KR20220085532A - Composition comprising Annona muricata leaf polysaccharides extracts as an active ingredient for attenuating toxicity of anticancer agent, and anti-cancer adjuvant - Google Patents

Abstract

The composition for reducing anticancer drug toxicity according to an embodiment of the present invention includes graviola leaf extract as an active ingredient. By using the anticancer agent toxicity reduction composition and anticancer adjuvant comprising the graviola leaf extract of the present invention as an active ingredient, the toxicity of the anticancer agent is reduced to prevent various side effects caused by the toxicity of the anticancer agent in the chemical treatment of cancer or There is a minimization effect.

Description

Composition comprising Annona muricata leaf polysaccharides extracts as an active ingredient for attenuating toxicity of anticancer agent, and anti-cancer adjuvant

The present invention relates to a composition for reducing the toxicity of an anticancer drug and an anticancer adjuvant comprising an extract of graviola leaf (Annona muricata leaf) as an active ingredient.

Cancer is a disease that causes about 7.6 million deaths per year worldwide, accounting for 13% of all deaths. According to the 2009 Statistical Yearbook of Causes of Death of the National Statistical Office, cancer-related deaths in Korea accounted for 28.3% of the total deaths, taking the first place among all causes of death. Currently, various methods such as surgery, radiation therapy, and gene therapy are used as a method of treating cancer, but one of the most used treatment methods is chemotherapy in which an anticancer agent is administered.

Chemotherapy is a systemic treatment. In most cases, when chemotherapy is administered by injection or orally, it spreads throughout the body along the bloodstream. Therefore, it is a treatment that acts on micrometastasis spread throughout the body rather than a local effect. Therefore, there are many systemic side effects and the degree is very severe compared to surgery or radiation therapy. Chemotherapy, but most anticancer drugs do not distinguish between normal cells and cancer cells, to selectively act on cancer cells by using the difference in drug sensitivity between normal cells and cancer cells, thus exhibiting dose-limiting toxicity. There is that problem.

Cisplatin, a representative anticancer drug, is widely used in clinical practice as a chemotherapeutic agent for the treatment of ovarian cancer, bladder cancer, lung cancer, head and neck cancer, and testicular cancer. Cisplatin is known to show anticancer effects by generating reactive oxygen species to attack cancer cells, inducing inter-intrastrand cross-linking of DNA in cancer cells, and formation of DNA adducts.

However, it is known that side effects such as hearing loss, neurotoxicity, and renal toxicity occur at more than a limited amount of the drug during the course of treatment, and liver toxicity is also frequently observed when high concentrations of cisplatin are administered.

These side effects by cisplatin are closely related to increased lipid peroxidation due to reactive oxygen species produced by cisplatin, inhibition of antioxidant enzyme activity in tissues, depletion of glutathione and disruption of intracellular calcium homeostasis. have.

Therefore, in order to reduce the side effects caused by the anticancer drug treatment and increase the treatment efficiency, it is necessary to develop an inhibitor capable of alleviating the toxicity of the anticancer drug administration.

Accordingly, the present inventors completed the present invention by confirming that the Graviola leaf extract alleviates the toxicity of anticancer drugs as a result of research on substances capable of alleviating the toxicity of anticancer drugs.

Republic of Korea Patent No. 10-1398076 (Registered on May 15, 2014)

The problem to be solved by the present invention is to provide a composition for reducing the toxicity of anticancer drugs comprising a graviola leaf extract as an active ingredient.

Another object is to provide an anticancer adjuvant comprising a graviola leaf extract as an active ingredient.

The composition for reducing anticancer drug toxicity according to an embodiment of the present invention includes graviola leaf extract as an active ingredient.

The anticancer agent may be cisplatin.

The graviola leaf extract may contain crude polysaccharide.

The extraction method of the graviola leaf extract comprises the steps of: hot air drying the graviola leaves; pulverizing the dried graviola leaves to obtain a powder; mixing distilled water with the obtained powder and hot water extraction to prepare an extract; Filtering the extract to prepare a filtrate, adding ethanol to the filtrate and centrifuging to produce a precipitate, and separating and drying the precipitate to obtain crude polysaccharide.

A health functional food composition according to another embodiment of the present invention includes graviola leaf extract as an active ingredient.

The anticancer agent may be cisplatin.

The graviola leaf extract may contain crude polysaccharide.

