KR20230065013A - Composition comprising herbal mixture extract for inhibiting anti-cancer drug resistance - Google Patents

Abstract

The present invention relates to a composition for suppressing anticancer drug resistance containing a mixed extract of Rhus verniciflua, Angelica gigas, and Morus alba as an active component. The mixed extract of Rhus verniciflua, Angelica gigas, and Morus alba according to the present invention inhibits anticancer drug resistance and has excellent anticancer effects against anticancer drug resistant cancer, so it can be usefully used as a composition to enhance the anticancer effect.

Description

Composition comprising herbal mixture extract for inhibiting anti-cancer drug resistance}

The present invention relates to a composition for inhibiting anticancer drug resistance comprising a mixed extract of dry lacquer, Angelica gigas, and Mortice bark as an active ingredient.

In recent years, Korea has emerged as a major factor threatening public health with a continuous increase in cancer incidence and cancer deaths due to changes in population and health behavior, environmental structure, rapid entry into an aging society, and changes in disease structure. According to data from the National Statistical Office, cancer is the number one cause of death in Korea, and about 28% of all deaths in Korea in 2012 died from cancer. Gastric cancer and colorectal cancer were found in that order. In addition, it is expected that the increase in colorectal cancer, prostate cancer, and breast cancer through westernization of lifestyle and the deaths from cancer due to aging will also increase.

When cancer is discovered, various treatment methods can be selected depending on the degree of progression. In the early stages of progression, most treatments are performed by removing cancer tissue through direct surgery through surgical methods, and radiation therapy is also used through the patient's choice. do. In the case of advanced cancer, traditional anticancer chemotherapy is performed alone or together with surgical operation, or targeted anticancer chemotherapy or hormone therapy that has recently been developed and has significant effects is sometimes treated.

Among the three standard therapies, anticancer chemotherapy refers to treatment through anticancer drugs designed to destroy cancer cells for cancer treatment, and is a systemic treatment method that uses anticancer drugs to attack cancer cells spread throughout the body. Anticancer chemotherapy is primarily used as a treatment for primary cancer, but it is expected that the effect of preventing cancer recurrence and increasing survival rate in patients by performing adjuvant therapy to suppress unpredictable micrometastasis after surgery with systemic therapy is expected. In order to increase the success rate of surgery, neoadjuvant therapy, which reduces tumor size or induces destruction of micrometastases, is sometimes performed. Palliative therapy for the relief of symptoms in terminal cancer patients is also performed as chemotherapy. However, existing traditional anticancer drugs affect not only cancer cells but also normal cells, are likely to cause various side effects, and often show resistance.

On the other hand, dry lacquer belongs to the lacquer tree Rhus verniciflua Stokes (According to Anacardiaceae), and according to Donguibogam, it has an insecticidal effect that removes blood and makes menstruation smooth and eliminates parasites.

Angelica gigas also refers to the dried root of Angelica gigas Nakai. According to Donguibogam, it has the effect of supplementing blood to treat lack of blood or excessive blood, harmonize menstruation, and stop pain. In addition, there are research reports on antibacterial activity, relief of menopausal disorders, deep vein thrombosis, and antioxidant activity.

In addition, Morus alba is a medicinal material made from the root bark of a mulberry tree (Morus alba L.) or a plant of the same genus. According to Donguibogam, it has an expectorant effect, an effect to stop coughing, an effect to remove stagnant blood, and a haemorrhagic effect. In addition, there are research reports on anti-inflammatory action, antioxidant effect, blood sugar and blood pressure lowering action.

As described above, various pharmacological effects of dried lacquer, angelica quinoa, and moth bark are known, but the anticancer effect of these mixed extracts has not been clarified, and there is no research on this.

Accordingly, the present inventors conducted research to develop a novel anticancer agent that can be applied to cancer cells resistant to existing anticancer agents, and completed the present invention by confirming that the mixed extract of dried chil, Angelica gigas, and moth bark had an anticancer drug resistance inhibitory effect.

An object of the present invention is to provide a composition for inhibiting anticancer drug resistance comprising a mixed extract of dried lacquer, Angelica gigas and Mortice bark as an active ingredient.

Another object of the present invention is to provide an anti-cancer adjuvant composition comprising a mixed extract of dried lacquer, Angelica gigas, and Mortice bark as an active ingredient.

