KR102349970B1 - Polysaccharide from Celosia cristata flower extract, method of extracting the same and immunostimulating composition comprising the same - Google Patents

Abstract

The present invention relates to a polysaccharide derived from a cockscomb flower extract, an extraction method thereof, and a composition for enhancing immunity comprising the same, and more particularly, includes xylose, rhamnose, fucose, arabinose, galactose, glucose, glucuronic acid and / Or it relates to a polysaccharide derived from a cockscomb flower extract further comprising a monosaccharide consisting of galacturonic acid, a method for extracting the polysaccharide, and a composition for enhancing immunity comprising the polysaccharide.

Description

Polysaccharide from Celosia cristata flower extract, method of extracting the same and immunostimulating composition comprising the same

The present invention relates to a polysaccharide derived from a cockscomb flower extract, an extraction method thereof, and a composition for enhancing immunity comprising the same, and more particularly, includes xylose, rhamnose, fucose, arabinose, galactose, glucose, glucuronic acid and / Or it relates to a polysaccharide derived from a cockscomb flower extract further comprising a monosaccharide consisting of galacturonic acid, a method for extracting the polysaccharide, and a composition for enhancing immunity comprising the polysaccharide.

Recently, various pharmacological activities have been observed in polymer fractions of hot water extracts of food ingredients and medicinal herbs. In particular, high immune enhancing activity of plant-derived polysaccharides has been reported, which has been demonstrated to have relatively low toxicity and broad-spectrum therapeutic properties.

In addition, these polysaccharides are currently considered to play a potential role as a material for functional foods. In recent decades, polysaccharides isolated from vegetable sources (mushrooms, algae, moss and higher plants) have received much attention in the biomedical field due to their broad therapeutic properties and relatively low toxicity (Non-Patent Document 1, Non-Patent Document 2) .

Cockscomb ( Celosia) cristata L.) is an edible herb of Celosia (Amaranthaceae), widely distributed in subtropical and tropical regions of the world, such as southern China, Africa, India and South America, especially its dry and mature seeds, which are used to treat hypertension, paralysis, cataract, diabetes and keratitis. Although it is known to cause the same disease, studies on the immune stimulation of flowers have not been conducted yet.

In the art related to extraction gaemaendeurami cockscomb (Celosia argenta ) Immune regulation and immune enhancing effect of seed extract (Non-Patent Document 3), a composition for immunomodulatory or immune enhancement containing as an active ingredient a mixed herbal extract composed of Gamanthrax outpost, garlic and radish (Patent Document 1), etc. are disclosed. has been

However, the prior art discloses only the immune enhancing effect of the extract of cockscomb seed or outpost, but does not disclose the immune enhancing effect of the polysaccharide derived from the cockscomb flower extract. It is being demanded.

Laid-open Patent Publication No. 10-2016-0005197

Clin. Microbiol. Rev. 2000, 13:523-533 Curr. Org. Chem. 2001, 5:939-950 Planta Medica 1997, 63:216-219

The present invention has been devised to solve the problems of the prior art as described above, and an object of the present invention is to provide a polysaccharide derived from a cockscomb flower extract.

Another object of the present invention is to provide a method for extracting polysaccharides derived from a cockscomb flower extract.

Another object of the present invention is to provide an immune-enhancing use of polysaccharides derived from cockscomb flower extract.

In order to achieve the above object, the present invention provides a polysaccharide derived from a cockscomb flower extract containing xylose.

In addition, the present invention provides a step of (a) filtering the extract obtained by hot water extraction of the washed cockscomb flower, (b) adding ethanol to the filtrate of the extract obtained by the filtration and mixing, followed by centrifugation, ( c) removing the protein of the precipitate precipitated by centrifugation, (d) dissolving the precipitate from which the protein has been removed in water, and (e) lyophilizing the precipitate dissolved in the water It provides a method for extracting polysaccharides derived from a cockscomb flower extract.

In addition, the present invention provides a composition for enhancing immunity comprising the polysaccharide derived from the extract of cockscomb flower.

According to the present invention, by including polysaccharides extracted from cockscomb flower extract, it stimulates protein receptors to activate macrophages, and for immune enhancement that can regulate immune decline by immune stimulation through the activation of macrophages compositions may be provided.

