0059] The next step is to prepare an extract by extracting the chopped cleavers with a solvent.

[0060] The solvent is preferably water, which can be water at 70°C to 100°C, more preferably 70°C to 90°C, more preferably 75°C to 85°C, more preferably 78°C to 82°C, more preferably 79°C to 81°C, and most preferably 80°C.

[0061] The weight ratio of cleavers to water during extraction can be 1:5 to 1:20, preferably 1:5 to 1:15, more preferably 1:8 to 1:12, even more preferably 1:9 to 1:11, even more preferably 1:9.5 to 1:10.5, and most preferably 1:10. Furthermore, the extraction can be performed for 2 to 12 hours, preferably 2 to 10 hours, more preferably 2 to 8 hours, even more preferably 3 to 6 hours, even more preferably 3 to 5 hours, even more preferably 3.5 to 4.5 hours, even more preferably 3.8 to 4.2 hours, and most preferably repeated 1 to 5 times within 4 hours, preferably repeated 2 to 4 times.

[0062] Based on the above method, cleavers extract can be prepared, and the cleavers extract fraction can be prepared by the following steps: separating the cleavers extract based on a specified molecular weight; and collecting the separated fraction.

[0063] In the method for preparing fractions of the *Galium affine* extract, the specified molecular weight for the step of separating the *Galium affine* extract based on a specified molecular weight can be 3000. Furthermore, the separation can be performed using size exclusion chromatography (SEC), ultrafiltration, dialysis, differential centrifugation, gradient centrifugation, etc., but is not necessarily limited to these methods.

[0064] In the step of collecting the separated fractions, the collected fractions may be high molecular weight fractions with a molecular weight of 3000 or higher.

[0065] As another embodiment, the present invention provides a method for preventing, improving or treating anticancer treatment side effects in subjects by administering an anticancer treatment side effect suppression composition containing the aforementioned *Galium aparine* extract or its fractions as an active ingredient.

[0066] The effects of the extract of Galium aparine or its fractions on inhibiting the side effects of cancer treatment are as described above.

[0067] The methods for preventing, improving, or treating diseases according to the present invention may specifically include: administering the composition in a pharmaceutically effective amount to a subject who has or may have oxidative stress due to anticancer treatment; decreased immunity or abnormal immune regulation; gastrointestinal damage such as vomiting, diarrhea, gastric bleeding, or gastric ulcers; or skin damage such as redness, edema, blisters, dermatitis, xerosis, keratosis, and pigmentation changes. The method of administration is as described above.

[0068] The prevention can refer to the suppression or delay of all behaviors that are side effects of anticancer treatment, such as oxidative stress, decreased immunity or abnormal immune regulation, gastrointestinal damage such as vomiting, diarrhea, gastric bleeding or gastric ulcers, and skin damage such as redness, edema, blisters, dermatitis, xerosis, keratosis and pigmentation changes, by administering the composition containing the present invention's cleavers extract or its fractions as an active ingredient to the subject.

[0069] The improvement or treatment can refer to all behaviors that improve or benefit the condition of a disease based on symptoms and disease progression, such as oxidative stress; decreased immunity or abnormal immune regulation; gastrointestinal damage such as vomiting, diarrhea, gastric bleeding or gastric ulcers; and skin damage such as skin redness, edema, blisters, dermatitis, xerosis, keratosis and pigmentation changes, by administering the composition to the subject containing the present invention's *Gnaphalium affine* extract or its fractions as an active ingredient.

[0070] In order to administer the compositions of the present invention, in addition to the active ingredients described above, pharmaceutically acceptable carriers, excipients, or diluents may be included. Examples of such carriers, excipients, and diluents include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, propylparaben, talc, magnesium stearate, and mineral oil.

[0071] The appropriate dosage of the composition of the present invention may vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, and the duration of administration, but can be appropriately selected by those skilled in the art. Specifically, the dosage of the *Gnaphalium affine* extract or its fractions may be 1-3000 mg / kg.

[0072] (III) Beneficial Effects

[0073] The extract of *Gnaphalium affine* and its high molecular weight fractions of 3000 or more according to the present invention are beneficial for use in pharmaceuticals, food, or cosmetics for the purpose of inhibiting the side effects of anticancer treatment due to their excellent effects in preventing, improving, or treating the side effects of anticancer treatment. These side effects include, for example, oxidative stress; decreased immunity or abnormal immune regulation; gastrointestinal damage such as vomiting, diarrhea, gastric bleeding, or gastric ulcers; and skin damage such as redness, edema, blisters, dermatitis, xerosis, keratosis, and pigmentation changes. Attached Figure Description

[0074] Figure 1 The graphs show the macrophage survival ability, NO and TNF-α production enhancement activity of hot water extracts of leaves and stems of *Galium aparine* at 4°C or 80°C, respectively, according to different concentrations, based on an embodiment of the present invention.

[0075] Figure 2 The results are the assay results of cytotoxicity and proliferative activity when macrophages are treated alone with the 80°C aqueous extract of Galium aparine and its GA-H (>3000) and GA-L (<3000) or when LPS-induced macrophages are treated with the substances.

[0076] Figure 3 This is a confirmation of the degree of NO production when macrophages are treated alone with the 80°C aqueous extract of Galium aparine and its GA-H (>3000) and GA-L (<3000), or when LPS-induced macrophages are treated with the aforementioned substances.

[0077] Figure 4This is a confirmation of the extent of TNF-α and IL-10 production when macrophages are treated alone with the 80°C aqueous extract of Galium aparine and its GA-H (>3000) and GA-L (<3000), or when LPS-induced macrophages are treated with the substances described herein.

[0078] Figure 5 This is a graph showing the activity of IL-6 and IL-12 generation for the crude polysaccharide fraction (GS-S) of high molecular weight polysaccharides isolated and recovered from Galium aparine.

[0079] Figure 6 This is the electrophoresis result of the crude polysaccharide fraction (GS-S) of high molecular weight polysaccharides isolated and recovered from Galium aparine.

[0080] Figure 7 This is the result of converting the cytokine production patterns of the high molecular weight fractions of Galium sarmentosum (GA-H) and GS-S into protein concentrations.

[0081] Figure 8 This is a confirmation result of the immunomodulatory activity of the high molecular weight fraction (GA-H) of Galium aparine extract in the tumor microenvironment.

[0082] Figure 9 This is a diagram showing cell proliferation when HaCaT cells or H2O2-treated HaCaT cells are treated with an 80°C aqueous extract of Galium aparine and its high molecular weight fraction (GA-H).

[0083] Figure 10 This is a graph analyzing the changes in the expression of Bax and Bcl genes in HaCaT cells treated with HaCaT cells or H2O2 under conditions of treatment with high molecular weight fractions of Galium sarmentosum.