Another object of the present invention is to provide a composition for enhancing anti-cancer effect comprising a mixed extract of dried lacquer, Angelica gigas, and Mortice bark as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting anticancer drug resistance or treating anticancer drug resistant cancer, comprising a mixed extract of dried lacquer, angelica quinoa and moth bark as an active ingredient.

In addition, the present invention provides an anti-cancer adjuvant composition comprising a mixed extract of dried lacquer, Angelica gigas, and Mortice bark as an active ingredient.

In addition, the present invention provides a food composition for enhancing anti-cancer effect comprising a mixed extract of dry lacquer, Angelica gigas and Mortice bark as an active ingredient.

The mixed extract of dried lacquer, Angelica gigas and Mortice bark according to the present invention suppresses anticancer drug resistance and has an excellent anticancer effect against anticancer drug resistant cancer, so it can be usefully used as a composition for enhancing the anticancer effect.

1 is a diagram showing the results of the MTT assay (A), colony formation assay (B), and adhesion-independent assay (C) on the effect of mixed extracts of dried cilantro, angelica quince and moth bark on HCC827 and HCC827GR cell lines.
Figure 2 shows the results of confirming the effect of the mixed extract of dried lacquer, Angelica quinoa and Mortalis epithelium on invasion (A, B) and metastasis (C, D) and related protein expression (E, F) of HCC827 and HCC827GR cell lines. It is also
Figure 3 is a diagram showing the results of confirming the effect of the mixed extract of geonchi, Angelica gigas and Mortice bark on the cell cycle of HCC827 and HCC827GR cell lines.
Figure 4 is a view showing the results of confirming the effect of the mixed extract of geonchi, Angelica gigas and Mortice bark on the expression of anticancer drug resistance-related proteins in HCC827 and HCC827GR cell lines.
Figure 5 shows the results of confirming the effect of the mixed extract of dried lacquer, Angelica gigas and Mortice bark on gefitinib-resistant lung cancer animal model design (A), cancer tissue volume / weight (B, D) and body weight (D) in the animal model. is the diagram shown.
Figure 6 shows the effect of mixed extracts of dried lacquer, Angelica quinoa, and Mortalis Morus on the expression (A, B) of PCNA and CD31, the expression of cell proliferation markers (C), and the distribution of capillaries (D) in gefitinib-resistant lung cancer animal models. It is a diagram showing the result of checking.
Figure 7 is a diagram showing the results of Western blot (A) and immunohistochemical analysis (B, C) of the effect of mixed extracts of dried cilantro, Angelica quinoa, and Mortice bark on protein expression in tissues of gefitinib-resistant lung cancer animal models. am.

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

The present invention provides a pharmaceutical composition for inhibiting anticancer drug resistance comprising a mixed extract of dry lacquer, Angelica gigas, and Mortice bark as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the treatment of anticancer drug-resistant cancer comprising a mixed extract of dried lacquer, Angelica gigas and Mortice bark as an active ingredient.

In the composition of the present invention, the mixed extract of dry lacquer, Angelica gigas, and Mortice bark, which are active ingredients, can be obtained by the following method.

First, the dried lacquer, angelica guinea pig, and moth bark are washed with water to remove foreign substances. Cultivated or commercially available dried lacquer, Angelica quinoa, and Mortar may be used without limitation. 10 to 30 times the volume of solvent is added to each of the dried lacquer, angelica quinoa and moth bark so that they are completely immersed. The extraction method can be impregnation or warming at room temperature. The extraction solvent is not limited thereto, but may use one or more solvents selected from water, alcohol having 1 to 4 carbon atoms, and mixed solvents thereof, and preferably water or ethanol. More preferably, water for dry lacquer or Angelica quail, and ethanol for moth bark may be used as an extraction solvent. The extracts are filtered and concentrated under reduced pressure to obtain extracts of dried chil, Angelica gigas, and Mortice bark, respectively. Thereafter, a mixed extract is obtained by mixing the extracts of dried lacquer, Angelica gigas, and Mortice bark.

