KR102294268B1 - Pharmaceutical composition containing Crepidiastrum denticulatum Houtt. Pak kawano) extract for immune-enhancing - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing immunity, comprising a Crepidiastrum denticulatum leaf extract as an active ingredient. Provided is a functional food composition for enhancing immunity which promotes generation of NO and induces generation of cytokine of IL-2, IL-10, and IFN-γ, thereby enhancing biological immunity.

Description

A health functional food composition for immune enhancement comprising a leaf extract of Igodeulbaegi as an active ingredient {Pharmaceutical composition containing Crepidiastrum denticulatum Houtt. Pak kawano) extract for immune-enhancing}

The present invention relates to a composition for enhancing immunity comprising a leaf extract of Igodeulbaegi as an active ingredient, and more specifically, it uses a natural raw material, Crepidiastrum denticulatum (Houtt.) Pak&kawano leaf extract, without toxicity and side effects. It relates to a health functional food composition for enhancing immunity that can be safely used.

Immunity is a phenomenon in which the internal environment of a living body defends against external factors, and there are three different components of immunity. Our body has skin, airways, and mucous membranes, which act as the first barrier against infection. However, when a virus enters the body through this barrier, our innate immune response is activated, and when this response is insufficient, the adaptive immune system is activated. Adaptive immunity is an antibody that contains cells and proteins, and it takes days or weeks for the body to make antibodies.

Among T cells, T helper (Th) cells have Th1 and Th2 cells. Th1 cells produce cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ), and Th2 cells contain interleukin-4 (IL -4) and interleukin-10 (IL-10). Th1 cells and TH2 cells act exclusively with each other, and the cytokines they produce play an important role in the immune response.

IL-2 was called T cell growth factor (TCGF) present in the lymphocyte culture medium because it can maintain the growth of T lymphocytes showing cytotoxicity for a considerable period of time. After that, using the recombinant IL-2, T cells, natural killer cells (NK cells), lymphocyte-activated killer (LAK) cells, B cell growth factors, stimulators of monocyte-type cells, etc. It was found to be active.

IFN-γ is an acid-labile interferon, type II interferon, and is distinguished from acid-stable interferon, type I interferon, and type I interferon includes α and β. IFN-γ production occurs when T cells and NK cells are stimulated by mitogens, antibodies, or antigens. IFN-γ is involved in MHC antigen expression, macrophage activation, NK cell activation, regulation of IgG isotype, antiviral activity, and nitric oxide synthase (NOS) induction.

IL-10 is produced by Th2 cells and interferes with cytokine production by Th1 cells. Therefore, this molecule was initially called cytokine synthesis inhibitory factor (CSIF). However, various activities were further revealed by recombinant IL-10. IL-10 is produced in relatively various cells such as Th2 cells, macrophages, activated T cells, and mast cells. The function also shows various inhibitory and stimulatory activities.

Crepidiastrum denticulatum (Houtt.) Pak&kawano) is a plant belonging to the genus Asteraceae, biennial, distributed in Korea, Japan, and China, growing in dry places in mountains and fields. The roots of the plant are thick and plump, so it has been used as laver tea and pickled pickles, like godeulppae, and served as an appetite stimulant to stimulate appetite in spring. In addition, it is rich in chicholic acid, which enhances the activity of liver detoxification and antioxidant enzymes, including quinone reductase, an enzyme that is an indicator of the cancer-preventing effect.

The stem of Igodeulppaegi is 30~120cm high, and the branches are very branched and colored in purple. Stem leaves are alternate phyllotaxis, spatula-shaped, the lower part wraps around the stem a little, and there are irregular tooth-like teeth on the edge. The upper leaves of the stem surround the stem in the shape of an ear with a rounded bottom. The root leaves wither when the flower blooms. The underside of the leaves are white as if they were painted with powder. Flowers are yellow in August-October, with head-shaped flowers hanging from the tips of branches and stems like corymbae. There are 2 flower wrapper leaves. The fruits are aqua, black-brown.

Recently, interest in immunity is growing again due to the novel coronavirus. By studying the efficacy of natural products such as Igodeul-ppae, a domestic natural resource traditionally known to improve the immunity of children with weakened immunity, the elderly, diabetes patients, and those with cardiovascular disease, it has few side effects and helps in the prevention and recovery of diseases. We want to develop a material that can be

Korean Patent Application No. 10-2012-0113915 Korean Patent Registration No. 10-1710303 Korean Patent Registration No. 10-1871115

An object of the present invention is to provide a functional food composition having an immune-enhancing function containing, as an active ingredient, Igodeulpagi extract, which is a natural resource, which can be safely used without toxicity and side effects.