[0084] Figure 11 This graph compares the expression levels of AMPK and phosphorylated AMPK (phospho-AMPK) in HaCaT cells treated with H2O2 and then treated with a high molecular weight fraction of Galium sarmentosum.

[0085] Figure 12 This figure compares the bone marrow cell proliferation efficacy of mouse bone marrow cells treated with different concentrations of *Galium aparine* extract or its high molecular weight fraction (GA-H).

[0086] Figure 13 This is a graph confirming the ability of mouse bone marrow cells to produce cytokines (IL-3, IFN-γ, GM-CSF, IL-5) under the condition of treatment with a high molecular weight fraction of Galium sarmentosum.

[0087] Figure 14This is a graph showing the effect of *Galium affine* extract or its high molecular weight fraction (GA-H) on the proliferation of AGS cells and the degree of inhibition of IL-8 production in AGS cells induced by TNF-α inflammation.

[0088] Figure 15 This is a graph showing the effect of the high molecular weight fraction of Galium sarmentosum (GA-H) on the activation of TNF-α-based NK-κ and MAPK signaling pathways in AGS cells.

[0089] Figure 16 This is a diagram showing the effect of the high molecular weight fraction of cleavers (GA-H) on the production of mucin 2 and trefoil factor 3 (TFF-3) derived from mucus-producing cells (LS174T cells).

[0090] Figure 17 This is a graph analyzing the expression levels of MMP-1 and TGF-β in HaCaT cells, a human keratinocyte cell line treated with hydrogen peroxide (H2O2), after treatment with a high molecular weight fraction of Galium sarmentosum (GA-H).

[0091] Figure 18 This is a graph analyzing the expression levels of filaggrin and β-defensin 2 in HaCaT cells, which belong to the human keratinocyte cell line, after treatment with the high molecular weight fraction of Galium sarmentosum (GA-H).

[0092] Figure 19 This is a graph analyzing the expression level of COLA1 in HaCaT cells, which belong to the human keratinocyte cell line, after treatment with the high molecular weight fraction of Galium sarmentosum (GA-H). Detailed Implementation

[0093] In the following description, to aid in understanding the invention, examples and other methods will be used. However, embodiments of the invention can be modified in many other ways, and the scope of the invention should not be construed as limited to the following examples. The embodiments of the invention are provided to provide a more comprehensive explanation of the invention to those skilled in the art.

[0094] Example 1: Preparation of Galium aparine extract and determination of extraction solvent

[0095] *Galium affine* was collected in Yangyang, Gangwon Province, South Korea at the end of May. Since water extraction primarily extracts water-soluble components, while ethanol extraction is better at extracting fat-soluble components, both methods were used in parallel. For ethanol extraction, 100 v / v%, 70 v / v%, 30 v / v%, and 10 v / v% ethanol were used. After washing, drying, and pulverizing the leaves and stems of *Galium affine*, 10 times their weight of solvent (water or ethanol) was added, and extraction was carried out at 80°C for 4 hours. After extraction, the mixture was filtered through a 0.45 µm pore size filter, concentrated under reduced pressure, and freeze-dried. The recovery rates after freeze-drying are shown in Table 1.

[0096] [Table 1]

[0097]

[0098] As shown in Table 1 above, the ethanol extracts with a recovery rate below 50 v / v% exhibit excellent recovery, while the extraction method with the highest recovery rate is the aqueous extract at 80°C. These results indicate that the useful components of *Galium aparine* are better extracted primarily from highly polar water.

[0099] Next, to determine the optimal extraction solvent temperature, extracts of *Galium aparine* leaves and branches were prepared at 4°C and 80°C using the same method described above. These extracts were then applied to macrophages to determine viability, the induction of macrophage and innate immune system stimulation activity, and the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). Specifically, Raw264.7 macrophage cells were cultured in DMEM medium (10% FBS) at 37°C and 5% CO2, and then injected at a rate of 1×10⁻⁶. 4 Cells / wells were seeded into 96-well plates, and samples prepared at different concentrations were processed and cultured for 48 hours. After culture, cell viability was determined using a WST-based cytotoxicity / proliferation assay kit (EZ-Cytox, DoGenBio, Seoul, South Korea) according to the manufacturer's instructions. NO and TNF-α produced in the culture supernatant were determined using individual assay kits (NO: Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. G7921; TNF-α: Biosciences, San Jose, CA, USA, Cat. No. 560478) according to the manufacturer's instructions.

[0100] The result, such as Figure 1 As shown, compared to the 4°C extract, the 80°C extract exhibited superior activity in enhancing macrophage survival and the production of NO and TNF-α. Therefore, the 80°C aqueous extract was deemed appropriate. On the other hand, although not specifically stated in this specification, there was no significant difference between water extraction at 80°C and the 100°C aqueous extract.

[0101] Example 2: Preparation and Characterization of Galium aparine Extract and Fractions of Different Molecular Weights

[0102] Fractions with molecular weights above and below 3000 were prepared using extracts and Amicon filters. Specifically, following the method of Example 1, 135 g of *Galium aparine* leaf and branch powder was extracted with hot water at 80°C, and the hot water extract was filtered using an Amicon filter with a pore size of 3000. After filtration, each fraction was freeze-dried, and the recovery rate was determined. A standard curve for asperulosidic acid was prepared after analysis using an HPLC system (waters 2695, 2996 models) equipped with a Water company photodiode array (PDA) detector. The column was set with Capcell PpakC 18 mg Ii (sample temperature 15°C, column temperature 30°C) as the analytical conditions, with a flow rate (1 ml / min) and injection volume (1 μL) and a run time of 40 minutes. The analysis was performed using H₂O containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (ACN) containing 0.1% TFA as the mobile phase. The standard curve is as follows: asperulosidic acid was injected into the HPLC at concentrations of 10 µg / mL, 25 µg / mL, 50 µg / mL, and 100 µg / mL, and the peak area for each concentration was determined. A standard curve was constructed with concentration (µg / mL) on the X-axis and peak area on the Y-axis. Based on this, the content of asperulosidic acid in the *Galium aparine* extract was analyzed. Furthermore, the protein content of the extract and fractions was determined using the Quick Start™ Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA; Cat. No. 5000201). The total carbohydrate content of the extract and fractions was analyzed in 96-well plates using the phenol-sulfuric acid method. Specifically, 50 µl of a 1 mg / mL sample was prepared for each well, followed by the addition of an equal volume of 5% phenol reagent. Then, 100 µl of 98% concentrated sulfuric acid was carefully added. After incubating the mixture at room temperature for 1 hour, the absorbance was measured at 490 nm. The total carbohydrate concentration was calculated by substituting the measured absorbance values ​​into a standard curve. Glucose was used as the carbohydrate indicator for constructing the standard curve. The experimental results are shown in Table 2.

