KR20190096824A - Composition for preventing, ameliorating or treating disease caused by side effect of anticancer agent comprising Sicyos angulatus extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating disease caused by side effects of an anticancer agent containing an extract of Sicyos angulatus as an active component. The extract of Sicyos angulatus significantly restores the survival of hematopoietic stem cells reduced by administration of anticancer agents, increases the expression level of hematopoietic related cytokines (IL-3, IL-6 and IFN-γ), relieves hematopoietic toxicity by anticancer agents of various mechanisms of action in an ex vivo model, promotes the growth and NO production of primary spleen immune cells, and thus relieves hematopoietic toxicity in an in vivo model. Therefore, the extract of Sicyos angulatus of the present invention can be used as a medicine, health functional food or anticancer adjuvant for preventing, alleviating or treating diseases caused by side effects of anticancer agents.

Description

Composition for preventing, ameliorating or treating disease caused by side effect of anticancer agent comprising Sicyos angulatus extract as effective component}

The present invention is Sicyos angulatus ) relates to a composition for preventing, ameliorating or treating a disease caused by side effects of an anticancer agent containing the extract as an active ingredient.

Cancer is the number one cause of death in Korea, and 30% of deaths in Korea's 50s and 60s are dying of cancer. Surgical surgery, radiation therapy, and chemotherapy are most commonly used to treat such cancers. Among them, there have been various attempts to develop anticancer drugs as chemotherapy, but until now, anticancer drugs have not been developed as a true therapeutic agent, and they are only supplementary treatment or short term life support.

Anticancer agents used as chemotherapy are cytotoxic to cells by interfering with the metabolic pathways of cancer cells and directly interacting with DNA to block DNA replication, transcription, and translation, or to inhibit the synthesis of nucleic acid precursors and to inhibit cell division. Indicates. However, current anticancer drugs have no specific selectivity for cancer, resulting in both therapeutic and toxic effects.The toxic effects of anticancer drugs can be attributed to the effects of bone marrow destruction such as leukocytes, platelets, erythrocytes, and follicular cell destruction. Caused by alopecia, ovaries and testicles, as a result of menstrual irregularity and male infertility, as a side effect due to the destruction of the mucous membrane cells of the digestive system, including stomatitis, nausea and food swallowing disorders and digestive disorders, diarrhea symptoms, renal toxicity due to tubular necrosis, Various side effects include peripheral neuritis and weakness caused by nervous system disorders, vascular disorders such as vascular pain and rashes, and skin and nail discoloration. Accordingly, there is an urgent need for the development of drugs that can increase the anticancer treatment effect while minimizing the side effects caused by the anticancer drugs.

On the other hand, Sicyos angulatus is a two or two year old vine plant belonging to the fruit family, the stem extends about 4-8m, and it grows around other objects with tendrils, and the running leaves are divided into 5-7 branches in the shape of palms. Male and female bloom in June-September, male flowers are light green and run as gunshot inflorescences. Female flowers have two pistils and one pistil. Fruits are clustered together and covered with hairy thorns. Native to North America, it is a naturalized plant in Korea.

Prior art related to the medical use of the thorn gourd extract as described in Korean Patent No. 1802970, a composition for preventing, ameliorating or treating liver disease comprising the thorn gourd extract or a fraction thereof as an active ingredient and administering the composition to a suspected liver disease individual It discloses a method for preventing or treating liver disease, comprising the step of, and the composition for the prevention, improvement or treatment of metabolic diseases comprising a barley gourd extract or fractions thereof as an active ingredient in Korean Patent No. 1793145, And it relates to a method for the prevention or treatment of metabolic diseases comprising the step of administering the composition to a suspicious subject of metabolic disease, but the prevention of diseases caused by side effects of anticancer drugs containing the thorn gourd extract of the present invention as an active ingredient, There is no disclosure of improved or therapeutic compositions.

The present invention is derived from the above demands, the present invention is Sicyos angulatus ) relates to a composition for preventing, improving or treating a disease caused by side effects of an anticancer agent containing an extract as an active ingredient, specifically, Sicyos of the present invention angulatus ) extracts were treated to bone marrow cells of mice whose hematopoietic stem cells were reduced by administration of docetaxel, thereby remarkably restoring the survival of hematopoietic stem cells, and hematopoietic related cytokines (IL-3, IL- 6 and IFN-γ) was confirmed to increase the expression level, and in the ex vivo model to alleviate hematopoietic toxicity by anti-cancer drugs of various mechanisms of action, promote the growth and NO production of primary spleen immune cells, in vivo By confirming that the model can alleviate hematopoietic toxicity, the present invention was completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by side effects of anticancer drugs containing Sicyos angulatus extract as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of diseases caused by side effects of anticancer drugs containing thorn gourd extract as an active ingredient.

