KR20230033937A - Composition for enchancing stimulatory activity comprising extracts of Platycodon grandiflorum and Chrysanthemum zawadskii - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity comprising, as an active ingredient, a mixed extract of Platycodon grandiflorum and Chrysanthemum zawadskii, wherein the mixed extract of Platycodon grandiflorum and Chrysanthemum zawadskii of the present invention has a suppressing effect of a significantly superior antioxidant activity, NO secretion activity of macrophages, and activity of inflammation-related substances compared to each extract of the Platycodon grandiflorum extract and the Chrysanthemum zawadskii extract, thereby being usefully used as a food or a pharmaceutical composition for enhancing immunity.

Description

Composition for enhancing immunity containing extracts of bellflower and gujeolcho {Composition for enchancing stimulatory activity comprising extracts of Platycodon grandiflorum and Chrysanthemum zawadskii}

Composition for enhancing immunity containing extracts of bellflower and gujeolcho {Composition for enchancing stimulatory activity comprising extracts of Platycodon grandiflorum and Chrysanthemum zawadskii}

The immune system can be divided into natural resistance, non-specific immune system and specific immune system. Natural resistance (first line of defense) refers to the anatomical and physiological factors that block all invaders, including microorganisms, regardless of their type, and non-specific immunity (second line of defense) is phagocytes that break through natural resistance and eliminate invaders entering the body. The bispecific immune system is a highly developed immune system with memory and the ability to distinguish between self and nonself. In addition, leukocytes constitute the second or third line of defense and break through the first line of defense to take charge of foreign substances entering the body. In the event of bacterial or viral infection or inflammatory reaction, the regulation of macrophage and lymphocyte activity determines the therapeutic effect of medicines. plays a pivotal role in

Immune function regulation is classified into immune function enhancement and immune function suppression. Immune function enhancement refers to contributing to enhancing the immune function of a weakened body, and may include enhancing normal immune function. Immune function suppression is the function of suppressing an excessive immune response.

Reactive oxygen species (ROS) such as superoxide anion (O ^2- ), hydrogen peroxide (H ₂ O ₂ ) and nitric oxide (NO) by activated macrophages Production is an important cytotoxic and cytostatic mechanism in non-specific immunity. In addition, macrophages are antigen presenting, tumoricidal, or microbicidal cells that play a central role in cell mediated or humoral immunity. NO produced from activated macrophages acts as a non-specific host defense mechanism, phagocytosis, and exhibits anti-proliferation activity of bacteria and cancer cells. In addition, macrophages have many lysosomes, which contain acid hydrolase and peroxidase. In addition, it has the property of strongly adhering to glass and plastic surfaces and actively eats microorganisms and tumor cells. The cells have cytokine receptors such as IFN-γ. They produce cytokines such as complement components, interferon, interleukin-1 and tumor necrosis factor, and their function can be enhanced by various cytokines produced from T-cells.

Therefore, many studies are currently being conducted to improve health through immunity enhancement worldwide, and recently, as side effects of synthetic compounds are emerging, research is focused on the development of materials that can effectively derive immunity enhancement from natural products. It is becoming.

Bellflower ( Platycodon grandiflorum ) is a perennial plant native to Korea, northeast China, and Japan, and grows in sunny mountain meadows. In addition, it is a perennial plant belonging to the bellflower family, and the root is thick, and the stem is solitary or foot-borne and has a height of 40 to 100 cm. Leaves are alternate or whorled, and are long egg-shaped or elliptical without petioles. Flowers bloom in June-August, and the flower color is blue-purple, bell-shaped, and blooms one by one at the end of the stem or branch. The system with white flowers is called Baekdoraji, and the root is used as a medicine or food for patients with hemoptysis. The root of bellflower has been used as an edible vegetable or medicinal, and the root is washed with water and dried or peeled and dried, and dried is called kyunggeun and is used medicinally. The root contains 2% of triterpenoid saponin and 0.03% of sterol [Saeki, T. et al., Planta Med. 65: 428 (1937)]. Other sugars include inulin and platycodinin (a polysaccharide composed of 10 fructose molecules). In oriental medicine, the main effect of bellflower is known to be good for symptoms such as cough, phlegm, sore throat, loss of sound, difficulty in urinating, diarrhea, and hemorrhage, which are closely related to lung disorders [Lee, KJ et al., Food Chem. Toxicol. 40: 517 (2002), Han, SB et al., Int. Immunopharmacol. 1: 1969 (2001), Choi, CY et al., Cancer Lett. 166: 17 (2001)]. The cancer cell proliferation inhibitory effect of bellflower extract [Lee Ji-young et al., Journal of the Korean Food Science Society, vol.30(1), 13-21, 1998] has been described, and only lymphocytes and solid cancers have been proven.

