KR102205078B1 - 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, ameliorating or treating diseases caused by side effects of anticancer agents containing a thorny medicinal extract as an active ingredient, and the thorny gourd ( Sicyos angulatus ) extract of the present invention is a reduction in hematopoietic stem cells by administration of an anticancer agent. Remarkably restores survival, increases the expression levels of hematopoietic cytokines (IL-3, IL-6 and IFN-γ), and alleviates hematopoietic toxicity by anticancer drugs of various mechanisms of action in an ex vivo model, It promotes the growth of primary splenic immune cells and NO production, and can alleviate hematopoietic toxicity in an in vivo model. Therefore, the prickly pear extract of the present invention can be used as a pharmaceutical, health functional food, or anticancer adjuvant for the prevention, improvement or treatment of diseases caused by side effects of anticancer drugs.

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 thorny leaf ( Sicyos angulatus ) It relates to a composition for preventing, improving or treating diseases caused by side effects of anticancer drugs containing extract as an active ingredient.

Cancer is the number one cause of death in Korea, and 30% of deaths in their 50s and 60s are dying of cancer. In general, surgical surgery, radiation therapy, and chemotherapy are most commonly used to treat these cancers. Among them, various attempts have been made to develop anticancer drugs as chemotherapy, but until now, anticancer drugs as true treatments have not been developed, and they are only a supplementary treatment or help to prolong life for a short time.

Anticancer drugs used as chemotherapy intervene in the metabolic pathways of cancer cells and act directly with DNA to block DNA replication, transcription, and translation processes, interfere with the synthesis of nucleic acid precursors, and inhibit cell division, thereby causing cytotoxicity to cells. Represents. However, current anticancer drugs do not have specific selectivity for cancer, so they exhibit both therapeutic and toxic effects at the same time, and the toxic effects of anticancer drugs are due to hemocytosis of leukocytes, platelets, red blood cells, etc. due to bone marrow destruction, and hair follicle cell destruction. Alopecia caused by symptoms of alopecia, causes of menstrual impurity and male infertility as a side effect on the ovaries and testes, stomatitis, nausea and vomiting and food dysphagia and digestive problems as a side effect of the destruction of mucous membrane cells of the digestive tract, diarrhea symptoms, kidney toxicity due to tubular necrosis Various side effects such as peripheral neuritis and weakness that occur due to nervous system disorders, vascular disorders such as vascular pain and rash, and discoloration of the skin and nails appear. Accordingly, there is an urgent need to develop a drug that can increase the effect of anticancer treatment while minimizing side effects caused by anticancer drugs.

On the other hand, Sicyos angulatus is a one or two-year-old vine plant belonging to the Cactus family. The stem extends to about 4 to 8 m, grows by wrapping other objects with tendrils, and the leaves running alternately are split into 5 to 7 branches in the shape of a palm. It is a male and female flower that blooms in June-September. The male flower is light green and hangs as a raceme, and the female flower is a head inflorescence and has 1 pistil. Fruits are clustered and covered with hairy thorns. It is a naturalized plant native to North America and naturalized to Korea.

As a prior art related to pharmaceutical use of prickly pear extract, in Korean Patent No. 1802970, a composition for preventing, ameliorating or treating liver disease containing prickly pear extract or a fraction thereof as an active ingredient, and administration of the composition to an individual with suspected liver disease It discloses a method for preventing or treating liver disease, comprising the step of, and a composition for the prevention, improvement or treatment of metabolic diseases comprising a thorn extract or a fraction thereof as an active ingredient in Korean Patent No. 1793145, And it discloses a method for preventing or treating metabolic diseases comprising administering the composition to an individual suspected of metabolic disease, but prevention of diseases caused by side effects of anticancer drugs containing the prickly pear extract of the present invention as an active ingredient, There is no disclosed composition for improvement or treatment.

The present invention is derived from the above requirements, the present invention is prickly leaf ( Sicyos angulatus ) relates to a composition for the prevention, improvement or treatment of diseases caused by side effects of anticancer drugs containing an extract as an active ingredient, and in detail, thorns of the present invention ( Sicyos angulatus ) extract was treated on the bone marrow cells of mice with reduced hematopoietic stem cells by the administration of docetaxel to remarkably restore the survival of hematopoietic stem cells, and hematopoietic-related cytokines (IL-3, IL- 6 and IFN-γ) was confirmed to increase, and it was confirmed in an ex vivo model that hematopoietic toxicity by anticancer drugs of various mechanisms of action was alleviated, and the growth of primary spleen immune cells and NO production were promoted, and in vivo By confirming that hematopoietic toxicity can be alleviated in the model, 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 agents containing an extract of Sicyos angulatus as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diseases caused by side effects of an anticancer agent containing a prickly pear extract as an active ingredient.