The anticancer adjuvant according to another embodiment of the present invention includes graviola leaf extract as an active ingredient.

By using the anticancer agent toxicity reduction composition and anticancer adjuvant comprising the graviola leaf extract of the present invention as an active ingredient, the toxicity of the anticancer agent is reduced to prevent various side effects caused by the toxicity of the anticancer agent in the chemical treatment of cancer or There is a minimization effect.

1 shows the cytotoxic effect on crude polysaccharides (Annona muricata leaf polysaccharides extracts, ALPS) of graviola leaf extract in Raw264.7 cells.
Figure 2 shows the measurement of cell viability after co-treatment of lung cancer cells A549, H460 and macrophage Raw264.7 cells with cisplatin and ALPS by concentration.
Figure 3 shows the effect of ALPS on Cisplatin-treated apoptosis in Raw264.7 cells (Raw264.7 cells were treated with ALPS 31.25 and 62.5 μg/ml in the presence or absence of 15 μM Cisplatin for 24 hours, Annexin V/ Measured by flow cytometry using PI staining.Resuspended cells were washed and immediately analyzed by flow cytometry using FACS VerseTM.Flow cytometry histograms are presented as mean ± SD of three independent experiments).
FIG. 4 shows macrophage Raw264.7 cells treated with ATLS at concentrations of 31.25 and 62.5 μg/ml, followed by combined treatment with 15 μM of Cisplatin, Pro-apoptotic protein Bax, Anti-apoptotic protein Bcl-2, and apoptosis signaling related The expression levels of the proteins cytochrome c , cleaved caspase-3, -8, -9 and PARP were analyzed.

Hereinafter, the present invention will be described in more detail.

Graviola (Annona muricata) of the present invention is generally known as soursop, and is a species widely used in countries with a tropical climate. It belongs to Magnoliales: Annonaceae and includes about 2,300 species. 130 genera are widely distributed in Central America, Mexico, Cuba and Brazil. The bark, seeds, and leaves have been consumed as traditional medicines since ancient times. In particular, in Africa, they are effective in improving headaches, hypertension, cystitis, diabetes, liver diseases, asthma, inflammation, tumors and skin diseases. In addition, it exhibits anticancer activity against graviola leaf extract, thereby inhibiting the growth of cancer cells such as colorectal cancer, liver cancer, and stomach cancer. In addition, graviola leaf and seed extracts contain large amounts of bioactive substances such as flavonoids, polyphenols, terpenoids, tannins, and stearic acid, which are effective in antioxidant and anti-inflammatory activity.

The composition for reducing anticancer drug toxicity according to an embodiment of the present invention includes graviola leaf extract as an active ingredient.

The extraction method of the graviola leaf extract is the extraction method of the graviola leaf extract, hot air drying the graviola leaves, grinding the dried graviola leaves to obtain a powder, and mixing distilled water with the obtained powder and Preparing an extract by hot water extraction, filtering the extract to prepare a filtrate, adding ethanol to the filtrate and centrifuging to produce a precipitate, and separating and drying the precipitate to obtain crude polysaccharide steps are provided.

The anticancer agent of the present invention is a generic term for drugs that act on various metabolic pathways of cancer cells to exhibit cytotoxicity or cytostatic effect on cancer cells. It can be classified into metabolic antagonists, plant alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones, and other drugs.

The anticancer agent is oxaliplatin, imatinib, docetaxel, pemetrexed, gefitinib, tegafur, capecitabine, erlotidib, doxyfluridine, paclitaxel, interferon alpha, gemcitabine, fludarabine, irinotecan, carboplatin, cisplatin, taxotier, doxorubicin, epirubicin, 5-fluorouracil, UFT, tamoxifen, goserelin, herceptin, anti-CD20 antibody, leuprolide (Lupron) and flutamide; , preferably cisplatin.

The cisplatin (Cisplatin, Cis-dichlorodiammineplatinum) is a representative anticancer agent and is widely used in clinical practice as a chemotherapeutic agent for the treatment of ovarian cancer, bladder cancer, lung cancer, head and neck cancer, testicular cancer, and the like. The cisplatin is known to exhibit anticancer effects by generating reactive oxygen species to attack cancer cells, inducing inter-intrastrand cross-linking of DNA in cancer cells, and formation of DNA adducts. It is known that side effects such as hearing loss, neurotoxicity, and renal toxicity occur at more than a limited amount of the drug, and liver toxicity is also frequently observed when high concentrations of cisplatin are administered.