The present invention also provides an anticancer adjuvant containing the thorn gourd extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating a disease caused by side effects of an anticancer agent containing an extract of thorn gourd as an active ingredient. The present invention not only significantly reduces a decrease in hematopoietic stem cell colonies induced by an anticancer agent, It has the effect of increasing the expression level of caine, relieves hematopoietic toxicity by anticancer drugs of various mechanisms of action, promotes the growth and NO production of primary spleen immune cells, and also reduces the hematopoietic toxicity in in vivo models, The thorn gourd extract of the present invention can be utilized as a medicine, health functional food or anticancer adjuvant for the prevention, improvement or treatment of diseases caused by anticancer side effects.

Figure 1 shows a decrease in bone marrow hematopoietic stem cells of mice by docetaxel treatment and a decrease in the number of hematopoietic stem cell colonies by docetaxel, and 25, 50 and 100 µg / ml spiny starch hot water extract (A); And the combination of spiny gourd ethanol extract (B) was confirmed that the spiny gourd hot water extract and spiny gourd ethanol extract effectively restore the decrease in the number of hematopoietic stem cell colonies. ## is a statistically significant decrease in the number of hematopoietic stem cell colony forming units (CFUs) in the docetaxel alone-treated group compared to the control without the docetaxel, p <0.01, *, * * Docetaxel alone compared to docetaxel and spinach hot water extract; Or CFU number of hematopoietic stem cells in the group treated with docetaxel and spinach ethanol extract was statistically significant, * is p <0.05 and ** is p <0.01.
Figure 2 shows a decrease in bone marrow hematopoietic stem cells of mice by docetaxel treatment and a decrease in the number of hematopoietic stem cell colonies by docetaxel and 25, 50 and 100 μg / ml of spinach leaf hydrothermal extract (A); And thorny leaf stem hot water extract (B) in combination, the result of confirming that the thorny leaf hot water extract and thorny leaf stem hot water extract effectively restore the decrease in the number of hematopoietic stem cell colonies. # Is a statistically significant decrease in the number of hematopoietic stem cell colony forming units (CFUs) in the docetaxel-only group compared to the control group not treated with the docetaxel, p <0.05, and *, ** Docetaxel and thorn leaf hot water extract compared to the docetaxel alone treatment group; Or CFU number of hematopoietic stem cells was statistically increased in the group treated with docetaxel and spinach hot water extract, * is p <0.05 and ** is p <0.01.
Figure 3 shows the reduction of bone marrow hematopoietic stem cells in mice by docetaxel treatment and the decrease in the number of hematopoietic stem cell colonies by docetaxel, and 25, 50 and 100 μg / ml of spinach hot water extract and fractions thereof (hexane, Hx; ethyl). Acetate, EtOAc; butanol, BuOH; water, Wa) was used in combination to confirm that spiny hot water extract, butanol (BuOH) fraction and water (Wa) fraction effectively recovered the decrease in the number of hematopoietic stem cells. #### is a statistically significant decrease in the number of hematopoietic stem cell colony forming units (CFUs) in the docetaxel-only group compared to the control group not treated with the docetaxel, and p <0.0001. *, ***, **** is docetaxel and thorny hot water extract compared to docetaxel alone treatment group; Butanol fraction of spiny hot water extract; Or water fraction of thorny hot water extract; and the CFU number of the hematopoietic stem cells in the combination treatment group was statistically significant, ** p <0.01, *** p <0.001, **** p <0.0001.
Figure 4 shows a decrease in the CFU number of hematopoietic stem cells by docetaxel treatment in a culture medium that promotes hematopoietic stem cell growth and differentiation of a specific lineage, and when the docetaxel and the spinach extract of the present invention are treated in combination, The results confirmed that the CFU number of hematopoietic stem cells can be effectively recovered. GF M3434 media promotes hematopoietic stem cell growth and differentiation in all lineages, GF M3534 media promotes the growth and differentiation of hematopoietic stem cells (monocytes, eosinophils and neutrophils) in the myeloid lineage SF M3436 medium is a medium for promoting the growth and differentiation of erythroid stem cells (erythrocytes). ###, #### is a statistically significant decrease in the number of CFUs of hematopoietic stem cells in the docetaxel-treated group compared to the control without the docetaxel, ### is p <0.001, ### # Is p <0.0001. *, **, ***, **** are statistically significant increase in the number of CFU of hematopoietic stem cells of the group treated with docetaxel and spinach hot water extract compared to the docetaxel alone treatment group, * is p <0.05 , ** is p <0.01, *** is p <0.001, and **** is p <0.0001.
FIG. 5 shows hematopoietic and immune function-related cytokines (IL-3, IL-6) when treated with raw splenocytes or Raw macrophage, mouse macrophage, Raw264.7 cells. , IFN- [gamma] and TNF- [alpha]) increased in a concentration dependent manner. ** and *** are statistically significant increase in cytokine expression in the group treated with spinach hot water extract compared to the control group (vehicle), ** is p <0.01, *** p <0.001. ConA is a positive control group in the experiment using spleen immune cells, Concanavalin A (Concanavalin A), LPS is a positive control group used in the experiment using Raw264.7 cells, lipopolysaccharide (lipopolysaccharide).
6 is a result confirming the effect of reducing the hematopoietic toxicity induced by anticancer drugs of different action mechanism of prickly pear hot water extract of the present invention. #, ##, #### are treated with anticancer agents of actinomycin D (A), doxorubicin (B), paclitaxel (C) or mitomycin (D), respectively, as compared to the control group without treatment with anticancer drugs The number of hematopoietic stem cell CFU in one group was statistically significantly reduced. # Is p <0.05, ## is p <0.01, and #### is p <0.0001. *, **, ***, **** are docetaxel and spiny bacterium compared to the group treated with anticancer agents of actinomycin D (A), doxorubicin (B), paclitaxel (C) or mitomycin (D), respectively. The number of hematopoietic stem cell CFU in the group treated with the hydrothermal extract was statistically significantly increased, with * for p <0.05, ** for p <0.01, *** for p <0.001, and **** for p < 0.0001.
Figure 7 shows the growth of spinal hot water extract of the present invention (A) primary splenocytes (A) and the production of nitric oxide (Nitric oxide, NO) which is an immune-related intracellular signaling material from spleen immune cells (B) This is to confirm that it can promote. *, **, *** are statistically significant increase in cell growth and nitric oxide production in the group treated with spinach hot water extract compared to the negative control group, where p <0.05, ** p <0.01 and *** are p <0.001.
8 is an animal model in which hematopoietic toxicity is induced by repeated intraperitoneal administration of docetaxel, and weight loss (A), peripheral blood of experimental animals (mouse) due to anticancer drug toxicity by daily oral administration of spinach hot water extract of the present invention. Can effectively restore the decrease in leukocyte count (B), absolute weight of spleen (C) and thymus (D), the major immune organs, or decrease in spleen index (SI; thymus index (TI)). The result is confirmed. G-CSF, a positive control group, is a granulocyte-colony stimulating factor agent used clinically as a neutropenia treatment. # Indicates that the hematopoietic toxicity model group (Model, M) treated with docetaxel alone compared to the normal control group (sham) statistically significantly reduced the absolute and relative weight of the thymus, p <0.05. * Indicates that the absolute and relative weights of the thymus were statistically significantly increased in the experimental group treated with H. pylori extract compared to the hematopoietic toxicity model group (Model, M), p <0.05.
9 is a spleen, thymus and bone marrow, which are major immune organs when daily oral administration of the spinach hot water extract of the present invention in an animal model (mouse) inducing hematopoietic toxicity by repeated intraperitoneal administration of docetaxel. Hematoxylin-eosin (H & E) tissue staining technique has been shown to effectively protect against tissue damage caused by chemotherapy in the microenvironment of bone marrow. R and W refer to the red and white pulp observed in the spleen and C and M refer to the cortex and medulla observed in the thymus. G-CSF, a positive control group, is a granulocyte-colony stimulating factor agent used clinically as a neutropenia treatment.