An object of the present invention is to provide a composition for increasing immunity that can be safely used without toxicity and side effects by using the extract of Korean iris, a natural resource. In order to achieve the above object, the present invention provides a functional food composition having an immune-enhancing function comprising the extract of Igodeul-ppaegi as an active ingredient.

The extraction method of a functional food composition having an immune enhancing function is performed by washing Igodeulbaegi with distilled water and then mixing with distilled water to extract hot water or at least one selected from the group consisting of alcohols having 1 to 5 carbon atoms or mixed solvents thereof. can be used for extraction.

The extract containing an extract soluble in any one of the water, methanol, ethanol, propanol, isopropanol, butanol, or a mixed solvent thereof as an active ingredient is a functional food for enhancing immunity by containing it in an amount of 0.01 to 99.9% by weight. A composition is provided. In addition, the health functional food composition for enhancing immunity is prepared by including the extract of the leaf extract in an amount of 0.1 to 2000 mg/kg body weight/day.

The composition may be prepared in one or more forms selected from tablets and capsules, soft capsules, granules, liquids, and beverages.

The extract of Igodeulbaegi having an immune enhancing function of the present invention is useful as a functional food composition useful for enhancing immunity by increasing mouse splenocyte proliferation and secretion of immune cytokines such as IL-2, IL-10, and IFN-γ in vitro. It has the effect of being used.

1 shows the results of confirming the toxicity to the extract of Igodeulbaegi.
Figure 2 is a change in IL-2 cytokine level of splenocytes with respect to the extract of Igodeulbaegi.
3 is a change in the IL-10 cytokine level of splenocytes with respect to the extract of Igodeulbaegi.
4 is a change in the level of IFN-γ cytokine in splenocytes with respect to the extract of Igodeulbaegi.
Figure 5 shows the concentrate of the hot water extract of the leaves of the present invention for the manufacture of health food.
6 shows a dried powder of the concentrate of the hot water extract of the leaves of the present invention for the manufacture of health food.

In order to help the understanding of the present invention, the configuration and effect of the present invention will be described in more detail through specific examples and comparative examples.

1. Preparation of Igodeulbaegi extract

1.1 Preparation of hot water extract

100 g of dried Igodeulbaegi leaves were washed with distilled water, and then 500 ml of distilled water was added, followed by heating and extraction at 100° C. in a reflux extractor for 3 hours. After filtration using filter paper (Whatman No. 41), the filtrate was concentrated under reduced pressure.

The residue remaining after filtration was extracted two more times in the same process using the same amount of distilled water again, filtered and concentrated under reduced pressure. The concentrated hot water extract was freeze-dried at 50° C. for 48 hours using a freeze dryer. According to the above method, the hot water extract was used as a sample in the following experimental example to obtain 10 g (10%) of the hot water extract from the leaves of Igodeulbaegi according to the extraction solvent.

2. Verification of cytotoxicity of extract

2.1 Macrophage Culture

Macrophage Raw 264.7 cells were cultured under the conditions of 37°C and 5% CO₂ using a growth medium containing DMEM medium, 10% FBS and 100 unit/ml PS. In order to solve the overdensity phenomenon caused by the proliferation of the cell number, subculture was performed every 48 hours to maintain an appropriate number of cells.

2.2 Measurement of cytotoxicity of extract

Raw 264.7 cells were aliquoted in a 24-well plate at 1×10 ⁵ cells/ml, cultured for 24 hours, and then treated with the extract of Igodeul-myeon in serum-free DMEM at each concentration (0.01, 0.1, 0.5, 1, 5 μg/ml). After 30 minutes, 5ug/ml of lipopolysaccharide (LPS) was treated and cultured for 24 hours. After 24 hours, it was washed with PBS, and 1ml of PBS was dispensed into each well, and 250μl of XTT was added and mixed by shaking lightly. After culturing in an incubator of 37°5% CO ₂ conditions for 2 hours, absorbance was measured at 450 nm using a microplate reader (Synergy HT, BioTek Instruments Inc.). As a result, it was confirmed that there was no cytotoxicity to the extracts of Igodeulbaegi (FIG. 1).

3. Measurement of Splenocyte Cytokine Secretion for Igodeul-baegi Extract

3.1. Splenocyte Extraction and Culture

Six-week-old Bablc/c male mice were killed by cervical dislocation, spleen removed aseptically, and crushed on a 100 mesh mesh to make single cells. Only the mononuclear cell layer was taken and centrifuged 3 times at 12,000 rpm for 5 minutes to prepare splenocytes. Splenocytes were diluted with RPMI 1640 medium containing 10% heat-inactivate FBS to 1 x 2 ⁵ cells/ml and dispensed in a 96-well plate by 100 μl.