The anticancer drugs have various intracellular targets depending on the drug. They block the DNA replication, transcription, and translation processes of cells or interfere with the action of proteins important for cell survival. through cell death. Since the metabolic pathway of these anticancer drugs is not specific to cancer cells and is the same for normal cells, damage to normal tissues, that is, toxicity, is inevitable when anticancer drugs are administered.

The toxicity of the anticancer agent in the present invention may be renal toxicity, hepatotoxicity, neurotoxicity, hematological toxicity, gastrointestinal toxicity, and lung toxicity.

In the present invention, the anticancer agent corresponds to any anticancer agent that has cancer suppression and therapeutic efficacy, and the cancer is not particularly limited in its type, but preferably testicular cancer, bladder cancer, prostate cancer, ovarian cancer, breast cancer, colorectal cancer, It may be any one selected from the group consisting of head and neck cancer, lung cancer, esophageal cancer, stomach cancer and cervical cancer.

The composition of the present invention has excellent efficacy in reducing the toxicity of anticancer agents in normal cells.

Accordingly, the present invention provides an anticancer adjuvant comprising a graviola leaf extract as an active ingredient.

The anticancer adjuvant refers to an agent that reduces the side effects of an anticancer agent or enhances the therapeutic effect of an anticancer agent. The anticancer adjuvant of the present invention is characterized by comprising graviola leaf extract as an active ingredient, and has excellent efficacy in reducing the toxicity of anticancer drugs.

The composition of the present invention may be a pharmaceutical composition comprising, as an active ingredient, a graviola leaf extract having inhibitory activity against the toxicity of an anticancer agent, and may further include a commonly used pharmaceutically acceptable carrier, diluent or excipient. have.

In addition, the dosage form of the composition may be used in the form of an acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds or in an appropriate group.

The composition of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the graviola leaf extract as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the kkujippong extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

In addition, the composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. In order to formulate a formulation for parenteral administration, the graviola leaf extract of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, and it is formulated as a unit dosage form in an ampoule or vial. The dosage of the graviola leaf extract, which is the active ingredient of the composition of the present invention, is not particularly limited, but it varies depending on the type, dosage, and period of administration of the anticancer agent, and it is preferred that about 1 to 500 times the weight of the anticancer agent is administered compared to the total weight of the anticancer agent. and more preferably about 1 to 200 times by weight. The composition of the present invention may be administered alone before or after administration of an anticancer agent, or may be mixed with an anticancer agent and administered together as an anticancer agent composition.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art.

In addition, the composition of the present invention may be a food composition comprising, as an active ingredient, a graviola leaf extract having inhibitory activity against the toxicity of an anticancer agent, and specifically may be a health functional food composition.

The function is to exhibit the effect of reducing the toxicity of the anticancer agent.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, and there is no particular limitation on the type of food to which the graviola leaf extract of the present invention can be added. For example, various beverages, chewing gum, tea, vitamin complexes, and health supplements are available.

In addition to the graviola leaf extract, other ingredients may be added to the food composition, and the type thereof is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the extract of the present invention or a fraction thereof in the preparation of food may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for health and hygiene purposes or for health control, the above amount may be used below the above range.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

<Materials and Methods>

1-1. Graviola Leaf Polysaccharide Extraction

Graviola leaves were provided and used by Todam (Cheonan, Korea). After drying graviola leaves in hot air at 60 °C for 24 hours, pulverize them with an experimental grinder (NSG-1002SS, Hanil, Seoul, Korea). extracted. After filtering the extract with filter paper (No 4, Whatman, Kent, UK), 70% ethanol was added to the filtrate, left at 4 ° C. for 12 hours, and centrifuged (3,200 rpm, 20 min) to immerse polysaccharide (Annona) muricata leaf polysaccharides extracts (ALPS) were isolated, freeze-dried and used in the experiment.