1 shows a process for extracting polysaccharides derived from a cockscomb flower extract according to an embodiment of the present invention.
Figure 2 shows the effect of polysaccharide (CCP) derived from cockscomb flower extract on cell survival and NO production.
3 shows the effect of polysaccharide (CCP) derived from cockscomb flower extract on the expression of TNF-α and IL-6.
Figure 4 shows the effect of polysaccharide (CCP) derived from cockscomb flower extract on the expression of iNOS and COX-2.
5 shows the effect of polysaccharide (CCP) derived from cockscomb flower extract on NF-κB activation and IkBα degradation.
6 shows the effect of polysaccharide (CCP) derived from cockscomb flower extract on MAPK phosphorylation.

The terminology used in the present invention is used to describe exemplary embodiments only, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In the present invention, terms such as "comprises" or "have" are intended to designate the existence of an embodied feature, step, element, or a combination thereof, and one or more other features, steps, element, or these It should be understood that it does not preclude the possibility of the existence or addition of combinations.

In the present invention, "immune enhancement" refers to a function of promoting an immune response to an antigen non-specifically in the initial activation process of immune cells or a function of enhancing immunity by increasing the activity of cells of the immune system.

In the present invention, "health functional food" means a food in which the functionality of general food is improved by adding polysaccharide derived from cockscomb flower extract of one embodiment of the present invention to general food. Functionality can be roughly divided into physical properties and physiological functionality. When the polysaccharide derived from the cockscomb flower extract of the present invention is added to general food, the physical properties and physiological functionality of general food will be improved, and the present invention provides food with such improved functions. It is comprehensively defined as 'health functional food'.

In the present invention, the term "pharmaceutical composition" refers to a drug used for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases in animals including humans, unless otherwise explicitly stated. It is to be understood that not only a single unit dosage form such as a tablet, capsule, or injection, but also a plurality of unit dosage forms administered in two or more divided doses can be encompassed and referred to.

A first embodiment of the present invention relates to a polysaccharide derived from a cockscomb flower extract comprising xylose.

In the polysaccharide derived from the cockscomb flower extract of the present invention, the polysaccharide is, in addition to the xylose, rhamnose, fucose, arabinose, mannose, galactose , may further include one or more monosaccharides selected from the group consisting of glucose, glucuronic acid, and galacturonic acid.

On the other hand, the non-patent document 3 Gamandrami ( Celosia) argenta ) seed extract consists only of rhamnose, fucose, arabinose, mannose, galactose, glucose, glucuronic acid, galacturonic acid, etc. (see page 216), which is included in the polysaccharide derived from the cockscomb flower extract of the present invention. There are differences that Xylose does not include.

In the polysaccharide derived from the cockscomb flower extract of the present invention, the polysaccharide is xylose 0.6 to 0.8 mol%, rhamnose 2.2 to 2.8 mol%, fucose 0.2 to 0.4 mol%, arabinose 5.6 to the total hydrocarbon of the extract 8.2 mol%, mannose 0.9-2.7 mol%, galactose 5.6-7.4 mol%, glucose 11.1-13.9 mol%, glucuronic acid 1.6-2.0 mol%, and galacturonic acid 53.7-65.5 mol%.

In the polysaccharide derived from the cockscomb flower extract of the present invention, the cockscomb flower extract further contains uronic acid, protein and polyphenol in addition to the polysaccharide, but 2-keto-3-deoxy-D-manno- May not contain octulosonic acid (2-keto-3-deoxy-D-manno-octulosonic acid)-like material (KDO-like material).

In the polysaccharide derived from the cockscomb flower extract of the present invention, the cockscomb flower extract is 50.5-51.1 wt% of the polysaccharide, 43-49.9 wt% of the uronic acid, 5.2-6 wt% of the protein, relative to the total weight of the cockscomb flower extract and 0.7 to 0.9 wt% of the polyphenol.

A second embodiment of the present invention is (a) washed cockscomb ( Celosia) cristata L.) filtering the extract obtained by hot water extraction of flowers, (b) adding ethanol to the filtrate of the extract obtained by the filtration and mixing, followed by centrifugation, (c) by the centrifugation Removing the protein of the precipitated precipitate, (d) dissolving the precipitate from which the protein has been removed in water, and (e) lyophilizing the precipitate dissolved in the water. It is about the extraction method.

