KR102590150B1 - Pharmaceutical composition for preventing or treating of lung cancer comprising medicinal herb complex extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating lung cancer containing a complex extract of herbal medicine as an active ingredient, specifically, complex extracts of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla, and Chrysanthemum chinensis. It relates to a pharmaceutical composition for preventing or treating lung cancer containing as an active ingredient. The complex extract of the present invention exhibits cytotoxicity against lung cancer cell lines, induces cell death by causing intracellular reactive oxygen species, inhibits substances that cause inflammation in the serum, and increases substances that regulate the immune response, thereby preventing lung cancer. It can be useful for prevention or treatment.

Description

Pharmaceutical composition for preventing or treating lung cancer comprising a complex extract of herbal medicine as an active ingredient {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OF LUNG CANCER COMPRISING MEDICINAL HERB COMPLEX EXTRACT}

The present invention relates to a pharmaceutical composition for preventing or treating lung cancer containing a complex extract of herbal medicine as an active ingredient, specifically, complex extracts of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla, and Chrysanthemum chinensis. It relates to a pharmaceutical composition and health functional food for preventing or treating lung cancer containing as an active ingredient.

The incidence of cancer patients in Korea is increasing every year as habits change toward a Western-style diet and various causative factors such as stress and environmental pollution in daily life increase. According to the Korea Institute of Oriental Medicine, the incidence of cancer patients in Korea is increasing by more than 5% every year, and the number of recent deaths due to cancer is high, accounting for 12% of the total mortality rate.

Although the current cancer treatment rate has increased significantly compared to the past, cancer is still one of the diseases that is difficult to completely conquer. In particular, when cancer occurs, treatment is carried out through surgery, radiation therapy, and chemotherapy, but there is a problem in that it is difficult to completely remove microscopic lesions that have infiltrated surrounding tissues or metastasized to lymph nodes.

As a new approach for cancer treatment, research is being actively conducted to develop carcinogenesis inhibitors or cancer treatments with low side effects and excellent efficacy from natural products with relatively low toxicity. According to various basic and clinical studies, oriental medicine prescriptions have anticancer effects by strengthening immune function and suppressing tumor growth, and have been shown to significantly reduce side effects such as hematopoiesis and immune function suppression, which are commonly observed in chemotherapy and radiation therapy.

Republic of Korea Patent Publication No. 10-2013-0040578

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating lung cancer containing complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis as active ingredients.

According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating lung cancer comprising complex extracts of Hwanggeum, portulaca, guaruin, Gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis as active ingredients.

According to another embodiment of the present invention, a health functional food for preventing or improving lung cancer containing complex extracts of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis as active ingredients is provided.

The complex extracts of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla and galleon chinensis are characterized in that they are hot water extracts.

The complex extract of golden, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, ilcheongung, perilla, and galleon chinensis contains 6 parts by weight of gold, 12 parts by weight of portulaca quince, 10 parts by weight of guaruin, 4 parts by weight of gilgyeong, and 4 parts by weight of ginseng. , It is characterized by mixing 4 parts by weight of Macmundong, 4 parts by weight of Schisandra chinensis, 4 parts by weight of Ilcheongung, 4 parts by weight of Jasoja, and 4 parts by weight of Five Baeja.

The complex extract is characterized by dissolving the mixed powder of hot water extraction of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Maekmundong, Schisandra chinensis, Ilcheongung, Perilla chinensis and Chrysanthemum chinensis in distilled water at a concentration of 1 to 10% (w/v).

The complex extract is characterized in that it is a lactic acid bacteria fermentation extract, and the lactic acid bacteria are selected from the group consisting of Streptococcus genus ( Steptococcus sp.), Lactobacillus genus ( Lactobascillus sp.), and Bifidobacterium sp. However, it is not limited to this.

The complex extract exhibits an apoptotic effect on lung cancer cells, but does not exhibit an apoptotic effect on normal liver cells.

The complex extract is characterized by increasing ROS levels in lung cancer cells and lung cancer cell mitochondria.

The complex extract is characterized by lowering the mitochondrial membrane potential in lung cancer cells.

The complex extract is characterized in that it has the effect of increasing TNF-α in the serum in individuals with cancer.

The complex extract is characterized by reducing the expression of intracellular p-AKT, p-Pi3k, and Blc-2 proteins and increasing the expression of C-casepase3, C-casepase7, C-casepase9, and Bad proteins.

