KR102590151B1 - Pharmaceutical composition for preventing or treating of pancreatic cancer comprising medicinal herb complex extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of pancreatic cancer containing a complex extract of herbal medicine as an active ingredient, and specifically, a complex extract of Sanbaek bark, Baekbokryeong, Hubbark, Goryangkang, ginseng, dermis, Corydalis, Yin-Yang Gwak, Sain, and Chrysanthemum bark. It relates to a pharmaceutical composition for preventing or treating pancreatic cancer containing it as an active ingredient. The main mechanism of action of the complex extract of the present invention was found to induce accumulation of reactive oxygen species in cancer cells, resulting in cell cycle arrest and mitochondrial dysfunctin, ultimately causing cell death through PI3K-AKT-MAPK signaling.

Description

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

The present invention relates to a pharmaceutical composition for the prevention or treatment of pancreatic cancer containing a complex extract of herbal medicine as an active ingredient, and specifically, a complex extract of Sanbaek bark, Baekbokryeong, Hubbark, Goryangkang, ginseng, dermis, Corydalis, Yin-Yang Gwak, Sain, and Chrysanthemum bark. It relates to a pharmaceutical composition and health functional food for the prevention or treatment of pancreatic cancer containing it as an active ingredient.

Pancreatic cancer is a tumor mass made up of cancer cells that arise in the pancreas. There are several types of pancreatic cancer, and pancreatic adenocarcinoma, which arises from pancreatic duct cells, accounts for about 90%, so pancreatic cancer generally refers to pancreatic ductal adenocarcinoma.

Pancreatic cancer has been known to be a relatively rare cancer, but recently, like other cancers, the number of patients with pancreatic cancer has been steadily increasing as lifestyles have become westernized. Looking at the outbreak status, there are 9.8 men and 8 women per 100,000 population, which is close to the level of more than 10 in developed countries. Currently, pancreatic cancer ranks 8th in cancer incidence and 5th in mortality. It occurs more often in men than in women, less often before the age of 40, and mainly occurs after the age of 50.

Pancreatic cancer has no special early symptoms and has sufficient reserve function, making it very difficult to detect cancer in the early stages. In addition, the pancreas is thin, about 2 cm thick, and is covered only with a capsule. It is also in close contact with the superior mesenteric artery, which supplies oxygen to the small intestine, and the portal vein, which transports nutrients absorbed from the intestines to the liver, making it easy for cancer to invade. In addition, early metastasis occurs in the nerve bundles and lymph nodes behind the pancreas, and pancreatic cancer cells in particular have a fast growth rate.

Symptoms of pancreatic cancer are non-specific and may include symptoms seen in various pancreatic diseases, with abdominal pain, loss of appetite, weight loss, and jaundice being the most common symptoms. It appears differently depending on the location and size of the tumor and the degree of metastasis. Most patients with pancreatic cancer develop abdominal pain and weight loss, and most patients with pancreatic head cancer develop jaundice. Cancer that occurs in the body and tail of the pancreas rarely shows symptoms in the early stages and is often discovered over time.

In addition, there are symptoms such as steatorrhea or gray stools showing the appearance of oily stools due to incomplete digestion of fat, pain after eating, vomiting, and nausea. Diabetes may develop new or existing diabetes may worsen, and clinical symptoms of pancreatitis may appear. . A small number of patients may develop gastrointestinal bleeding, mental disorders such as depression or emotional anxiety, and superficial thrombophlebitis, and in rare cases, symptoms such as weakness, dizziness, chills, muscle cramps, and diarrhea may appear.

If a malignant tumor develops in the pancreas, the prognosis is poor, with a 5-year survival rate of only 8%. Only 30% of all pancreatic cancer patients are eligible for surgery in stage 1 (where cancer cells are located only in the pancreas) or stage 2 (where cancer cells have metastasized to surrounding tissues or lymph nodes). Surgery is not possible for stage 3 patients whose cancer has progressed significantly and stage 4 patients whose cancer has spread distantly to the liver or lungs. Fortunately, even if you are in stage 1 or 2 and undergo surgery, the 5-year survival rate is low at 20%.

Republic of Korea Patent No. 10-0950564

The purpose of the present invention is to provide a pharmaceutical composition for the prevention or treatment of pancreatic cancer containing complex extracts of Sangbaek bark, Baekbokryeong, Hubak, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Cory bark as active ingredients.

According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating pancreatic cancer is provided, comprising as an active ingredient a complex extract of Sanbaek bark, Baekbokryeong, Hubbark, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydalis as active ingredients.

According to another embodiment of the present invention, there is provided a health functional food for preventing or improving pancreatic cancer containing as effective ingredients extracts of Sanbaek bark, Baekbokryeong, Daebaek, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydera bark complex.

The complex extracts of Morus bark, Baekbokryeong, Daeguak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum bark are characterized in that they are hot water extracts.