1-2. Macrophage culture

RAW264.7 cells, a macrophage cell line derived from mice, were purchased from the Korea Cell Line Bank (Seoul, Korea) and used for cell culture with 100 unit/mL penicillin and 100 unit/mL streptomycin and 10% fetal bovine serum (FBS). Dulbecco'smodified Eagle's medium (DMEM; Lonza, _Walkersville , MD, USA) containing

1-3. Macrophage viability assessment

Raw264.7 macrophages were aliquoted in a 96-well plate at a concentration of 1×10 ⁴ cells/well, and incubated for 15 hours at 37°C, 5% CO ₂ in an incubator to allow the cells to completely adhere and ALPS to phosphate buffered saline (PBS). ; WelGene, Daegu, Korea) and combined treatment at concentrations of 15.125, 31.25, 62.5, 125, 250, 500 and 1,000 μg/μl and left for 24 hours to confirm cytotoxicity. , H460 and macrophage Raw264.7 cells were left in cisplatin and ALPS for 24 hours.

For cell viability evaluation, 30 μl of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich Co.) solution was added to 5 mg/ml per well. and reacted for 4 hours. In order to dissolve Formazan produced by the addition of MTT reagent, 100 μl of dimethyl sulfoxide (Sigma-Aldrich Co.) was added and the absorbance was measured at 570 nm using a Microplate reader after 1 hour, and the absorbance value of the control (Medium only) Cell viability was confirmed based on .

1-4. Cytotoxicity assessment

To analyze apoptosis, FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA) was used. After washing the cells with PBS, using Annexin Vbinding buffer, the cells were suspended at a concentration of 1×10 ⁶ cells/μl. 100 μl (1×10 ⁵ cells) of cells were collected from this solution, aliquoted into a 5 μl culture tube, and stained at room temperature for 20 minutes by adding Annexin V-FITC (5 μl) and PI-PE (5 μl). was carried out. After adding Annexin V binding buffer (400 μl) to the stained mixture, flow cytometer (BD FACS VerseTM; BD Biosciences) analysis was performed. Data obtained after analysis were evaluated using FlowJo software (V10, Tree Star, Ashland, OR, USA).

1-5. Western blot analysis

Raw264.7 macrophages were dispensed in a 6-well plate at a concentration of 1×10 ⁶ cells/well, and then completely adhered for 15 hours. Cisplatin at a concentration of 15 μM and ALPS at a concentration of 31.25 and 62.5 μg/dL were used in combination for 24 hours. processed. After culturing cells were collected, washed with PBS, NP40 cell lysis buffer (Biosource, Seoul, Korea) was added, and then the cell lysate was separated by centrifugation at 13,000 rpm for 15 minutes. The separated cell lysate was subjected to protein quantification using a BCA protein detection kit (Thermo Scientific, Rockford, IL, USA), and 10 μg of cell lysate per well was loaded on a 10% polyacrylamide gel, respectively, and denatured separation was performed by SDS-PAGE. did This was transferred to a polyvinylidene difluoride membrane (Millipore Merck KGaA, Darmstadt, Germany), and the membrane was left at room temperature for 1 hour in 12 μl of a blocking solution (5% skim milk) to prevent non-specific binding of the antibody. Then, it was washed three times with TBST (20 nM tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 10 minutes each, Pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2, Cytochrome c , Cleaved-caspase To measure the expression levels of 3, 8, 9, PARP and β-actin, the primary antibody (Cell Signaling, Danvers, MN, USA) was diluted 1:2,000 and reacted at 4 °C for 15 hours, followed by 10 TBST Washed 3 times per minute. Thereafter, the secondary antibody (Goat-anti rabbit lgG, Calbiochem, La Jolla, CA, USA) was diluted 1:5,000 and reacted for 1 hour, followed by printing using Electrochemiluminescence (ECL; Millipore Merck KGaA) reagent for development.

1-6. statistical processing

The results obtained from the above toxicity alleviation effect experiment on cisplatin of macrophage extracts of graviola leaf extract were confirmed by one-way ANOVA using statistical software (Prism, version 4.03; GraphPad Software, San Diego, CA, USA). Then, it was analyzed by Tukey's multiple comparison test. Significance between samples was compared at P<0.05, P<0.01, and P<0.001 levels using Student's t-test.

<Result>

<Experimental Example 1> Evaluation of cytotoxicity mitigation in macrophages and lung cancer cells of ALPS

To evaluate the effect of graviola leaf ALPS on alleviating macrophage cytotoxicity, macrophage Raw264.7 was treated with ALPS at concentrations of 15.125, 31.25, 62.5, 125, 250, 500 and 1,000 μg/μl, followed by MTT method The macrophage viability was evaluated through (FIG. 1).