The mixing ratio of the herbal medicine is preferably a weight ratio of dry coating: Angelica: Mortice bark = 1 to 10: 1 to 10: 1, more preferably 1 to 5: 1 to 5: 1, but is not limited thereto. In one embodiment of the present invention, it was prepared by mixing dried lacquer, angelica guinea pig, and moth bark at a weight ratio of 2:1:1. The content is a range having the effect presented in the present invention, and there is a possibility that the effect may be exerted differently depending on the mixing ratio of the raw material. The mixing ratio can be controlled to an appropriate level so that effects such as inhibition of anticancer drug resistance by the active ingredient are appropriately implemented.

In the present invention, the extract includes all of an extract obtained by extraction treatment, a diluted solution thereof, a concentrated solution, a dried product of the extract, and a purified product thereof. The extraction method is not particularly limited, and may be extracted through various extraction methods known in the art, such as heat extraction, pressure extraction, ultrasonic extraction, reflux circulation extraction, ultrasonic extraction, and supercritical extraction. The extraction may be performed using a conventional method in the art, such as cooling, warming, or heating using the solvent.

The mixed extract of dried lacquer, Angelica gigas and Mortice bark according to the present invention has an excellent effect of suppressing anticancer drug resistance and increasing anticancer effect in anticancer drug resistant cancer cells and animal models, and thus can be usefully used as a pharmaceutical composition and food composition.

In the present invention, the anticancer agent is an EGFR inhibitor, and the EGFR inhibitor is gefitinib, erlotinib, osimertinib, cetuximab, afatinib , neratinib, panitumumab, dacomitinib, lapatinib, necitumumab, mobocertinib, or vandetanib, Not limited to this.

The composition according to the present invention can be used for the treatment of anticancer drug-resistant cancer, and applicable cancer diseases include all general cancer diseases, for example, gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, and pancreatic cancer. , skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, proximal anal cancer, fallopian tube carcinoma, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease ), esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal pelvic cancer, blood It may be cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma, preferably lung cancer, but not limited thereto.

The composition of the present invention may further contain at least one known active ingredient having an anti-cancer effect together with the mixed extract of dried lacquer, Angelica gigas, and Mortice bark.

The composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. In addition, it may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods. Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included in the composition 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, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. In addition, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogeratin and the like may be used.

As used herein, the term "administration" means providing a given composition of the present invention to a subject by any suitable method.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the subject, the severity of the disease, the drug type, the route and duration of administration, and can be appropriately selected by those skilled in the art. For a desirable effect, the mixed extract of the present invention may be administered in an amount of 1 mg/kg to 10000 mg/kg per day, and may be administered once or several times a day.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of cancer.

In addition, the present invention provides a food composition for preventing or improving anticancer drug-resistant cancer, comprising a mixed extract of dry lacquer, Angelica gigas, and Mortice bark as an active ingredient.

In addition, the present invention provides a food composition for enhancing anti-cancer effect comprising a mixed extract of dried lacquer, Angelica gigas, and Mortice bark as an active ingredient.

The food composition may preferably be in the form of a health functional food.

In the present invention, 'health functional food' refers to food having bioregulatory functions such as disease prevention or improvement, biodefense, immunity, recovery after illness, aging inhibition, etc., and should be harmless to the human body when taken for a long period of time. The mixed extract of dry lacquer, Angelica gigas and Mortice bark of the present invention may be added to functional health food for the purpose of preventing or improving cancer. When the mixed extract of dried cilantro, Angelica quinoa and Mortice rind of the present invention is used as a food additive, the mixed extract of dried cilantro, Angelica quinoa and Mortice rind can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. can The mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the mixed extract of dried lacquer, angelica quasi, and sandalwood bark of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages; and the like. In addition, the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides an anti-cancer adjuvant composition comprising a mixed extract of dried lacquer, Angelica gigas, and Mortice bark as an active ingredient. The composition may be in the form of a pharmaceutical composition or food composition, and more specifically, may be an anti-cancer pharmaceutical or anti-cancer food supplement.

In the present invention, "anti-cancer adjuvant" refers to an agent that can be used adjuvantly to enhance the effect of a cancer treatment commonly used in the art, and by using the adjuvant according to the present invention, the effect of anti-cancer treatment can be enhanced. can

Hereinafter, preferred examples, experimental examples, and manufacturing examples are presented to aid understanding of the present invention. However, the following examples, experimental examples, and preparation examples are provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples, experimental examples, and preparation examples.