As a result of confirming the therapeutic efficacy of the complex extract of the present invention against lung cancer cells A549 and A549/PTX, the present inventors confirmed that it exhibits an excellent cell killing effect on lung cancer cells.

Although it showed somewhat weak anticancer efficacy in anticancer resistant A549/PTX cells, overall, the complex extract of the present invention has the ability to kill lung cancer cells. Cell test results show that the complex extract of the present invention destroys the function of mitochondria in lung cancer cells by promoting the accumulation of active oxygen in lung cancer cells, suppressing metastasis and colony formation of lung cancer cells and at the same time inducing death of lung cancer cells. appear.

To prove this, an in vivo nonclinical experiment was conducted using mice. The complex extract of the present invention had no significant toxic reaction or effect on body temperature changes in other organs of the body, and was found to significantly inhibit cancer cell growth targeting cancer tissues in the mouse body. Furthermore, as a result of measuring the content of inflammatory substances contained in serum, it was found that the content of TNF-a, which induces cancer cell death, was significantly higher in the mouse group treated with the complex extract of the present invention compared to the control group. As a result, the complex extract of the present invention has excellent anti-cancer efficacy in lung cancer, and is expected to be of great help in clinical anti-cancer projects as a lung cancer treatment. However, since the high concentration of the complex extract of the present invention is highly toxic, it is considered necessary to adjust the concentration appropriately depending on the situation during clinical use.

Figure 1 is a diagram showing the cell viability measurement results of the complex extracts of the present invention, Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Maekmundong, Schisandra chinensis, Ilcheongung, perilla and galleon chinensis.
Figure 2 is a diagram showing the results of measuring cell migration and cell colony formation ability of the complex extract of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Maekmun-dong, Schisandra chinensis, Ilcheongung, perilla and galleon chinensis complex extract of the present invention.
Figure 3 is a diagram showing the results of measuring the effect of the complex extracts of the present invention, including Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla, and Chrysanthemum chinensis, on ROS levels in A549 and A549-PTX cells.
Figure 4 is a diagram showing the measurement results of A549 and A549-PTX cell damage induction of the complex extracts of the present invention, Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla perilla, and Chrysanthemum chinensis.
Figure 5 is a diagram showing the results of A549 and A549-PTX apoptosis measurement of the complex extracts of the present invention, Hwanggeum, Portulaca hyeon, Guaruin, Gilgyeong, Ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis.
Figure 6 is a diagram showing the results of measuring changes in the expression of A549 and A549-PTX apoptosis proteins of the complex extracts of the present invention, Hwanggeum, Portulaca vulgaris, Guaruin, Gilgyeong, Ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis.
Figure 7 shows changes in body temperature and feeding amount after heterogeneous transplantation of A549 and A549-PTX cells into mice of the complex extract of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, Ilcheongung, perilla and galleon chinensis of the present invention. This is a drawing showing the results.
Figure 8 shows the results of measuring tumor changes by L2 after heterologous transplantation of A549 and A549-PTX cells of the complex extract of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, Ilcheongung, perilla, and gallberry complex extract of the present invention. It is a drawing.
Figure 9 is a diagram showing the results of pathological analysis of mouse tumor tissue of the complex extracts of the present invention, Hwanggeum, Portulaca vulgaris, Guaruin, Gilgyeong, Ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis.
Figure 10 is a diagram showing the results of pathological analysis of mouse organ tissues of the complex extracts of the present invention, Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Maekmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis.

In this specification, when a member is said to be located “on” another member, this includes not only the case where a member is in contact with another member, but also the case where another member exists between the two members.

In this specification, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

The present invention provides a pharmaceutical composition for preventing or treating lung cancer containing complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis as active ingredients.

Gold exhibits excellent antioxidant properties without affecting the survival rate of cells due to the flavonoid components baicalin, baicalein, and woogonin, and has the effect of preventing photoaging caused by ultraviolet rays.