Gujeolcho ( Chrysanthemum zawadskii var. latilobum Kitamura ) is a perennial plant belonging to the Asteraceae family and is wild throughout Korea. In the private sector, it has been mainly used as a treatment for women's diseases such as coldness, menstrual pain, and menstrual irregularities.

Accordingly, the present inventors have made various attempts to find a material that can enhance the human body's immunity from various natural materials. The present invention has been completed.

One object of the present invention is to provide a composition for enhancing immunity comprising a mixed extract of bellflower and gujeolcho.

In addition, another object of the present invention is to provide a pharmaceutical or food composition for enhancing immunity comprising the composition.

Another object of the present invention is to provide a method for enhancing the immunity of an individual by administering the composition to the individual.

In one aspect, the present invention provides a composition for enhancing immunity comprising a mixed extract of bellflower and gujeolcho.

In the present invention, the bellflower ( Platycodon grandiflorum ) refers to a perennial plant of the bellflower family, which is native to Korea, northeastern China, and Japan and grows in the meadows of Sanya where sunlight is well exposed. Any parts such as flowers, leaves, stems, roots, and outposts may be used for the bellflower, but preferably any one of flowers, leaves, stems, and roots, more preferably roots.

In the present invention, the Gujeolcho refers to Chrysanthemum zawadskii var. latilobum Kitamura as a resource plant native to Korea, and specifically, Gujeolcho ( Chrysanthemu zawadskii (or sibirium) var. latilobum ), or a related plant of the same genus Pocheon Gujeolcho ( Chrysanthemum zawadskii var. tenuisectum ), Halla Gujeolcho ( Chrysanthemum zawadskii subsp. coreanum ), Nakdong Gujeolcho ( Chrysanthemum zawadskii subsp. Naktongense ) and Rock Gujeolcho ( Chrysanthemum zawadskii var. And, the gujeolcho may use any part such as flowers, leaves, stems, roots, and outposts, but preferably uses any one of flowers, leaves, and stems, more preferably leaves and stems. .

In the present invention, "extract" refers to a liquid component obtained by immersing a desired substance in various solvents, then extracting and fractionating a substance at room temperature or at elevated temperature for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It means the result of solid content, etc. In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.

The mixed extract of bellflower and gujeolcho of the present invention may be prepared by mixing bellflower and gujeolcho after preparing an extract or preparing extracts of bellflower and gujeolcho, respectively. Therefore, in the present invention, both the mixture of bellflower extract and Gujeolcho extract prepared by these methods, and the mixed extract of bellflower and Gujeolcho are named and used as the mixed extract of bellflower and Gujeolcho.

The mixed extract of bellflower and Gujeolcho can be obtained by extraction with water or various organic solvents. At this time, the organic solvent used is not particularly limited thereto, as long as an extract having an immune enhancing effect can be obtained, but may be preferably water, a polar solvent or a non-polar solvent, more preferably water and 1 to 1 carbon atoms. It may be a lower alcohol of 4 (ethanol, methanol, propanol or butanol, etc.), a mixed solvent thereof, and the like, and ethanol may be used most preferably.