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the hot water extraction may be performed at 90 to 110° C., preferably at 95 to 100° C., for 1 to 2 hours, preferably for 80 to 100 minutes.

In step (b) of the method for extracting polysaccharide derived from the cockscomb flower extract of the present invention, 3 to 5 times, preferably 4 times the volume of ethanol is added to the filtrate of the extract compared to the volume of the filtrate, and the ethanol The concentration may be 80-95% (v/v).

In the method for extracting polysaccharides derived from the cockscomb flower extract of the present invention, the removal of the protein may be performed by treating a Sevag reagent containing n-butanol and chloroform in a volume ratio of 1:4, but is limited thereto. it is not

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the centrifugation may be performed at 2500 to 3500 rpm, preferably at 3000 rpm for 5 to 15 minutes, preferably for 10 minutes, but is not limited thereto.

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the freeze-drying (lyophilization) is sufficient to be performed by a method widely known in the art, and detailed description thereof will be omitted.

The extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention may further include the step of pulverizing the freeze-dried polysaccharide to obtain a polysaccharide in powder form.

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the polysaccharide may include xylose.

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the polysaccharide is rhamnose, fucose, arabinose, and mannose. It may further include one or more monosaccharides selected from the group consisting of galactose, glucose, glucuronic acid and galacturonic acid.

In the extraction method of the polysaccharide derived from the cockscomb flower extract of the present invention, the polysaccharide is 0.6-0.8 mol% of xylose, 2.2-2.8 mol% of rhamnose, 0.2-0.4 mol% of fucose, arabinose, relative to the total hydrocarbon of the extract 5.6-8.2 mol%, mannose 0.9-2.7 mol%, galactose 5.6-7.4 mol%, glucose 11.1-13.9 mol%, glucuronic acid 1.6-2.0 mol%, and galacturonic acid 53.7-65.5 mol%.

A third embodiment of the present invention relates to a composition for enhancing immunity comprising a polysaccharide derived from a cockscomb flower extract.

Innate immunity is the first response to infection, and it is known that the innate immune system contains macrophages, monocytes and granulocytes. However, macrophages perform different functions according to different signals through differentiated activation.

In the activation of macrophages, immune stimulants generally act on phagocytes, such as macrophages. It is a compound that can be activated to ameliorate a non-specific immune response. In general, most immune stimulants activate the function of macrophages after binding specifically to cell surface receptors. For example, toll-like receptors are known to be expressed on macrophages and other cells.

In addition, these polysaccharides are currently considered to play a potential role as materials for functional foods, one of the mechanisms of action of these substances appears to include non-specific stimulation of the immune system, and in fact, immune stimulation of plant polysaccharides, anti-tumor , the underlying mechanism of bactericidal and other therapeutic effects occurs through stimulation of immune cells and modulation of the complement system.

Consequently, modulation of innate immunity has a significant impact on the host's ability to respond rapidly and robustly to a variety of pathogens and is dependent on a variety of substances that interact with TLR signaling, including NF-kB and mitogen-activated protein kinase (MAPK). Several downstream signaling pathways are triggered by this to generate reactive oxygen species (ROS), nitric oxide, etc.

Cytokines/chemokines also induce production; macrophages are multifunctional cells and play important roles in the host defense system. After activation of macrophages, they produce various inflammatory mediators and cytokines such as NO and TNF-a.

The polysaccharide included in the composition for enhancing immunity of the present invention may include xylose.

The polysaccharide included in the composition for enhancing immunity of the present invention is rhamnose, fucose, arabinose, mannose, galactose, glucose, glucuronic acid It may further include one or more monosaccharides selected from the group consisting of (glucuronic acid) and galacturonic acid.

On the other hand, the non-patent document 3 Gamandrami ( Celosia) argenta ) seed extract consists only of rhamnose, fucose, arabinose, mannose, galactose, glucose, glucuronic acid, galacturonic acid, etc. (see page 216), which is included in the polysaccharide derived from the cockscomb flower extract of the present invention. There are differences that Xylose does not include.

The polysaccharide contained in the composition for enhancing immunity of the present invention is based on the total hydrocarbon of the extract, xylose 0.6-0.8 mol%, rhamnose 2.2-2.8 mol%, fucose 0.2-0.4 mol%, arabinose 5.6-8.2 mol%, mannose 0.9-2.7 mol%, galactose 5.6-7.4 mol%, glucose 11.1-13.9 mol%, glucuronic acid 1.6-2.0 mol%, and galacturonic acid 53.7-65.5 mol%.