The present invention is Sicyos angulatus ) relates to a pharmaceutical composition for preventing or treating a disease caused by side effects of an anticancer agent containing the extract as an active ingredient.

The spiny gourd extract may be prepared by a method comprising the following steps, but is not limited thereto.

(1) extracting by adding an extraction solvent to the thorn foil;

(2) filtering the extract of step (1); And

(3) preparing the extract by concentrating under reduced pressure and drying the filtered extract of step (2).

The extraction solvent in step (1) is preferably water, lower alcohols of C ₁ ~ C ₄ or mixtures thereof, but is not limited thereto.

In the above production method, the extraction of the thorn extract can be used for all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction. The extraction solvent is preferably extracted by adding 1 to 20 times the volume of dried spiny foil, more preferably 3 to 10 times. Extraction temperature is preferably 20 ~ 50 ℃ but is not limited thereto. In addition, the extraction time is preferably 10 to 100 hours, more preferably 24 to 96 hours, most preferably 72 hours is not limited thereto. In the above method, the decompression concentration in step (3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The thorn gourd extract may be extracted using an outpost, leaf or stem area, and is not particularly limited.

The cancer is preferably any one selected from lung cancer, breast cancer, liver cancer, stomach cancer, colon cancer, colon cancer, skin cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, thyroid cancer, kidney cancer, fibrosarcoma, melanoma and hematological cancer. However, the present invention is not limited thereto and any cancer that can be diagnosed is not limited.