In addition, the method for obtaining the extract is not particularly limited thereto, as long as an extract having an immune enhancing effect can be obtained. The extraction method includes cold needle extraction, ultrasonic extraction, or reflux cooling extraction method, and it is preferable to perform the ultrasonic extraction method.

The ultrasonic extraction is a method of extracting useful components from various natural materials at room temperature by physical force rather than chemical force using a vibration element having a frequency of 20,000 times or more per second, solvent extraction, vegetable oil extraction, or steam distillation extraction. Unlike, it has the advantage of being able to extract at high concentration without destroying the active ingredients contained in natural materials at room temperature.

As one specific example, the ultrasonic extraction can be carried out for 24 hours to 72 hours at room temperature using each or a mixture of dried bellflower and gujeolcho powder using a 60% to 80% ethanol solution. Preferably, it is carried out at room temperature for 36 hours to 60 hours using a 65% to 75% ethanol solution, and more preferably for 48 hours at room temperature using a 70% ethanol solution. If the value is out of the lower limit, the active ingredient of the present invention contained in the powder of bellflower and gujeolcho is not sufficiently extracted, and if it exceeds the upper limit, there is no significant difference in the amount of extraction of the active ingredient, impurities other than the active ingredient are extracted, and the efficiency of the process is improved. will decrease

In the present invention, "immunity" is a self-defense system that exists in the body, and refers to a process in which the human body recognizes various substances or living things invading from the outside as foreign substances to itself, removes them, and metabolizes them, and "immunity enhancement" means to enhance immune function.

In the present invention, the "active ingredient" refers to a component that exhibits the desired activity alone or that can exhibit activity together with a non-active carrier.

The active ingredient of the composition of the present invention, the combined extract of bellflower and gujeolcho, exhibits at least 1.6 times increased antioxidant effect compared to each of the extracts of bellflower and gujeolcho, increases the NO secretion activity of macrophages by at least 45 times, COX-2, etc. It has the effect of suppressing the expression of inflammation-related factors. Therefore, the mixed extract of bellflower and gujeolcho according to the present invention has a superior immune enhancing effect compared to each extract of bellflower and gujeolcho.

As one specific aspect, the composition of the present invention may be provided as a pharmaceutical composition for enhancing immunity.

In the present invention, the "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and diluents or excipients such as lubricants, wetting agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and surfactants It can be formulated and used as a parenteral formulation using. In addition, it may be formulated and used in the form of external preparations, suppositories, external preparations for the skin, and sterile injection solutions.

In addition, according to each formulation, pharmaceutically acceptable carriers such as buffers, analgesics, solubilizers, isotonic agents, stabilizers, and bases may be prepared by further including carriers known in the art. As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and characteristics of an active ingredient to be injected without stimulating living organisms. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more. The carrier may be a non-naturally occurring carrier.

The composition of the present invention can be administered in a pharmaceutically effective amount. The pharmacologically effective amount means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. It may be determined according to the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, it is generally 0.01 mg to 5000 mg per day per 1 kg of the body weight of the administered subject, and may be administered once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist, but is not limited thereto.

The mixed extract of bellflower and Gujeolcho included in the pharmaceutical composition is not particularly limited thereto, but is 0.00001 to 100% by weight, preferably 0.0001 to 15% by weight, more preferably 0.001 to 15% by weight, and more based on the total weight of the composition. It may preferably be included in 0.001 to 10% by weight.

In addition, the pharmaceutical composition of the present invention may be used alone or in combination with other pharmaceutically active compounds exhibiting an immune enhancing effect, or in a suitable set.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by those skilled in the art.

In the present invention, the "administration" means introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration of the composition of the present invention can be oral or parenteral as long as it can reach the target tissue. It can be administered via any route.

The administration method of the pharmaceutical composition according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration.

The pharmaceutical composition according to the present invention may be prepared in various formulations depending on the desired administration method.

The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose.

As another aspect, the present invention provides an immune enhancement method comprising the step of administering a composition containing a mixed extract of bellflower and gujeolcho.