The complex extract of Morus bark, Baekbokryeong, Hubbark, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Green bark contains 12 parts by weight of Moruscopi, 8 parts by weight of Baekbokryeong, 7 parts by weight of Hubbark, 4 parts by weight of Goryanggang, 4 parts by weight of ginseng, It is characterized in that it is mixed in a ratio of 4 parts by weight of dermis, 3 parts by weight of corydalis, 3 parts by weight of yin-yang gwak, 2.8 parts by weight of sine, and 2 parts by weight of green skin.

The complex extract is characterized by dissolving the mixed powder of thermal water extraction of Sanbaek bark, Baekbokryeong, Hubak, Goryanggang, ginseng, dermal bark, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum bark 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 pancreatic cancer cells, but does not exhibit an apoptotic effect on normal liver cells.

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

The complex extract is characterized by increasing mitochondrial membrane permeability in pancreatic cancer cells.

The complex extract is characterized by arresting the cell cycle of pancreatic cancer cells in the G2/M phase.

The complex extract is characterized as having the effect of increasing IL-6 in serum in subjects with cancer.

The complex extract is characterized by increasing phosphorylation of intracellular AKT, PI3K, ERK and P38 proteins.

The complex extracts of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Chrysanthemum bark of the present invention exhibit excellent anti-pancreatic cancer efficacy within the corresponding concentration (3-10%), and at the same time, they exert excellent anti-pancreatic cancer efficacy against normal cells. It is considered non-toxic. The main mechanism of action of the complex extract of the present invention was found to induce accumulation of reactive oxygen species in cancer cells, resulting in cell cycle arrest and mitochondrial dysfunctin, ultimately causing cell death through PI3K-AKT-MAPK signaling.

The results of in vivo experiments showed that PA2 administration did not affect the body temperature and body weight of xenograft model mice and did not cause damage to normal organs except tumor tissue. It also inhibited the growth of tumor size, weight, and volume while inhibiting the growth of IL. It was found that it has an anti-cancer function dependent on -6.

Figure 1 is a diagram showing the results of an analysis of the inhibitory effect on cancer cell activity of the complex extracts of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermal, Corydalis, Yin Yang Gwak, Sain, and Coryalis of the present invention.
Figure 2 is a diagram showing the results of an analysis of the inhibitory action on pancreatic cancer cell migration and colony formation of the complex extracts of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermal, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum bark of the present invention.
Figure 3 is a diagram showing the results of analyzing the effects of the complex extracts of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, Corydalis, Corydalis, Yin-Yanggwak, Sain, and Chrysanthemum bark of the present invention on reactive oxygen species and mitochondrial MMPs in PANC1 pancreatic cancer cells.
Figure 4 is a diagram showing the results of the inhibitory action on PANC1 pancreatic cancer apoptosis and cell cycle of the complex extracts of Sangbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermal, Corydalis, Yin Yang Gwak, Sain and Chrysanthemum bark of the present invention.
Figure 5 This figure shows the results of confirming the close relationship with the expression of the corresponding cyclin protein in the cells of the complex extracts of the present invention, Sangbaekpi, Baekbokryeong, Hubak, Goryanggang, Ginseng, Corydalis, Corydalis, Yin Yang Gwak, Sain and Chrysanthemum bark.
Figure 6 is a diagram showing the results of analyzing the effect on the expression of apoptosis-related proteins of the complex extracts of the present invention, Sangbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Chrysanthemum bark.
Figures 7 and 8 are diagrams showing the results of analyzing the effect on active oxygen of the complex extracts of Sanbaek bark, Baekbokryeong, Hubbark, Goryanggang, ginseng, dermal bark, Corydalis, Yin Yang Gwak, Sain and Green bark complex according to the present invention.
Figure 9 is a diagram showing the results of analyzing the effect on apoptosis and cell cycle of the complex extract of Sanbaek bark, Baekbokryeong, Hubak, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Cory bark of the present invention.
Figure 10 is a diagram showing the results of analyzing the effect of the complex extracts of the present invention, Sangbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydera ginseng on PANC1 pancreatic cancer cell signaling.
Figures 11 and 12 are diagrams showing the results of the PA2 anti-cancer efficacy analysis using a mouse model of the complex extracts of the present invention, Sanbaekpi, Baekbokryeong, Hubak, Goryanggan, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydera bark.
Figure 13 is a diagram showing the results of analyzing the effect on the organs of mice of the complex extract of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Chrysanthemum bark of the present invention.

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 the prevention or treatment of pancreatic cancer, comprising as active ingredients a complex extract of Sanbaek bark, Baekbokryeong, Hubbark, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Cory bark.

The Morus bark is the root bark of a plant from the genus Mulberry, a deciduous tree belonging to the Moraceae family. It can be used by peeling off the root bark and drying it in winter. It has a slightly unique smell, a sweet taste, and is cold in nature, so it has antipyretic and anticonvulsant properties. , It is used for anti-allergy and anti-inflammation, and can be used to treat symptoms such as asthma, high blood pressure, and edema. In addition, the extract of the Mori alba bark can increase hair follicles and show a hair growth effect.