In addition, the present invention provides an anticancer adjuvant containing a prickly pear extract as an active ingredient.

The present invention relates to a composition for preventing, ameliorating or treating diseases caused by side effects of anticancer agents containing a thorn extract as an active ingredient, remarkably recovering the decrease in hematopoietic stem cell colonies caused by anticancer agents, as well as hematopoietic cytotoxicity It has the effect of increasing the expression level of kine, alleviates hematopoietic toxicity by anticancer drugs of various mechanisms of action, promotes the growth of primary splenic immune cells and NO production, and has the effect of alleviating hematopoietic toxicity in in vivo models. The prickly pear extract of the present invention can be used as a drug for preventing, improving or treating diseases caused by side effects of anticancer drugs, health functional foods, or anticancer adjuvants.

Figure 1 shows the reduction of bone marrow hematopoietic stem cells of mice by docetaxel treatment and the reduction of the number of hematopoietic stem cell colonies by the docetaxel with docetaxel and hot water extracts of 25, 50 and 100 µg/ml of prickly pear outpost (A); And it is the result of confirming that the hot water extract of Prickly Pear and the ethanol extract of Prickly Pear extract effectively recovers the decrease in the number of hematopoietic stem cell colonies by treatment with the ethanol extract (B) of Prickly Pear. ## indicates that the number of hematopoietic stem cell colony forming units (CFU) in the docetaxel alone-treated group was statistically significantly reduced compared to the control group not treated with docetaxel, p<0.01, *, * * Is docetaxel and prickly pear hot water extract compared to docetaxel alone treatment group; Alternatively, the number of CFU of hematopoietic stem cells was statistically significantly increased in the group treated with docetaxel and prickly pear ethanol extract in combination, * = p<0.05, ** = p<0.01.
Figure 2 shows the reduction of the bone marrow hematopoietic stem cells of the mouse by docetaxel treatment and the reduction of the number of hematopoietic stem cell colonies by the docetaxel, with docetaxel and hot water extract of 25, 50, and 100 µg/ml prickly leaf leaves (A); And it is a result of confirming that the hot-water extract of thorny leaves and the hot-water extract of thorny leaves stems effectively recovers the reduction in the number of hematopoietic stem cell colonies by using a combination treatment with the prickly pear stem hot water extract (B). # Is that the number of hematopoietic stem cell colony forming units (CFU) in the group treated with docetaxel alone compared to the control group not treated with docetaxel (Control) was statistically significantly reduced, p<0.05, *, ** Silver docetaxel and hot water extract of prickly pear leaves compared to the group treated with docetaxel alone; Alternatively, the number of CFU of hematopoietic stem cells was statistically significantly increased in the group treated with docetaxel and prickly pear stem hot water extract, * = p<0.05, ** = p<0.01.
Figure 3 shows the reduction of bone marrow hematopoietic stem cells in mice by docetaxel treatment and the reduction of the number of hematopoietic stem cell colonies by the docetaxel. Docetaxel and 25, 50, and 100 ㎍ / ㎖ of prickly pear hot water extract and its fractions (hexane, Hx; ethyl) Acetate, EtOAc; butanol, BuOH; water, Wa) in combination treatment to confirm that the prickly pear hot water extract, butanol (BuOH) fraction and water (Wa) fraction effectively recover the reduction in the number of hematopoietic stem cell colonies. #### indicates that the number of hematopoietic stem cell colony forming units (CFU) in the docetaxel alone-treated group was statistically significantly decreased compared to the control group not treated with docetaxel, p<0.0001, * *, ***, **** are docetaxel and prickly pear hot water extract compared to docetaxel alone treatment group; Butanol fraction of hot water extract of prickly pear; Alternatively, the number of CFU of hematopoietic stem cells was statistically significantly increased in the group treated with the water fraction of hot water extract of Prickly Pear; ** for p<0.01, *** for p<0.001, **** for p <0.0001.