Portulaca quince is named because its leaves resemble horse teeth and its smooth medicinal properties are similar to those of amaranth. The one with large leaves is called Donicho, and the one with small leaves is called Seochihyeon. This is also an expression of its appearance, and because the leaves do not fade for a long time, it is also called Jangmyeong (long life). It is also called the Five Elements Plant, meaning the five elements because its leaves are blue, its stems are red, its flowers are yellow, its roots are white, and its seeds are black. It is named so because it has a . This medicine is soft, has a peculiar odor, is viscous, has a salty taste, and is cold in nature. Portulaca quince has antipyretic, detoxification, and hemostatic effects, so it is used for bacterial dysentery, boils, hemorrhoids, cervical lymphadenitis, eczema, prawns, uterine bleeding, and urinary incontinence. Pharmacological effects such as antibacterial action, intestinal peristalsis due to enhancement of uterine smooth muscle contraction, and diuretic action have been reported.

Guaruin refers to the seeds of Trichosanthes kirilowii Maximowicz or Trichosanthes kirilowii Max. var. japonica Kitamura of the cucurbit family. Guaruin is known to be effective in reducing fever, constipation, boils, and promoting milk secretion. It is effective in treating acute bronchitis, pleurisy, and pneumonia due to its pharmacological action, and its fruit, Guarru, has been reported to have an inhibitory effect on Escherichia coli, Shigella, dysenteric bacteria, and sarcoma ascites cancer.

Gilgyeong (桔梗) is a medicinal herb made by removing the roots or pericarp of Bellflower (Platycodon grandiflorum A. De Candolle) of the Campanula family. It has a slight odor and a bitter taste, but its properties are flat and not biased towards one side. Gilgyeong acts on the lungs to treat symptoms of excessive seawater and phlegm and difficulty in breathing. It clears the lungs, relieves stuffy chest, and relieves cold energy in the stomach, stopping coughing and removing phlegm. It is also used when there is edema throughout the body and the amount of urine is small due to difficulty in urinating. It is used for sore throat, cough due to cold, phlegm, nasal congestion, asthma, bronchitis, pleurisy, headache, chills, tonsillitis, etc. Other names include Gilgyeonggeun, Gyeongcho, Gogyeong, Gogilgyeong, Liyeo, Bangdo, Baekyak, and Fuho. etc.

Ginseng is a representative medicinal plant and is a perennial herb of the cotyledonous plant, Araliaceae family. It is widely cultivated for medicinal purposes. Since ancient times, ginseng has been said to be the best medicine for immortality, longevity, and godliness. The root of ginseng grown in Korea is a large root, consisting of a primary root and 2 to 5 slow roots, and is light yellow-white in color. It is a plant with strong branching, and the shape of its roots varies depending on age, and is harvested when the roots are 4 to 6 years old. Most of the raw materials used for red ginseng, a Korean exclusive product, are 5-6 year old. The main root (trunk) of a 6-year-old root is about 7 to 10 centimeters long, its diameter is about 2.5 centimeters, the root length is 34 centimeters, and it weighs about 100 grams. Ginseng sprouts every year from the head connected to the end of the underground stem, and the stem and leaves die in the fall. Organic ginseng has long, thin roots and weighs less than regular ginseng.

Macmundong is a perennial herb belonging to the Liliaceae family and refers to the tuber root of Liriope platyphylla or Ophiopogon japonica, and is also called pulse. In Korea, Macmundong is mainly used for medicinal purposes. Macmundong has a sweet taste and a slightly cool and mild nature, so it is a medicine that strengthens the respiratory system.

Schisandra chinensis is a vine-like deciduous tree distributed from 200 to 1,600 meters above sea level and growing abundantly in Jiri Mountain, Songnisan Mountain, and Taebaek Mountain. The height is 6~9m, and the leaves are 7~10cm long and 3~5cm wide, have hairs on the veins on the back, have small tooth-like teeth on the edges, and are alternate in a wide oval shape. The flowers are slightly reddish-yellow-white, about 1.5 cm in diameter, and bloom in clusters of 3 to 5, one at a time, in the leaf axils of new, short branches. The fruit has a strong sour taste, ripens to red in August to September, is 0.6 to 1.2 cm long, and has several hanging downwards in the shape of grape clusters. It is used for ornamental purposes and the fruit is used for medicinal purposes.

Ilcheongung is a perennial plant of the umbel family and is called by other names Cheongung (川芎), Gunggungi, and Waecheongung. It is effective in pain relief, antispasmodics, foaming, and blood flow.