Example 1. Preparation of Mixed Extracts of Dried Chil, Angelica Quail and Morus Morus

After washing the dried lacquer, Angelica quinoa, and Mortar bark, respectively, immersing the dried lacquer and Angelica quinoa in water, and 30% ethanol in 30% ethanol for water extraction, turning on the heater (set at 105 ° C) and boiling the water, and organic solvent extraction Extraction was performed for 3 hours in a heater (set at 80° C.). The extract was filtered, and the filtrate was concentrated under reduced pressure, and then dried to obtain an extract.

The extracts of dried lacquer, angelica quince, and sandal bark obtained through the above process were mixed in a weight ratio of 2: 1: 1 based on dry weight to obtain a mixed extract, which was named SH005S7.

Experimental Example 1. Confirmation of growth inhibitory effect on cancer cells and drug-resistant cell lines of mixed extracts of geonchi, Angelica gigas and Morus burdock

In order to confirm the growth inhibition of lung cancer cells and drug-resistant cell lines of the mixed extract of dried cilantro, angelica quinoa and moth bark, MTT assay was performed using gefitinib-resistant HCC827GR and parental lung cancer cell line HCC827 according to a conventionally known method. was performed.

More specifically, a cancer cell line cultured in a 96-well plate was treated with the mixed extract of dried cilantro, Angelica gigas, and moth cortex prepared in Example 1 at various concentrations (50 to 500 μg/ml), and then treated at 37° C. and 5% for 72 hours. It was cultured under CO ₂ conditions. Thereafter, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, USA) reagent was treated in each well and incubated for 4 hours, and the supernatant was removed. 100 μl of dimethyl sulfoxide (DMSO) was added. Finally, absorbance was measured at a wavelength of 590 nm using an ELISA microplate reader (VERSAMAX, Molecular Devices, USA), and the absorbance value was compared with a control group that was not treated with anything. The results are shown in A of FIG. 1 .

As shown in A of FIG. 1 , it was confirmed that the mixed extract of dried cilantro, angelica cinnamon, and sandalwood extract according to the present invention exhibited excellent anticancer activity not only against lung cancer cells but also against anticancer drug-resistant cell lines.

Next, a clonogenic assay was performed on the same cell line in order to confirm the effect of the mixed extract of dried lacquer, Angelica gigas, and Morus burdock on colony formation of cancer cells.

More specifically, the cancer cell line cultured in a 6-well plate was treated with the mixed extract of dried cilantro, Angelica gigas, and moth cortex prepared in Example 1 at various concentrations (0, 50, 100 μg/ml), and then cultured at 37° C. for 5 weeks. It was cultured under % CO ₂ conditions. Thereafter, colonies were stained with crystal violet (Sigma, USA) and the number was measured. The results are shown in B of FIG. 1 .

As shown in B of FIG. 1 , it was confirmed that the mixed extract of dried cilantro, angelica cinnamon, and moth epidermis according to the present invention had an excellent anticancer effect of inhibiting colony formation in not only lung cancer cells but also anticancer drug resistant cell lines.

Next, an anchorage-independent assay was performed according to a conventionally known method in order to verify the effect of the mixed extracts of dried lacquer, Angelica gigas, and Morus epiphyllum on the adhesion dependence of cancer cells.

More specifically, after culturing HCC827 and HCC827GR on a soft agar plate, they were treated with the mixed extract of dried cilantro, Angelica gigas, and Mortice bark prepared in Example 1 at a concentration of 500 μg/ml at two-day intervals, and cultured for a total of 15 days. On the 15th day, the cells were stained with 0.5% crystal violet, and the number of colonies was measured using an optical microscope. The results are shown in C of FIG. 1 .

As shown in FIG. 1C , it was confirmed that the mixed extract of dried cilantro, angelica cinnamon, and moth epidermis according to the present invention exhibits an effect of significantly inhibiting adhesion dependence not only in lung cancer cells but also in anticancer drug-resistant cell lines.

Experimental Example 2. Confirmation of Effects of Mixed Extracts of Geonchil, Angelica Quail and Morus Morus on Invasion and Metastasis of Cancer Cells and Drug-Resistant Cell Lines

In order to confirm the anticancer effect of the mixed extracts of dried lacquer, angelica quinoa and moth baekpi on lung cancer cells and drug-resistant cell lines, a transwell assay was performed.