In the composition for enhancing immunity of the present invention, the cockscomb flower extract further includes uronic acid, protein and polyphenol in addition to the polysaccharide, but 2-keto-3-deoxy-D-manno-octulo May not contain sonic acid (2-keto-3-deoxy-D-manno-octulosonic acid)-like material (KDO-like material).

In the composition for enhancing immunity of the present invention, the cockscomb flower extract contains 50.5-51.1 wt% of the polysaccharide, 43-49.9 wt% of the uronic acid, 5.2-6 wt% of the protein and the total weight of the cockscomb flower extract It may contain 0.7 to 0.9% by weight of polyphenol.

In the composition for enhancing immunity of the present invention, the composition may be a health functional food composition or a pharmaceutical composition.

In the composition for enhancing immunity of the present invention, the health functional food composition may include the polysaccharide derived from the cockscomb flower extract alone as an active ingredient, and additional ingredients, i.e., food It may further include an acceptable carrier, excipient, diluent or accessory ingredient.

The health functional food composition of the present invention, in addition to the active ingredients, nutrients, vitamins, electrolytes, flavoring agents, colorants, thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, It may further contain sub-components or additives such as stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages.

In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline From the group consisting of cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline It may be one or more selected, but is not limited thereto.

In the composition for enhancing immunity of the present invention, the pharmaceutical composition contains a pharmaceutically acceptable excipient in addition to the polysaccharide derived from the cockscomb flower extract as an active ingredient, such that it is a powder, granules, tablets, pills, capsules, suspensions, emulsions, It may be a composition that can be used for pharmaceutical purposes by being formulated in the form of any one oral preparation selected from spray, pasta, and syrup, or parenteral preparations such as injection, patch, spray, ointment, warning, etc. The formulation is not particularly limited.

When the pharmaceutical composition is formulated to include pharmaceutically acceptable excipients in addition to polysaccharides derived from cockscomb flower extract, commonly used fillers, extenders, binders, wetting agents, disintegrants, and diluents such as surfactants or surfactants are used to prepare the pharmaceutical composition. can be

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose, gelatin, Any one or more selected from sorbitol, xylitol, mannitol, mannitol, ramnitol, inositol, erythritol, paratinose, and quercitol may be used.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, suspensions, emulsions, and lyophilized preparations.

Hereinafter, the present invention will be described in detail through examples. However, these examples are only for describing the present invention in detail, and the scope of the present invention is not limited by these examples.

<Example 1> (Extraction of polysaccharide derived from cockscomb flower extract)

Cockscomb ( Celosia ) purchased from oriental herbal medicine (Jecheon, Korea) cristata L.), washed thoroughly under running water, placed in a water heater, boiled at 100°C for 90 minutes, and extracted with hot water.

After filtration of the hot water extract obtained above through filter paper (Whatman No. 41), ethanol (80% (v/v)) of 4 times the volume of the filtrate was added to the filtrate of the extract obtained by filtration. After mixing, centrifugation was performed for 10 minutes at a rotation speed of 3000 rpm.

Proteins were removed by treating the precipitate precipitated by the centrifugation with Seva's reagent (Sevag, Lackman, & Smolens, 1938) containing n-butanol and chloroform in a volume ratio of 1:4.

The protein-removed precipitate was dissolved in water and then freeze-dried in a freeze dryer (Samwon Industrial, Seoul) for 7 days to obtain a freeze-dried polysaccharide. Thereafter, the freeze-dried polysaccharide was powdered to extract the polysaccharide derived from the flower extract of cockscomb in powder form.

<Comparative Example 1> (Extraction of polysaccharides derived from the flower extract of Cockscomb)

Instead of the cockscomb flower, the cockscomb ( Celosia) argentea L.), polysaccharides derived from the flower extract of Caesaroniasis in powder form were extracted in the same manner as in Example 1, except that flowers of argentea L. were used.

<Experimental Example 1> (Ingredient analysis of cockscomb flower extract)

Cockscomb ( Celosia) cristata L.), washed thoroughly under running water, put in a pot, boiled at 100° C. for 90 minutes, and analyzed the components of the extract extracted with hot water. Table 1 shows the results.