Baekbongryeong (Poria cocas) is a mushroom from the Basidiomycete family of Aspergillus spp. It is a medicinal mushroom that has been used as an herbal medicine ingredient since ancient times. What is used medicinally is the sclerotia that this mushroom creates on underground pine roots, and fruiting bodies are rarely formed on the surface of the sclerotia when they are exposed to the ground. Sclerotia are potato-shaped or oval lumps with a diameter of 10 to 30 cm. The surface is dark brown and covered with a hard, wrinkled shell. As a diuretic, sedative, and tonic, it is used to treat edema, mental instability, palpitations, and indigestion. In addition, in terms of skin beauty, it has the effect of detoxifying, preventing freckles, and protecting (stabilizing) the skin.

Hubak has effects such as purifying energy (circulating energy), moisturizing (removing dampness), sojeok (removing odor), and pyeongcheon (stopping coughing). Stagnation of moisture), tightness in the chest and abdomen due to gas, abdominal pain and nausea, gastric congestion (food remains in the stomach and intestines without being digested), abdominal flatulence (bloated stomach) It is used to treat coughs caused by constipation, constipation, and phlegm (a condition in which fluid accumulates in the body due to the inability to dissolve normally).

Goryanggang is a perennial herb belonging to the ginger family and about 1 meter tall. The rhizome is dug up in the fall and dried. In traditional medicine, it is said to be effective in treating abdominal pain, vomiting, and diarrhea. It is also highly effective in treating abdominal pain and diarrhea caused by eating cold food, drinks, or fruit. It is also said to be effective in treating weak kidney function, loss of stamina due to poor circulation, diarrhea, and dysentery.

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.

Dermis is a medicinal herb made from dried tangerine peel. It is known to be spicy, non-toxic, and warm. When it enters the human body, it acts on the stomach, nose, and lungs to exert its effects.

Corydalis is a medicinal herb that facilitates the circulation of both blood and energy. It has analgesic, sedative, and antispasmodic effects by facilitating the circulation of energy and blood for the pain accompanying symptoms of blood or energy blockage. It has pharmacological effects such as increasing coronary blood flow and suppressing stomach ulcers. Therefore, Corydalis is used in Oriental medicine as a sedative, analgesic, and antispasmodic for hemostasis, anti-ulcer, etc., and is also prescribed for pain caused by nerve stimulation, such as abdominal pain, back pain, stomach pain, and headache. Corydalis is also prescribed for menstrual irregularities or dysmenorrhea caused by poor blood circulation in the body (Kim Dae-geun et al., Herbal Medicine, Shinilsangsa, pp.274-275, 2005).

Yin-Yang Gwak consists of a stem and leaves. The stem is thin and cylindrical, and the surface is yellow-green or light yellow and shiny. It has no odor, has a spicy and sweet taste, and is warm in nature. In Oriental medicine, it is used for erectile dysfunction, oil wells, cold uterus, cold extremities, skin paralysis, Guanwasa, forgetfulness, hemiplegia, weak waist and knees, high blood pressure, and polio. Pharmacological effects include promoting semen secretion, lowering blood pressure, increasing coronary blood flow, lowering blood sugar, lowering cholesterol, improving immune function, anti-tussive, expectorant, sedative, anti-bacterial, anti-inflammatory, and activating femoral bone growth and protein polysaccharide synthesis in chickens. there is.

Amomum xanthioides Wallich is a perennial herb belonging to the Zingiberaceae family and is distributed overseas in southern China, Southeast Asia, and India. The shape of the medicine is oval or oval with three blunt edges, 15 to 20 mm in length and 10 to 15 mm in diameter. In Donguibogam, it is recorded that it is used when the stomach hurts and is bulging and the food you ate does not go down and you feel nauseous and vomit.

“Citrii Unshiu Immaturi Pericarpium” is the unripe fruit peel of Citrus unshiu Markovich or Citrus reticulata Blanco, a tangerine tree belonging to the Rutaceae family. It is used when experiencing stress, stiffness in the side, or indigestion after eating food. It is known to be used for lumps in the chest, hemorrhoids, etc.