[0103] [Table 2]

[0104]

[0105] As shown in Table 2 above, the freeze-drying results showed that the total dry weight of the hot water extract at 80°C was 46.575 g, with a recovery rate of 34.5% relative to the raw material, and the content of the standard substance asperulosidic acid was 1.24%. The fraction with a molecular weight greater than 3000 (GA-H) showed a dry weight of 14.175 g (recovery rate 10.5%), with an asperulosidic acid content of 0.92%. The fraction with a molecular weight less than 3000 (GA-L) showed a dry weight of 33.075 g (recovery rate 24.5%), with an asperulosidic acid content of 1.37%. This result indicates that asperulosidic acid is concentrated in a higher concentration in GA-L (molecular weight <3000), rather than being completely fractionated according to different molecular weights. Therefore, GA-H (molecular weight >3000) also showed a high percentage of asperulosidic acid (0.92%), and approximately 18% of the asperulosidic acid was considered to have disappeared due to adhesion to the amitan filter. On the other hand, the results of testing the protein content of the extract and each fraction showed that the 80℃ extract contained 1.32±0.08% protein, GA-H contained 3.77±0.17% protein, but GA-L contained no protein.

[0106] Example 3: Antioxidant Effect Analysis

[0107] To confirm the ability to remove reactive oxygen species (ROS) aimed at reducing oxidative stress and skin damage caused by radiation and anticancer drug therapy, the scavenging abilities of DPPH and ABTS free radicals were evaluated. The DPPH free radical scavenging ability was measured by the change from purple to light color upon reduction due to the antioxidant. The ABTS free radical scavenging ability was measured by the extent to which the antioxidant effectively eliminated ABTS free radical cations. These tests were performed using corresponding assay kits (EZ-ABTS Antioxidant Assay Kit and EZ-DPPH Antioxidant Assay Kit: DoGenBio Co., Seoul, South Korea). When oxidative stress is reduced, damage to normal cells is reduced, thereby alleviating the side effects of anticancer treatment and alleviating skin aging and inflammation caused by oxidative damage.

[0108] [Table 3]

[0109]

[0110] As shown in Table 3 above, different fractions of *Galium aparine* exhibited varying antioxidant effects, and the antioxidant effect was not directly proportional to the content of cypermethrin. In particular, the high molecular weight fraction GA-H showed excellent antioxidant activity, indicating that other high molecular weight components besides cypermethrin also played an important role. In contrast, the low molecular weight fraction GA-L, despite having a relatively high cypermethrin content, showed lower antioxidant activity compared to the high molecular weight fraction. According to existing research reports, the IC50 values ​​for DPPH and ABTS free radical scavenging capacity of cypermethrin... 50 The values ​​were approximately in the range of 200-300 µg / ml and about 100-200 µg / ml, respectively, which showed antioxidant activity similar to that of the low molecular weight fraction.

[0111] Example 4: Cytotoxicity and Proliferative Activity

[0112] To determine the effects of the samples on macrophages, the cytotoxicity and cell proliferation activity of Raw264.7 mouse macrophages treated with the samples alone were examined, as well as the cytotoxicity and proliferation activity of Raw264.7 mouse macrophages induced by lipopolysaccharide (LPS) in the samples. Raw264.7 cells were cultured in DMEM medium (10% FBS) at 37°C and 5% CO2. Raw264.7 cells were then cultured at a rate of 1×10⁻⁶ cells / cells. 4 Cells / wells were seeded into 96-well plates, and samples prepared at different concentrations were processed. After 48 hours of incubation, LPS (final concentration 0.5 µg / ml) was added. Following incubation, the effect on cell viability was determined using a WST-based cytotoxicity / proliferation assay kit (EZ-Cytox, DoGenBio, Seoul, South Korea) according to the manufacturer's instructions. The results are as follows: Figure 2 As shown.

[0113] When macrophages were treated alone, the 80°C extract and GA-H (molecular weight >3000) induced cell proliferation, but not GA-L (molecular weight <3000) cells. For the 80°C extract, cell proliferation was induced at concentrations of 20–300 µg / ml, with the optimal concentration being approximately 100 µg / ml, exhibiting 118.9% proliferative activity. For GA-H (molecular weight >3000), cell proliferation was induced at concentrations of 4–100 µg / ml, with the optimal concentration being approximately 20 µg / ml, exhibiting 123.1% proliferative activity. On the other hand, for GA-L (molecular weight <3000), no cytotoxicity or proliferative activity was observed at concentrations below 500 µg / ml, which were suitable for experimental use. Extracts at 80°C and fractions with a molecular weight of 3000 or higher (GA-H) exhibit cell proliferation at specific concentrations. At these concentrations, the sample is considered to have the best cell viability, proliferation stimulation, and cell protection effects.

[0114] Compared to the culture medium treatment group alone, the LPS (1µg / ml) treatment group showed approximately 36% inhibition of proliferation activity, and no significant changes were observed in any of the sample treatment groups at concentrations below 500µg / ml.

[0115] In summary, it was confirmed that all fractions containing the extract had no cytotoxic effects, and the cleavers component exhibiting cell proliferation activity was GA-H, which is composed of high molecular weight molecules with a molecular weight of over 3000.

[0116] Example 5: Analysis of the effects on the production of NO and inflammation-regulating cytokines

[0117] Radiation and anticancer drug therapy can also damage normal cells, thereby triggering an inflammatory response. When macrophages recognize pathogens or receive inflammatory signals, they generate nitric oxide (NO) based on inducible NO synthase (iNOS), directly removing microorganisms or binding to reactive oxygen species (ROS) to form potent reactive oxygen species that damage proteins and DNA. This plays a crucial role in the activation of the innate immune system and inducing an inflammatory response to strengthen immune defense mechanisms. TNF-α, as a major mediator of the inflammatory response promoted by radiation and anticancer drug therapy, is excessively produced and is a representative cause of worsening post-treatment side effects. In particular, TNF-α secreted by macrophages can activate the inflammatory response and cause tissue damage. IL-10, as a representative anti-inflammatory cytokine, plays a role in regulating the inflammatory response and inhibiting excessive immune system responses. Therefore, this experiment evaluated the efficacy of a sample that could inhibit the side effects of radiation and anticancer agents using lipopolysaccharide (LPS)-induced Raw264.7 mouse macrophages. LPS is known to induce a strong inflammatory response in macrophages, and Raw264.7 cells are a suitable cell line for studying this inflammatory model. The production activities of nitric oxide (NO) as an inflammatory mediator and TNF-α and IL-10 as cytokines were assessed.

[0118] Specifically, Raw264.7 cells (1×10⁻⁶) 4 Cells / wells were seeded into 96-well plates, and samples prepared at different concentrations were processed. After two hours of incubation, inflammatory inducing substances were added to achieve a final LPS concentration of 100 to 500 ng / ml. After 24 hours of incubation, the concentrations of NO (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. G7921), TNF-α (BD Biosciences, San Jose, CA, USA, Cat. No. 560478), and IL-10 (BD Biosciences, San Jose, CA, USA, Cat. No. 555252) produced in the culture supernatant were measured according to the manufacturer's instructions using a kit. The results are as follows: Figure 3 and Figure 4 As shown.