The anticancer agents include antitumor antibiotics, topoisomerase inhibitors, platinum anticancer agents, taxane anticancer agents or receptor tyrosine kinase inhibitors, This includes, but is not limited to, all anticancer drugs that can be used clinically, pharmaceutically and biomedically.

The antitumor antibiotics are actinomycin D, bleomycin sulfate, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, doxorubicin, idarubicin ), Mitomycin, mitomycin-C, and mitramycin; The topoisomerase inhibitor is irinotecan, irinotecan, camptothecin, novobiocin, epirubicin, epirubicin, dactinomycin, amsacrine At least one selected from teniposide and etoposide; The platinum-based anticancer agent is at least one selected from the group consisting of cisplatin, carboplatin, and oxaliplatin; The taxane-based anticancer agent is paclitaxel or docetaxel; The tyrosine kinase inhibitor is preferably at least one selected from gefitinib, erlotinib, and apatinib, but is not limited thereto.

The disease caused by the side effects of the anticancer agent is preferably one or more selected from the group consisting of hematopoietic toxicity, anemia and neutropenia, but is not limited thereto.

As used herein, the term 'hematopoietic toxicity' refers to damage or disorder of organs or cells of the hematopoietic system, resulting in hematopoietic dysfunction, in particular hematopoietic disorder or dysfunction of erythrocytes or immune system cells such as leukocytes. Preferably, hematopoiesis or function of the immune system in the bone marrow is affected by hematopoietic toxicity. Thus, the hematopoietic toxicity generally includes myelotoxicity, anemia and neutropenia, and more preferably is induced by or as a result of the administration of a chemical compound or drug.

The composition of the present invention contains 0.1 to 99.9% by weight of the spiny gourd extract or fraction of the present invention based on the total weight of the composition as an active ingredient, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select a skin external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease of the patient, the daily dosage is the amount of thorn extract 0.01 to 2,000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times per day. The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In addition, the present invention ( Sicyos) angulatus ) relates to a health functional food composition for the prevention or improvement of diseases caused by side effects of anticancer drugs containing the extract as an active ingredient.

The extraction solvent of the spiny gourd extract is preferably water, a lower alcohol of C ₁ ~ C ₄ or a mixture thereof, but is not limited thereto. The anticancer agent includes, but is not limited to, an antitumor antibiotic, a phase isomerase inhibitor, a platinum anticancer agent, a taxane anticancer agent, or a receptor tyrosine kinase inhibitor, and includes all anticancer agents that can be used clinically, pharmaceutically, and biomedically. The disease caused by side effects of the anticancer agent is characterized in that any one or more selected from the group consisting of hematopoietic toxicity, anemia and neutropenia.

The nutraceutical composition of the present invention may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. There is no particular limitation on the type of dietary supplement. Examples of foods to which the thorn extract may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, teas, Drinks, alcoholic beverages and vitamin complexes, etc., includes all of the health functional foods in the conventional sense. The health beverage comprising the composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

The health functional food composition of the present invention, in addition to the active ingredient, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives , Glycerin, alcohol, carbonation agent used in the carbonated beverage may further contain. In addition it may further contain a pulp for the production of fruit juices or vegetable drinks. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected from 0.01-2 weight part with respect to 100 weight part of compositions of this invention.

In addition, the present invention ( Sicyos) angulatus ) relates to an anticancer adjuvant containing an extract as an active ingredient.

The anticancer adjuvant is characterized by suppressing at least one anticancer drug side effect selected from hematopoietic toxicity, anemia and neutropenia caused by the administration of an anticancer agent.

The anticancer adjuvant may contain one or more active ingredients exhibiting the same or similar functions in addition to the thorn gourd extract. The anticancer adjuvant may be administered orally or parenterally during clinical administration, and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection, or thoracic injection during parenteral administration. It can be administered by internal injection and can be used in the form of a general pharmaceutical formulation.

The anticancer adjuvant may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers. The daily dosage of the anticancer adjuvant is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once or several times a day, but the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The anticancer adjuvant of the present invention may be administered in various parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Example  One. Barbed  Preparation of Extract

(1) Preparation of Prickly Pear Hot Water Extract

In order to prepare the thorn gourd extract which is an active ingredient of the present invention, the thorn gourd was harvested in the wild, and the dried thorn gourd was dried in a shade without light, and then, 100 g of dry thorn gourd starch was pulverized, and 2 liter of water and Put together in a round flask and mix. A water bath connected to a reflux extraction device with a cooling tube was heated and repeatedly extracted two times, once every two hours. The extracted spiny hot water extract was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (Vacuum pump, GAST). The filtered spiny hot water extract was concentrated under reduced pressure using a rotary evaporator (YEYELA), and the concentrated spiny hot water extract was lyophilized, and then homogenized with a mortar to obtain spiny hot water extract. It was stored in a sealed plastic container and stored in a 4 ° C. cold store until the experiment. In the present invention, thorny hot water extract means that extracted by using thorny starch.