The complex extract of Morus bark, Baekbokryeong, Hubbark, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Green bark contains 12 parts by weight of Moruscopi, 8 parts by weight of Baekbokryeong, 7 parts by weight of Hubbark, 4 parts by weight of Goryanggang, 4 parts by weight of ginseng, It is characterized in that it is mixed in a ratio of 4 parts by weight of dermis, 3 parts by weight of corydalis, 3 parts by weight of yin-yang gwak, 2.8 parts by weight of sine, and 2 parts by weight of green skin. Preferably, after grinding each of Sanbaekpi, Baekbokryeong, Hubak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Cheongpi, distilled water (DW) is added at a volume of 10 times the volume of each grind, and sterilized in a high-pressure steam sterilizer (autoclave). ) was extracted for 2 hours and then 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 12 parts by weight of Morus bark, 8 parts by weight of Baekbokryeong, 7 parts by weight of Haebaek, 4 parts by weight of Goryanggang, 4 parts by weight of ginseng, 4 parts by weight of dermis, 3 parts by weight of Corydalis, 3 parts by weight of Yin Yang Gwak, and 2.8 parts by weight of Sain. and 2 parts by weight of green bark.

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 paracasei .

According to the present inventor's experiments, it was confirmed that when the complex extract was further fermented using Lactobacillus paracasei strain, it exhibited a superior pancreatic cancer cell killing effect compared to the unfermented complex extract. However, although not shown herein, when fermented with Lactobacillus plantarum strain, which is a strain of the same Lactobacillus genus, it did not increase the pancreatic cancer cell killing effect like the Lactobacillus paracasei strain. .

The complex extract exhibits an apoptotic effect on pancreatic cancer cells, but does not exhibit an apoptotic effect on normal liver cells. According to the present inventor's experiments, the complex extract exhibits an apoptotic effect on pancreatic cancer cells, PANC1, but does not exhibit an apoptotic effect on normal liver cells, WRL68.

The complex extract is characterized by increasing ROS levels in pancreatic cancer cells and pancreatic 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 pancreatic cancer cells by increasing the specific ROS level of pancreatic cancer cells and pancreatic cancer cell mitochondria.

The complex extract is characterized by increasing the mitochondrial membrane permeability in pancreatic cancer cells, and the complex extract of the present invention significantly increases the accumulation of reactive oxygen species in pancreatic cancer cells and increases the permeability of the mitochondrial membrane, thereby inhibiting cancer cell activity. Shows effect.

The complex extract is characterized by arresting the cell cycle of pancreatic cancer cells in the G2/M phase. As a result of detecting the expression of cell cycle-related proteins cyclin A, B, D, E and cell cycle inhibitory protein p21, the contents of cell cycle inhibitory protein p21 and G2/M phage arrest-related cyclin B were increased, while G0/G1 phage-related proteins were high. It was confirmed that the cyclin A, cyclin D, and cyclin E contents were significantly lowered.

The complex extract is characterized as having the effect of increasing IL-6 in serum in subjects with cancer. Interleukin-6 (IL-6) is a cytokine that plays an important role in acute phase response, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6 signals through a cell surface hetero-dymeric receptor complex composed of a ligand-binding subunit (IL-6Rα) and a signaling subunit (gp130). Since this increase in cytokines is considered a marker of immune system activity in the body, the complex extract of the present invention has the effect of activating the individual's immune system.

In addition, the complex extract is characterized by increasing phosphorylation of intracellular AKT, PI3K, ERK, and P38 proteins. As a result, it can be seen that the complex extract of the present invention increases the content of intracellular reactive oxygen species, which causes mitochondrial damage and induces death of PANC1 pancreatic cancer cells through cell cycle arrest of cancer cells.

The pharmaceutical composition of the present invention may further include a carrier, excipient, or diluent in addition to the complex extracts of Sanbaek bark, Baekbokryeong, Hubbark, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum bark complex.

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, there is provided a health functional food for preventing or improving cancer containing as active ingredients extracts of Sanbaek bark, Baekbokryeong, Dauphin, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydalis as active ingredients. .

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 pancreatic cancer containing the above-mentioned Morus bark, Baekbokryeong, Dauphin, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum complex extract as active ingredients are available in beverages, pills, tablets, capsules ( capsule), 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 Morus ginseng, Baekbongryeong, Daebi, Goryangkang, and ginseng

Sufficiently dried Morus bark, Baekbokryeong, Hubak, Goryanggang, 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 a high-pressure steam sterilizer (autoclave), and filtered three times. . Afterwards, distilled water (DW) was removed using a vacuum concentrator, and each extracted powder was separated by freeze-drying. The powders obtained were mixed with 12g of Morus bark, 8g of Baekbokryeong, 7g of green leaf custard, 4g of Goryanggang, and 4g of ginseng, and were used by dissolving them in distilled water (DW).

<Example 2> Preparation of complex extracts of Morus bark, Baekbokryeong, Daebi, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Green bark

After sufficiently drying the dermis, corydalis, yin-yang, sine, and green peel, each was pulverized, 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 powders obtained were mixed with 12g of Morus bark, 8g of Baekbokryeong, 7g of green leaf, 4g of Goryanggang, 4g of ginseng, 4g of dermis, 3g of Corydalis, 3g of Yin Yang Gwak, 2.8 g of Sain, and 2 g of green bark, and were used by dissolving in distilled water (DW). The present inventor designated the complex extracts of Sanbaek bark, Baekbokryeong, Hubble, Goryanggang, ginseng, dermal bark, Corydalis, Yin Yang Gwak, Sain and Green bark complex as PA2 and used them in the following experiments.