Perilla seeds are the seeds of perilla seeds and contain a lot of unsaturated fatty acids. In addition to the above-mentioned effects such as suppressing inflammation, antioxidant effects, and anticancer effects in the human body, they also play roles such as activating antibacterial effects and increasing phagocytosis. It is known that

A gallbladder is an insect gall (bug nest made by aphids) of the Rhus chinensis family, which has an irregular sac shape and is hollow. The chemical components of gallnuts include gallotannin, resin, starch, and tannin, and in oriental medicine, it is mainly used for diseases such as astringency, hemostasis, detoxification, diarrhea, stomach ulcers, hematochezia, hematuria, burns, and stomatitis. In addition, gallnut has excellent antibacterial activity against Staphylococcus aureus, Streptococcus, Pneumococcus, Typhus, Shigella bacillus, and Pseudomonas aeruginosa, and as a result of toxicity tests, it is known to be toxic when administered intraperitoneally in mice.

The complex extract of golden, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, ilcheongung, perilla, and galleon chinensis contains 6 parts by weight of gold, 12 parts by weight of portulaca, 10 parts by weight of guaruin, 4 parts by weight of gilgyeong, and 4 parts by weight of ginseng. , It is characterized by mixing 4 parts by weight of Macmundong, 4 parts by weight of Schisandra chinensis, 4 parts by weight of Ilcheongung, 4 parts by weight of Jasoja, and 4 parts by weight of Five Baeja. Preferably, after grinding each of golden, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, Ilcheongung, perilla, and starberry, distilled water (DW) is added in a volume 10 times the volume of each grind, and then sterilized in a high-pressure steam sterilizer ( It was extracted for 2 hours in an autoclave and filtered three times. Afterwards, distilled water (DW) is removed using a vacuum concentrator and freeze-dried to separate each extracted powder to produce powder. The powders obtained were 6 parts by weight of gold, 12 parts by weight of portulaca quince, 10 parts by weight of guaruin, 4 parts by weight of gilgyeong, 4 parts by weight of ginseng, 4 parts by weight of Macmundong, 4 parts by weight of Schisandra chinensis, 4 parts by weight of Ilcheongung, and 4 parts by weight of Perilla. It can be prepared by mixing 4 parts by weight of starfish and 4 parts by weight.

The complex extract is characterized in that it is a lactic acid bacteria fermentation extract, and the lactic acid bacteria are selected from the group consisting of Streptococcus genus ( Steptococcus sp.), Lactobacillus genus ( Lactobascillus sp.), and Bifidobacterium sp. However, it is not limited thereto, and is preferably Lactobacillus plantarum .

According to the present inventor's experiments, it was confirmed that when the complex extract was further fermented using Lactobacillus plantarum strain, it exhibited a better lung cancer cell killing effect compared to the unfermented complex extract.

The complex extract exhibits an apoptotic effect on lung cancer cells and resistant lung cancer cells, but does not exhibit an apoptotic effect on normal liver cells. According to the present inventor's experiments, the complex extract shows an apoptotic effect on lung cancer cells A549 and A549-PTX, a paclitaxel-resistant lung cancer cell, but does not show an apoptotic effect on normal liver cells QSG-7701. It is characterized by not

The complex extract is characterized by increasing ROS levels in lung cancer cells and lung cancer cell mitochondria. It is known that cancer cells produce higher levels of ROS than normal cells to induce continuous proliferation, and it has recently been reported that derivatives that produce ROS induce specific ROS stress in cancer cells, leading to apoptosis of cancer cells. There is a bar. Therefore, the complex extract of the present invention can effectively induce death of lung cancer cells by increasing the specific ROS level of lung cancer cells and lung cancer cell mitochondria.

The complex extract is characterized by lowering the mitochondrial membrane potential in lung cancer cells, and the complex extract of the present invention can induce apoptosis of cancer cells not only through the generation of ROS but also through disruption of the mitochondrial membrane potential. .

The complex extract is characterized in that it has the effect of increasing TNF-α in the serum in individuals with cancer.

TNF-α (Tumor necrosis factor-α) is a substance directly involved in the inflammatory response. It attracts immune cells that cause inflammation, such as macrophages and neutrophils, while increasing their activity to induce an inflammatory response. Therefore, the complex extract of the present invention can have the effect of alleviating inflammation by reducing TNF-α in the serum.