At this time, the content of neutral sugar, uronic acid, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO)-like substance and protein was determined by protein analysis dye (Bio-Rad Laboratories, Hercules , CA, USA), using the phenol-sulfuric acid method (Int J Anal Chem 1956;28:350-356), the m-hydroxybiphenyl method (Anal Biochem 1973;54:484-489), modified thiobarbi thiobarbituric acid method (Anal Biochem 1978;85:595-601) and Bradford method (Anal Biochem 1976;72:248-254).

ingredient content (wt%) neutral sugar 50.8±0.3 uronic acid 44.4±1.4 protein 5.6±0.4 KDO-like substances - polyphenols 0.8±0.1

<Experimental Example 2> (Sucrose analysis of polysaccharides derived from flower extracts)

Table 2 below shows the results of the sugar analysis of the polysaccharides derived from the cockscomb flower extract extracted in Example 1 and the polysaccharides derived from the cockscomb flower extract extracted in Comparative Example 1.

The sugar analysis was performed using HPLC (Shimadzu Corporation, Kyoto, Japan) equipped with SPD-20A UV/VIS detector and Acclaim ^™ 120 C18 column (Thermo Fisher Scientific)) 1-phenyl-3-methyl-5-pyrazolone (PMP) was measured by the derivatization method (Anal Biochem 1989;180:351-357).

Optimal analysis conditions of the column were maintained at a temperature of 30°C. Elution of the PMP derivative was performed with a mixture of 82% phosphate buffer (0.1 M, pH 6.7) and 18% acetonitrile at a flow rate of 1 mL/min, and the UV absorbance of the eluent was monitored at 245 nm.

High performance size exclusion chromatography (HPSEC) of polysaccharide samples was performed using an Agilent 1260 Infinity LC system (Agilent Technologies Co., Ltd., Palo Alto, USA) according to the method described in Carbohyd polym 2014;111:72-79. did

In addition, in Table 2 below, the unit is mole %, and the mole % was calculated from the total hydrocarbons detected.

Ingredients (sugar) Example 1 Comparative Example 1 rhamnose 2.5±0.3 5.1±0.7 fucose 0.3±0.1 1.1±0.2 arabinos 6.9±1.3 5.3±0.4 xylose 0.7±0.1 - mannose 1.8±0.9 1.5±0.3 galactose 6.5±0.9 6.1±0.6 glucose 13.0±1.9 14.2±1.3 glucuronic acid 1.8±0.2 2.6±0.4 galacturonic acid 59.6±5.9 57.3±4.3

As shown in Table 2 above, it can be seen that the polysaccharide derived from the cockscomb flower extract does not contain xylose, unlike the polysaccharide derived from the comparative cockscomb flower extract.

<Experimental Example 3> Immune enhancing effect of polysaccharide derived from cockscomb flower extract

1. Materials and Methods

(One). material

E. coli lipopolysaccharide (LPS, serotype 055: B8), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA) , ammonium persulfate, Triton X-100 and Trizma base were purchased from Sigma-Aldrich (USA).

Dulbescco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (USA), COX-2, iNOS, phospho-JNK, phospho-p38, phospho-ERK, NFκB p65 , The primary antibody against IκBα was purchased from Cell Signaling (USA).

The ELISA kit for quantifying IL-6 and TNF-α was purchased from BD Science (USA), the PVDF membrane was purchased from GE Healthcare Life Sciences (Brazil), and Bradford reagent was purchased from Bio-Rad (Bio-Rad Laboratories, Inc.). .), and a Pierce High Capacity Endotoxin Removal Spin Column was purchased from Thermo Fisher Scientific (USA).

The test ball cockscomb ( Celosia) cristata L.) flower extract-derived polysaccharide (hereinafter, CCP) was extracted in Example 1 as a sample, and as a control, it was derived from Celosia argentea L. flower extract prepared in Comparative Example 1. of polysaccharide 25 μg/mL was used as a sample.

(2). cell culture

RAW 264.7 cells were purchased from Korea Cell Line Bank (Seoul). Cells were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin in an incubator maintained at 37° C. with a humidified and 5% CO _{2 environment.}

(3). Nitric oxide (NO) measurement

The concentration of NO in the cell culture medium was measured by Griess reagent using sodium nitrite as a standard. RAW 264.7 cells were treated with polysaccharide (CCP) derived from cockscomb flower extract for 18 h, and then the supernatant (100 μL) was transferred to a 96-well plate.