More specifically, after culturing the lung cancer cell line in the upper chamber coated with matrigel, the mixed extract of dried cilantro, Angelica gigas and Mortice bark prepared in Example 1 at concentrations of 250 and 500 μg/ml was treated and maintained for 48 hours. After culturing, invasion assay was performed. In the case of the metastasis assay, the method is almost the same as the invasion assay, but the chamber is not coated with Matrigel.

In addition, Western blotting assay was performed to confirm the effect of mixed extracts of dried lacquer, Angelica gigas, and Morus burdock on protein expression.

More specifically, HCC827 and HCC827GR were treated with the mixed extract of dry lacquer, Angelica gigas, and Mortice bark prepared in Example 1 at a concentration of 200 μg/ml or 500 μg/ml for 72 hours. After separating proteins from cells using RIPA buffer (Biosesang, Republic of Korea), E-cadherin, Vimentin, and Snail. The amount of protein expressed in cells was compared using MMP-9 or GAPDH antibody (Cells signaling, USA). The above experimental results are shown in FIG. 2 .

As shown in Figure 2, the mixed extract of geonchil, Angelica gigas and Mortice bark according to the present invention inhibited the ability to invade and metastasize not only in lung cancer cells but also in anticancer drug-resistant cell lines, and Vimentin, Snail. It was confirmed that the protein expression of MMP-9 was significantly inhibited.

Experimental Example 3. Confirmation of Cell Cycle Progression Inhibitory Effect on Cancer Cells and Drug-Resistant Cell Lines of Mixed Extracts of Geonchil, Angelica Quail and Morus Morus

BrdU assay was performed to measure the cell cycle progression inhibitory effect of the mixed extracts of dried cilantro, Angelica gigas, and Morus quincea on lung cancer cells and drug-resistant cell lines.

More specifically, HCC827 and HCC827GR were diluted to a cell count of 1.5×10 ² per well in a 6-well plate and cultured for 24 hours at 37° C. under 5% CO ₂ conditions. Thereafter, 2 ml of a diluted medium of each concentration (200, 500 μg/ml) of the mixed extract of dry lacquer, Angelica gigas, and Mortice bark prepared in Example 1 was added to each well and cultured for 72 hours. BrdU (10 mM) was added per well 1 hour prior to harvesting the cells. The BrdU labeling solution was removed from the cells, washed twice with phosphate buffered saline (PBS, Welgene, Republic of Korea), and then treated with trypsin to harvest the cells. Pellets were released for 30 minutes with a solution of 2N HCl dissolved in DW to which 0.5% Triton X-100 was added, left at room temperature, and then neutralized with 0.1 M sodium tetraborate for 2 minutes. Then, the cells were incubated with α-BrdU (Santa Cruz, USA) antibody for 1 hour, washed with phosphate buffered saline (PBS), and then Goat anti-Mouse igG-FITC (Sigma, USA) was attached. After washing twice with phosphate-buffered saline (PBS) and blocking light in propidium iodide buffer (PI buffer, Sigma, USA) containing 50 μg/ml RNase A (Sigma, USA) for 30 minutes, leave it at room temperature and After transferring to a polystyrene round-bottom tube, the cell cycle was analyzed using a BD FACS Calibur, FACS calibur flow cytometer, BD Biosciences. The results are shown in Figure 3.

As shown in FIG. 3, the S phase was significantly increased in lung cancer cells treated with the mixed extract of dried chil, Angelica gigas, and Mortice bark according to the present invention, as well as in anticancer drug-resistant cell lines, and through this, the extract according to the present invention inhibited cell cycle progression. The inhibitory effect was confirmed.

Experimental Example 4. Verification of anticancer drug resistance effect and effect on ErbB / HER family signal transduction system of mixed extract of dried cilantro, Angelica gigas and Mortice bark

Gefitinib is an anticancer drug that exhibits significant anticancer effects on non-small lung cancer cells by inhibiting its activation as an EGFR inhibitor, but as the recurrence rate increases with resistance, the need for a new drug that can overcome drug resistance has emerged. The target protein that causes gefitinib resistance is currently known as MET, and it seems that overexpression of ErbB3/HER3 as well as overexpression of MET has an effect. Based on the results of Experimental Examples 1 to 3, the protein expression level was analyzed by western blotting to confirm whether the mixed extract of dried cilantro, angelica guinea pig, and moth epiphyllum according to the present invention affects the ErbB/HER family. The results are shown in FIG. 4 .