In addition, the complex extract reduces the expression of p-AKT, p-Pi3k, and Blc-2 proteins in A549 and A549-PTX cells, and increases the expression of C-casepase3, C-casepase7, C-casepase9, and Bad proteins. It is characterized by According to the present inventor's experiments, when treated with the complex extract of the present invention, the expression of p-AKT, p-Pi3k, and Blc-2 proteins in A549 and A549-PTX cells was reduced, and the expression of C-casepase3, C-casepase7, and C -It was confirmed that the expression of casepase9 and Bad proteins was increased. Caspase 7, similar to other types of caspases, exists as an inactive proenzyme and is cleaved by caspase-8 or caspase-9, which is activated by apoptotic signal stimulation, and changes into the active form. Caspase 7 is an enzyme that is activated in the final stage of the cell death process and directly decomposes matrix proteins, so an increase in Caspase 7 protein within cells can be seen as promoting cell death.

Therefore, the complex extract of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Maekmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis of the present invention can also be used as an anti-inflammatory pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a carrier, excipient, or diluent in addition to the complex extracts of Hwanggeum, portulaca, guaruin, gilgyeong, ginseng, Maekmundong, Schisandra chinensis, Ilcheongung, perilla, and Chrysanthemum chinensis.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select the injection method for external application on the skin or intraperitoneal, rectal, intravenous, muscle, subcutaneous, intrauterine dura, or intracerebrovascular injection.

The pharmaceutical composition of the present invention may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycero gelatin, etc. can be used.

In addition, according to another embodiment of the present invention, providing a health functional food for preventing or improving cancer containing complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla and Chrysanthemum chinensis as active ingredients. do.

The characteristics of the active ingredients of the above-mentioned health functional food are the same as those of the active ingredients of the pharmaceutical composition, and detailed descriptions are omitted to avoid redundant explanation.

Health functional foods for preventing or improving lung cancer containing the above golden, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, Ilcheongung, perilla seed, and Chrysanthemum chinensis as active ingredients are beverages, pills, tablets, and capsules. , it can be manufactured with any one selected from powders, or by adding it as an ingredient in food, and can be manufactured appropriately according to conventional methods.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients.

Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Example 1> Preparation of complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong and ginseng

Sufficiently dried goldenrod, portulaca, guaruin, gilgyeong, and ginseng were each pulverized, then distilled water (DW) was added at 10 times the volume of each pulverized material, extracted for 2 hours in an autoclave, and then filtered three times. . Afterwards, distilled water (DW) was removed using a vacuum concentrator, and each extracted powder was separated by freeze-drying. The obtained powder was mixed with 6g of goldenrod, 12g of portulaca quince, 10g of guaruin, 4g of ginseng, and 4g of ginseng, and dissolved in distilled water (DW) before use.

<Example 2> Preparation of complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, perilla and galleon chinensis

After sufficiently drying, pulverize Macmundong, Schisandra chinensis, Ilcheongung, Jasoja, and Chrysanthemum chinensis, add 10 times the volume of distilled water (DW) to each grind, extract for 2 hours in a high-pressure steam sterilizer (autoclave), and filter three times. did. Afterwards, distilled water (DW) was removed using a vacuum concentrator, and each extracted powder was separated by freeze-drying. The powder obtained was mixed with 6g of gold, 12g of portulaca quince, 10g of guaruin, 4g of gilgyeong, 4g of ginseng, 4g of maekmundong, 4g of Schisandra chinensis, 4g of Ilcheongung, 4g of perilla seed, and 4g of shipwreck and used by dissolving in distilled water (DW). The present inventor designated the complex extract of Hwanggeum, Portulaca vulgaris, Guaruin, Gilgyeong, Ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis as LC2 and used them in the following experiments.

<Example 3> Preparation of lactic acid bacteria fermentation complex extract

The Lactobacillus plantarum strain used in the present invention was purchased from the Korea Culture Center of Microorganisms (KCCM) and used in experiments. The received strain was inoculated into a slope medium and cultured in an incubator at 37°C for 24 hours, and when colonies were formed, it was inoculated into a liquid medium and used.

1.0% (v/v) of Lactobacillus plantarum was inoculated into 100 mL of the complex extract of Example 2, fermented at 35°C for 48 hours, filtered, and freeze-dried to obtain Hwanggeum, Portulaca, Guarin, and Gilgyeong. , Ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis mixed fermentation complex extract powders were isolated.