4 is a reduction in the number of CFU of hematopoietic stem cells by docetaxel treatment in a culture medium that promotes the growth and differentiation of hematopoietic stem cells of a specific lineage and when the docetaxel and the prickly pear hot water extract of the present invention are co-treated, This is the result of confirming that it can effectively recover the decrease in the number of CFUs in hematopoietic stem cells. GF M3434 medium promotes the growth and differentiation of hematopoietic stem cells of all lines, and GF M3534 medium promotes the growth and differentiation of hematopoietic stem cells (monocytes, eosinophils, and neutrophils) of the myeloid lineage. , SF M3436 medium is a medium that promotes the growth and differentiation of hematopoietic stem cells (erythrocytes) of the erythroid family. ###, #### indicates that the number of CFU of hematopoietic stem cells in the group treated with docetaxel alone was statistically significantly reduced compared to the control group not treated with docetaxel, ### being p<0.001, ### # Is p<0.0001. *, **, ***, **** indicates that the number of CFU of hematopoietic stem cells in the group treated with docetaxel and prickly pear hot water extract in combination compared to the group treated with docetaxel alone was statistically significantly increased, and * is p<0.05 , ** is p<0.01, *** is p<0.001, **** is p<0.0001.
5 is a primary splenocyte or mouse macrophage, Raw264.7 cells, when treated with hot water extract of the present invention, hematopoietic and immune function related cytokines (IL-3, IL-6 , IFN-γ and TNF-α) was confirmed that the concentration-dependent increase in the expression. **, *** indicates that the cytokine expression was statistically significantly increased in the group treated with Prickly Pear Hot Water Extract compared to the control group (vehicle) not treated with anything, ** indicates p<0.01, *** p<0.001. ConA is a positive control in an experiment using splenic immune cells, Concanavalin A, and LPS is a positive control used in an experiment using Raw264.7 cells, and is a lipopolysaccharide.
6 is a result of confirming the effect of alleviating hematopoietic toxicity induced by anticancer agents of different mechanisms of action of the prickly pear hot water extract of the present invention. #, ##, #### were treated alone with anticancer drugs such as actinomycin D (A), doxorubicin (B), paclitaxel (C), or mitomycin (D) compared to the control group (Control) not treated with anticancer drugs. The number of CFU of hematopoietic stem cells in one group was statistically significantly decreased, with # being p<0.05, ## being p<0.01, #### being p<0.0001. *, **, ***, **** are docetaxel and prickly pear compared to the group treated with an anticancer agent such as actinomycin D (A), doxorubicin (B), paclitaxel (C) or mitomycin (D) alone. It was found that the number of hematopoietic stem cells CFU of the group treated with hot water extract increased statistically significantly, * for p<0.05, ** for p<0.01, *** for p<0.001, **** for p< Is 0.0001.
Figure 7 is the growth of primary splenocytes (A) and production of nitric oxide (NO), which is an immune-related intracellular signaling material from splenocytes, in which the prickly pear hot water extract of the present invention (B) This is the result of confirming that it can promote *, **, *** indicates that the cell growth and nitric oxide production in the group treated with hot water extract of Prickly Pear compared to the negative control group (vehicle) increased statistically significantly, * indicates p<0.05, ** p<0.01, *** is p<0.001.
8 is an animal model in which hematopoietic toxicity was induced by repeatedly administering docetaxel intraperitoneally, by daily oral administration of the prickly pear hot water extract of the present invention, weight loss (A) of the experimental animal (mouse) due to anticancer drug toxicity (A), peripheral blood The reduction in leukocyte count (B), the absolute weight of the spleen (C) and thymus gland (D), which are major immune organs, or the relative weight of the body weight (spleen index, SI; thymus index, TI) can be effectively restored. This is the result of confirmation. G-CSF is a positive control group, and is a granulocyte-colony stimulating factor formulation used clinically as a therapeutic agent for neutropenia. # Indicates that the hematopoietic toxicity model group (Model, M) treated with docetaxel alone compared to the normal control group (sham) statistically significantly decreased the absolute and relative weight of the thymus, p<0.05. * Indicates that the absolute and relative weight of the thymus were statistically significantly increased compared to the hematopoietic toxicity model group (Model, M) in the experimental group treated with hot water extract of Prickly Pear, p<0.05.
9 shows the main immune organs, spleen, thymus, and bone marrow when daily oral administration of the prickly pear hot water extract of the present invention in an animal model (mouse) in which hematopoietic toxicity was induced by repeatedly intraperitoneal administration of docetaxel. This is the result of confirming that it can effectively protect against tissue damage caused by administration of anticancer drugs in the microenvironment of (bone marrow) with the hematoxylin-eosin (H&E) tissue staining technique. R and W refer to the red pulp and white pulp observed in the spleen, and C and M refer to the cortex and medulla observed in the thymus. G-CSF is a positive control group, and is a granulocyte-colony stimulating factor formulation used clinically as a therapeutic agent for neutropenia.