In the present invention, "individual" may mean all animals, including humans, whose immune function is reduced or likely to be reduced. The animal may be not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but are not limited thereto.

Specifically, the enhancement method of the present invention may include administering the composition in a pharmaceutically effective amount to a subject at risk of or having reduced immunity. The administration method is as described above.

The enhancement may mean any action that suppresses or delays a decrease in immunity by administering the composition for enhancing immunity according to the present invention to a subject, or any action that improves or benefits immunity.

The pharmaceutical composition containing the mixed extract of bellflower and gujeolcho is as described above.

For administration, the composition of the present invention may contain pharmaceutically acceptable carriers, excipients or diluents in addition to the above-described active ingredients. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

An appropriate dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, and the time, but can be appropriately selected by those skilled in the art. Specifically, the dose of the mixed extract of bellflower and gujeolcho may be 50 ~ 500mg / Kg.

As another specific aspect, the present invention provides a food composition for enhancing immunity comprising a mixed extract of bellflower and gujeolcho as an active ingredient.

The immune-enhancing effect of the mixed extract of bellflower and gujeolcho is as described above.

When the composition of the present invention is used as a food additive, the bellflower and Gujeolcho mixed extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment), and may further include food additives acceptable in food science. Since the composition of the present invention uses an extract derived from a natural product as an active ingredient, there is no problem in terms of stability, so there is no great limitation on the mixing amount.

The food composition of the present invention may include all food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

The term "functional food" of the present invention refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body in accordance with the Act on Health Functional Foods No. 6727, and "functional" refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological functions.

In addition, the term "health functional food" of the present invention refers to food manufactured and processed by using specific ingredients as raw materials or by extracting, concentrating, refining, mixing, etc. specific ingredients contained in food ingredients for the purpose of health supplementation. It refers to food designed and processed by the above ingredients to sufficiently exert bioregulatory functions such as biodefense, regulation of biorhythm, prevention and recovery of disease, etc. It can perform functions related to recovery, etc.

There are no restrictions on the types of foods that can be used for the composition of the present invention for enhancing immunity. In addition, the composition containing the extracts of bellflower and gujeolcho of the present invention as an active ingredient can be prepared by mixing suitable other auxiliary ingredients and known additives that may be contained in food according to the selection of those skilled in the art. Examples of food that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and Vitamin complexes, etc., can be prepared by adding the extract and its fractions according to the present invention to juices, teas, jellies, juices, etc. prepared as main components.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplements, Seasoned foods (e.g. soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickles, etc.) , beverages (eg fruit, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (eg ramen soup, etc.)

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional components, like conventional beverages. In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, and glycerin. , alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages.

Since the mixed extract of bellflower and gujeolcho of the present invention is made of natural medicinal plants as raw materials, even when used as a pharmaceutical composition or food composition, side effects may be less than those of general synthetic compounds, so it can be safely included and usefully used.

The mixed extracts of bellflower and gujeolcho of the present invention exhibit increased antioxidant activity, macrophage NO secretion activity, and inhibitory activity of inflammation-related substances compared to the respective extracts. In particular, the mixed extract of bellflower and gujeolcho of the present invention increases the production of nitric oxide in a concentration-dependent manner, thereby contributing to the enhancement of immunity through the induction of the activation reaction of immune cells. Therefore, the mixed extract of bellflower and gujeolcho is edible, non-toxic, and has an excellent immune enhancing effect, so it can be usefully used as a pharmaceutical and food composition for enhancing immunity.