As shown in Figure 4, it was confirmed that the basal level of MET was higher in HCC827GR with gefitinib resistance than the parental cell, HCC827, and ErbB3/HER3 also showed relatively high expression. In addition, it was confirmed that the activation of EGFR, the activation of MET and ErbB3/HER3, and the expression level decreased as the concentration of the mixed extract of dried lacquer, Angelica gigas, and Mortice bark according to the present invention increased. In addition, the activation of AKT and ERK, which are involved in cell survival, was also reduced by the treatment of the mixed extract of dried lacquer, angelica quince, and moth bark according to the present invention, and through this, changes in the signal transduction system for growth inhibition were confirmed.

Experimental Example 5. Verification of anticancer activity in animal models

The following experiment was performed to confirm whether the mixed extracts of dried cilantro, angelica quarry, and moth bark showed anticancer activity in gefitinib-resistant lung cancer animal models.

5-week-old nude (Nu/Nu) mice (Nara Biotech) were used as experimental animals, and gefitinib-resistant lung cancer cell line HCC827GR was subcutaneously injected at a number of 2×10 ⁶ per mouse. When the cancer was formed in the form of millet at the site of the subcutaneous injection, each mouse was randomly divided into groups, and then the mixed extract of dried cilantro, angelica chinensis and moth bark prepared in Example 1 was orally administered daily at a concentration of 500 mg / kg for 39 days. In the case of the control group, saline was orally administered. The body weight and arm size of the mice were measured three times a week. After the experiment was completed, blood collected from the heart of each mouse was sacrificed and whole blood was analyzed using a hematology analyzer (Drew Scientific, USA). Toxicity was analyzed. Tissues were cut into appropriate sizes and partially dissolved in RIPA buffer to perform western blotting assays. The isolated proteins were classified into Ki-67 and PCNA (Abcam, UK), which are cell proliferation markers, p-EGFR, EGFR, p-HER3, HER3 (cell signaling, USA), which are ErbB/HER family members, and p, which is the cause of gefitinib resistance. -MET, MET (cells signaling, USA) antibodies were attached to confirm the amount of protein expressed in cells. The other part was fixed with 4% formaldehyde, and the fixed tissue was embedded in paraffin and used for histological observation analysis. Some of the tissues were stained with hematoxylin and eosin (H&E), others were stained with PCNA and the angiogenic marker CD31 (Abcam, UK), and others were stained with p-EGFR, EGFR, p-MET, MET, p -Immunohistochemistry was performed using HER3, HER3 or Vimentin (cell signaling, USA) antibodies. Blood analysis results are shown in Tables 1 and 2, and other experimental results are shown in FIGS. 5 to 7.

As shown in Tables 1 and 2, the mixed extract of dried lacquer, Angelica quinoa and Mortice bark according to the present invention did not cause renal toxicity and hepatotoxicity, and did not affect the number or specific gravity of various blood cells beyond the normal range. Confirmed.

In addition, as shown in Figure 5, the mixed extract of dry lacquer, Angelica gigas, and Mortice bark according to the present invention did not affect the total weight change of mice in gefitinib-resistant animal models, but reduced the volume and weight of cancer tissues. It was confirmed that the effect was shown.

In addition, as shown in FIG. 6, as a result of tissue staining with α-PCNA and α-CD31 antibodies, it was confirmed that the group administered with the mixed extract of dried cilantro, angelica radishes, and moth bark according to the present invention significantly decreased (Fig. 6 A and B) . In addition, cell proliferation markers (Ki-67, PCNA) were decreased in the group administered with the mixed extract of dry lacquer, Angelica gigas, and Mortice bark according to the present invention (FIG. 6C), and photographs of cancer tissues and tissues near cancer tissues were analyzed. As a result, it was confirmed that the distribution of capillaries was also significantly decreased in the group administered with the mixed extract of dried lacquer, Angelica gigas, and Mortice bark according to the present invention (Fig. 6D).