The present invention is thorny leaf ( Sicyos angulatus ) It relates to a pharmaceutical composition for the prevention or treatment of diseases caused by side effects of anticancer drugs containing the extract as an active ingredient.

The prickly pear extract may be prepared by a method including the following steps, but is not limited thereto.

(1) extracting by adding an extraction solvent to the thorny leaf;

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

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

The extraction solvent in step (1) is preferably water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof, but is not limited thereto.

In the above manufacturing method, extraction of the prickly pear extract may use 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 the dried thorny leaf, more preferably 3 to 10 times. The extraction temperature is preferably 20 ~ 50 ℃, but is not limited thereto. Further, the extraction time is preferably 10 to 100 hours, more preferably 24 to 96 hours, and most preferably 72 hours, but is not limited thereto. In the above method, the vacuum concentration in step (3) is preferably a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

The prickly pear extract may be extracted using an outpost, leaf, or stem, and is not particularly limited.

The cancer is preferably any one cancer 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 blood cancer. However, the present invention is not limited thereto, and any cancer that can be diagnosed is not limited thereto.

The anticancer agents include antitumor antibiotics, topoisomerase inhibitors, platinum-based anticancer agents, taxane-based anticancer agents, or receptor tyrosine kinase inhibitors, It is not limited thereto, and includes all anticancer drugs that can be used clinically, pharmacologically, and biomedically.

The antitumor antibiotics include actinomycin D, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, and rubicin. ), mitomycin, mitomycin-C, and at least one selected from mitomycin; The topoisomerase inhibitor is irinotecan, camptothecin, novobiocin, epirubicin, dactinomycin, and amsacrine. , Teniposide (teniposide) and one or more selected from 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 preferably at least one selected from gefitinib, erlotinib, and afatinib, but is not limited thereto.

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

The term'hematopoietic toxicity', as used herein, refers to a hematopoietic dysfunction, in particular a hematopoietic disorder or damage or disorder of an organ or cell of the hematopoietic system, which results in a dysfunction of red blood cells or immune system cells, such as white blood cells. Preferably, hematopoietic or immune system function in the bone marrow is affected by hematopoietic toxicity. Therefore, the hematopoietic toxicity generally preferably includes myelotoxicity, anemia and neutropenia, and more preferably, it is specifically induced by the administration of a chemical compound or drug, or a result of such administration.

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

The composition of the present invention may be in various oral or parenteral dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives are included. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

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

The composition according to the present 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 the effective dose level is the type of disease, severity, and drug activity of the patient. , Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

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

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

The extraction solvent of the prickly pear extract is preferably water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof, but is not limited thereto. The anticancer agents include, but are not limited to, antitumor antibiotics, phase isomerase inhibitors, platinum anticancer agents, taxane anticancer agents or receptor tyrosine kinase inhibitors, and include all anticancer agents that can be used clinically, pharmacy, and biomedically. The disease caused by side effects of the anticancer agent is characterized in that at least one selected from the group consisting of hematopoietic toxicity, anemia, and neutropenia.

The health functional food composition of the present invention may be added as it is, or used with other foods or food ingredients, and may be appropriately used according to a conventional method. There is no particular limitation on the kind of the health functional food. Examples of foods to which the prickly meal extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the usual sense are included. Health beverages comprising the composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like ordinary beverages. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio 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.

In addition to the active ingredients, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives. , Glycerin, alcohol, carbonated beverages, etc. may further contain. In addition, it may further contain pulp for the manufacture of fruit juice or vegetable beverage. These ingredients may be used independently or in combination. Although the proportion of these additives is not very important, it is generally selected from 0.01 to 2 parts by weight based on 100 parts by weight of the composition of the present invention.

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

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

The anticancer adjuvant may contain one or more active ingredients exhibiting the same or similar function in addition to the prickly pear extract. The anticancer adjuvant can be administered orally or parenterally at the time of clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or chest It can be administered by intramuscular injection, and can be used in the form of a general pharmaceutical formulation.