Griess reagent (100 μL) was added to each well and incubated for 15 min at room temperature. Absorbance was measured with a microplate reader on a 550 nm reader (MULTISKAN GO, Thermo Scientific, USA).

(4). cell viability

Cell viability of polysaccharide samples in RAW 264.7 cells was used with MTT assay. Briefly, RAW 264.7 cells were seeded in 96 well plates at a density of 2x104 cells/well. After 12 h, cells were treated with various concentrations of CCP (25-200 μg/mL) or LPS (50 ng/mL) for 24 h.

The medium was then removed and treated with MTT reagent for 2 hours. After removing the medium, the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 540 nm with a microplate reader.

(5). Measurement of cytokines

RAW 264.7 cells (1×10 ⁵ cells/well) were seeded in 24-well plates. After 12 hours, CCP (25-100 μg/mL) or LPS (50 ng/mL) was treated, and culture was maintained for 18 hours. The release of IL-6 and TNF-α from the cell supernatant was analyzed using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

(6). Western blot analysis

RAW 264.7 cells (1×10 ⁶ cells/well) were seeded in 6 well plates. After 12 hours, the cells were treated with CCP (25-100 μg/mL) or LPS (50 ng/mL) and incubated for 18 hours.

Cells were harvested in ice-cold lysis buffer (PRO-PREP Protein Extraction Solution, iNtRON biotechnology Co. Ltd., Korea), and the cell lysate was centrifuged at 13,000 rpm and 4° C. for 1 hour, and then obtained from the supernatant.

Protein concentration was measured using the Bradford assay kit (Sigma-Aldrich, USA). Equal amounts of protein (40 μg/lane) were blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA).

Proteins were separated on a 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK). ?gu? The protein was present at 4° C. for 2 hours.

Protein bands were detected using ECL (Enhanced Chemiluminescence) reagents, signals were captured using Davinch-Chemi™ imaging system (Young Wha Scientific Co. Ltd, Seoul, Korea), and densitometry data were compared to β-actin was normalized to .

The intensity of each band was quantitatively measured using Chemidoc (Bio-rad Laboratories, Inc.), and quantitatively measured using Image J software.

2. Results

(One). Effect of CCP on cell survival and NO production

Cell viability was measured to determine whether the immunostimulatory effect of CCP on NO production was due to cytotoxicity. As shown in Fig. 2(A), no cytotoxicity was observed when the cells were exposed to CCP for 24 hours. Therefore, the following experiments were performed without cytotoxicity in the range of 25-200 μg/mL CCP.

The production of NO was measured by NO assay. NO production was increased by CCP in the concentration range of 25-200 μg/mL of CCP or LPS (50 ng/mL) after 18 hours of treatment (see Fig. 2(B)).

Figure 2 (A) is the result of evaluating the cell viability by MTT analysis after 24 hours of processing RAW 264.7 macrophages with LPS (50 ng/mL) or (25-200 μg/mL) CCP for 24 hours. it has been shown

2(B) shows the results of NO analysis performed with LPS (50 ng/mL) or CCP (25-200 μg/mL) for 18 hours, and all values were expressed as mean±SD (** *p < 0.001 vs control).

(2). Effect of CCP on expression of TNF-α and IL-6

Proinflammatory cytokines such as TNF-α and IL-6 are major indicators of macrophage activation in RAW 264.7 cells and are also potent modulators of inflammation.

The secretion levels of TNF-α and IL-6 were measured in RAW 264.7 cells stimulated with CCP or LPS. 3 , CCP concentrations (25-100 μg/mL) and LPS (50 ng/mL) increased cytokine secretion levels.

These results show that CCP stimulates macrophages in a concentration-dependent manner, as in NO production in FIG. 2 . CCP and LPS were measured by ELISA kit in the supernatant after 18 hours of treatment.

As shown in Figure 3, it can be seen that CCP increases the release of cytokines (IL-6 (A); TNF-α (B)), at this time cytokine production was determined by ELISA analysis, All values are expressed as mean±SD (*** p<0.001 vs control).

(3). Effect of CCP on expression of iNOS and COX-2

Inducible nitric oxide synthase (iNOS) is an enzyme that synthesizes NO from L-arginine, and is expressed in various cell types, including large cells. Cyclooxygenase (COX-2) is involved in the conversion of arachidonic acid to prostaglandin E2.