[0119] The result, such as Figure 3As shown, the strongest NO inhibition effect was observed when the fraction with a molecular weight less than 3000 (GA-L) was simultaneously treated with LPS. For the 80℃ extract and the fraction with a molecular weight greater than 3000 (GA-H), since the samples themselves also promote NO production, the NO inhibition effect is likely to be low when simultaneously treated with LPS. The enhanced NO production due to individual sample treatment is thought to be due to the interaction of the components of the fraction with specific signal transduction receptors such as Toll-like receptors (TLRs), thereby inducing the production of various inflammatory cytokines, including NO. Therefore, it was determined that the main reason why the fraction with a molecular weight greater than 3000 (GA-H) showed a lower NO production inhibition effect compared to the 80℃ extract is that this fraction contains a relatively small amount of components with a molecular weight less than 3000 and the antioxidant activity of the components contained in the low molecular weight fraction (GA-L) is not significant (see Table 3).

[0120] Experimental results regarding TNF-α production ( Figure 4 The 80°C extract and the high molecular weight fraction (GA-H) with a molecular weight of over 3000 stimulate macrophages to produce TNF-α through the sample itself. When inflammation was induced with LPS, TNF-α production was inhibited at concentrations of 20-500 µg / ml for both the 80°C extract and GA-H. Although GA-L did not directly contribute to TNF-α production based on the sample alone, when treated with LPS, it exhibited an overall pattern similar to the regulatory effect of NO production, showing an inhibitory effect on TNF-α production.

[0121] Results of IL-10 generation detection ( Figure 4 While individual samples did not induce IL-10 production in the extracts and all fractions, concentration-dependent increases in IL-10 production were observed in both the 80°C extract and GA-H when inflammation was induced with LPS. In particular, the significant increase in LPS-stimulated IL-10 production in both the 80°C extract and GA-H suggests that these components can induce an anti-inflammatory response to suppress inflammation. This result is similar to reports of LPS-stimulated IL-10 production not being spontaneously induced in plant extracts such as curcumin, but rather induced based on LPS stimulation.

[0122] Therefore, considering the results of the two cytokines (cytokines) and NO, the 80°C extract and its GA-H are expected to promote cancer cell removal while suppressing excessive inflammation and reducing side effects by regulating the immune response after anticancer treatment. Thus, it is judged to be a component that can play a positive role in recovery and recurrence prevention after cancer treatment. Simultaneously, this result may be related to the regulation of the immune response based on M1 / M2 macrophage differentiation and the regulation of NF-κB and MAPK signaling pathways. M1 macrophages induce inflammatory responses by secreting inflammatory cytokines such as TNF-α, which helps in the removal of external pathogens or cancer cells, while M2 macrophages secrete anti-inflammatory cytokines such as IL-10 and suppress inflammation, thus contributing to tissue repair. Therefore, if the function of the 80°C extract of *Galium affine* and its GA-H in regulating the M1 / M2 balance can be confirmed, it is possible that the 80°C extract and its GA-H of the present invention can suppress excessive inflammation and help maintain the anticancer immune response.

[0123] Example 6: Immunostimulatory activity and its indicators and useful components

[0124] Example 5 above confirmed that the 80°C extract and GA-H possess the activity of autonomously stimulating macrophages to produce TNF-α and NO. This result implies that the 80°C extract and GA-H have immunostimulatory activities, including macrophage activation, indicating that their components are concentrated in the high molecular weight fraction (GA-H) with a molecular weight of over 3000. Therefore, further research was conducted on the components in the Galium aparine extract that possess immunostimulatory activity.

[0125] Specifically, to separate the crude proteoglycan components contained in GA-H, an ethanol precipitation method was used. The 80°C extract of *Galium aparine* was dissolved in distilled water to adjust the ethanol concentration to 80%, and gently stirred at room temperature for 12 hours to separate the precipitate and ethanol-soluble fractions. After stirring, the precipitate, which is the crude polysaccharide component, was recovered by centrifugation (15000 rpm / 20 min). This precipitate was then dissolved in distilled water and dialyzed. The obtained material after dialysis was freeze-dried to obtain high-molecular-weight crude proteoglycan fractions (GS-S). To confirm that the GS-S component in the 80°C extract is a useful component with immunostimulatory activity, the production activities of IL-6 and IL-12 were detected after stimulating macrophages. The experimental methods were the same as those used for TNF-α, and various cytokine assay kits from the same company were used. The results are as follows: Figure 5 As shown, the IL-6 and IL-12 generating activities of GS-S exhibited similar activities at concentrations approximately 10 times lower than those of the 80°C extract, suggesting its potential as a useful component.

[0126] To confirm the composition of GS-S as a component of high-molecular-weight crude proteoglycans, protein analysis (QuickStart™ Bradford Protein Assay Kit) was performed, confirming that 1g of GS-S contains approximately 230µg of protein. Furthermore, 12% SDS-PAGE electrophoresis confirmed a protein band of approximately 35kDa. Figure 6 Furthermore, the results of converting protein concentrations to determine the cytokine production patterns of the macromolecular fractions (GA-H) and GS-S both showed a protein concentration-dependent cytokine production pattern. Figure 7 Additionally, although the results are not shown, the results for the 80°C extract also show the same state.

[0127] The above results strongly indicate that the immunostimulatory component in *Galium affine* that directly influences cytokine production is a proteoglycan containing a protein of 35 kDa. Therefore, the useful immunostimulatory component in *Galium affine* could be a proteoglycan containing a protein of approximately 35 kDa. For subsequent immune-related experiments, a high molecular weight fraction (GA-H) with a molecular weight of over 3000, possessing both antioxidant and immunostimulatory activities, should be used.

[0128] Example 7: Immunomodulatory Activity in the Tumor Microenvironment

[0129] The results of the above embodiments confirm that, compared to the GA-L fraction containing a high concentration of asperulosidic acid, the immunomodulatory fraction of *Galium aparine* primarily acts on the high molecular weight GA-H fraction containing proteoglycans with a molecular weight of 35 kDa. In particular, antioxidant and immunomodulatory functions play important roles in the relief of side effects from radiotherapy and anticancer agents; therefore, the immunomodulatory activity of the high molecular weight fraction of *Galium aparine* (GA-H) containing 35 kDa proteoglycans in the tumor microenvironment has been demonstrated.