The anticancer adjuvant may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using 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, and it is preferable to administer once to several times a day, but the weight, age, sex, health status of the patient, The range varies according to diet, administration time, administration method, excretion rate, and severity of disease. The anticancer adjuvant of the present invention can be administered in various parenteral formulations at the time of actual clinical administration.When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It is prepared. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example One. Thorn Preparation of extract

(1) Preparation of hot water extract of prickly pear

In order to prepare the active ingredient of the present invention, prickly pear extract, prickly leaf was collected from the wild. After drying the collected prickly leaf in a shade where there is no light, 100 g of prickly leaf outpost in the shade was pulverized, and 2 liters of water and Put them together in a round flask and mix. A water bath connected to a reflux extraction device with a cooling tube was heated, and extraction was repeated twice for 2 hours each time. The extracted prickly pear hot water extract was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (GAST). Using a rotary evaporator (EYELA), the filtered hot water extract of prickly pear was concentrated under reduced pressure, the concentrated hot water extract of prickly pear was lyophilized, and homogenized with a mortar to obtain a hot water extract of prickly pear. This was put in a sealed plastic container and stored in a cold storage at 4°C until the experiment. In the present invention, the hot water extract of prickly pear refers to the extract using prickly pear outpost.

In addition, the prickly leaf or stem was extracted with hot water in the same manner as the prickly leaf outpost to obtain the prickly leaf hot-water extract and the prickly leaf stem hot-water extract, and stored in a sealed plastic container before the experiment and stored in a low temperature storage at 4°C.

(2) Preparation of ethanol extract of thorn meal

After mixing 10.0 g of crushed prickly pear outpost and 70% (v/v) ethanol in an Erlenmeyer flask, ultrasonic extraction (Branson 5210) was repeatedly extracted twice for a total of 1 hour. The extracted ethanol extract of prickly pear was filtered under reduced pressure using a paper filter paper (Whatman No. 2) and a vacuum pump (GAST). The filtered prickly pear ethanol extract was concentrated under reduced pressure using a rotary evaporator (EYELA), and the concentrated prickly pear ethanol extract was vacuum-dried (vacuum dry oven, WiseVen) or freeze-dried (Freeze Dryer, IlshinBioBase). Ethanol extract of gourd was obtained. The obtained ethanol extract of prickly pear was put in a sealed plastic container and stored in a refrigerator at 4°C until the experiment. In the present invention, the ethanol extract of Prickly Pear refers to the extract using Prickly Pear outpost.

In addition, the leaves or stems of thorns were ethanol-extracted in the same manner as in the above thorns meal to obtain ethanol extracts of thorns leaves and stalks of thorns, and stored in a sealed plastic container until the experiment and stored in a storage at 4°C.

(3) Preparation of solvent fraction of hot water extract of thorny leaf

① Preparation of thorny leaf hexane fraction (Hx)

300 g of hot water extract of prickly pear 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 interfacial layer, and the hexane layer was separated and concentrated under reduced pressure at 0.07 MPa to obtain a hexane fraction.

② Preparation of thorny leaf ethyl acetate fraction (EtOAc)

After the hexane fractionation, 1.5 L of ethyl acetate was added to the remaining fraction to perform solvent fractionation twice. The ethyl acetate fraction was obtained by concentrating under reduced pressure at a vacuum degree of 0.07 MPa.

③ Preparation of thorny leaf butanol fraction (BuOH)

After the ethyl acetate fractionation, 1.5 L of water saturated butanol was added to the remaining fraction to perform solvent fractionation twice. The butanol fraction was obtained by concentrating under reduced pressure at 0.07 MPa in vacuum.

④ Preparation of water fraction (Wa)

After the butanol fractionation, the remaining fraction was concentrated under reduced pressure of 80 to 90% at a vacuum degree of 0.07 MPa, and then freeze-dried to obtain.

Example 2. Confirmation of hematopoietic toxicity of docetaxel using mouse bone marrow cells and alleviation effect of hematopoietic toxicity of prickly pear hot water extract ( ex vivo )

Ex vivo experiments were performed to determine the effect of docetaxel anticancer drug, known to have strong hematopoietic toxicity side effects, on the growth and differentiation of hematopoietic stem cells in mouse bone marrow cells, although it is currently frequently administered to solid cancer patients in tumor clinical trials.

