KR20240056274A - A composition for improving, preventing and treating of myocardial damage comprising Saururus Chinensis extract - Google Patents

Abstract

The present invention relates to a composition for improving, preventing or treating myocardial damage caused by cardiotoxic drugs. By containing Saururus chinensis extract as an active ingredient, it is possible to improve, prevent or treat myocardial damage caused by cardiotoxic drugs. Additionally, since it is non-toxic, it can be consumed in food form.

Description

A composition for improving, preventing and treating myocardial damage comprising Saururus Chinensis extract as an active ingredient {A composition for improving, preventing and treating of myocardial damage comprising Saururus Chinensis extract}

The present invention relates to a composition that contains Saururus chinensis extract as an active ingredient and can improve, prevent, or treat myocardial damage caused by cardiotoxic drugs.

Treatment with anticancer drugs has contributed to increasing the patient's survival rate, but side effects include memory decline, cognitive decline, and cardiotoxicity.

Specifically, anticancer drugs cause cardiotoxicity in approximately 41% of patients, leading to myocardial cell death, and a typical example is atrioventricular blockage.

Myocardium is the muscle that makes up the heart and is the main body of heart contraction, and is metabolically extremely active. In these myocardium, mitochondria exist in more than 30% of the volume of individual fibers, so reserves of high-energy phosphate are very low, so aerobic metabolism is essential.

Cardiomyocyte death proceeds as adenosine triphosphate (ATP) levels are very low and anaerobic glycolysis is virtually halted. Recently, the increase in side effects due to the administration of treatments for various diseases has become a problem, especially doxorubicin, a quinone-based anticancer drug that exhibits an inhibitory effect on signaling between cells, and sodium nitrorfuside (disodium), a vasodilator.

Drugs such as nitroferricyanide (SNP) increase intracellular reactive oxygen species and decrease mitochondrial function, leading to the death of cardiomyocytes, which can lead to decreased cardiac function and various heart diseases.

However, mammalian cardiomyocytes have limited proliferation potential and do not regenerate sufficiently when severely damaged.

Treatment methods for damaged myocardium include removal of irreversibly damaged cells through an inflammatory response after reperfusion injury, but complete healing is fundamentally difficult because regeneration of myocardial cells is difficult. Therefore, the best way is to protect heart cells against damage-causing factors and prevent heart cell loss.

Meanwhile, Saururus chinensis is a functional raw material for industrial health functional foods and is a traditional medicine used in the treatment of edema, jaundice, jaundice, staghorn, carbuncle, etc. as it has moist heat, cleansing, and detoxifying effects. Recently, there has been a lot of research on Saururus chinensis. Studies have shown its antioxidant, antibacterial, and whitening effects, but there is no known effect of trifolium prickly pear on the side effects of treatment with anticancer drugs.

Republic of Korea Patent No. 2213913 Republic of Korea Patent No. 1489478

The purpose of the present invention is to provide a food composition that can improve or prevent myocardial damage caused by cardiotoxic drugs by containing Saururus chinensis extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition that contains Saururus chinensis extract as an active ingredient and can prevent or treat myocardial damage caused by cardiotoxic drugs.

In addition, another object of the present invention is to provide a food composition for companion animals for improving or preventing myocardial damage caused by cardiotoxic drugs, containing Saururus chinensis extract as an active ingredient.

In addition, another object of the present invention is to provide a combination preparation for anticancer treatment containing Saururus chinensis extract and an anticancer agent as active ingredients.

The food composition for improving or preventing myocardial damage of the present invention to achieve the above object may contain Saururus chinensis extract as an active ingredient.

The myocardial damage may be induced by myocardial cell death by cardiotoxic drugs.

The cardiotoxic drug may be an anticancer agent that provides cancer cell damage, cancer cell growth inhibitory activity, or cancer metastasis inhibitory activity.

The anticancer agent may be one or more types selected from the group consisting of chemical anticancer agents and targeted anticancer agents.

The chemical anticancer agent is selected from the group consisting of doxorubicin, epirubicin, mitoxantrone, idarubicin, daunorubicin, and valrubicin; The anticancer drugs in the table above are osmertinib, erlotinib, gefitinib, crizotinib, ceritinib, alectinib, and brigatinib ( brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, lapatinib (lapatinib), vandetanib, afatinib, ruxolitiib, tofacitinib, axitinib, lenvatinib, nintedanib , may be selected from the group consisting of pazopanib, regorafenib, sorafenib, sunitinib, dasatinib, and axitinib. .

The trifoliate extract may be extracted with water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof.