As can be seen in FIG. 1 , it was confirmed that there was no cytotoxicity of ALPS.

In addition, cell viability was confirmed after co-treatment of lung cancer cells A549, H460 and macrophage Raw264.7 cells with cisplatin and ALPS by concentration ( FIG. 2 ).

As can be seen in FIG. 2 , it can be seen that the cell viability decreased in lung cancer cells, but the cell viability decreased by cisplatin was increased by ALPS in macrophages.

In addition, whether ALPS alleviates apoptosis of macrophages caused by Cisplatin was evaluated through Annexin V/PI staining analysis (FIG. 3).

As can be seen in FIG. 3 , it can be confirmed that the increased apoptosis of macrophages caused by Cisplatin is reduced by ALPS treated with concentrations of 31.25 and 62.5 μg/ml.

As a result, it was confirmed that ALPS alleviates the toxicity of cisplatin in macrophages and increases the cell viability, which confirms that ALPS inhibits the toxicity of cisplatin in macrophages and suppresses apoptosis.

<Experimental Example 2> Evaluation of apoptosis inhibition of ALPS

In the present invention, Western blot analysis was performed to confirm the effect of ALPS on apoptosis of Cisplatin.

Macrophage Raw264.7 cells were treated with ALPS at concentrations of 31.25 and 62.5 μg/ml, and then 15 μM of Cisplatin was co-treated to confirm the expression levels of Pro-apoptotic protein Bax and Anti-apoptotic protein Bcl-2 after 24 hours. (Fig. 4A).

As can be seen in FIG. 4A , it can be seen that the expression level of the pro-apoptotic protein Bax causing apoptosis was decreased, and the expression level of the anti-apoptotic protein Bcl-2 that inhibits apoptosis was increased.

In addition, it was confirmed that the Cytochrome c release phenomenon that occurs in the mitochondria when apoptosis occurs (Fig. 4B).

As can be seen in FIG. 4B , it can be confirmed that the expression level of cytochrome c increased by Cisplatin was decreased by ALPS.

In addition, macrophage Raw264.7 cells were treated with ALPS at concentrations of 31.25 and 62.5 μg/ml, and then treated with Cisplatin 15 μM to measure the expression levels of Cleaved-caspase 3, 8, 9 and PARP 24 hours later ( Figure 4C).

As can be seen in Figure 4C, the expression levels of cleaved caspase 3, 8, 9 and PARP increased the most in the Cisplatin alone treatment group, but the expression levels of cleaved-caspase 3, 8, 9 and PARP were significantly increased in the ALPS and Cisplatin combination treatment group. It can be confirmed that it decreases, and it can be confirmed that the concentration further decreases as the concentration increases.

As a result, when the composition for reducing the toxicity of anticancer drugs and the anticancer adjuvant comprising the graviola leaf extract according to the present invention as an active ingredient is used, the toxicity of anticancer drugs is reduced, and various It has the effect of preventing or minimizing side effects.

Claims (8)

A pharmaceutical composition for reducing the toxicity of anticancer drugs comprising a graviola leaf extract as an active ingredient. According to claim 1,
The anticancer agent,
A pharmaceutical composition for reducing anticancer drug toxicity, characterized in that it is cisplatin. According to claim 1,
The graviola leaf extract is a pharmaceutical composition for reducing toxicity of anticancer drugs, characterized in that it contains crude polysaccharide. According to claim 1,
The extraction method of the graviola leaf extract,
Step of hot air drying graviola leaves,
pulverizing dried graviola leaves to obtain a powder;
Mixing distilled water with the obtained powder and extracting with hot water to prepare an extract;
Filtering the extract to prepare a filtrate;
adding ethanol to the filtrate and centrifuging to produce a precipitate; and
A pharmaceutical composition for reducing toxicity of anticancer drugs, characterized in that it comprises the step of isolating and drying the precipitate to obtain crude polysaccharides. A health functional food composition for reducing the toxicity of anticancer drugs comprising graviola leaf extract as an active ingredient. 6. The method of claim 5,
The anticancer agent,
A health functional food composition for reducing anticancer drug toxicity, characterized in that it is cisplatin. 6. The method of claim 5,
The graviola leaf extract is a health functional food composition for reducing the toxicity of anticancer drugs, characterized in that it contains crude polysaccharide. An anticancer supplement containing graviola leaf extract as an active ingredient.

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