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

The Lactobacillus paracasei 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.

100 mL of the complex extract of Example 2 was inoculated with 1.0% (v/v) of Lactobacillus paracasei strain, fermented at 35°C for 48 hours, filtered, and freeze-dried to produce Morus lily, Baekbokryeong, Dauphin, and Goji. The combined fermentation complex extract powder of Ryanggang, ginseng, dermal, corydalis, yin-yang, sain, and green bark was isolated.

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

100 mL of the complex extract of Example 1 was inoculated with 1.0% (v/v) of Lactobacillus paracasei strain, fermented at 35°C for 48 hours, filtered, and freeze-dried to produce Morus L., Baekbongryeong, Huak, and Goji. Liangjiang and ginseng mixed fermentation complex extract powder was separated.

<Experimental example>

1. Cell culture

PANC-1 (pancreatic cancer cells) & WRL68 (human normal liver cells) cells were incubated with 10% fetal bovine serum (Hyclone, South Logan, UT, USA) and 1% antimicrobial-antimycotic (Gibco, Invitrogen, Carlsbad). , CA, USA) were cultured in Dulbecco's Modified Eagles medium (DMEM) or RPMI 1640 culture medium at 37°C and 5% CO ₂ conditions. The cells were subcultured after reaching 80% confluency, and cells from passage 3 were used in the experiment.

2. Pretreatment of composite materials

After natural precipitation of PA2 complex extract at 4℃, the supernatant was centrifuged at 12000rpm. The supernatant was filtered through a 0.2μm filter and treated with cells at various concentrations (0, 1, 3, 5, 10%) to measure cell viability.

3. Cell viability assay

5,000 cells contained in 100 ul of culture medium (1% FBS) were distributed in 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 PA2 complex extract at various concentrations (0, 1, 3, 5, and 10%) for 24 hours and then treated with the cell activation reagent MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added. 4 hours after MTT addition, the cell culture medium was removed, 100ul of DMSO solution was added to dissolve the MTT contained in the cells, and the OD value was measured at a wavelength of 490nm. The quantity of living cells was calculated according to the measured OD value, and the cytotoxic activity at each concentration was evaluated.

4. Cell migration and colony forming assay

After inoculating PANC1 cells in a 24 well plate (2.5Х105 cells per well), scratch the cells. The dead cells floating on the upper layer were removed, replaced with new culture medium, treated with PA2 complex extract, and cancer cell metastasis was measured by observing the change in the size of the space where the scratch was created 24 hours later.

PANC1 cells were inoculated into a 6-well plate (1.5Х103 cells/well) and cultured for 1 week with PA2 complex extract treatment. After 1 week, the cultured cells were fixed with paraformaldehyde, washed twice with 1ХPBS, then stained with crystal violet, and the stained cell clones were imaged.

5. Measurement of intracellular and intramitochondrial oxygen oxygen content

DHE and MitoSOX are used as fluorescence detectors to identify changes in intracellular (DHE) and intramitochondrial (MitoSOX) oxygen radical levels, respectively. PANC1 cells were inoculated into a 24 well plate (2Х10 ⁴ cells/well) and treated with PA2 complex extract for 24 hours. Each cell was fixed with 1% formalin solution and then stained with a fluorescence detector (processed according to the fluorescence detector user manual). The effect of PA2 complex extract on accumulation of reactive oxygen species in cancer cells was measured by observing the intracellular fluorescence level using a fluorescence microscope.

6. Mitochondrial MMP measurement

JC-1 dye is mainly used to detect cellular MMP (mitochondrial membrane potential). PANC1 cells were treated with PA2 complex extract for 24 hours and then stained with JC-1 dye. The intracellular JC-1 dye color change was observed using a fluorescence microscope (Red: normal intracellular MMP level; Green: indicates decreased intracellular MMP level).

7, Measurement of cell activity and intracellular calcium content

Calcein-AM is mainly a dye that can simultaneously measure cell activity and intracellular calcium content. The higher the fluorescence intensity, the greater the amount of living cells, and can simultaneously indicate the intracellular calcium content. PA2 complex extract was treated with PANC1 cells for 24 hours, stained with Calcein-AM, and intracellular Calcein-AM fluorescence intensity was measured using a fluorescence microscope.

8. Cell cycle measurements

To measure the cell cycle, a cell cycle measurement kit (PI/RNase buffer) was used. PANC1 cells were treated with PA2 complex extract for 24 hours and then fixed with 70% ETOH (cold). The effect of PA2 complex extract on the cell cycle was measured by quantifying the cell content of each stage using FACS.