The lower alcohol having 1 to 4 carbon atoms may be 20 to 80% methanol, ethanol, butanol, or propanol.

In addition, the pharmaceutical composition for preventing or treating myocardial damage of the present invention to achieve the above-described other purposes may contain Saururus chinensis extract as an active ingredient.

In addition, the food composition for companion animals for improving or preventing myocardial damage of the present invention to achieve the above-described other object may contain Saururus chinensis extract as an active ingredient.

In addition, the combination preparation for anticancer treatment for preventing cardiac toxicity caused by the anticancer agent of the present invention to achieve the above-described other purpose may contain Saururus chinensis extract and an anticancer agent as active ingredients.

The combination preparation for anticancer treatment may be a mixture of Saururus chinensis extract and anticancer agent, or the Saururus chinensis extract and anticancer agent may be formulated separately and administered simultaneously or sequentially.

The composition for improving, preventing or treating myocardial damage caused by cardiotoxic drugs of the present invention is non-toxic and has an excellent effect for improving, preventing or treating myocardial damage caused by cardiotoxic drugs, making it a competitive food composition and even a health functional food. Alternatively, it can be used as a pharmaceutical composition.

Figure 1 is a graph measuring the survival rate of H9C2 cardiomyocytes when treated with doxorubicin after treatment with the extract of Trifolium prickly pear prepared according to Example 1 of the present invention.
Figure 2 is a graph measuring the survival rate of MDA-MB-231 breast cancer cells when treated with doxorubicin after treatment with the Trifolia extract prepared according to Example 1 of the present invention.
Figure 3a is a graph measuring the survival rate of H9C2 cardiomyocytes when treated with osmertinib after treatment with the Triacium chinensis extract prepared according to Example 1 of the present invention; Figure 3b is a graph measuring the survival rate of H9C2 myocardial cells when treated with erlotinib after treatment with the P. chinensis extract prepared according to Example 1 of the present invention; Figure 3c is a graph measuring the survival rate of H9C2 myocardial cells when treated with gefitinib after treatment with the Triacium vulgaris extract prepared according to Example 1 of the present invention.
Figure 4a is a graph measuring the survival rate of A549 lung cancer cells when treated with osmertinib after treatment with the Triacium chinensis extract prepared according to Example 1 of the present invention; Figure 4b is a graph measuring the survival rate of A549 lung cancer cells when treated with erlotinib after treatment with the Trifolia extract prepared according to Example 1 of the present invention; Figure 4c is a graph measuring the survival rate of A549 lung cancer cells when treated with gefitinib after treatment with the Trifolia extract prepared according to Example 1 of the present invention.
Figure 5a is a graph showing the heart beats per minute of the Control group, Dox group, P. ginseng extract group, Berberine 20 group, and Berberine 40 group; Figure 5b is a graph showing the RR interval of the Control group, Dox group, Trifolia extract group, Berberine 20 group, and Berberine 40 group; Figure 5c is a graph showing the QT intervals of the Control group, Dox group, P. chinensis extract group, Berberine 20 group, and Berberine 40 group; Figure 5d is a graph showing the ST segment of the Control group, Dox group, Trifolia extract group, Berberine 20 group, and Berberine 40 group.
Figure 6a is a graph showing CK (creatin kinase) in the Control group, Dox group, P. ginseng extract group, and Berberine 20 group; Figure 6b is a graph showing the LHD (lactate dehydrogenase) of the Control group, Dox group, Triacium chinensis extract group, and Berberine 20 group; Figure 6c is a graph showing AST (aspartate aminotransferase) in the Control group, Dox group, P. ginseng extract group, and Berberine 20 group; Figure 6d is a graph showing ALT (alanine aminotransferase) in the Control group, Dox group, Triacium chinensis extract group, and Berberine 20 group.
Figure 7 is a diagram measuring the degree of fibrosis of heart tissue in the Control group, Dox group, and P. ginseng extract group.
Figure 8 is a diagram measuring the degree of myocardial cell apoptosis in the Control group, Dox group, and P. ginseng extract group.

The present invention relates to a composition that contains Saururus chinensis extract as an active ingredient and can improve, prevent, or treat myocardial damage caused by cardiotoxic drugs.

The myocardial damage is induced by cardiomyocyte cell death by a cardiotoxic drug, and the cardiotoxic drug may be an anticancer agent that provides cancer cell damage, cancer cell growth inhibitory activity, or cancer metastasis inhibitory activity.