Finally, as shown in FIG. 7, as a result of western blot and immunohistochemical analysis, as in the cell model, activation of EGFR, activation of MET and HER3, and protein amount in the group administered with the mixed extract of Geonchi, Angelica gigas and Morus moth according to the present invention, It was confirmed that the activation of AKT and ERK involved in cell survival was reduced.

Through the above experimental results, it was confirmed that the mixed extract of dry lacquer, angelica quince and moth bark has an excellent anticancer effect to suppress anticancer drug resistance, and thus can be used in the field of medicine and health functional food.

Hereinafter, formulation examples of the pharmaceutical composition and food composition of the present invention are described, but are not intended to limit the present invention, but are only specifically described .

Formulation Example 1. Preparation of pharmaceutical composition

1-1. manufacture of powders

20 mg of mixed extract of dried cilantro, angelica quince and moth bark

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

1-2. manufacture of tablets

10 mg of mixed extract of dried cilantro, angelica quince and moth bark

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

1-3. Manufacture of capsules

10 mg of mixed extract of dried cilantro, angelica quince and moth bark

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

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

1-4. Manufacture of injectables

10 mg of mixed extract of dried cilantro, angelica quince and moth bark

Mannitol 180 mg

Sterile Distilled Water for Injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above component content per 1 ampoule (2 ml) according to the conventional method for preparing injections.

1-5. Manufacture of Liquids

20 mg of mixed extract of dried cilantro, angelica quince and moth bark

Isomerized sugar 10 g

5 g mannitol

Purified water . Appropriate amount

According to the conventional liquid preparation method, each component is dissolved in purified water, lemon flavor is added in an appropriate amount, the above components are mixed, and purified water is added to adjust the total volume to 100 ml, and then filled in a brown bottle. Sterilize to prepare a liquid formulation.

Formulation Example 2. Preparation of food composition

2-1. Manufacture of health food

100 mg mixed extract of dried cilantro, angelica quince and moth bark

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for relatively healthy food in a preferred embodiment, the mixing ratio may be arbitrarily modified. , Granules can be prepared and used in the preparation of health food compositions according to conventional methods.

2-2. Manufacture of health drinks

100 mg mixed extract of dried cilantro, angelica quince and moth bark

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g iron lactate

Zinc oxide 3.5 g

Nicotinamide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

water quantity

After mixing the above ingredients according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 l container, sealed and sterilized, and then refrigerated. It is used for preparing the health drink composition of the present invention.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Claims (9)

A pharmaceutical composition for inhibiting anti-cancer drug resistance comprising a mixed extract of dried cilantro, Angelica gigas and Mortice bark as an active ingredient.
The pharmaceutical composition for inhibiting anticancer drug resistance according to claim 1, wherein the mixed extract is extracted with at least one solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.
The pharmaceutical composition for inhibiting anticancer drug resistance according to claim 1, wherein the mixed extract of dried lacquer, Angelica quinoa, and Mortice rind is a mixture of dried lacquer, Angelica quinoa, and Mortalis in a weight ratio of 1 to 10:1 to 10:1.
According to claim 1, wherein the anti-cancer agent is characterized in that the EGFR inhibitor, anti-cancer drug resistance inhibition pharmaceutical composition.
The method of claim 4, wherein the EGFR inhibitor is gefitinib, erlotinib, osimertinib, cetuximab, afatinib, neratinib ), panitumumab, dacomitinib, lapatinib, necitumumab, monosertinib, and vandetanib. To, a pharmaceutical composition for inhibiting anti-cancer drug resistance.
A pharmaceutical composition for the treatment of anticancer drug-resistant cancer, comprising a mixed extract of dried cilantro, angelica chinensis, and moth bark as an active ingredient.
A food composition for preventing or improving anti-cancer drug-resistant cancer, comprising a mixed extract of dried cilantro, angelica chinensis, and moth bark as an active ingredient.
A food composition for enhancing anti-cancer effect, comprising a mixed extract of dried cilantro, angelica quince, and moth bark as an active ingredient.
An anti-cancer adjuvant composition comprising a mixed extract of dried cilantro, angelica quince, and moth bark as an active ingredient.

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