9. western blot

Total intracellular protein was extracted, electrophoresed on a 12% SDS gel, and then transferred using nitrocellulose membranes. After blocking with 5% skim milk for 30 minutes, blotting was performed with the primary antibody required for the study, and TTBS (10 mM After washing 5 times with Tris-HCl (pH 7.5), 150 mM NaCl (Tris-buffered saline, TBS), 0.2% Tween 20), blotting was performed again with 2nd antibody at 1:2000, and the corresponding stain was detected using a chemiluminescence detection system. Protein content was analyzed.

10. Construction and treatment of in vivo tumor model using mice

Normal Balb/C mice (6-8 weeks old) were divided into two groups (10 mice/group) and then subcutaneously inoculated with PANC1 cells at 2x107/mice. After the subcutaneously inoculated tumors grew to a certain size (4-5 days), they each ingested a fixed amount of water and PA2 complex extract (Method: Only 5 mice were raised in each cage and kept from 18:00 to 6:00 AM every day. No water (water intake for 12 hours)). The next day, at 6:00 AM, 20 ml of DW and PA2 complex extract were provided to the control group (DW) and PA2 treatment group, respectively, to ensure that all mice in each group consumed sufficient water and PA2 complex extract. After drug treatment for 7 days, the size of the tumor was measured and changes in body weight and body temperature that occurred during the treatment were frequently checked.

11. Measurement of inflammatory factor content in serum

Serum INF-γ, TNF-, IL-6 and IL-10 contents of mice treated for 7 days (control group and PA2 treated group) were measured using respective measurement kits. INF-γ symbolizes changes in the body's T cell immune system, IL-6 and TNF- are representative cytokines that promote inflammatory responses, and IL-10 is a representative cytokine symbolizing anti-inflammatory and T-regular cell responses in the body. The change can be seen to some extent to reflect the immune response occurring in the body.

<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 pancreatic cancer cells, PANC-1, for 24 hours, respectively, and then the survival rate of PANC-1 cells was measured. did. 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 85 ± 1.2 15±1.6 8 ± 1.2 84±3.8

Referring to [Table 1], it was confirmed that the composite extract of Example 2 had a better pancreatic cancer cell killing effect than the composite extract of Example 1. Therefore, the complex extract (PA2) of Example 2 shows an excellent cell killing effect on pancreatic cancer cell lines and can be used to prevent or treat pancreatic cancer.

In addition, according to [Table 1], it was confirmed that Example 3, in which PA2 was fermented with Lactobacillus paracasei strain, exhibited a better cell killing effect compared to Example 2, while compared 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 (PA2) of Example 2 below. did.

2. Analysis of inhibitory effect of PA2 complex extract on cancer cell activity

The PA2 complex material was subjected to precipitation, centrifugation, and filtration, and the supernatant from which foreign substances were removed was used for cell experiments (Figure 1A). To measure the effect of the composite on cellular activity, human pancreatic cancer cells PANC1 and normal human hepatocytes WRL68 were treated with various concentrations of 1, 3, 5, and 10% (w/w) for 24 hours. As a result of measuring the effect of the composite on the MTT assay, it was found that the composite significantly inhibited pancreatic cancer cell activity at concentrations of 3%, 5%, and 10% (Figure 1C). At the same time, it was determined that treatment at the corresponding concentration had no effect on normal cell activity (Figure 1B). As a result, it can be seen that the PA2 complex extract has anticancer efficacy.

3. Analysis of the inhibitory effect of PA2 complex extract on pancreatic cancer cell migration and colony formation

To further reveal the anticancer efficacy of the PA2 complex extract, cell migration and colony forming abilities, which are important characteristics of cancer cells, were measured. As a result of treating PANC1 cells with 10% of the PA2 complex extract for 48 hours, the migration of cancer cells was significantly inhibited (Figure 2 A, C), and the colony forming phenomenon was also significantly reduced (Figure 2 B and D). As a result, it was confirmed that treatment with the PA2 complex extract significantly reduces the metastasis of pancreatic cancer and the proliferation of cancer cells.

4. Analysis of the effect of PA2 complex extract on reactive oxygen species and mitochondrial MMPs in PANC1 pancreatic cancer cells

Mass accumulation of active oxygen is one of the important factors that induce cancer cell death and is a method used in the evaluation of many anti-cancer drugs. To determine whether the anticancer efficacy of the PA2 complex extract is related to intracellular oxygen radicals, the PA2 complex extract was treated with PANC1 pancreatic cancer, and the oxygen content inside cells and mitochondria and mitochondrial MMPs were evaluated. Treatment with the PA2 complex extract was found to significantly increase not only intracellular oxygen radicals (Figure 3A) but also mitochondrial oxygen oxygen content (Figure 3B). At the same time, as a result of dye (JC-1) staining used for mitochondrial MMP detection, PA2 complex It can be seen that the extract increases mitochondrial MMP (mitochondrial membrane permeability) in cancer cells (Figure 3C).