In addition, the spiny leaf or stem was subjected to hydrothermal extraction in the same manner as the spiny starch starch to obtain the spiny leaf hydrothermal extract and the spiny leaf stem hydrothermal extract, which were stored in a sealed plastic container until stored in a 4 ° C. low temperature reservoir.

(2) Preparation of Spiny Bak Ethanol Extract

10.0 g of pulverized barley starch and 70% (v / v) ethanol were mixed and mixed in an Erlenmeyer flask, and ultrasonic extraction (Branson 5210) was repeatedly extracted twice for 1 hour. The extracted spiny ethanol extract was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (GAST). The filtered spiny ethanol extract was concentrated under reduced pressure using a rotary evaporator (YEYELA), and the concentrated spiny ethanol extract was vacuum dried (vacuum dry oven, WiseVen) or freeze-dried (Freeze Dryer, IlshinBioBase), Park ethanol extract was obtained. The obtained spiny ethanol extract was placed in a sealed plastic container and stored in a 4 ° C. low temperature refrigerator until the experiment. In the present invention, thorny ethanol extract means that it is extracted by using thorny starch.

In addition, the thorny leaf or stem was extracted with ethanol in the same manner as the thorny outpost, to obtain the thorny leaf ethanol extract and thorny stem ethanol extract, and stored in a sealed plastic container until 4 ℃ low temperature storage until the experiment.

(3) Preparation of Solvent Fraction of Prickly Pear Extract

① Preparation of spiny hexane fraction (Hx)

300 g of thorny hot water extract was suspended in 0.8 L of distilled water, and then 1.2 L of hexane was added to perform solvent fractionation twice. There was no interface layer, and after separation of the hexane layer, the resultant was concentrated under reduced pressure at a vacuum degree of 0.07 MPa to obtain a hexane fraction.

② Preparation of spiny ethyl acetate fraction (EtOAc)

After the hexane fraction, 1.5 liters of ethyl acetate was added to the remaining fractions, and the solvent fractionation was performed twice. Ethyl acetate fractions were obtained by concentration under reduced pressure at 0.07 MPa vacuum.

③ Preparation of spinach butanol fraction (BuOH)

After the ethyl acetate fraction, 1.5 L of saturated butanol was added to the remaining fractions, and the solvent fraction was performed twice. The butanol fraction was obtained by concentrating under reduced pressure at a vacuum degree of 0.07 MPa.

④ Spiny water fraction (Wa) preparation

After the butanol fraction, the remaining fractions were obtained by concentrating under reduced pressure 80-90% at a vacuum degree of 0.07 MPa and then lyophilizing.

Example 2 Confirmation of the Hematopoietic Toxicity of Docetaxel and the Hematopoietic Toxicity Effect of Prickly Pear Water Extract Using Mouse Bone Marrow Cells ( ex vivo )

Ex vivo experiments were performed to determine the effects of docetaxel anticancer drugs, which are frequently administered to solid cancer patients in the tumor clinic but have strong side effects of hematopoietic toxicity, on the growth and differentiation of hematopoietic stem cells in mouse bone marrow cells.

Specifically, bone marrow nucleated cells are isolated from the mouse femur, and then 20 nM docetaxel is added to a mouse-derived murine stem cell factor (rm stem cell factor), interleukin-3. culturing the bone marrow cells for 7-10 days in medocult GF M3434 medium containing (rm IL-3), human-derived recombinant IL-6 (rh IL-6) and erythropoietin (rh erythropoietin) Thereby inducing growth and differentiation of hematopoietic stem cells. Colony Forming Unit assay (CFU assay) of hematopoietic stem cells was performed to observe the effect of docetaxel anticancer agent on the growth and differentiation of mouse hematopoietic stem cells.

In addition, mouse bone marrow cells were treated with 20 nM docetaxel in combination with 25, 50, and 100 μg / ml of thorny gourd extracts to determine whether thorny gourd extracts could alleviate hematopoietic toxicity caused by anticancer drugs. The difference in the effects of the solvents (70% ethanol and water) used for the extraction of thorns and the parts of the thorns (outposts, leaves and stems) used for extraction were compared.

As a result, the number of mouse hematopoietic stem cell CFU was statistically significantly decreased by docetaxel, and the spinach starch hydrothermal extract and ethanol extract were able to mitigate the decrease in the CFU number of hematopoietic stem cells by docetaxel, but were more visible than the spinach ethanol extract. Park hot water extract was confirmed to be more effective (Fig. 1).