The levels of iNOS and COX-2 protein expression in RAW 264.7 cells were investigated for their effect on inflammatory proteins by treating them with CCP or LPS (50 ng/mL) and then examining them by Western blot analysis.

As shown in Figure 4, treatment with different concentrations (25-100 μg/mL) can increase iNOS and COX-2 protein expression levels compared to the control (untreated). In addition, LPS treatment was performed as a positive control, and LPS showed strong levels of iNOS and COX-2 protein expression compared to the control.

Detection of the housekeeping gene β-actin was performed in the same blot as the internal control. These results can be concluded that CCP activates macrophages by upgrading the expression of proinflammatory media enzymes such as iNOS.

Figure 4 shows CCP-induced iNOS and COX-2 protein expression, RAW 264.7 cells were treated with CCP or LPS (50 ng/mL) for 18 hours, and protein was detected by Western blot analysis.

(4). Effect of CCP on NF-κB activation and IkBα degradation

As NF-κB is an essential transcription factor in the inflammatory process and is essential for the activation of iNOS, expression and phosphorylation levels of the NF-κB subunit p65 were measured in RAW 264.7 cells using western blot analysis.

The expression level of the p65 subunit was upregulated in cells by CCP stimulation. CCP at 100 μg/mL significantly stimulated p65 expression and phosphorylation. IκBα, when inactive, is a major protein involved in NF-κB activation coupled to inhibitors of NF-κB.

IκBα is degraded when phosphorylated by the IκBα kinase complex, and the effect of CCP on the phosphorylation of IκBα in RAW 264.7 cells was investigated. The phosphorylation level of IκBα was increased in CCP-stimulated cells compared to untreated cells.

Consequently, CCP significantly increased the phosphorylation level of IκBα in a dose-dependent manner. These results indicate that CCP has regulatory activity of the NF-κB pathway in macrophages (see FIG. 5 ).

Figure 5 shows CCP stimulation of NF-κB signal in RAW 264.7 cells. RAW 264.7 cells were treated with CCP or LPS (50 μg/mL) for 30 min, and IκB-α and NF-κB in RAW 264.7 macrophages. was detected by a protein-specific antibody, β-actin was used as a control, and the detection method was measured by Western blot.

(5). Effect of CCP on MAPK phosphorylation

MAPK is associated with cell signaling pathways, where endogenous or extracellular stimulation induces inflammation and includes generation of inflammatory mediators. was related to the MAPK signaling pathway was investigated.

As shown in FIG. 6 , the phosphorylation levels of p38, JNK and ERK were all significantly increased for 30 minutes after CCP treatment. These results suggest that CCP can stimulate TLR and Dectin-1 signals such as LPS by increasing the phosphorylation level of MAPK in macrophages.

(6) Conclusion

When PAMP and DAMP meet, macrophages can detect both internal and external stimuli as well as tissue damage. Macrophages promptly initiate a host immune response and through swallowing and degradation of foreign stimuli and cellular debris. It plays an important pro-inflammatory role.

Potential signaling pathways involved in macrophage activation by plant polysaccharides include complement receptor 3 (CR3), mannose receptor (MR), scavenger receptor (SR), destin-1 and toll-like receptor 4 (TLR4) do. The present inventors evaluated the activation of macrophages through the above-mentioned receptors.

Cytotoxicity was evaluated to evaluate the activation of polysaccharide macrophages. CCP did not show cytotoxicity at the maximum concentration (25-200 μg/mL) in RAW 264.7 cells. In addition, we evaluated the activity of macrophages through the release of cytokines when CCP regulates NO secretion.

Consequently, polysaccharides increased the levels of NO production and secretion of cytokines. The levels of iNOS and COX-2 proteins, which affect NO production and cytokines, also increased the levels of the protein. In addition, by confirming the activity of CCP macrophages by Western blotting, it was determined whether the activity of NF-κB was affected.

In addition, NF-κB binds to IκB and is inactivated, whereas NF-κB is activated by phosphorylation of IκB. This results in translocation of NF-κB from the cytoplasm to the nucleus. According to the present invention (FIG. 5), CCP was shown to bring the translocation of NF-κB to the nucleus and lowered the phosphorylation level of IκB.