[0130] Specifically, to recreate the cancer microenvironment in vitro, cancer cells (colon26) and macrophages were cultured simultaneously, and the effects of the GA-H fraction of *Galium aparine* on the production of cytokines such as TNF-α, IL-12, and IL-10 were evaluated. Macrophages, through interaction with cancer cells, polarize into inflammatory M1 or anti-inflammatory M2 cytokines based on environmental stimuli, thereby secreting different cytokines. M1 macrophages play an important role in cancer cell removal and immune stimulation by secreting inflammatory cytokines such as TNF-α and IL-12. M2 macrophages contribute to inflammation suppression and tissue protection by secreting anti-inflammatory cytokines such as IL-10. For the levels of TNF-α, IL-12 and IL-10 generated, their respective TNF-α (BD Biosciences, San Jose, CA, USA, Cat. No. 560478), IL-12 (BD Biosciences, Cat. No. 550582) and IL-10 (BD Biosciences, Cat. No. 555252) kits were used.

[0131] like Figure 8 As shown, for GA-H treatment at concentrations of 2–200 µg / ml, the highest levels of TNF-α and IL-12 production were induced at a specific concentration of 2 µg / ml. For IL-10, concentration-dependent IL-10 production activity was observed below 200 µg / ml. This result is attributed to the distinct immunostimulatory and inhibitory activities of the components constituting GA-H. In other words, it indicates that at high concentrations, the production of IL-10, as an anti-inflammatory component, is activated, potentially inhibiting the production of TNF-α and IL-12. Therefore, the reduction in TNF-α and IL-12 production at high concentrations could be a result of a combination of immunomodulatory effects and activation of anti-inflammatory pathways at high concentrations. In summary, the GA-H fraction maintains an inflammatory response at low concentrations while anti-inflammatory feedback is activated at high concentrations, suggesting the ability to regulate macrophage differentiation from M1 to M2. Furthermore, it strongly suggests that the *Galium aparine* extract may also exhibit similar activities to the GA-H fraction.

[0132] Example 8: Proliferation and DNA Repair Capacity of DNA-Damaged Cells

[0133] 8-1. Protective effect of H2O2 treatment on HaCaT cells

[0134] Radiation and anticancer therapy generate reactive oxygen species (ROS), leading to DNA damage in normal cells and activating cell suicide pathways. Therefore, in this experiment, HaCaT cells were exposed to oxidative stress via H2O2 treatment, inducing DNA damage and cell suicide. To evaluate the cytoprotective effects of *Galium aparine* extract and its high-molecular-weight fractions, H2O2-treated HaCaT cell lines were co-cultured with 500-0.16 µg / ml *Galium aparine* extract for 24 hours, and cell viability was measured using EZ-Cytox.

[0135] like Figure 9 As shown, HaCaT cells were treated with either the 80°C hot water extract of *Galium affine* or its molecular weight fraction (GA-H) with a molecular weight greater than 3000, and the effects on cell proliferation were analyzed. The 80°C extract and the GA-H induced cell proliferation in normal HaCaT cells. This indicates that active ingredients such as antioxidants or polysaccharides contained in the *Galium affine* extract and its GA-H stimulate cell proliferation by promoting cell metabolism or activating cell survival signaling pathways. Furthermore, this proliferative effect suggests that the high molecular weight components contained in the 80°C extract and GA-H can bind to receptors on the cell surface, thereby acting as a mechanism for activating cell growth factor signaling. In conclusion, this demonstrates the potential for use of *Galium affine* extract and its GA-H for purposes such as preventing skin damage that aids in skin regeneration and tissue recovery after radiation or anticancer therapy, and promoting wound healing.

[0136] On the other hand, the results of detecting the effects of extracts such as *Galium affine* in HaCaT cells treated with H2O2 (… Figure 9 In H2O2-damaged HaCaT cells (40% survival), compared with H2O2-treated HaCaT cells, the extract of Galium aparine and GA-H showed higher cell proliferation effects at concentrations above 4 µg / ml, and compared with the extract at 80℃, GA-H showed higher proliferative activity.

[0137] These results indicate that fractions with a molecular weight of over 3000 isolated from the cleavers extract (GA-H) can be used as a composition with the dual effects of preventing damage to normal cells that may occur after radiation and anticancer drug treatment, and promoting tissue recovery.

[0138] 8-2. Analysis of Expression Changes in Bax and Bcl Genes

[0139] The Bax gene, acting as a pro-apoptotic gene, promotes cell suicide by inducing apoptosis when cell damage cannot be repaired. In contrast, the Bcl gene, acting as an anti-apoptotic gene, promotes cell survival. Therefore, by analyzing the expression changes of Bax and Bcl genes in HaCaT cells treated with H2O2 using a high molecular weight fraction of cleavers garnet extract (GA-H), the effects on the survival and repair of cells treated with radiation or anticancer agents were evaluated.

[0140] Experimental results showed that H2O2 treatment in HaCaT cells increased the expression of the Bax gene and inhibited the expression of the Bcl-2 gene, thereby inducing cell suicide due to oxidative stress. Figure 10 However, in the group treated with the high molecular weight fraction of *Galium affine* extract (GA-H), Bax expression was inhibited and Bcl-2 expression was enhanced, thus confirming that *Galium affine* extract plays a role in inhibiting cell suicide and promoting cell survival. Previous experiments showed that in experiments with co-cultured macrophages and cancer cells, the high molecular weight fraction of *Galium affine* extract (GA-H) promoted the removal of cancer cells while inducing the production of M1 cytokines such as TNF-α and IL-12, and simultaneously protected normal tissues and induced inflammation suppression through anti-inflammatory cytokines such as IL-10. This result is consistent with the cell survival effect achieved by altering Bax / Bcl gene expression in this experiment and supports the conclusion that the high molecular weight fraction of *Galium affine* extract (GA-H) effectively inhibits damage to normal cells caused by radiation and anticancer therapy and induces cytoprotective effects. In particular, the high molecular weight fraction (GA-H) of *Galium affine* extract inhibited cell suicide by suppressing increased Bax and Bcl-2 expression in normal cells, suggesting that it may help alleviate side effects by reducing damage to normal cells that may occur during cancer treatment. This, along with similar results to those reported in existing studies regarding the alleviation of side effects from anticancer agents and the promotion of tissue recovery, demonstrates a similar mechanism by which natural components such as curcumin protect normal cells by modulating inflammatory responses and oxidative stress.

[0141] In summary, the above results demonstrate that the *Galium aparine* extract and its high molecular weight fraction (GA-H) of the present invention inhibit cell suicide caused by oxidative stress due to radiation exposure and anticancer agent treatment, and promote the survival of normal cells, thereby exhibiting effects such as preventing skin damage, promoting wound healing, and inhibiting inflammation. This indicates its potential use as a food composition and cosmetic composition capable of reducing side effects during cancer treatment and improving the quality of life for patients.