The anticancer agent may be one or more types selected from the group consisting of chemical anticancer agents and targeted anticancer agents. The chemical anticancer drugs cause cardiotoxicity by producing free radicals and induce cardiomyocyte cell death, specifically doxorubicin, epirubicin, mitoxantrone, and idarubicin. It may be selected from the group consisting of idarubicin, daunorubicin, and valrubicin. In addition, the targeted anticancer agent performs anticancer activity through a mechanism of inhibiting tyrosine kinase, which is involved in tumor neovascularization and tumor cell proliferation mechanisms. Tyrosine kinase, which is involved in the survival of cardiomyocytes, is Since myocardial toxicity and myocardial cell death are induced due to inhibition of kinase, specifically osmertinib, erlotinib, gefitinib, crizotinib, and ceritinib. ), alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib (ibrutinib), cabozantinib, lapatinib, vandetanib, afatinib, ruxolitiib, tofacitinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, sunitinib, dasatinib, and Exiti It may be selected from the group consisting of axitinib.

Hereinafter, the present invention will be described in detail.

The composition of the present invention that can improve, prevent or treat myocardial damage contains Saururus chinensis extract as an active ingredient.

The whole plant of Saururus chinensis is used as an antidote or to treat beriberi, and is used as a diuretic in Japan. Additionally, in oriental medicine, the entire plant is dried and used when the body is swollen and urinating is difficult, and it is also prescribed for beriberi, jaundice, and hepatitis.

The trichomes are mixed with an extraction solvent at a weight ratio of 1:5 to 25, preferably 1:8 to 15, extracted at 80 to 110°C for 5 to 15 hours, preferably 7 to 10 hours, and then extracted for 50 to 60 hours. The extract is prepared by performing reduced pressure concentration at ℃. If the weight ratio of Tripodia anchovya and the extraction solvent is outside the above range, a small amount of the active ingredient of Tripodia sinensis may be extracted into the extract.

If the extraction temperature is below the above lower limit, a small amount of the active ingredients of trichomes may be extracted, and if the extraction temperature is above the above upper limit, toxic substances may be generated.

The extraction solvent for extracting each of the above extracts is water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The lower alcohol may include 20 to 80% methanol, ethanol, butanol, or propanol.

The extraction solvent is not particularly limited, but hot water extracts extracted with water are preferred for improving, preventing, or treating myocardial damage caused by cardiotoxic drugs.

The Trifolia extract of the present invention can be used not only as a composition for improving, preventing, or treating myocardial damage caused by cardiotoxic drugs, but also as a combination agent.

The combination preparation for anticancer treatment of the present invention is intended to prevent cardiac toxicity caused by anticancer drugs, and may contain Saururus chinensis extract and anticancer drugs as active ingredients.

The combination preparation for anticancer treatment may be a mixture of Saururus chinensis extract and anticancer agent, or the Saururus chinensis extract and anticancer agent may be formulated separately and administered simultaneously or sequentially.

The anticancer agent is not particularly limited as long as it provides activity to inhibit cancer cell migration or cancer metastasis, but is preferably composed of doxorubicin, osmertinib, erlotinib, and gefitinib. One or more types selected from the group may be mentioned.

The term ‘extract’ used in this specification when referring to Trifolium vulgaris includes not only the crude extract obtained by processing an extraction solvent, but also the processed product of the Trillium vulgaris extract. For example, the P. chinensis extract can be prepared in powder form by additional processes such as reduced pressure distillation and freeze-drying or spray-drying.

In addition, in the broad sense of the word, the extract of the present invention includes the processed products of the Camellia sinensis, such as the Camellia sinensis powder, which are formulated to enable administration to animals. Although the present invention conducted experiments with trichomes, those skilled in the art would be able to predict that the desired effect can be achieved even in the form of trichomes processed products.

Meanwhile, in this specification, the term ‘containing as an active ingredient’ means containing a sufficient amount to achieve the efficacy or activity of the extract of Trifolium prickly pear. As an example, the Triacium vulgaris extract is used at a concentration of 10 to 1500 ㎍/㎖, preferably 100 to 1000 ㎍/㎖. Since the extract of Trifolia sinensis is a natural product and has no side effects on the human body even when administered in excessive amounts, the upper quantitative limit of the extract of Trifolia sinensis contained in the composition of the present invention can be selected within an appropriate range by a person skilled in the art.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants or flavoring agents can be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed by Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, as an appropriate method in the field.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and is preferably administered orally. .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention can be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or can be prepared by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

In addition, the present invention provides a food composition for improving, preventing, or treating myocardial damage caused by cardiotoxic drugs, containing an extract of Trifolia sinensis as an active ingredient.