Specifically, after separating bone marrow nucleated cells from the mouse femur, 20 nM of docetaxel was added to the mouse-derived recombinant stem cell factor (recombinant murine stem cell factor, rm stem cell factor), interleukin-3 (rm IL-3), human-derived recombinant IL-6 (rh IL-6) and erythropoietin (rh erythropoietin) containing medocult GF M3434 (methocult GF M3434) culture medium containing the bone marrow cells for 7-10 days Thus, growth and differentiation of hematopoietic stem cells were induced. 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, it was confirmed that mouse bone marrow cells were treated with 20 nM docetaxel and prickly pear extract at concentrations of 25, 50 and 100 µg/ml in combination to confirm that the prickly pear extract could alleviate hematopoietic toxicity induced by anticancer agents. The difference in the effect according to the solvent (70% ethanol and water) used for extraction of prickly pear and the part of prickly pear used for extraction (outpost, leaf and stem) was compared.

As a result, the number of mouse hematopoietic stem cells CFU was statistically significantly reduced by docetaxel, and the hot water extract and ethanol extract of prickly pear can alleviate the decrease in the number of CFU of hematopoietic stem cells by docetaxel, but more visible than the ethanol extract of prickly pear. It was confirmed that the extract of Pak hot water was more effective (FIG. 1).

In addition, it was confirmed that the hot-water extract of prickly pear leaves and the hot-water extract of prickly pear stem had an effect of alleviating hematopoietic toxicity by anticancer agents at a level similar to that of the prickly pear outpost extract (FIG. 2).

Example 3. In mouse bone marrow cells Thorn Hydrothermal Extract and its Fraction Hematopoietic toxicity Check the relief effect ( ex vivo )

GF M3434 for four solvent fractions (hexane (Hx), ethyl acetate (EtOAc), butanol (BuOH) and water (Wa) fractions) derived from the hot-water extract of prickly pear and hot-water extract of prickly pear, the active substances of the present invention. The effect of alleviating hematopoietic toxicity induced by docetaxel was compared and evaluated using the culture medium (FIG. 3). Docetaxel and prickly pear hot water extract (50, 100, 200 µg/ml), or a fraction thereof (1, 10, 100 µg/ml) were treated in combination, and then the CFU number was counted. The hexane and ethyl acetate fractions showed strong cytotoxicity as the concentration increased, but the butanol and water fractions showed a hematopoietic toxicity similar to the hot water extract of prickly pear. In the case of the butanol and water fractions, the activity of the butanol and water fractions was more effective than that of the prickly pear hot water extract, because the activity of the butanol and water fractions was similar to the cytoprotective effect found at the 50 ㎍/mL concentration of hot water extract even at a low concentration of 1 μg/mL. It was determined to be more excellent in (Fig. 3).

Example 4. Various Differentiation medium Used Thorn Hydrothermal Induced anticancer drug of extract Hematopoietic toxicity Relaxation Confirm( ex vivo )

In the hematopoietic toxicity test system induced by docetaxel anticancer drug using a culture medium that promotes the growth and differentiation of hematopoietic stem cells of a specific lineage, the alleviating effect of the prickly pear hot water extract was confirmed. The culture medium used for the hematopoietic stem cell CFU test and the combination of growth factors defining the characteristics of each medium and the cell lines forming colonies are shown in Table 1 below.

Culture medium used in the CFU test, growth factors constituting the medium, and different colony types 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 Myeloid family CFU-GM, CFU-G, CFU-M SF M3436 cytokines including rh EPO BFU-E of the Erythroid family

As a result of confirming whether the hematopoietic toxicity alleviation effect of the prickly pear hot water extract is limited to the growth and differentiation of hematopoietic stem cells of a specific lineage, the prickly pear hot water extract is not only the total hematopoietic stem cells (Fig. 4A) and myeloid lineage (Fig. It was confirmed that the hematopoietic toxicity alleviation effect was also found in the differentiation of hematopoietic stem cells of the (erythroid) line (FIG. 4C).

These results mean that the prickly pear 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 changes in the expression of hematopoietic and immune-related cytokines by thorny leaf hot water extract through culture of primary splenocytes and mouse macrophages (Raw264.7)

Primary splenocytes isolated from mouse spleen and Raw264.7 cells, which are mouse macrophages, were cultured by selecting an appropriate culture medium. After 72 hours after treating cells with the active substance of the present invention, the extract of prickly pear at a concentration of 1-100㎍/㎖, the amount of expression of cytokines related to hematopoietic and immune function in the spleen immune cells and Raw264.7 cells was commercially determined. The cytokine concentration in the medium was measured using an Enzyme Linked ImmunoSorbent Assay (ELISA) kit available as an enzyme. In this 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 used as positive controls. It was used in the experiment.