[0142] Example 9: Function of AMPK activity in cell damage due to stress

[0143] AMP-activated protein kinase (AMPK) regulates cellular energy balance and is associated with DNA repair. AMPK can be activated under cellular stress to promote cell survival. In cancer cells, the AMPK pathway is often abnormally inhibited or regulated; therefore, AMPK activators inhibit cancer cell growth and proliferation. In normal cells, they have functions of DNA repair and promoting cell survival. Representative drugs under investigation include metformin and aminoimidazole carboxamide ribonucleotide (AICAR). Curcumin, resveratrol, and epigallocatechin gallate (EGCG) from green tea have been reported to have antioxidant and anti-inflammatory effects, while also inhibiting the repair capacity of cancer cells and protecting normal cells.

[0144] Therefore, this experiment investigated the effect of the high molecular weight fraction (GA-H) of *Galium aparine* extract on AMPK activation in H2O2-damaged HaCaT cell lines. Specifically, HaCaT cells were grown to 70% confluence in 6-well plates, samples were added, and the cells were cultured for 24 hours. After culture, the cells were washed twice with PBS, and a protease and a phosphatase inhibitor (Sigma-Aldrich, USA) were added to RIPA lysis buffer (Thermo Fisher Scientific, USA). The lysed cells were cultured at 4°C for 30 minutes, then centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected. The collected proteins were quantified using the Bradford protein quantification method. After separating an equal volume (20 µg) of protein by 10% SDS-PAGE, the protein was transferred to a polyvinylidene fluoride membrane (PVDF membrane, EMD Millipore, USA) and then blocked with 5% skim milk (BD Biosciences, San Jose, CA, USA) at room temperature for 1 hour. Then, primary antibodies against phosphorylated AMPK (Thr172, Cell Signaling Technology, Danvers, MA, USA; Cat. No. 2535) and AMPK (Cell Signaling Technology, Danvers, MA, USA; Cat. No. 2532) were reacted at 4°C for 12 hours at a 1:1000 dilution. Following the initial antibody reaction, cell membranes were washed three times with PBS-T, and the HRP-conjugated secondary antibody (Cell Signal Transduction Technology, Danfoss, MA, USA; Cat. No. 7074) was diluted 1:3000 and reacted at room temperature for 1 hour. Western blot signal was detected using ECL solution (Thermo Fisher Scientific, USA), and the expression levels of AMPK and phosphorylated AMPK (phospho-AMPK) were compared.

[0145] As a result, the H2O2 treatment group inhibited AMPK phosphorylation by inducing oxidative stress, thereby inhibiting AMPK phosphorylation due to cell damage and stress response. However, when the high molecular weight fraction of Galium aparine extract (GA-H) was added, AMPK phosphorylation significantly increased. Figure 11Therefore, the high molecular weight fraction (GA-H) of *Galium aparine* extract is considered to have the function of overcoming stress and activating survival and recovery mechanisms by increasing AMPK phosphorylation. On the other hand, constant AMPK expression in all applicable groups in the experiment means that the basic stock of AMPK did not change, but the phosphorylation level was regulated by the influence of the high molecular weight fraction (GA-H) of *Galium aparine* extract. Therefore, the high molecular weight fraction (GA-H) of *Galium aparine* extract should be interpreted as having the function of regulating AMPK activity through phosphorylation, rather than affecting the total expression level of AMPK.

[0146] In summary, the restoration and activation of AMPK phosphorylation inhibited by H2O2 treatment by the high molecular weight fraction (GA-H) of cleavers extract indicates that the high molecular weight fraction (GA-H) of cleavers extract has a protective effect against oxidative stress.

[0147] Example 10: Effects on hematopoietic cell proliferation

[0148] To evaluate the efficacy of *Galium affine* extract or its high-molecular-weight fraction for alleviating the side effects of radiation therapy and anticancer agents, the proliferative activity of mouse bone marrow cells was determined. Since bone marrow cells are vulnerable to damage after radiation and anticancer treatment, this study aimed to evaluate not only the alleviation of side effects but also the potential immunomodulatory effects by analyzing the toxicity and proliferation-promoting effects of *Galium affine* extract or its high-molecular-weight fraction (GA-H) on bone marrow cells. Bone marrow cells were harvested from the mouse femur and cultured with *Galium affine* extract or its high-molecular-weight fraction (GA-H) for 5 days. Cell proliferation activity was then measured using the EZ-cytox kit. Specifically, 6-week-old BALB / c mice were anesthetized, and both femurs were dissected under sterile conditions. After femoral dissection, the ends were severed, and bone marrow cells were flushed with PBS and recovered from the bone. The recovered bone marrow cells were screened through a 40µm cell strainer (BD Biosciences, San Jose, CA, USA) to prepare a homogeneous cell suspension. The recovered bone marrow cells were adjusted in RPMI-1640 medium to a cell concentration of 1 x 10⁶ cells / well in 96-well plates. 6Cells / wells. Multiple concentrations of *Galium aparine* extract or its high molecular weight fraction (GA-H) were added to bone marrow cells. The control group received no treated samples and was cultured under the same conditions. All experimental groups were cultured for 5 days under identical conditions. After 5 days of culture, for cell proliferation activity, EZ-Cytox reagent was added to each well, and the reaction was carried out at 37°C for two hours. The absorbance was measured at 450 nm using an ELISA reader (Bio-Rad, Hercules, CA, USA). Cell proliferation activity was calculated for each group based on the ratio to the control group.

[0149] The experimental results showed that the extract of *Galium affine* (80℃ extract (Ext.)) exhibited significant proliferative activity at concentrations up to 20 µg / ml, while the polymer fraction (GA-H) showed significant proliferative activity at concentrations above 4 µg / ml. Figure 12 Therefore, the components used to induce the bone marrow cell proliferation activity of *Galium affine* extract are believed to be mainly present in the GA-H fraction.

[0150] On the other hand, the proliferation of bone marrow cells ultimately leads to their differentiation into various types of blood cells, including hematopoietic stem cells (HSCs), immune cells, and platelets. This cell differentiation is regulated by various cytokines produced by immune cells (mainly T cells, B cells, macrophages, etc.) and cell-cell interactions. In particular, IL-3, IFN-γ, GM-CSF, and IL-5 play important roles in immune responses and cell growth. Therefore, after co-culturing splenocytes and GA-H, the production capacity of cytokines was examined. Specifically, mouse spleens were aseptically removed, and splenocytes were adjusted to 2x10⁶ cells / cm² using RPMI-1640 medium. 5 After determining the cell / ml concentration, GA-H was added to achieve a final concentration of 0.8–500 µg / ml. Cells were cultured at 37°C in a 5% CO2 incubator for 48–72 hours. After culture, the amounts of various cell differentiation-related cytokines produced in each culture supernatant were analyzed using each ELISA assay kit (BD Biosciences).

[0151] The experimental results showed that the production of cytokines associated with bone marrow cell differentiation in spleen cell culture medium due to GA-H treatment tended to be similar to that of bone marrow cell proliferation activity. Figure 13In other words, the optimal concentration of GA-H, a cytokine associated with differentiation from spleen cells to bone marrow cells, is shown to be 20–100 µg / ml.