The food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may contain not only extract of Trifolium prickly pear as an active ingredient, but also ingredients commonly added during food production, including, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the extract of the present invention.

The present invention provides a health functional food comprising a food composition for improving, preventing, or treating myocardial damage containing the extract of Trifolium prickly pear as an active ingredient. Health functional foods are foods that are manufactured by adding extracts of Cypress chinensis to food ingredients such as beverages, teas, spices, gums, and confectionery, or by encapsulating, powdering, or suspending them, and bringing about specific health effects when consumed. However, unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of ginseng extract in such health functional foods cannot be uniformly specified as it varies depending on the type of health functional food being targeted, but it can be added within the range that does not damage the original taste of the food, and is usually 0.01% for the target food. to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets, or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule, or beverage.

In addition, the present invention provides the use of a P. chinensis extract for the production of medicine or food for improving, preventing or treating myocardial damage. As mentioned above, the extract of Trifolia sinensis can be used to improve, prevent, or treat myocardial damage.

Additionally, the present invention provides a method for improving, preventing, or treating myocardial damage, comprising administering an effective amount of an effective amount of an extract of Trifolia chinensis to a mammal.

As used herein, the term “mammal” refers to a mammal, preferably a human, that is the subject of treatment, observation or experiment.

As used herein, the term “effective amount” means the amount of an active ingredient or pharmaceutical composition that is believed by a researcher, veterinarian, physician, or other clinician to induce a biological or medical response in a tissue system, animal, or human, which means that It includes amounts that induce relief of symptoms of a disease or disorder. The effective amount and frequency of administration of the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, and can be determined based on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, and general health. It can be adjusted according to various factors, including condition, gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. In the prevention, treatment or improvement method of the present invention, the composition containing the extract of Trifolia sinensis as an active ingredient may be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, or topically. , can be administered in a conventional manner via intraocular or intradermal routes.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

Example 1.

The aerial parts of Trifolium vulgaris and water were mixed at a weight ratio of 1:10, and extracted under reflux at 80°C for 8 hours to obtain a Trifolium vulgaris extract.

<Test Example Ⅰ> In vitro

Prepared in Examples After filtering the extract, the filtrate was concentrated under reduced pressure below 60°C and used.

Test Example 1. Measurement of H9C2 cardiomyocyte survival rate_doxorubicin

To measure the cell viability of H9C2 cardiomyocytes (ATCC, Manassas, VA, USA), ¹ 24 hours later (Day 1), the extract samples were diluted in medium to concentrations of 50, 100, 200, and 400 ug/mL and then treated with 100 uL of each cell. 24 hours later (Day 2), except for the negative control group (Cont.), cells were treated with 100 uL of Doxorubicin HCl (Apexbio, Houston, TX, USA) at a concentration of 2 uM. After 48 hours (Day 4), to measure cell viability, 10 uL of WST-1 solution (Roche, Pleasanton, CA, USA) was added to the medium and incubated at 37°C for 2 hours. Afterwards, optical density (OD) values were measured at 450 nm and 650 nm (Reference) using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA). Cell viabilit was calculated as OD ₄₅₀ - OD ₆₅₀ , and the negative control group was normalized to 100% and the 0.1% Triton X-100 treated group was normalized to 0%. Data were expressed as mean ± standard deviation from three independent experiments, and significance between groups was calculated using students' t-test (GraphPad). ###, p < 0.001 (vs. negative control); *, p < 0.05 (vs. Dox group); ***, p < 0.001 (vs. Dox group)

Figure 1 is a graph measuring the survival rate of H9C2 cardiomyocytes when treated with doxorubicin after treatment with the extract of Trifolium prickly pear prepared according to Example 1 of the present invention.

As shown in Figure 1, compared to the negative control group not treated with Doxorubicin, the Dox group treated with Doxorubicin (group treated only with Doxorubicin) had a decrease in cells of about 40%, while the cells of the Dox group treated with Doxorubicin (group treated only with Doxorubicin) were reduced by about 40%, while the cells of the Dox group treated with Doxorubicin (group treated only with Doxorubicin) were reduced by about 40%. It was confirmed that pretreated H9c2 cells were protected from the toxicity of doxorubicin and showed a relatively high cell survival rate.

In particular, it was confirmed that H9c2 cells pretreated with a concentration of 400 ug/ml of Chrysanthemum sinensis extract showed a similar level of cell viability as cells not treated with doxorubicin (negative control).

These results mean that pretreatment with Trifolia extract protects cardiomyocyte H9c2 from the toxicity of doxorubicin, an anticancer drug.