As a result, prickly pear hot water extract increases the expression of cytokines (IL-3, IL-6) that promote differentiation from primary splenic immune cells to myeloid cells in the upper stage of the hematopoiesis process, and at the same time In splenic immune cells and Raw264.7 macrophages, an increase in cytokines (INF-γ and TNF-α) that improves immune response to viruses and anti-tumor functions was confirmed (FIG. 5).

Example 6. Confirmation of hematopoietic toxicity alleviation efficacy of Prickly pear hot water extract in a hematopoietic toxicity model induced by anticancer agents of various mechanisms of action ( ex vivo )

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

As a result, the prickly pear hot water extract inhibits actinomycin D (DNA binding, Fig. 6A), doxorubicin (topoisomerase) activity, which are anticancer agents of different mechanisms of action, Fig. 6B), and paclitaxel. It was confirmed that the decrease in the number of CFU of hematopoietic stem cells induced by (paclitaxel, inhibition of cell division, Fig. 6C) and mitomycin C (mitomycin C, DNA cross-linking, Fig. 6D) can be effectively restored.

Example 7. The effect of hot water extract of Prickly Pear on the growth of primary splenocytes and the production of immune function-regulating cell signaling substances (NO) ( in vitro )

When the primary splenocytes of mice were treated with hot water extract at a concentration of 0 to 200 μg/ml, the growth of splenic immune cells and the regulation of immune function were produced by nitric oxide (NO). Confirmation of production. The growth and cell viability of splenic immune cells were determined using an automatic cell counter (ADAM-MC), and the concentration of NO in the culture medium was determined using a Thermo Scientific NO measurement kit.

As a result, it was confirmed that the hot water extract of prickly pear can promote the growth of spleen immune cells and the production of NO in a concentration-dependent manner (FIG. 7).

Example 8. Anticancer drug-induced Hematopoietic toxicity In animal models Thorn Hydrothermal Extract of Hematopoietic toxicity Check the relief effect ( in vivo )

Ex vivo In order to confirm that the hematopoietic toxicity alleviation effect of the prickly pear hot water extract identified in the test system is effective in the in vivo animal model, hematopoietic toxicity when the prickly pear hot water extract is administered orally in a mouse animal model that induces hematopoietic toxicity by repeatedly injecting an anticancer agent. Check if this is alleviated.

Specifically, C57BL/6 mice were purchased and subjected to an acclimatization period for one week, and then randomly separated into 6 groups using an online randomization program (www.randomizer.org) (experiment day 0, groups 1-6). On the first day of the experiment, a 26 gauge injection needle and a capillary tube coated with 10% (w/v) ethylenediaminetetraacetic acid dipotassium salt (K ₂ -EDTA) was used from the saphenous vein of the hind limb of each group. After collecting the peripheral blood, the white blood cell (WBC) level was measured using a hemocytometer (HemaVet) to confirm the baseline value of white blood cells before inducing hematopoietic toxicity. On the 2nd to 4th day of the experiment (for 3 days), Group 1 was given a 0.9% (w/v) saline solution containing 5% (v/v) ethanol and 2% (v/v) polysorbate 80. Intraperitoneal administration, groups 2 to 6, dissolved docetaxel in 0.9% saline containing 5% (v/v) ethanol and 2% (v/v) polysorbate 80 in order to induce hematopoietic toxicity. After that, intraperitoneal administration was performed daily at a dose of 30mg/kg/d. Lastly, for 7 days from the day after administration of docetaxel (experiment day 5 to 11), mice belonging to group 1 (normal control group, sham) and group 2 (hematopoietic toxicity model group, Model, M) were treated with sterile distilled water. To mice belonging to groups 3 to 5, hot water extract of prickly pear dissolved in sterile water was orally administered at 30, 100, and 300 mg/kg/d doses, respectively. Group 6 was a positive control, and human recombinant G-CSF (Granulocyte-Colony Stimulating Factor) was injected subcutaneously to the back of the neck at a dose of 61.5 μg/kg on the 5th and 7th days of the experiment. All dosage and administration volumes were determined based on the weight of the experimental animals measured daily. Peripheral blood was collected from the replicated vein every 2-3 days from the day after the administration of docetaxel was terminated, and then the leukocyte count was checked using a hemocytometer.