[0152] Therefore, the high molecular weight fraction (GA-H) of cleavers extract can have a powerful effect on the differentiation of bone marrow cells and the activation of immune cells, and increase the immune response.

[0153] Example 11: Gastric Inflammation Suppression Experiment

[0154] 11-1. The effect of AGS cell proliferation and the inhibition of IL-8 production in TNF-α-induced inflammation-induced AGS cells.

[0155] Radiation therapy and anticancer agents act on rapidly dividing cells, thus inhibiting the production of hematopoietic cells, white blood cells, red blood cells, and platelets in the bone marrow. This increases the risk of weakened immunity (neutropenia), anemia, and bleeding, and damages the epithelial cells of the digestive tract, causing gastric inflammation and resulting in side effects such as nausea, vomiting, diarrhea, and even gastric bleeding and ulcers. Furthermore, these treatments can promote the production of reactive oxygen species (ROS) and increase the expression of inflammatory cytokines, thereby exacerbating gastrointestinal inflammation. Therefore, inhibiting gastric inflammation in gastrointestinal cells can reduce the side effects of anticancer therapy and help improve patients' quality of life.

[0156] Therefore, experiments were conducted to determine whether *Galium aparine* extract and its high-molecular fractions had a direct inhibitory effect on gastric inflammation. The cell line used in the experiments was the gastric adenocarcinoma (AGS) cell line, a gastric epithelial cell line. As a human-derived gastric adenocarcinoma cell line, AGS possesses characteristics of gastric epithelial cells, making it very useful in the study of gastrointestinal inflammation. TNF-α (20 ng / ml) was utilized for inflammation induction in AGS cells. IL-8 is a potent inflammatory cytokine that primarily induces and activates neutrophils, amplifying the inflammatory response. Therefore, the inhibition of IL-8 production based on TNF-α is an important mechanism for regulating the inflammatory response and preventing excessive tissue damage. N-acetylcysteine ​​(NAC) was used as a positive control for inhibiting gastritis. NAC is a glutathione precursor that not only promotes glutathione production but also possesses potent antioxidant and anti-inflammatory activities. It inhibits TNF-α-induced inflammatory responses by removing reactive oxygen species (ROS). In this experiment, 3200 µg / ml (19.6 mM) was used. As an indicator of gastric inflammation inhibition, the production of IL-8, a cytokine, was detected using an ELISA kit.

[0157] like Figure 14 As shown, treatment of TNF-α with AGS reduced IL-8 production with both *Galium aparine* extract and GA-H, but showed almost no change with GA-L treatment. As expected, GA-H with a molecular weight >3000 exhibited the highest IL-8 inhibitory activity. Based on these results, it can be confirmed that *Galium aparine* extract and its GA-H inhibit IL-8 production, indicating a potential to block or modulate inflammatory responses, with the active ingredients concentrated in the high-molecular-weight GA-H fraction.

[0158] The above results indicate that *Galium aparine* extract and its high-molecular-weight fractions not only modulate the inflammatory microenvironment in specific cancers such as gastric cancer, playing a crucial role in inhibiting cancer growth and metastasis, but also reduce inflammatory responses to alleviate inflammatory side effects, and enhance the efficacy of anticancer therapy while inhibiting damage to normal tissues. Therefore, *Galium aparine* extract and its high-molecular-weight fractions can be used as a tool to help reduce side effects and promote post-treatment patient recovery by simultaneously using radiation and anticancer therapy.

[0159] 11-2. Effects of activating TNF-α-based NK-κ and MAPK signaling pathways in AGS cells

[0160] To elucidate the mechanism by which AGS cells exhibit anti-inflammatory activity, the effects of GA-H derived from *Gnaphalium affine* extract on the activation of TNF-α-based NF-κ (Nuclear Factor-kappa B) and mitogen-activated protein kinase (MAPK) signaling pathways were investigated using Western blot. Specifically, after adding TNF-α (20 ng / ml) to cultured AGS cells to induce an inflammatory response, GA-H was applied, and the cells were cultured for an additional 30 minutes. Cells were then collected and lysed using lysis buffer. Protein concentration was determined using a BCA assay, and proteins were separated by SDS-PAGE. Finally, the proteins were transferred to a PVDF membrane (Fisher Scientific, T2234) via Western blot. To detect the NF-κB pathway, p-p65 (Cell Signal Transduction Technology, #3033) antibody was used, and to detect the MAPK pathway, p-p38 (Cell Signal Transduction Technology, #9211), p-JNK (Cell Signal Transduction Technology, #9251), and p-ERK (Cell Signal Transduction Technology, #9101) antibodies were used. Then, HRP-conjugated secondary antibodies were applied, and the results were developed using the ECL kit (Dogen bio).

[0161] Experimental results confirm that for TNF-α, the NF-κB (p-p65) and MAPK (p-p38, p-JNK, p-ERK) pathways are activated, and the GA-H of the *Gnaphalium affine* extract inhibits these pathways to reduce the inflammatory response. Figure 15 In particular, the inhibition of the NF-κB and p38 pathways suggests that, since GA-H plays an important role in regulating inflammation in inflammatory diseases or anticancer treatments, the inhibition of major inflammatory response-related pathways of GA-H is an important mechanism that can help alleviate the side effects caused by anticancer treatments or radiation therapy.

[0162] 11-3. Effects on mucosal and related protein production in LS174T cells

[0163] Radiation and anticancer drugs reduce the function of goblet cells, leading to decreased mucus production or the production of mucus with a poorer consistency. Mucus produced by goblet cells in the mucous membranes of the digestive and respiratory organs is composed of water, proteins, electrolytes, and various carbohydrates, protecting the organ linings from physical, chemical, and microbial damage. In particular, when gastrointestinal mucus production is suppressed due to anticancer treatment, the intestinal wall becomes fragile, potentially leading to side effects such as bleeding ulcers or diarrhea. Furthermore, reduced mucus production in the respiratory organs causes the mucous membranes to dry out, making them more susceptible to infection. Mucus plays a crucial role not only in its protective function but also in regulating the immune response. Because mucus contains antimicrobial proteins and immunoglobulins, it prevents pathogen invasion and modulates the immune response. If the mucus layer is damaged due to radiation and anticancer treatment, this protective function is weakened, increasing the risk of infection and inflammation.

[0164] Therefore, the effect of GA-H on the extract of *Gnaphalium affine* produced from mucosa-associated proteins of mucus-producing cells (LS174T cells) was experimentally investigated, specifically in 6-well plates, at a concentration of 0.5 × 10⁻⁶. 6 After 24 hours of LS174T cell plating, the final GA-H concentration was adjusted to 0.8-500 µg / ml, and the cells were cultured for another 12 hours. Following culturing, the LS174T cells were washed with PBS and lysed using a freeze-thaw method. The supernatant was recovered by centrifugation (10000 rpm / 10 min / 4°C), and protein concentration was determined. Each sample (10 μg protein) was electrophoresed on an SDS-PAGE gel (250 V for 30 min) and then transferred to a PVDF membrane. After reaction with anti-MUC2 (Santa Cruz Biotechnology, sc-7314) and a secondary antibody-HRP, the membrane was visualized using an ECL kit.