3.2 Measurement of Cytokine Secretion for Igodeul-baegi Extract

The concentration of cytokines (IL-2, IL-10, IFN-γ) secreted by splenocytes was measured using an ELISA method. The amount of generated cytokines was measured using a Duoset sandwich ELISA Mouse kit (R&D system, USA).

In a 96-well plate for ELISA, the primary antibody characterized for each cytokine measurement was diluted in PBS, aliquoted in 100 μl, treated for one day, and the next day, after washing the primary antibody with washing buffer, the other To fill the space, a buffer was added and treated for 2 hours, and then washed with a washing buffer.

Put 100 μl of standard and sample into each well and react for 2 hours. After the reaction is over, wash with washing buffer. Dispense 100 μl of diluted buffer into each well and process for 2 hours. After this process, the plate was washed with a washing buffer, and 100 μl of a substrate that helps color development was added and reacted. The absorbance was measured in an ELISA reader 570 nm, and the amount of cytokines generated in the cells was calculated using a standard curve.

2, 3, and 4 show the secretion amounts of IL-2, IL-10 and IFN-γ cytokines from splenocytes for the extracts of Igo-deulbaegi. As shown in FIG. 2 , in the case of IL-2 secretion, it was confirmed that the secretion amount increased to 10.09±5.50 pg/ml compared to the control (1.87±2.42 pg/ml) at a concentration of 5 μg/ml of the hot water extract.

3 shows the effect of Igodeulbaegi hot water extract on IL-10 secretion in rat splenocytes. Cells were treated only with water and treated for 24 hours. Data values represent duplicates of the mean ± from each experiment. As shown in FIG. 3, IL-10 production was 7.90±1.15 pg/mL in the control, whereas it was 9.88±7.90 pg/mL at the concentration of 1ug/mL and 10.98±5.50 pg/mL at the concentration of 5ug/mL. It was confirmed that the secretion amount was significantly increased compared to the control.

Figure 4 shows the effect of Igodeulbaegi hot water extract on the amount of IFN-γ secretion in rat splenocytes. Cells were treated only with water and treated for 24 hours. Data values represent duplicates of the mean ± from each experiment. As shown in FIG. 4 , IFN-γ production was 40.50±80.64 pg/ml in the control, 65.83±39.98 at the concentration of 1ug/mL of the hot water extract, and 106.00±23.52 pg/ml at the concentration of 5ug/mL compared to the control. It was confirmed that the secretion amount was significantly increased.

Summarizing the above results, the present invention relates to a health functional food composition for immune enhancement comprising a leaf extract of oleifera leaf extract as an active ingredient, and according to the present invention, cytokine production of IL-2, IL-10 and IFN-γ By inducing it, it can exhibit the effect of enhancing biological immunity.

4. Manufacture of health food using Igodeulpagi extract

A pharmaceutical composition or health food composition for enhancing immunity using the leaf extract of Igodeulbaegi as an active ingredient may be prepared in the form of tablets and capsules, soft capsules, granules, and liquids. 5 shows the concentrate of the hot water extract of the leaves of Igodeulbaegi of the present invention for manufacturing health food, and FIG. 6 shows the dried powder of the concentrate of the extract of the leaves of Igodeulbaegi of the present invention.

According to another embodiment of the present invention, the composition may be prepared as a beverage additive. The immune-enhancing pharmaceutical or health food is a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, transdermal agent so that the composition comprising the leaf extract of E. , suppositories or sterile injectable solutions. In addition, the health functional food composition for enhancing immunity is prepared by including the extract of the leaf extract in an amount of 0.1 to 2000 mg/kg body weight/day.

The functional food composition for enhancing immunity containing the extract of the present invention as an active ingredient improves immunity by increasing mouse splenocyte proliferation and secretion of immune cytokines such as IL-2, IL-10 and IFN-γ in vitro. The activating effect was confirmed, and it can be usefully used as a functional food composition useful for immune enhancement. In addition, by replacing manufacturing raw materials with plants that live in nature, it is possible to expect reduction in manufacturing cost and import substitution and export effects through industrialization.

Claims (5)

A health functional food composition for enhancing immunity comprising the hot water extract of Crepidiastrum denticulatum (Houtt.) Pak&kawano) leaves as an active ingredient delete The health functional food composition for enhancing immunity according to claim 1, wherein the health functional food composition for enhancing immunity is prepared by containing the extract of the leaf extract in an amount of 0.1 to 2000 mg/kg body weight/day. The health functional food composition for enhancing immunity according to claim 1 or 3, wherein the health functional food composition for enhancing immunity is formulated as a powder, granule, tablet, capsule, suspension, emulsion, or syrup. delete

Images