<Comparative Example> Production of lactic acid bacteria fermentation complex extract

1.0% (v/v) of Lactobacillus plantarum was inoculated into 100 mL of the complex extract of Example 1, fermented at 35°C for 48 hours, filtered, and freeze-dried to produce Hwanggeum, Portulaca, Guarin, and Gilgyeong. and ginseng mixed fermentation complex extract powder were separated.

<Experimental example>

1. Cell culture

Human lung cancer cell lines A549 and A549PTX cells and normal hepatocyte WRL 6 cells distributed from the American Type Culture Collection were used in the experiment. Cells were cultured in Dulbecco's Modified Eagles medium (DMEM) containing 10% fetal bovine serum (Hyclone, South Logan, UT, USA) and 1% antimicrobial-antimycotic (Gibco, Invitrogen, Carlsbad, CA, USA). ) Cultured in culture medium at 37°C and 5% CO ₂ conditions.

2. Cytotoxicity assay

5,000 cells contained in 100ul of culture medium were inoculated into a 96-well plate (Nunc, Weisbaden, Germany) and cultured for 24 hours at 37°C and 5% CO ₂ conditions. To confirm cytotoxicity, cultured cells were treated with LC2 at concentrations of 0%, 0.5%, 1%, 2%, and 5% for 24 hours and then used as samples. In subsequent experiments, LC2 was used as samples at concentrations of 0%, 0.5%, 1%, 2%, and 5%. Samples treated for 24 hours at a concentration of 5% were used.

3. DHE, MitoSOX, Calcein-AM, JC-1 staining and measurement

To measure the effect on ROS levels in A549 and A549-PTX cells, DHE, MitoSOX, Calcein-AM, and Stained with JC-1 and observed under a fluorescence microscope.

4. In vivo testing

For in vivo tests for stability and toxicity of LC2, 8-week-old male BALB/c mice were used and reared in a clean room. The rearing conditions were brightly lit for 12 hours from 7 a.m. to 7 p.m., and the lights were turned off for the next 12 hours. Diet and water intake were measured.

5. Effects on dietary intake

The average daily dietary intake was measured for 7 days, and there was no difference between each group in any week of measurement. There was no difference in average daily dietary intake over 7 days.

6. Hematoxylin-eosin staining

Tissues were collected, fixed in 10% neutral buffered formalin solution, and paraffin-embedded tissue sections were prepared. The tissue was cut into 4μm thick pieces, washed with paraffin, stained with hematoxylin, fractionated with alcohol hydrochloric acid, and neutralized with aqueous ammonia. It was colored in running water, stained with eoxi, dehydrated with alcohol, and sealed. On the microscope, the nucleus was stained blue and the cytoplasm was stained red.

7. Statistical analysis

To confirm the reproducibility of the experimental results, the experimental results below are expressed as the average value ± standard deviation, and each experiment was performed three times. Statistical analysis of experimental results was performed using Graphpad Prism V5.01. One-way ANOVA was performed using Dunnett's method using software.

<Experiment results>

1. Confirmation of toxicity of complex extract to liver cancer cells

The complex extract powders of the above Examples and Comparative Examples were dissolved in distilled water (DW) at a concentration of 10% (w/v), and then treated with human lung cancer cells, A549, for 24 hours, respectively, and the survival rate of the A549 cells was measured. Cell viability was expressed as a percentage relative to the untreated control.

control group Example 1 Example 2 Example 3 Comparative example Cell survival rate (%) 100 74 ± 1.5 18 ± 2.4 11 ± 2.7 71±3.8

Referring to [Table 1], it was confirmed that the composite extract of Example 2 had a better lung cancer cell killing effect than the composite extract of Example 1. Therefore, the complex extract (LC2) of Example 2 shows an excellent cell killing effect on lung cancer cell lines and can be used to prevent or treat lung cancer.

In addition, according to [Table 1], it was confirmed that Example 3, in which LC2 was fermented with Lactobacillus plantarum strain, showed a better cell killing effect compared to Example 2, while in comparison In the case of the example, it was confirmed that, unlike Example 3, it did not affect the cell death effect.

Example 3 showed a superior cell killing effect compared to Example 2, but the difference was not significant and considering the convenience of extract preparation, the present inventors conducted additional experiments on the complex extract (LC2) of Example 2 below. did.