In addition, through Calcein-Am staining (a dye for measuring intracellular calcium content, widely used to simultaneously evaluate cell activity), PA2 complex extract lowers PANC1 cell activity. These results show that the PA2 complex extract significantly increases the accumulation of reactive oxygen species within pancreatic cancer cells and inhibits cancer cell activity by increasing the permeability of the mitochondrial membrane.

5. Analysis of the inhibitory effect of PA2 complex extract on PANC1 pancreatic cancer apoptosis and cell cycle

Inhibition of cell activity ultimately induces cell death. It can be inferred that the PA2 complex extract inhibits the activity of PANC1 pancreatic cancer cells, which is evidence that it can induce cancer cell death.

To measure the effect of PA2 complex extract on PANC1 pancreatic cancer apoptosis, cells treated with PA2 complex extract for 24 hours were observed under a fluorescence microscope through Annexin-V/PI staining. As a result, cells treated with PA2 complex extract were found to be more effective than control cells. It was observed that Annexin-V-FITC (apoptosis marker) and PI (dead cell nuclear marker) positive cells increased (Figure 4A).

At the same time, as a result of measuring the effect of the PA2 complex extract on the cell cycle, it was found that treatment with the PA2 complex extract arrested the PANC1 cell cycle in G2/M phage (Figure 4B).

Cell cycle arrest is known to be closely related to the expression of the corresponding cyclin protein in the cell, and to elucidate the inhibitory mechanism of PA2 complex extract on the PANC1 pancreatic cancer cell cycle, cell cycle-related proteins cyclin A, B, D, and E were examined. As a result of measuring the expression of cell cycle inhibitory protein p21, the contents of cell cycle inhibitory protein p21 and cyclin B related to G2/M phage arrest were increased, while the contents of G0/G1 phage related proteins cyclin A, cyclin D, and cyclin E were significantly lowered. This can be seen (Figure 5A). Figures 5B-F are quantitative displays of the results of Figure 5A. As a result, it can be seen that treatment with PA2 complex extract inhibits the cell cycle of PANC1 pancreatic cancer cells, thereby lowering cancer cell proliferation and causing cancer cell death.

6. Mitochondrion-dependent death mechanism of PA2 complex extract

Initial research results showed that treatment with PA2 complex extract upregulated mitochondrial MMP expression, ultimately leading to death of pancreatic cancer cells. In order to determine the anticancer effect mechanism of the PA2 complex extract, we first verified the expression of apoptosis-related proteins that occur depending on mitochondrial signals. As a result, the expression of apoptosis-promoting proteins Bax and c-caspase-3 increased with treatment of PA2. While the amount was increased, the protein content of Bcl2, an anti-apoptotic agent, was found to be decreased (Figure 6A). Figures 6B-D are quantitative representations of the results of Figure 6A.

7. Effect of PA2 complex extract on intracellular oxygen radicals

Previous research results have demonstrated that treatment with PA2 complex extract increases the content of reactive oxygen species within PANC1 pancreatic cancer cells. To determine the effect of reactive oxygen species on the anti-cancer efficacy of PA2 complex material, pretreatment with NAC, a reactive oxygen radical scavenger, was performed. PANC1 cells were treated with PA2 complex extract for 24 hours.

Referring to Figure 7, it was confirmed that treatment with NAC, an active oxygen inhibitor, significantly inhibited cancer cell metastasis (Figure 7 A, C) and colony forming ability (Figure 7 B, D).

As a result, it was found that factors such as intracellular reactive oxygen species (Figure 8A), mitochondrial reactive oxygen species (Figure 8B), mitochondrial MMP (Figure 8C), and cell activity (Figure 8D) were significantly lowered with NAC pretreatment compared to PA2 treatment. You can.

It was observed that both PA2 effects on apoptosis (Fig. 9 A) and cell cycle (Fig. 9 B-D) were alleviated by NAC pretreatment, and reactive oxygen species accumulated within cells are an important factor in the anticancer efficacy of PA2 complex extract. You can see that it is.

8. Analysis of the effect of PA2 complex extract on PANC1 pancreatic cancer cell signal

The death of cancer cells is controlled by various intracellular signals. In particular, AKT, PI3K, and MAPK, which are greatly affected by active oxygen, are known to be important signals of cell death caused by active oxygen. Additionally, this study showed that PA2 complex extract induced death of PANC1 pancreatic cancer cells by arresting the cell cycle.

Based on this previous research, it is expected that the anticancer efficacy mechanism of PA2 complex extract most likely includes proteins related to cell cycle inhibition and signals driven by the accumulation of active oxygen radicals.

As a result of analysis of related protein expression through western blotting, PA2 complex extract improved the expression of cell cycle inhibitory proteins p21 and p16 in PANC1 pancreatic cancer cells, and at the same time, it increased the expression of various kinases such as AKT, PI3K, ERK, and P38, which are regulated by reactive oxygen species. It was shown to increase phosphorylation (Fig. 10A).