Test Example 2. MDA-MB-231 Breast cancer cell survival rate measurement_doxorubicin

Cell survival rate was measured in the same manner as Test Example 1, using MDA-MB-231 breast cancer cells (ATCC, Manassas, VA, USA) instead of H9C2 cardiomyocytes. ###, p < 0.001 (vs. negative control)

Figure 2 is a graph measuring the survival rate of MDA-MB-231 breast cancer cells when treated with doxorubicin after treatment with the Trifolia extract prepared according to Example 1 of the present invention.

As shown in Figure 2, compared to the negative control group not treated with Doxorubicin, the survival rate of MDA-MB-231 cells, which are triple negative breast cancer cells, was reduced by about 40% in the Dox group treated with Doxorubicin, prepared according to Example 1 It was confirmed that MDA-MB-231 cells pretreated with P. ginseng extract also showed a cell survival rate similar to that of the Dox group.

These results mean that the Trifolia extract does not inhibit the cancer cell killing effect of the anticancer drug doxorubicin.

Test Example 3. Measurement of H9C2 cardiomyocyte survival rate_osmertinib, erlotinib, and gefitinib

In order to determine the cell survival rate of cardiomyocytes from the toxicity of anticancer drugs other than doxorubicin, we measured the cardiomyocyte cell protection effect of Triacium chinensis extract against representative anticancer drugs of the TKI (Tyrosine kinase inhibitor) class, which are well known to cause cardiomyocyte toxicity.

To measure the cell viability of H9C2 cardiomyocytes (ATCC, Manassas, VA, USA), ¹ 24 hours later (Day 1), the extract sample was diluted in medium at concentrations of 50, 100, and 200 ug/mL and then treated with 100 uL of each cell. 24 hours later (Day 2), except for the negative control group, cells were treated with 100 uL of Osimertinib, Erlotinib, and Gefitinib at concentrations of 10, 40, and 30 uM, respectively. After 48 hours (Day 4), to measure cell viability, 10 uL of WST-1 solution (Roche, Pleasanton, CA, USA) was added to the medium and incubated at 37°C for 2 hours. Afterwards, optical density (OD) values were measured at 450 nm and 650 nm (Reference) using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA). Cell viability was calculated as OD ₄₅₀ - OD ₆₅₀ , and the negative control group was normalized to 100% and the 0.1% Triton X-100 treated group was normalized to 0%. Data were expressed as mean ± standard deviation from three independent experiments, and significance between groups was calculated using students' t-test (GraphPad). ###, p < 0.0001 (vs. negative control); ***, p < 0.001 (vs. Dox group); ****, p < 0.0001 (vs. Dox group)

Figure 3a is a graph measuring the survival rate of H9C2 cardiomyocytes when treated with osmertinib after treatment with the Triacium chinensis extract prepared according to Example 1 of the present invention; Figure 3b is a graph measuring the survival rate of H9C2 myocardial cells when treated with erlotinib after treatment with the P. chinensis extract prepared according to Example 1 of the present invention; Figure 3c is a graph measuring the survival rate of H9C2 myocardial cells when treated with gefitinib after treatment with the Triacium vulgaris extract prepared according to Example 1 of the present invention.

As shown in Figures 3A to 3C, when treated with Osmertinib, the cell survival rate was about 40%, when treated with Erlotinib, the cell survival rate was about 30%, and when treated with Gefitinib, the cell survival rate was about 80%.

On the other hand, it was confirmed that the H9c2 cells pretreated with the Triacium vulgaris extract prepared according to Example 1 showed a relatively high cell survival rate compared to the group treated with each anticancer agent alone.

However, when H9c2 cells pretreated with a 200 ug/ml concentration of P. chinensis extract were treated with Osmertinib, the cell survival rate was confirmed to be similar to that of the group treated only with Osmertinib.

These results indicate that pretreatment with P. chinensis extract protects cardiomyocyte H9c2 not only from doxorubicin but also from other anticancer drugs.

Test Example 4. Measurement of A549 lung cancer cell viability_osmertinib, erlotinib, and gefitinib

Three types of TKIs anticancer drugs, consisting of osmertinib, erlotinib, and gefitinib, are mainly applied clinically for the treatment of non-small cell lung cancer. Therefore, an experiment was conducted to determine whether the extract of P. ginseng inhibits the non-small cell lung cancer cell killing effect of three types of TKIs anticancer drugs.