2, Measurement of cell viability of LC2

To measure the effect of LC2 on cell vitality, LC2 was centrifuged at 8000 r/min, the supernatant was filtered, and used for cell treatment (Figure 1A). Treatment of normal liver cells QSG-7701, lung cancer cells A549, and paclitaxel-resistant lung cancer cells A549-PTX for 24 hours at different LC2 drug concentrations (0%, 1%, 2%, 3%, 5%, and 10%). As a result, LC2 did not affect normal QSG-7701 cells (Figure 1B), and it was confirmed that it induced apoptosis in lung cancer cells A549 and lung cancer resistant cells A549-PTX (Figures 1C, D).

3. Measurement of cell migration and cell colony formation ability of LC2

To measure the effect of A549 and A549-PTX on cell regeneration and colony formation ability, treatment with LC2 drug (3% and 5% concentrations) for 24 h resulted in a significant decrease in cell migration ability (Figure 2A, C, D ). Additionally, the ability to form cell colonies was significantly reduced (Figure 2B, E, F). These results indicate that LC2 can reduce the migration and colony forming abilities of A549 and A549-PTX cells.

4. Measurement of the effect of LC2 on ROS levels in A549 and A549-PTX cells

To measure the effect on ROS levels in A549 and A549-PTX cells, LC2 drug (at concentrations of 3% and 5%) was treated for 24 h. As a result of fluorescence microscopy detection after DHE staining, the ROS level in A549 and A549-PTX cells increased (Figures 3A, 3B), and as a result of MitoSOX staining, the mitochondria within A549 and A549-PTX cells were damaged as the drug concentration increased, and the ROS level in A549 and A549-PTX cells increased significantly. increased (Figure 3C and Figure 3D).

5. Measurement of A549 and A549-PTX cell damage induction of LC2

To measure damage to A549 and A549-PTX cells and particles, the cells were treated with LC2 drug (3% and 5% concentrations) for 24 h. As a result, as a result of JC-1 staining, the membrane potential of A549 and A549-PTX cells was significantly lowered (Figures 4A and 4B). Additionally, when the survival rate was measured by calcium staining, the survival rate of A549 and A549-PTX cells was significantly lowered (Figures 4C and 4D). LC2 induces damage in A549 and A549-PTX cells.

6. Measurement of A549 and A549-PTX apoptosis in LC2

To determine whether A549 and A549-PTX cells were killed, they were treated with LC2 drug (3% and 5% concentration) for 24 h, and PI/Annexin V was measured using a fluorescence microscope. As a result of staining, A549 and A549- Death of PTX cells was induced (Figures 5A and 5B).

7. Measurement of changes in expression of A549 and A549-PTX apoptosis proteins in LC2

To measure the death mechanism of A549 and A549-PTX cells, Western blot was performed after treatment with LC2 drug (5% concentration) for 0, 3, 6, 12, and 24 h. As a result, the complex extract of the present invention reduces the expression of p-AKT, p-Pi3k, and Blc-2 proteins in A549 and A549-PTX cells, and increases the expression of C-casepase3, C-casepase7, C-casepase9, and Bad proteins. An increase was confirmed (Figure 6A, statistical analysis bar graph B-O). Therefore, it was confirmed that LC2 can cause death in A549 and A549-PTX cells through the AKT-Pi3k signaling pathway.

8. Measurement of changes in body temperature and feeding amount after heterologous transplantation of A549 and A549-PTX cells into mice

To measure the effect on the health of laboratory mice after LC2 treatment, BALB/c healthy laboratory mice of the same week of age were divided into a control group and a LC2 treatment group, 4 each, and A549 and A549-PTX cells were heterogeneously transplanted. Afterwards, the mice in the control group were given normal drinking water every day, and the LC2 treatment group was given LC2.

There was no difference in body temperature and body weight between the two groups (Figure 7C, D), and as a result of statistical analysis 7 days after drug intake, there was no difference in water intake between the two groups of mice (Figure 7E). This result showed that LC2 was It can be seen that there are no significant side effects on general physiological activities such as weight, body temperature, and water intake.