Figures 10B-G are quantitative representations of the results of Figure 10A. As a result, it can be seen that the PA2 complex extract increases the content of intracellular reactive oxygen species, which causes mitochondrial damage and induces death of PANC1 pancreatic cancer cells through cell cycle arrest of cancer cells.

Additionally, this action was shown to ultimately induce mitochondria-dependent death by activating intracellular PI3K-AKT-MAPK signals. As a result, PA2 complex extract can be regarded as a substance that acts as an anticancer agent that inhibits the cell cycle and causes cancer cell death by inducing the accumulation of reactive oxygen species in cancer cells and causing mitochondrial damage.

9. Analysis of PA2 anticancer efficacy using mouse model

In order to analyze the actual anticancer efficacy of the PA2 complex extract, a xenograft model using mice was used. After administering the PA2 complex extract to the two groups (control, PA2), the mouse body weight (Fig. 11A) and body temperature (Fig. 11B) were measured, and both the control and treatment groups showed no change. As a result, it was confirmed that the PA2 complex extract had no side effects in vivo.

As a result of administration of PA2 complex extract for 7 days, the size of the subcutaneous tumor in the PA2 treatment group's xenograft model was significantly smaller than that of the control group (Fig. 12A), and the tumor weight (Fig. 12B), diameter (Fig. 12C), and It was confirmed that factors such as volume (Fig. 12D) were reduced compared to the control group, confirming that tumor growth was suppressed.

10. Analysis of the effects of PA2 complex extract on other organs

It is widely known that the side effects of anticancer substances cause damage not only to cancer cells but also to normal organs. After administration of the PA2 complex extract, pathological analysis (H&E staining) was performed on various organs in the mouse, and it was found that there was no difference between the two groups in organs such as liver, spleen, lung, and kidney. appeared, and no significant pathological changes were detected in all organs (Figure 13).

At the same time, as a result of pathological analysis of the tumors in the PA2 complex extract treatment group, it was observed that the cell content within the tumor in the PA2 treatment group was significantly reduced compared to the control group.

11. Analysis of the effect of PA2 complex extract on inflammatory factors in mouse serum

Inflammatory factors in serum are one of the important factors that cause the death of various cells, and the increase in cytokines during anticancer treatment is also considered a marker of the body's immune system activity. The PA2 complex extract used in this study significantly increased the IL-6 content in mouse serum, but had no effect on the content of IFN-r, IL-10, and TNF-a (Fig. 14).

This result suggests that the PA2 complex extract has the function of regulating the body's IL-6-related immune system, and at the same time, it is believed that there is no significant correlation with T cell immunity or Treg cell (regulatory T cell) immunity.

Claims (13)

A pharmaceutical composition for the prevention or treatment of pancreatic cancer comprising extracts of Morus bark, Baekbokryeong, Daeguak, Goryangkang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Corydera bark complex extract as active ingredients.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, characterized in that the complex extracts of Sanbaek bark, Baekbokryeong, Daeguak, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Chrysanthemum bark are hot water extracts.
According to paragraph 1,
The complex extract of Morus bark, Baekbokryeong, Hubbark, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain and Green bark contains 12 parts by weight of Moruscopi, 8 parts by weight of Baekbokryeong, 7 parts by weight of Hubbark, 4 parts by weight of Goryanggang, 4 parts by weight of ginseng, A pharmaceutical composition for preventing or treating pancreatic cancer, characterized in that it is mixed in a ratio of 4 parts by weight of dermis, 3 parts by weight of corydalis, 3 parts by weight of yin-yang gwak, 2.8 parts by weight of sine, and 2 parts by weight of green bark.
According to paragraph 1,
The complex extract is a pancreatic cancer prevention method, characterized in that the mixed powder of thermal water extraction of Sanbaek bark, Baekbokryeong, Hubbark, Goryanggang, ginseng, dermis, Corydalis, Yin Yang Gwak, Sain, and Blueberry bark was dissolved in distilled water at a concentration of 1 to 10% (w/v). or therapeutic pharmaceutical compositions.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, wherein the complex extract is a lactic acid bacteria fermentation extract.
According to clause 5,
A pharmaceutical composition for preventing or treating pancreatic 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 pancreatic cancer, wherein the complex extract exhibits an apoptotic effect on pancreatic cancer cells, but does not exhibit an apoptotic effect on normal liver cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, wherein the complex extract increases ROS levels in pancreatic cancer cells and mitochondria of pancreatic cancer cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, wherein the complex extract increases mitochondrial membrane permeability in pancreatic cancer cells.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, wherein the complex extract stops the cell cycle of pancreatic cancer cells in the G2/M phase.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, which has the effect of increasing serum IL-6 in a subject with cancer.
According to paragraph 1,
A pharmaceutical composition for preventing or treating pancreatic cancer, wherein the complex extract increases phosphorylation of intracellular AKT, PI3K, ERK and P38 proteins.
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