1 is a result of analyzing the cytotoxicity of the bellflower extract according to an embodiment of the present invention for each concentration of the macrophage cell line RAW 264.7 cells.
Figure 2 is a result of analyzing the cytotoxicity of the macrophage cell line RAW 264.7 cells of the gujeolcho extract according to an embodiment of the present invention by concentration.
Figure 3 is a result of analyzing the cytotoxicity of the bellflower extract according to an embodiment of the present invention to lung cancer cell line A549 cells by concentration.
Figure 4 is a result of analyzing the cytotoxicity of the lung cancer cell line A549 cells of the gujeolcho extract according to an embodiment of the present invention by concentration.
Figure 5 is a result of comparing the antioxidant effects of gujeolcho extract, bellflower extract, and these mixed extracts according to an embodiment of the present invention.
Figure 6 is an experimental result on the immune enhancing efficacy of gujeolcho extract and bellflower extract according to an embodiment of the present invention.
Figure 7 is a result of comparing the immune enhancing effect according to the amount of NO secretion of gujeolcho extract, bellflower extract, and these mixed extracts according to an embodiment of the present invention.
8 and 9 are the results of comparative analysis of the expression levels of COX-2 and β-actin of Gujeolcho extract and bellflower extract according to an embodiment of the present invention by concentration.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Preparation Example 1: Preparation of extracts of bellflower and Gujeolcho

Roots and leaves of bellflower grown for 3 to 6 years, and leaves and stems of Gujeolcho were purchased from Gyeongdong Market, washed, dried, and pulverized with a grinder, respectively. Bellflower powder was ultrasonically extracted in 80% ethanol and 1% acetic acid solution for 6 hours to prepare a bellflower extract, and Gujeolcho powder was ultrasonically extracted in 50% ethanol for 2 hours to prepare a Gujeolcho extract.

Example 1: Evaluation of cytotoxicity of mixed extracts of bellflower and gujeolcho according to the present invention

1-1. RAW 264.7 Intracellular Toxicity Assessment

Macrophage cell line RAW 264.7 cells were cultured in a 96-well plate at a concentration of 2 X 10 ⁴ cells/well, and after 24 hours of culture, the bellflower extract and Gujeolcho extract of Preparation Example 1 were prepared by concentration (0 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, 500 μg/ml, 1000 μg/ml, 2000 μg/ml) respectively. After 24 hours of treatment, 20 μl of MTS reagent was added to each well and reacted at room temperature for 30 minutes. After completion of the reaction, absorbance was measured in the 490 nm wavelength region. At this time, the above process was repeated a total of 3 times and the average value was measured to analyze the cytotoxicity of the extracts of bellflower and gujeolcho.

As a result, as can be seen in Figures 1 and 2, it was confirmed that the bellflower extract and gujeolcho extract of Preparation Example 1 did not have cytotoxicity to the macrophage cell line RAW 264.7 cells.

1-2. Assessment of A549 intracellular toxicity

Lung cancer cell line A549 cells were cultured in a 96-well plate at a concentration of 2 X 10 ⁴ cells/well, and after 24 hours of culture, the bellflower extract and Gujeolcho extract of Preparation Example 1 were prepared by concentration (0 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, 500 μg/ml, 1000 μg/ml, 2000 μg/ml) respectively. After 24 hours of treatment, 20 μl of MTS reagent was added to each well and reacted at room temperature for 30 minutes. After completion of the reaction, absorbance was measured in the 490 nm wavelength region. At this time, the above process was repeated a total of 3 times, and the average value was measured to analyze the cytotoxicity of the extracts of bellflower and gujeolcho.

As a result, as can be seen in Figures 3 and 4, it was confirmed that the bellflower extract and gujeolcho extract of Preparation Example 1 had no cytotoxicity to lung cancer cell line A549 cells.

Example 2: Polyphenol content analysis of bellflower extract and Gujeolcho extract

In a 96-well plate, 40 μl of each of the extracts of Preparation Example 1 of 1000 μg/ml of Bellflower and Gujeolcho was added, and 20 μl of Folin-Ciocalteu's phenol reagent (FC reagent) at a normal concentration of 1 was added to each well. In addition, 60 μl of 20% Na ₂ CO ₃ solution was added to each well and reacted for 30 minutes in a dark room. After completion of the reaction, absorbance was measured at 700 nm, and at this time, the above process was repeated a total of three times, and the polyphenol content was analyzed by comparing the average value with a gallic acid standard curve.