9. Measurement of tumor changes by L2 after heterogeneous transplantation of A549 and A549-PTX cells

To measure the effect on tumor growth after LC2 treatment, four normal BALB/c mice were selected from each group and A549 and A549-PTX cells were subcutaneously transplanted into the experimental mice. Control group mice were given normal drinking water every day and LC2 treated. The group consumed LC2. Seven days after ingestion, tumor tissue was extracted and its weight and diameter were measured. As a result of statistical analysis, it was found that the weight and size of the tumor in the LC2 treatment group were significantly lower than the control group, thereby suppressing tumor formation (Figures 8A-F).

10. Pathological analysis of mouse tumor tissue after LC2 treatment

To further explore the effect on mouse tumor suppression efficacy, A549 and A549-PTX cell tumors in the control group and LC2 treatment group were analyzed by Hematoxylin and Eosin staining (H&E) staining. As a result, it was confirmed through nuclear staining of the tumor that the size of the nuclei of the LC2 treatment group was reduced compared to the control group. We demonstrated that LC2 can inhibit tumor growth (Figures 9A, B).

11. Pathological analysis of mouse organ tissues after LC2 treatment

As a result of comparing the LC2-treated group and the control group, it was found that there was no infiltration of inflammatory cells and no lesions in the liver, spleen, lung, and kidney (Figure 10A). This result shows that LC2 is not toxic.

To further explore the cause of LC2 suppressing tumor growth, blood was collected from mouse eyes 7 days after LC2 administration and changes in inflammatory factors in serum were measured using the Elisa method. As a result, immunity was observed in the LC2-treated group and the control group. There was no significant change in the cell regulatory factors INF-γ, IL-6, and IL-10, and the inflammatory factor TNF-α appeared to increase significantly.

As a result, it was confirmed that LC2 can cause changes in inflammatory factor content.

Claims (12)

A pharmaceutical composition for preventing or treating lung cancer containing complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla chinensis and Chrysanthemum chinensis as active ingredients.
According to paragraph 1,
A pharmaceutical composition for preventing or treating lung cancer, wherein the complex extract of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla and Chrysanthemum chinensis is a hot water extract.
According to paragraph 1,
The complex extract of golden, portulaca, guaruin, gilgyeong, ginseng, maekmundong, Schisandra chinensis, ilcheongung, perilla, and galleon chinensis contains 6 parts by weight of gold, 12 parts by weight of portulaca, 10 parts by weight of guaruin, 4 parts by weight of gilgyeong, and 4 parts by weight of ginseng. , A pharmaceutical composition for preventing or treating lung cancer, characterized in that 4 parts by weight of Macmundong, 4 parts by weight of Schisandra chinensis, 4 parts by weight of Ilcheongung, 4 parts by weight of Perilla and 4 parts by weight of Chrysanthemum chinensis.
According to paragraph 1,
The complex extract is a lung cancer treatment, characterized in that the mixed powder of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla chinensis and Five chinensis hot water extract powder was dissolved in distilled water at a concentration of 1 to 10% (w/v). Pharmaceutical composition for prevention or treatment.
According to paragraph 1,
A pharmaceutical composition for preventing or treating lung cancer, wherein the complex extract is a lactic acid bacteria fermentation extract.
According to clause 5,
A pharmaceutical composition for preventing or treating lung cancer, wherein the lactic acid bacteria are selected from the group consisting of Steptococcus sp., Lactobascillus sp., and Bifidobacterium sp..
According to paragraph 1,
A pharmaceutical composition for preventing or treating lung cancer, wherein the complex extract exhibits an apoptotic effect on lung cancer cells, but does not exhibit an apoptotic effect on normal liver cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating lung cancer, wherein the complex extract increases ROS levels in lung cancer cells and lung cancer cell mitochondria.
According to paragraph 1,
The complex extract is a pharmaceutical composition for preventing or treating lung cancer, characterized in that it lowers the mitochondrial membrane potential in lung cancer cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating lung cancer, wherein the complex extract has the effect of increasing serum TNF-α in subjects with cancer.
According to paragraph 1,
The complex extract is used for lung cancer prevention or Pharmaceutical composition for therapeutic use.
A health functional food for preventing or improving lung cancer containing complex extracts of Hwanggeum, portulaca vulgaris, guaruin, gilgyeong, ginseng, Macmundong, Schisandra chinensis, Ilcheongung, Perilla perilla, and Chrysanthemum chinensis as active ingredients.