MAPK and NF-kB, downstream of the MyD88-dependent pathway, play important regulatory roles in proinflammatory mediators. MyD88 can activate responses such as cytotoxicity, activation of MAPK and NF-κB to activate transcription of inflammatory genes or cytoskeletal proteins.

In this study, CCP increased ERK, JNK and p-38 in a concentration-dependent manner (Fig. 6). Therefore, CCP is highly likely to stimulate MyD88 signaling, and it is predicted that MyD88 signaling is either a TLR signal or a Dectin-1 signal without a CR3 or SR signal.

The mechanism of action of CCP is expected to activate macrophages by stimulating membrane protein receptors, and it is considered as an immune enhancer because it modulates immunosuppression by immune stimulation through the activation of macrophages, and has various useful pharmacological activities. can be

<Example 2> Preparation of health functional food composition for enhancing immunity

100 mg of polysaccharide (CCP) derived from cockscomb flower extract extracted in Example 1

15 g vitamin C

100 g vitamin E (powder)

iron lactate 19.75 g

3.5 g zinc oxide

3.5 g of nicotinic acid amide

0.2 g vitamin A

0.25 g of vitamin B1

Vitamin B2 0.3g

water metering

After mixing the above ingredients according to a conventional health drink manufacturing method, stirring and heating at 85° C. for 1 hour, the resulting solution is filtered and injected into a sterilized 2L container, sealed and sterilized, and then stored in a refrigerator, cockscomb flower extract A health functional food (beverage) for enhancing immunity containing the derived polysaccharide was prepared.

<Example 3> Preparation of health functional food composition for enhancing immunity

100 mg of polysaccharide (CCP) derived from cockscomb flower extract extracted in Example 1

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

According to a conventional health food manufacturing method, the above ingredients were mixed and then granulated to prepare a health functional food (granule) for enhancing immunity containing polysaccharides derived from cockscomb flower extract.

<Example 4> Preparation of pharmaceutical composition for enhancing immunity

10 mg of polysaccharide (CCP) derived from cockscomb flower extract extracted in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients were mixed and filled in a gelatin capsule to prepare a pharmaceutical composition (capsule) for enhancing immunity containing polysaccharide derived from cockscomb flower extract.

<Example 5> Preparation of pharmaceutical composition for enhancing immunity

10 mg of polysaccharide (CCP) derived from cockscomb flower extract extracted in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

The above components were mixed and compressed according to a conventional tablet manufacturing method to prepare a pharmaceutical composition (tablet) for enhancing immunity containing polysaccharides derived from cockscomb flower extract.

Although described with reference to preferred embodiments of the present invention as described above, those of ordinary skill in the art can view the present invention within the scope without departing from the spirit and scope of the present invention as set forth in the following claims. It will be understood that various modifications and variations of the invention are possible.

Claims (15)

delete delete delete delete delete delete (a) filtering the extract obtained by hot water extraction of the washed cockscomb ( Celosia cristata L. ) flowers at 90-110° C. for 1-2 hours;
(b) adding ethanol at a concentration of 80 to 95% (v/v) of 3 to 5 times the volume of the filtrate to the filtrate of the extract obtained by the filtration and mixing, followed by centrifugation;
(c) removing proteins by treating the precipitate precipitated by centrifugation with Sevag reagent containing n-butanol and chloroform in a volume ratio of 1:4;
(d) dissolving the protein-removed precipitate in water; and
(e) freeze-drying the precipitate dissolved in water; in the polysaccharide extraction method comprising; 0.2-0.4 mol%, arabinose 5.6-8.2 mol%, mannose 0.9-2.7 mol%, galactose 5.6-7.4 mol%, glucose 11.1-13.9 mol%, glucuronic acid 1.6-2.0 mol% and galacturonic acid 53.7-65.5 A method for extracting polysaccharides, characterized in that it contains mole %. The method according to claim 7, wherein the polysaccharide comprises 50.5-51.1 wt%, uronic acid 43-45.8 wt%, protein 5.2-6 wt%, and polyphenol 0.7-0.9 wt%, based on the total weight of the extract. Polysaccharide extraction method. A polysaccharide extracted by the polysaccharide extraction method of claim 7. delete delete delete delete A health functional food composition for improving immune function, comprising the polysaccharide of claim 9 as an active ingredient. delete

Images