To measure the cell survival rate of A549 lung cancer cells (ATCC, Manassas, VA, USA, mainly applied with the above anticancer drugs), a non-small cell lung cancer cell line, ³ . 24 hours later (Day 1), the extract sample was diluted in medium at concentrations of 50, 100, and 200 ug/mL and then treated with 100 uL of each cell. 24 hours later (Day 2), except for the negative control group, cells were treated with 100 uL of Osimertinib, Erlotinib, and Gefitinib at concentrations of 10, 40, and 30 uM, respectively. 72 hours later (Day 5), to measure cell viability, 10 uL of WST-1 solution (Roche, Pleasanton, CA, USA) was added to the medium and incubated at 37°C for 2 hours. Afterwards, optical density (OD) values were measured at 450 nm and 650 nm (Reference) using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA). Cell viabilit was calculated as OD ₄₅₀ - OD ₆₅₀ , and the negative control group was normalized to 100% and the 0.1% Triton X-100 treated group was normalized to 0%. Data were expressed as mean ± standard deviation from three independent experiments, and significance between groups was calculated using students' t-test (GraphPad). ####, p < 0.0001 (vs. negative control)

Figure 4a is a graph measuring the survival rate of A549 lung cancer cells when treated with osmertinib after treatment with the Triacium chinensis extract prepared according to Example 1 of the present invention; Figure 4b is a graph measuring the survival rate of A549 lung cancer cells when treated with erlotinib after treatment with the Trifolia extract prepared according to Example 1 of the present invention; Figure 4c is a graph measuring the survival rate of A549 lung cancer cells when treated with gefitinib after treatment with the Trifolia extract prepared according to Example 1 of the present invention.

As shown in Figures 4a to 4c, the survival rate of A549 lung cancer cells in the osmertinib group, erlotinib group, and gefitinib group treated with each of the three types of anticancer drugs compared to the negative control group that was not treated with each of the three types of anticancer drugs. It decreased to about 30%, about 60%, and about 68%, and the survival rate of A549 lung cancer cells pretreated with the extract of Trifolium prickly pear prepared according to Example 1 was that of the osmertinib group, erlotinib group, and gefitinib group treated with only the above three anticancer drugs. It was confirmed that it was similar to .

These results mean that the Trifolia extract does not inhibit the non-small cell lung cancer cell killing effect of the anticancer drugs osmertinib, erlotinib, and gefitinib.

<Test Example Ⅱ> In vivo

animal testing

C57BL/6 mice (6 weeks old, Korea, male) weighing 18 to 22 g were used in the experiment. They were raised in acrylic cages (45 And constant temperature (20-24 ℃) and humidity (45-65%) were maintained. We monitored them for a week to help them adapt to the changed environment, maintained their sleep cycle, and checked for abnormal behavior.

The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Food Research Institute, and only healthy animals with consistent body weight changes were selected and classified by random placement method.

To create a mouse model of myocardial toxicity caused by an anticancer drug (Doxorubicin), doxorubicin was administered intraperitoneally (IP; intraperitoneal) to mice once a week for 4 weeks (4 times in total) until the total cumulative drug dose reached 20 mg/kg. ) was used in the experiment 1 week after injection (5 weeks from the first dose of Doxorubicin). Negative control group (Control) administered 200 uL of 0.9% saline, Dox group administered 200 uL Dox solution at 5 mg/kg a total of 4 times, and 400 mg/kg daily from the first day of Doxorubicin administration to two days before dissection. They were divided into three groups: the Trifolia extract group, which was administered the extract orally, the Berberine 20 group, which was orally administered 20 mg/kg berberine chloride daily from the first day of doxorubicin administration to two days before dissection, and the Berberine 40 group, which was orally administered 40 mg/kg berberine chloride daily. Four animals were used in each experimental group.

The day before the dissection, the electrocardiogram was measured using an electrocardiogram device (Heart Monitoring ECGenie, Mouse Specifics Inc.). After the dissection, heart tissue was collected, fixed in 4% formaldehyde solution, and tissue analysis was performed (n=4). Data are expressed as mean ± standard error from four mice, and significance between groups was calculated using students' t-test (GraphPad). #, p < 0.05 (vs. Cont. group); ###, p < 0.001 (vs. Cont. group); *, p < 0.05 (vs. Dox group); **, p < 0.01 (vs. Dox group); ***, p < 0.001 (vs. Dox group)

Test Example 5. Measurement of heart rate per minute and extensibility in mouse model of myocardial toxicity caused by anticancer drugs

Figure 5a is a graph showing the heart beats per minute of the Control group, Dox group, Chrysanthemum sinensis extract group, Berberine 20 group, and Berberine 40 group; Figure 5b is a graph showing the RR interval of the Control group, Dox group, P. chinensis extract group, Berberine 20 group, and Berberine 40 group; Figure 5c is a graph showing the QT intervals of the Control group, Dox group, P. ginseng extract group, Berberine 20 group, and Berberine 40 group; Figure 5d is a graph showing the ST segment of the Control group, Dox group, Trifolia extract group, Berberine 20 group, and Berberine 40 group.