In addition, it was confirmed that thorny leaf hot water extract and thorny leaf stem hot water extract also had a hematopoietic toxicity-reducing effect by anticancer drugs at a level similar to that of thorn gourd starch extract (FIG. 2).

Example  3. In mouse bone marrow cells Barbed Hydrothermal  Extracts and their Fraction Hematopoietic toxicity  Identify mitigation effects ex vivo )

GF M3434 for four kinds of solvent fractions (hexane (Hx), ethyl acetate (EtOAc), butanol (BuOH) and water (Wa) fractions) derived from the thorny hot water extract and thorny hot water extract, which are the active substances of the present invention. The culture medium was used to compare the hematopoietic alleviation effect induced by docetaxel (Fig. 3). Docetaxel and spinach hot water extract (50, 100, 200 µg / ml), or fractions thereof (1, 10, 100 µg / ml) were co-treated and the CFU number was counted. Hexane and ethyl acetate fractions showed strong cytotoxicity with increasing concentrations, but butanol and water fractions showed similar hematopoietic toxicity-releasing effects similar to prickly water extract. In the case of butanol and water fractions, the activity of butanol and water fractions was more effective than that of prickly water extracts, even at low concentrations of 1 μg / ml, which showed a similar effect to the cytoprotective effect observed at 50 μg / ml of the hydrothermal extract. Was judged to be better (Fig. 3).

Example  4. various Eruption Badges  Used Barbed Hydrothermal  Anticancer Induction of Extracts Hematopoietic toxicity Alleviation  Confirm( ex vivo )

A culture medium that promotes the growth and differentiation of hematopoietic stem cells of a specific strain was used to confirm the mitigating effect of Pseudothermal hot-water extract in hematopoietic toxicity test system induced by docetaxel anticancer drugs. The culture line used for the hematopoietic stem cell CFU test and a combination of growth factors that define the characteristics of each medium and cell lines forming colonies are shown in Table 1 below.

Culture medium used for the CFU test, growth factors constituting the medium and colony types differentiated badge Growth factor Colony type GF M3434 rh EPO, rm SCF, rh IL6, rm IL3 All hematopoietic stem cells including BFU-E, CFU-GM, CFU-G, CFU-M, CFU-GEMM GF M3534 rm SCF, rh IL6, rm IL3 CFU-GM, CFU-G, CFU-M of Myeloid Lines SF M3436 cytokines containing rh EPO BFU-E of the Erythroid strain

As a result of confirming that the hematopoietic mitigating effect of spiny hot water extract is limited to the growth and differentiation of hematopoietic stem cells of a specific strain, the spiny hot water extract is not only an entire hematopoietic stem cell (FIG. 4A) and a myeloid (myeloid) line (FIG. 4B) Hematopoietic stem cell differentiation of the (erythroid) line (FIG. 4C) was confirmed to be effective in reducing hematopoietic toxicity.

These results indicate that thorny hot water extract can be used for the purpose of preventing or treating neutropenia (FIG. 4B) and anemia (FIG. 4C) induced by anticancer agents.

Example 5 Confirmation of Hematopoietic and Immune-Related Cytokines Expression by Spinal Bacteria Extract from Cultured Primary Splenocytes and Mouse Macrophages (Raw264.7)

Primary splenocytes isolated from mouse spleen and Raw264.7 cells, which are mouse macrophage, were cultured by selecting the appropriate culture medium. After 72 hours of treatment with the hydrated hot water extract, an active substance of the present invention, at a concentration of 1-100 μg / ml, commercial expression of hematopoietic and immune function-related cytokines was expressed in splenic immune cells and Raw264.7 cells. The cytokine concentration in the medium was measured using an Enzyme Linked ImmunoSorbent Assay (ELISA) kit available. In Example 5, Concanavalin A (Con A) and lipopolysaccharide (LPS), which are known to increase cytokine production in primary splenic immune cells and Raw264.7 cells, were positive cells. It was used for the experiment.

As a result, thorny hot water extract increases the expression of cytokines (IL-3, IL-6) that promote differentiation into myeloid cells at the upper stages of the hematopoiesis process in primary spleen immune cells, An increase in cytokines (INF-γ and TNF-α), which enhance the immune response and antitumor function to viruses in splenic immune cells and Raw264.7 macrophages, was confirmed (FIG. 5).

Example 6 Confirmation of Hematopoietic Toxicity Efficacy of Prickly Pear Water Extract in Hematopoietic Toxicity Model Induced by Anticancer Agents of Various Mechanisms of Action ( ex vivo )

It was confirmed that thorny hot water extract can alleviate hematopoietic toxicity induced by anticancer drugs with different mechanisms of action. The medium used was SF M3436 medium that induces hematopoietic stem cell growth and differentiation of erythroid family.