As a result, in the model group (Model, Group 2), a rapid weight loss and a decrease in leukocyte count were observed due to the administration of docetaxel, and in the group administered the active substance, prickly pear hot water extract (groups 3 to 5), dose-dependently decreased. It was confirmed that the body weight was recovered (Fig. 8A). In addition, it was confirmed that the number of leukocytes increased in the group to which the prickly pear hot water extract was administered as compared to the model group on the 9th and 12th days of the experiment (FIG. 8B). After the end of the experiment (on the 12th day of the experiment), mice belonging to each group were sacrificed through inhalation of carbon dioxide (CO ₂ ), and the spleen and thymus, the main immune organs, were excised, and then the absolute organ weight of each organ. ) And the absolute weight divided by the body weight of each experimental animal and the relative weight (spleen index, SI; thymus index, TI) was measured.

As a result, it was confirmed that both the absolute and relative weights of the spleen (FIG. 8C) and thymus (FIG. 8D) increased dose-dependently compared to the model group in the group to which the hot water extract was administered. When G-CSF was administered as a positive control group, the white blood cell count increased compared to the model group on the 9th day of the experiment, but 3 days after stopping G-CSF (the 12th day of the experiment), the white blood cell count decreased to a level similar to that of the model group. It could be observed (Fig. 8B). In addition, in the case of the G-CSF administration group, a significant difference could not be observed in the weight measurement values of the spleen and thymus, which are immune organs, compared to the model group (FIGS. 8C and 8D ).

The effect of alleviating hematopoietic toxicity by hot water extract of prickly pear was observed through hematoxylin-Eosin (H&E) staining in the tissues of the spleen, thymus and bone marrow, which are major immune organs. Specifically, after the end of the experiment (on the 12th day of the experiment), mice belonging to each group were sacrificed through carbon monoxide inhalation, and the spleen, thymus, and hind femur were collected and then immersed in formalin fixative to fix the tissue. The fixed tissue was observed through a tissue microscope after paraffin embedding, tissue excision, and H&E staining.

As a result, due to the administration of docetaxel, necrosis and bleeding of cells in the spleen tissue of the model group, atrophy of the tissue in the thymus tissue, and the phenomenon that the boundary between the cortex and the medulla becomes unclear. In the bone marrow, hypocellularity was observed due to the decrease in hematopoietic cells. It was confirmed that such immune tissue damage according to the administration of an anticancer drug was significantly reduced when the extract of hot water thorn was administered orally (FIG. 9).

Claims (11)

Prickly pear ( Sicyos angulatus ) A pharmaceutical composition for the prevention or treatment of hematopoietic toxicity or neutropenia due to side effects of anticancer drugs containing water or ethanol extract as an active ingredient. delete The method of claim 1, wherein the anticancer agent is antitumor antibiotics, topoisomerase inhibitor, platinum anticancer agent, taxane anticancer agent, and receptor tyrosine kinase inhibitor. inhibitor), a pharmaceutical composition for the prevention or treatment of hematopoietic toxicity or neutropenia due to side effects of anticancer agents, characterized in that at least one selected from. The method of claim 3, wherein the antitumor antibiotics are actinomycin D, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, and Is at least one selected from idarubicin, mitomycin, mitomycin-C, and mitomycin; The topoisomerase inhibitor is irinotecan, camptothecin, novobiocin, epirubicin, dactinomycin, and amsacrine. , Teniposide (teniposide) and one or more selected from 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 one or more selected from gefitinib, erlotinib, and afatinib. Pharmaceutical composition for treatment. delete Prickly pear ( Sicyos angulatus ) health functional food composition for preventing or improving hematopoietic toxicity or neutropenia caused by side effects of anticancer drugs containing water or ethanol extract as an active ingredient. The method of claim 6, wherein the anticancer agent is antitumor antibiotics, topoisomerase inhibitors, platinum anticancer agents, taxane anticancer agents or receptor tyrosine kinase inhibitors. inhibitor), a health functional food composition for preventing or improving hematopoietic toxicity or neutropenia caused by side effects of anticancer drugs. The method of claim 7, wherein the antitumor antibiotics are actinomycin D, bleomycin sulfate, daunomycin, daunorubicin, doxorubicin, and Is at least one selected from idarubicin, mitomycin, mitomycin-C, and mitomycin; The topoisomerase inhibitor is irinotecan, camptothecin, novobiocin, epirubicin, dactinomycin, and amsacrine. , Teniposide (teniposide) and one or more selected from 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 one or more selected from gefitinib, erlotinib, and afatinib. Health functional food composition for improvement. delete Prickly pear ( Sicyos angulatus ) An anticancer adjuvant that contains water or ethanol extract as an active ingredient, and improves diseases caused by side effects of at least one anticancer agent selected from hematopoietic toxicity and neutropenia caused by administration of an anticancer agent. delete

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