As shown in FIGS. 5A to 5D, the heart rate per minute of the Control group is about 760, while the heart rate of the Dox group is lowered to about 670, and the group administered the P. chinensis extract of Example 1 is about 760 beats per minute. It was confirmed that the heart rate was 740 beats, similar to the berberine 20 group.

A decrease in heart rate is accompanied by delays in the RR interval, QT interval, and ST segment on the electrocardiogram. The results of Figures 5b to 5d also confirmed that the three factors increased in the Dox group were restored to a level similar to that of the Control group, Berberine 20 group, and Berberine 40 group by administration of P. ginseng extract.

These results mean that the abnormal cardiac function caused by doxorubicin can be alleviated by the administration of P. chinensis extract.

Test Example 6. Evaluation of blood and hepatotoxicity indicators related to myocardial toxicity in mouse model of myocardial toxicity caused by anticancer drugs

Blood was obtained through orbital blood collection before dissection, and serum was used for analysis. The myocardial toxicity indicators, such as creatin kinase (CK) and LHD (lactate dehydrogenase), and the liver damage indicators, such as AST (aspartate aminotransferase, GOT) and ALT (alanine aminotransferase, GPT), were measured.

Figure 6a is a graph showing CK (creatin kinase) in the Control group, Dox group, Panax chinensis extract group, and Berberine 20 group; Figure 6b is a graph showing the LHD (lactate dehydrogenase) of the Control group, Dox group, P. ginseng extract group, and Berberine 20 group; Figure 6c is a graph showing AST (aspartate aminotransferase) in the Control group, Dox group, P. ginseng extract group, and Berberine 20 group; Figure 6d is a graph showing ALT (alanine aminotransferase) in the Control group, Dox group, P. ginseng extract group, and Berberine 20 group.

As shown in Figures 6a to 6d, serum CK and LDH, which increased due to damage to heart tissue due to the toxicity of doxorubicin, decreased to the levels of the Control group and the Berberine 20 group in the group administered the P. chinensis extract of Example 1. Confirmed.

In addition, it was confirmed that the group administered the P. chinensis extract of Example 1 showed no statistical significance compared to the Control group and the Berberine 20 group in serum AST, an indicator of liver damage.

Test Example 7. Measurement of myocardial tissue fibrosis in mouse model of myocardial toxicity caused by anticancer drugs

After dissection, heart tissue was collected, fixed in 4% formaldehyde solution, and the degree of fibrosis of the heart tissue was confirmed through Massion's Trichrome staining. Fibrotic myocardial cells are stained blue, and it is known that cumulative use of the anticancer drug doxorubicin causes death and fibrosis of myocardial cells, ultimately making normal cardiac function impossible.

Figure 7 is a diagram measuring the degree of fibrosis of heart tissue in the Control group, Dox group, and P. ginseng extract group.

As shown in Figure 7, fibrosis occurred in the cardiomyocytes of the Dox group administered with doxorubicin, and it was confirmed that no traces of fibrosis were found in the myocardial cells of the Chrysanthemum sinensis extract group administered with doxorubicin and the Chrysanthemum sinensis extract of Example 1. .

These results mean that administration of P. chinensis extract can prevent, treat, or inhibit fibrosis of myocardial cells caused by doxorubicin.

Test Example 8. Measurement of myocardial cell death in mouse model of myocardial toxicity caused by anticancer drugs

After dissection, heart tissue was collected, fixed in 4% formaldehyde solution, and the degree of death of myocardial cells was confirmed through TUNEL staining. The nuclei of cardiomyocytes killed by the anticancer drug doxorubicin are stained brown, and the nuclei of normal cardiomyocytes are stained blue. Cumulative use of the anticancer drug doxorubicin induces the death of cardiomyocytes.

Figure 8 is a diagram measuring the degree of myocardial cell death in the Control group, Dox group, and P. ginseng extract group.

As shown in Figure 8, it was confirmed that death of cardiomyocytes occurred in the Dox group administered with doxorubicin, and in the group administered with Doxorubicin and the Chrysanthemum sinensis extract of Example 1, no traces of death of cardiomyocytes were found. Confirmed.

These results mean that administration of P. ginseng extract can prevent, treat, or inhibit the death of cardiomyocytes caused by doxorubicin.