As a result, the thorny hot water extract was treated with anti-cancer agent actinomycin D (DNA binding, Fig. 6A), doxorubicin (topoisomerase activity inhibition, Fig. 6B), paclitaxel (paclitaxel, inhibition of cell division, Figure 6C) and mitomycin C (mitomycin C, DNA cross-linking, Figure 6D) was confirmed that it can effectively restore the decrease in the number of CFU of hematopoietic stem cells induced.

Example 7 Effects of Prickly Pear Extracts on Growth of Primary Splenocytes and Production of Immune Regulating Cell Signaling Substances (NO) in vitro )

When mice were treated with 0 ~ 200 μg / ml concentration of spinach hot water extract, the primary splenocytes were treated with nitric oxide (NO), a cell signaling material that regulates the growth and immune function of spleen immune cells. The production was confirmed. Growth and cell viability of spleen immune cells was determined using an automatic cell counter (ADAM-MC) and the concentration of NO in culture medium was determined using a Thermo Scientific NO measurement kit.

As a result, it was confirmed that thorny hot water extract can promote NO production with growth of splenic immune cells in a concentration-dependent manner (FIG. 7).

Example  8. Anticancer Drugs Hematopoietic toxicity  In animal models Barbed Hydrothermal  Of extract Hematopoietic toxicity  Identify mitigation effects in vivo )

Ex vivo In order to confirm that the hematopoietic toxic effect of the prickly pear hot water extract confirmed in the test system is effective in the in vivo animal model, hematopoietic toxicity is obtained by oral administration of the prickly pear hot water extract in a mouse animal model that induces hematopoietic toxicity by repeatedly injecting anticancer drugs. Check if this is alleviated.

Specifically, C57BL / 6 mice were purchased and subjected to a period of purification for one week, and then randomly divided into six groups using an online random program (www.randomizer.org) (day 0 on experimental day, groups 1 to 6). On the first day of the experiment, a 26-gauge needle and a capillary tube coated with 10% (w / v) ethylenediaminetetraacetic acid dipotassium salt (K ₂ -EDTA) were obtained from the saphenous vein of mice in each group. After the peripheral blood was collected, the white blood cell (WBC) level was measured using a hematology analyzer (HemaVet) to determine the baseline value of the white blood cell (baseline) before inducing hematopoietic toxicity. On Days 2-4 of the experiment (3 days), Group 1 contained 0.9% (w / v) saline containing 5% (v / v) ethanol and 2% (v / v) polysorbate 80. Intraperitoneally administered, Groups 2 to 6 were dissolved in 0.9% saline containing 5% (v / v) ethanol and 2% (v / v) polysorbate 80 to induce hematopoietic toxicity. Thereafter, daily intraperitoneal administration was performed at a 30 mg / kg / d dose. Lastly, mice belonging to Group 1 (normal control group, sham) and Group 2 (model hematopoietic model group, Model, M) for 7 days from the day following docetaxel administration (test day 5-11) were sterilized with sterile water. To, mice belonging to groups 3 to 5 were orally administered every 30, 100, 300 mg / kg / d dose of spiny hot water extract dissolved in sterile water, respectively. Group 6 was a positive control group injected with human recombinant G-CSF (Granulocyte-Colony Stimulating Factor) subcutaneously in the back of the nape of the neck at the dose of 61.5㎍ / kg on the 5th day and 7th day of the experiment. All doses and dose volumes were determined based on the weight of the experimental animals measured daily. Peripheral blood was collected from the cloned vein at 2-3 days interval after the end of docetaxel administration and the leukocyte count was checked using a hemocytometer.

As a result, a rapid weight loss and a decrease in leukocyte count were observed due to docetaxel administration in the model group (Model 2), and a dose-dependent decrease in the group administered with the active ingredient prickly water extract (groups 3 to 5). It was confirmed that the recovered body weight was recovered (FIG. 8A). In addition, it was confirmed that the level of leukocytes increased in the group administered with thorn gourd hot water extract compared to the model group on the 9th and 12th day of the experiment (FIG. 8B). After the end of the experiment (day 12), mice in each group were sacrificed by inhalation of carbon dioxide (CO ₂ ), the spleen and thymus, which are the major immune organs, and then the absolute organ weight of each organ. ) And the absolute weight divided by the weight of each experimental animal (spleen index, SI; thymus index, TI) was measured.

As a result, it was confirmed that both the absolute weight and the relative weight of the spleen (FIG. 8C) and the thymus (FIG. 8D) were dose-dependently increased in comparison with the model group. When G-CSF was administered as a positive control group, the leukocyte count increased compared to the model group on the 9th day of the experiment, but after 3 days of stopping G-CSF (day 12 on the test day), the leukocyte count decreased to the same level as the model group. Was observed (FIG. 8B). In addition, in the G-CSF-administered group, no significant difference in the weight measurements of the spleen and thymus, which is an immune organ, was observed compared to the model group (FIGS. 8C and 8D).