[0165] As a result, the amount of trefoil factor 3 (TFF-3), which plays a role in the production of mucin 2 and mucosal regeneration, was significantly enhanced in the GA-H treatment groups at 4-100 µg / ml. Figure 16 Based on these results, it can be confirmed that GA-H has the function of activating mucosal regeneration and protection in the context of cell or tissue damage caused by radiation and anticancer drug therapy.

[0166] Example 12: Protective effect against skin damage

[0167] Radiation testing and anticancer drug therapy have diverse effects on skin cells, and skin damage may occur as a side effect. Radiation and anticancer drugs affect not only cancer cells but also normal cells; skin cells, in particular, are sensitive to external stimuli and may experience various side effects. Radiation therapy may cause radiation dermatitis (such as redness, swelling, blisters, and ulcers), dry skin, itching, and pigmentation changes, while anticancer drug therapy may cause side effects such as dry skin, xerosis, keratinization, and pigmentation changes. Because these treatments induce damage to skin cells caused by oxidative stress and inflammation, they can induce apoptosis in severe cases. Therefore, skin cell damage caused by radiation testing and anticancer drug therapy may reduce patients' quality of life, necessitating appropriate management and preventative measures.

[0168] Therefore, the protective function of the high molecular weight fraction (GA-H) of *Galium affine* extract was confirmed. Specifically, HaCaT cells, a human keratinocyte cell line, were treated with hydrogen peroxide (H2O2), and the expression of matrix metalloproteinase-1 (MMP-1) and transforming growth factor-β (TGF-β) was detected in cells under oxidative stress. MMP-1, as an enzyme involved in the degradation of the extracellular matrix (ECM), promotes the degradation of collagen in skin tissue, thus playing an important role in delaying skin aging and wound healing. TGF-β, as an important growth factor regulating tissue regeneration, promotes cell proliferation and ECM synthesis, thus contributing to the recovery of skin damage. Western blotting experiments were performed to determine the expression of MMP-1 and TGF-β based on GA-H. Western blotting was performed according to the method described above, and the cultured cells after sample treatment were cultured for 24 hours for analysis. The primary antibody for MMP-1 expression was purchased from Novus Biologicals (H00004312-D01P), and the antibody for TGF-β was purchased from Proteintech (21898-1-AP).

[0169] The experimental results showed that the increased MMP-1 expression after H2O2 treatment was significantly inhibited by GA-H treatment (at a concentration below 4 µg / m), thus showing results almost similar to the normal control group, and demonstrating that GA-H can alleviate collagen breakdown and skin damage. Figure 17TGF-β was significantly increased in the GA-H treatment groups of 4-20 µg / m, indicating that GA-H can promote tissue regeneration and wound healing.

[0170] Furthermore, the expression of key proteins involved in skin barrier function and antimicrobial defense mechanisms via GA-H in HaCaT cells was investigated to determine whether it could contribute to skin protection. Filagrin, a major protein constituting the Natural Moisturizing Factor (NMF), plays a role in skin moisturization and protection. β-defensin 2, an antimicrobial peptide, possesses antibacterial activity. Retinol was used as a positive control, and experiments were performed using Western blotting. Western blotting was performed as described above, and the sample-treated cells used for analysis were cultured for 24 hours. Antibodies for filaggrin expression were purchased from Santa Cruz Biotechnology (sc-166376), and antibodies for β-defensin 2 were purchased from Abcam (ab63982).

[0171] The experimental results confirmed increased expression of filaggrin and β-defensin 2 in the retinol-treated groups (positive control group). Furthermore, in GA-H treatment (0.8-20 µg / ml), the expression of filaggrin and β-defensin 2 was significantly increased not only compared to the normal control group but also compared to the positive control group. Figure 18 ).

[0172] Therefore, the GA-H of cleavers extract can be used as a skin protectant or therapeutic adjuvant to prevent or alleviate skin side effects that may occur during radiation and anticancer treatment.

[0173] On the other hand, to confirm whether the GA-H of *Gnaphalium affine* extract could be applied to the formulation of anticancer drug side effects, the effect on the expression of type I collagen α1 (COLA1) in HaCaT cells was evaluated. COLA1, as a major component of type I collagen, plays a crucial role in skin elasticity, durability, and wrinkle prevention. Therefore, collagen synthesis is essential for maintaining healthy skin, especially for the recovery of skin damaged by aging or anticancer treatment. Specifically, the analysis of COLA1 was the same as the aforementioned Western blot, using an α1-I type anti-collagen (Anti-Collagen Type I Alpha1, COL1A1) antibody (Abcam, Cambridge, UK; Cat. No. ab34710) as the primary antibody.

[0174] The experimental results showed that COLA1 expression was increased in the retinol treatment group used as a positive control. As expected, retinol promotes collagen synthesis, which helps to strengthen skin elasticity and improve wrinkles. At all applicable concentrations in the experiment, GA-H stimulation promoted higher COLA1 expression. Figure 19 This result demonstrates the potential for GA-H to be used as a product for protecting skin damage after anticancer treatment.

Claims

1. A composition for inhibiting the side effects of anticancer treatment, characterized in that, It contains cleavers extract or its fractions as active ingredients.

2. The composition for inhibiting the side effects of anticancer treatment according to claim 1, characterized in that, The fractions of the *Galium affine* extract are GA-H with a molecular weight of 3000 or higher.

3. The composition for inhibiting the side effects of anticancer treatment according to claim 1, characterized in that, The anti-cancer treatment is either radiation therapy or chemotherapy.

4. The composition for inhibiting the side effects of anticancer treatment according to claim 1, characterized in that, The side effects of the anticancer treatment include one or more of the following symptoms: Oxidative stress; Decreased immunity or abnormal immune regulation; Digestive tract damage such as vomiting, diarrhea, gastric bleeding, or gastric ulcers; Skin lesions characterized by redness, swelling, blisters, dermatitis, xerosis, keratinization, or pigmentation changes.

5. The composition for inhibiting the side effects of anticancer treatment according to claim 1, characterized in that, The cleavers extract is obtained by extracting the leaves and branches of cleavers with hot water at 75 to 85°C for 3 to 6 hours.

6. The composition for inhibiting the side effects of anticancer treatment according to claim 1, characterized in that, The composition for inhibiting the side effects of anticancer treatment can be used as a pharmaceutical, cosmetic, or food product for inhibiting the side effects of anticancer treatment.

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