Below, a formulation example of a composition containing the powder of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1. Preparation of powder

500 mg of extract powder obtained in Example 1

100 mg lactose

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2. Preparation of tablets

300 mg of extract powder obtained in Example 1

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3. Preparation of capsules

200 mg of extract powder obtained in Example 1

3 mg crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4. Preparation of injections

600 mg of extract powder obtained in Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above ingredients per ampoule according to the usual manufacturing method for injections.

Formulation Example 5. Preparation of liquid formulation

4 g of extract powder obtained in Example 1

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100g by adding purified water, and then fill in a brown bottle and sterilize. to produce a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of extract powder obtained in Example 1

Vitamin mixture dosage

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients relatively suitable for granules in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above ingredients are mixed according to a typical granule manufacturing method, and then the granules are mixed. It can be prepared and used to manufacture a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of extract powder obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g plum concentrate

1 g taurine

Add purified water to make a total of 900 mL.

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, stored in the refrigerator, and then refrigerated. Used for manufacturing the functional beverage composition of the invention.

The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, country of demand, and intended use.

Claims (14)

A food composition for improving or preventing myocardial damage, comprising an extract of Saururus chinensis as an active ingredient. The food composition for improving or preventing myocardial damage according to claim 1, wherein the myocardial damage is induced by cardiomyocyte cell death caused by a cardiotoxic drug. The food composition for improving or preventing myocardial damage according to claim 2, wherein the cardiotoxic drug is an anticancer agent that provides cancer cell damage, cancer cell growth inhibitory activity, or cancer metastasis inhibitory activity. The food composition for improving or preventing myocardial damage according to claim 3, wherein the anticancer agent is at least one selected from the group consisting of chemical anticancer agents and targeted anticancer agents. The method of claim 4, wherein the chemical anticancer agent consists of doxorubicin, epirubicin, mitoxantrone, idarubicin, daunorubicin, and valrubicin. selected from the group; The anticancer drugs in the table above are osmertinib, erlotinib, gefitinib, crizotinib, ceritinib, alectinib, and brigatinib ( brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, lapatinib (lapatinib), vandetanib, afatinib, ruxolitiib, tofacitinib, axitinib, lenvatinib, nintedanib , characterized by being selected from the group consisting of pazopanib, regorafenib, sorafenib, sunitinib, dasatinib, and axitinib. A food composition for improving or preventing myocardial damage. The food composition for improving or preventing myocardial damage according to claim 1, wherein the Triacium vulgaris extract is extracted with water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The food composition for improving or preventing myocardial damage according to claim 6, wherein the lower alcohol having 1 to 4 carbon atoms is 20 to 80% methanol, ethanol, butanol, or propanol. A pharmaceutical composition for preventing or treating myocardial damage, comprising an extract of Saururus chinensis as an active ingredient. The pharmaceutical composition for preventing or treating myocardial damage according to claim 8, wherein the myocardial damage is induced by cardiomyocyte cell death caused by a cardiotoxic drug. The pharmaceutical composition for preventing or treating myocardial damage according to claim 9, wherein the cardiotoxic drug is an anticancer agent that provides cancer cell damage, cancer cell growth inhibitory activity, or cancer metastasis inhibitory activity. The pharmaceutical composition for preventing or treating myocardial damage according to claim 10, wherein the anticancer agent is at least one selected from the group consisting of chemical anticancer agents and targeted anticancer agents. The method of claim 11, wherein the chemical anticancer agent is a group consisting of doxorubicin, epirubicin, mitoxantrone, idarubicin, daunorubicin, and valrubicin. is selected from; The anticancer drugs in the table above are osmertinib, erlotinib, gefitinib, crizotinib, ceritinib, alectinib, and brigatinib ( brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, ibrutinib, cabozantinib, lapatinib (lapatinib), vandetanib, afatinib, ruxolitiib, tofacitinib, axitinib, lenvatinib, nintedanib , characterized by being selected from the group consisting of pazopanib, regorafenib, sorafenib, sunitinib, dasatinib, and axitinib. A pharmaceutical composition for preventing or treating myocardial damage. A combination preparation for anticancer treatment to prevent cardiac toxicity caused by anticancer drugs containing Saururus chinensis extract and anticancer drugs as active ingredients. The method of claim 13, wherein the combination preparation for anticancer treatment is a mixture of Saururus chinensis extract and anticancer agent, or the Saururus chinensis extract and anticancer agent are formulated separately and administered simultaneously or sequentially. Combination agent for anti-cancer treatment.