Example 3: Analysis of flavonoid content of bellflower extract and gujeolcho extract

Into a 96-well plate, 25 μl of each extract of the bellflower root and Gujeolcho of Preparation Example 1 at a concentration of 1000 μg/ml was added, and 125 μl of double distilled water was added to each well. Then, 8 μl of 5% NaNO ₂ was added to each well and reacted for 5 minutes. After completion of the reaction, 15 μl of 10% AlCl ₃ was added to each well and reacted for another 6 minutes. After completion of the reaction, 50 μl of 1M NaOH and 27 μl of double distilled water were added to each well. The solution in each well was mixed well and the absorbance was measured at 510 nm. At this time, the above process was repeated a total of 3 times and the average value was measured to analyze the flavonoid content.

Example 4: Antioxidant enhancement effect of mixed extracts of bellflower and gujeolcho

In a 96-well plate, 80 μl of each of the extracts of bellflower and Gujeolcho of Preparation Example 1 and their mixed extracts by concentration (125 μg / ml, 250 μg / ml, 500 μg / ml, 1000 μg / ml, 2000 μg / ml) put in each 80 μl of 0.2 mM DPPH solution was added to each well, and reacted in the dark for 30 minutes. After completion of the reaction, the absorbance was measured at 517 nM. At this time, the above process was repeated a total of three times, and the average value was measured to analyze the DPPH radical scavenging activity of the extracts of Bellflower and Gujeolcho. The results are shown in FIG. 5 .

As can be seen in Figure 5, compared to the DPPH radical scavenging activity of each of the bellflower extract and the gujeolcho extract, it can be confirmed that the mixed extract of bellflower and gujeolcho is about 1.6 times more active than the bellflower extract and about 2.8 times more than the extract of gujeolcho. there was.

Example 4: Immune-enhancing effect of the extract according to the present invention

2-1. Analysis of immunity enhancement efficacy of extracts of bellflower and gujeolcho

Macrophage cell line RAW 264.7 cells were cultured in a 24-well plate at a concentration of 2 X 10 ⁵ cells/well, and after 24 hours of culture, interferon-γ was treated at a concentration of 10 μg/ml, followed by preparation of Preparation Example 1. Extracts of bellflower and gujeolcho were treated at each concentration (62.5 μg/ml, 125 μg/ml, 250 μg/ml). After 24 hours of treatment, 80 μl of the culture medium from each well was transferred to a 96-well plate, and 80 μl of Griess reagent was added to each well of the 96-well plate, followed by reaction at room temperature for 15 minutes. After completion of the reaction, the absorbance was measured at 540 nm, and at this time, the above process was repeated a total of three times, and the average value was compared with the NaNO ₂ standard curve to analyze the immunity enhancing efficacy.

As can be seen in Figure 6, compared to the NO (Nitric Oxide) secretion activity of the untreated group, it was confirmed that the activity of the Gujeolcho extract treatment group increased by about 45 times and the activity of the bellflower extract treatment group increased by about 135 times.

2-2. Analysis of immune-enhancing synergistic efficacy of Gujeolcho and bellflower extracts

Macrophage cell line RAW 264.7 cells were cultured in a 24-well plate at a concentration of 2 X 10 ⁵ cells/well, and after 24 hours of culture, interferon-γ was treated at a concentration of 10 μg/ml, followed by preparation of Preparation Example 1. The extracts of bellflower and Gujeolcho of the present invention were prepared by concentration (Gujeolcho: 0 μg / ml, 62.5 μg / ml, 125 μg / ml, 250 μg / ml, Bellflower: 0 μg / ml, 31.25 μg / ml, 62.5 μg / ml, 125 μg/ml, 250 μg/ml, 500 μg/ml) and treated after mixing. After 24 hours of treatment with the bellflower extract, 80 μl of the culture medium from each well was transferred to a 96-well plate, and 80 μl of Griess reagent was added to each well of the 96-well plate, followed by reaction at room temperature for 15 minutes. After completion of the reaction, absorbance was measured at 540 nm. At this time, the above process was repeated a total of 3 times, and the average value was compared with the NaNO ₂ standard curve to analyze the synergistic effect of enhancing immunity.