The hematopoietic mitigating effect of H. pylori water extract was observed through hematoxylin-Eosin (H & E) staining in the tissues of the spleen, thymus and bone marrow, which are the main immune organs. Specifically, after the end of the experiment (day 12 of the experiment), mice belonging to each group were sacrificed by inhalation of carbon monoxide, the spleen, thymus and hind limbs were collected, and the tissues were fixed in soaked formalin fixative. Fixed tissues were observed by tissue microscopy after paraffin embedding, tissue dissection and H & E staining.

As a result, necrosis and bleeding of the cells in the spleen tissue of the model group caused by docetaxel administration, and the atrophy of the tissue in the thymus tissue and the boundary between the cortex and the medulla were unclear. In bone marrow, hypocellularity was observed due to a decrease in hematopoietic cells. These immune tissue damages according to the anticancer drug administration was confirmed that significantly reduced when oral administration of prickly hot water extract (Fig. 9).

Claims (11)

Sicyos angulatus ) pharmaceutical composition for the prevention or treatment of diseases caused by side effects of anticancer drugs containing the extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is extracted with water, lower alcohols of C ₁ to C ₄ or a mixture thereof as a solvent. According to claim 1, wherein the anticancer agent (antitumor antibiotics), topoisomerase inhibitor (topoisomerase inhibitor), platinum-based anticancer agent, taxane-based anticancer agent and receptor tyrosine kinase inhibitor (receptor tyrosine kinase Inhibitors) pharmaceutical composition for the prevention or treatment of diseases caused by side effects of anti-cancer drugs, characterized in that at least one selected from. According to claim 3, wherein the antitumor antibiotics (actinomycin D), bleomycin sulfate (bleomycin sulfate), daunomycin (daunomycin), daunorubicin (donorubicin), doxorubicin (doxorubicin), At least one selected from idarubicin, mitomycin, mitomycin-C, and mitramycin; The topoisomerase inhibitor is irinotecan, irinotecan, camptothecin, novobiocin, epirubicin, epirubicin, dactinomycin, amsacrine At least one selected from teniposide and etoposide; The platinum-based anticancer agent is at least one selected from the group consisting of cisplatin, carboplatin, and oxaliplatin; The taxane-based anticancer agent is paclitaxel or docetaxel; The tyrosine kinase inhibitor (receptor tyrosine kinase inhibitor) is at least one selected from gefitinib (gefitinib), erlotinib (erlotinib) and apatinib (afatinib) pharmaceutical composition for the prevention or treatment of diseases caused by anti-cancer drug side effects . The pharmaceutical composition for preventing or treating a disease caused by side effects of the anticancer agent according to claim 1, wherein the disease caused by side effects of the anticancer agent is one or more selected from hematopoietic toxicity, anemia and neutropenia. Sicyos angulatus ) health functional food composition for the prevention or improvement of diseases caused by side effects of anticancer drugs containing the extract as an active ingredient. The anticancer agent according to claim 6, wherein the anticancer agent is antitumor antibiotics, topoisomerase inhibitor, platinum anticancer agent, taxane anticancer agent or receptor tyrosine kinase inhibitor. Inhibitor) health functional food composition for the prevention or improvement of diseases caused by side effects of anticancer drugs. According to claim 7, wherein the antitumor antibiotics (actinomycin D), bleomycin sulfate (bleomycin sulfate), daunomycin (daunomycin), daunorubicin (donorubicin), doxorubicin (doxorubicin), At least one selected from idarubicin, mitomycin, mitomycin-C, and mitramycin; The topoisomerase inhibitor is irinotecan, irinotecan, camptothecin, novobiocin, epirubicin, epirubicin, dactinomycin, amsacrine At least one selected from teniposide and etoposide; The platinum-based anticancer agent is at least one selected from the group consisting of cisplatin, carboplatin, and oxaliplatin; The taxane-based anticancer agent is paclitaxel or docetaxel; The tyrosine kinase inhibitor (receptor tyrosine kinase inhibitor) is at least one selected from gefitinib (gefitinib), erlotinib (erlotinib) and afatinib (afatinib) health function for the prevention or improvement of diseases caused by side effects of anti-cancer drugs Food composition. According to claim 6, wherein the disease caused by the side effects of the anticancer agent health functional food composition for preventing or improving the disease caused by the side effects of the anticancer agent, characterized in that at least one selected from hematopoietic toxicity, anemia and neutropenia. Sicyos angulatus ) Anticancer adjuvant containing extract as an active ingredient. 11. The anticancer adjuvant of claim 10, wherein the anticancer adjuvant ameliorates a disease caused by at least one anticancer side effect selected from hematopoietic toxicity, anemia and neutropenia caused by administration of the anticancer agent.

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