As can be seen in Figure 7, compared to the bellflower extract or gujeolcho extract treatment group, it was confirmed that the NO secretion of the bellflower and gujeolcho mixed extract treatment group was high. In particular, it was confirmed that the mixture of 250 mg/mL of bellflower extract and 250 mg/mL of Gujeolcho extract increased the NO secretion by about 4 times compared to the group treated with Gujeolcho extract of the same concentration and by about 1.25 times compared to the group treated with bellflower extract.

Example 3: Respiratory inflammatory inhibitory activity of the extract according to the present invention

3-1. Analysis of inflammation-related biomarker (protein) changes in lung cancer cells

The human lung cancer cell line A549 and the macrophage cell line RAW264.7 were cultured in a 100 mm cell culture dish at a concentration of 50 X 10 ⁴ cell/mL, respectively. After 24 hours of cultivation, the bellflower extract and Gujeolcho extract of Preparation Example 1 were treated at different concentrations (125 μg/ml, 250 μg/ml). After 24 hours of treatment with these extracts, 200 μl of a cell lysis buffer was treated and the supernatant was obtained by centrifugation (14000 RPM, 10 minutes). The supernatant was quantified through a protein quantification test. 8 μg of the quantified protein was put into a 1.5 microtube, and 5 μl of sample buffer reagent containing β-mercaptoethanol was added, followed by reaction at 95° C. for 5 minutes. After completion of the reaction, proteins were separated by size by polyacrylamide gel electrophoresis at 125 V for 90 minutes, and proteins were transferred to a PVDF membrane using transfer turbo equipment (25 V for 17 minutes). PVDF membrane was treated with 3% BSA blocking buffer and reacted for 1 hour. After completion of the reaction, each primary antibody (COX 2, β-actin) was treated at 4° C. and reacted for 12 hours. After completion of the reaction, the secondary antibody was treated and reacted for 1 hour. After completion of the reaction, 1 mL of a solution in which horseradish peroxidase (HRP) reagent and HRP substrate reagent were mixed in a 1:1 ratio was treated, and fluorescence expression was measured to analyze protein expression changes. The results of this are shown in Figures 8 and 9.

As can be seen in Figures 8 and 9, it was confirmed that the COX 2 expression level of the bellflower extract-treated group was reduced by about 40% compared to the expression level of the non-treated group (cont).

3-2. Analysis of inflammation-related biomarker (mRNA) changes in lung cancer cells

The human lung cancer cell line A549 was cultured in a 100 mm cell culture dish at a concentration of 50 X 10 ⁴ cell/mL, respectively. After 24 hours of incubation, the bellflower extract, Gujeolcho extract, and mixed extracts thereof of Preparation Example 1 were treated by concentration (62.5 μg/ml, 125 μg/ml, 250 μg/ml), respectively. After 24 hours of these extracts, RNA was extracted from each cell culture dish using an RNA extraction kit, and the concentration and purity of the extracted RNA were measured using a nucleic acid concentration measuring instrument 'NaNo drop'. Based on the measured concentration, each RNA was quantified and synthesized into cDNA using a cDNA synthesis kit. 7 μl of double distilled water, 1 μl of forward primer, 1 μl of reverse primer, 10 μl of syber green reagent, and 1 μl of cDNA were added to each well of the PCR plate, and the change in fluorescence expression was measured using a qPCR machine. At this time, the above process was repeated a total of three times, and the change in mRNA transcription amount was analyzed by measuring the average value.

Claims (2)

A food composition for enhancing immunity comprising a mixed extract of bellflower and gujeolcho as an active ingredient.
A pharmaceutical composition for enhancing immunity comprising a mixed extract of bellflower and gujeolcho extract as an active ingredient.

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