KR101790657B1 - Extraction of polysaccharides from pine nut cake and composition comprising pine nut extract for enhancement of immunity - Google Patents

Abstract

The present invention relates to a composition for reinforcing immune functions comprising a pine nut cake extract as an active ingredient and, more specifically, to a composition, a pharmaceutical composition, a food composition, a health functional food and a feed additive for reinforcing immune functions comprising a pine nut cake extract, and a method for manufacturing pine nut cake fractions. The pine nut cake extract of the present invention has no cytotoxicity with respect to macrophage and can increase the generation amount of NO and cytokine inducing immune reactions, and the generation amount of chemokine in quantity, thereby having an excellent immune boosting effect.

Description

TECHNICAL FIELD The present invention relates to a composition for enhancing immunity comprising an extract of Fowl Bark as an active ingredient,

More particularly, the present invention relates to a composition for enhancing immunity, a composition for food, a food composition, a health food, a feed additive, And a method for producing the same.

Recently, immigration has been increasing due to overworking of the environment, overwork caused by lack of sleep, lack of exercise, irregular eating, long-term stress and epidemic viral diseases caused by accelerated environmental pollution and excessive work. Regular exercise, eating, and enough sleep to increase the immunity, but the situation is not enough immune strengthening injections, immunity enhancers, vitamins and other health functional foods to replace the immune system is forced to maintain. In addition, this immune function enhances the activity of immune cells to remove tumor cells in advance, thereby lowering the incidence of cancer cancer treatment is likely to develop, so people's interest in immune enhancement is steadily continuing.

The immune response is divided into nonspecific immunity and specific immunity due to the defense of immune cells against external invaders such as fungi or viruses or tumor cells in the body. These two kinds of immune functions affect various immune cells and their factors . In non-specific immunity, dendritic cells and macrophages are typical antigen presenting cells (APCs) that respond non-specific to foreign substances and cause phagocytosis, Is an early immune defense immune cell that stimulates T cells and other immune cells. At this time, APCs stimulate other cells with chemicals such as nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor-α Interleukin (IL) consisting of a short peptide chain, RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1 alpha (macrophage inflammatory protein 1- alpha) secrete cytokines such as. These factors initiate antibody production in B cells and differentiation into immortalized T cells (cytotoxic T cells, killer T cells, helper T cells) and stimulate more immune cells to amplify the immune response (Lee et al , 2014; Jeon et al. , 2013; Noh et al , 2006).

NO is produced when three types of nitric oxide synthase (NOS) convert L-arginine into citrulline, which is known to be involved in neurotransmitters, vasodilation, and immune responses in vivo (Cleman , 2001). In particular, calcium-independent inducible NO synthase (iNOS) produces NO only when specific stimuli are given and its expression is regulated by cytokines. In addition, iNOS has been shown to inhibit MAPK (mitogen-activated protein kinases) such as JAK / STAT (janus kinase / signal transducer and activator of transcription) and p38, extracellular signal-regulated kinase (Erk) activated protein kinase signaling pathway (Chae et al , 2002).

Currently, immune enhancers are marketed as immunity enhancers and immunosuppressants that directly or indirectly regulate the immune response. The immunity enhancer is a drug that contains immunostimulants such as beta-glucan, resveratrol, polysaccharides derived from mushrooms, lectins, chitin, heparin, carrageenan and fucoidan derived from seaweed, and directly promotes the activity of immune- To increase the production of immunity. The adjuvant is a vaccine additive for amplifying the efficacy of the administered vaccine. Current European and US approved immunostimulants include aluminum salts, MF59, AS03 and AS04. The immunoadjuvants administered with the vaccine induce the dendritic cells and macrophages to easily absorb the antigen of the vaccine, while promoting the secretion of several cytokines and NO (Lee, 2012). Currently, metal salts, cytokines, attenuated cell components and lipid particle type immunoadjuvants are being developed, but there are still problems such as stability of the preparation, preservation of the quality of the vaccine, control of half-life, and various side effects Therefore, the importance of searching for bio-derived substances with an immune enhancing effect is increasing (Sohn et al , 2004).

Pinus pinus koraiensis) is a larch conifers Pine Neck Inn handaeseong alpine plants distributed in northeastern China, Japan, Korea and other East Asian gwonin (Larix leptolepis ) and Ligust pine ( Pinus rigida ) Next, as of 2010, it is one of the major economic species that accounts for 3.4% of the total forest area of the Korean peninsula. The wood is currently used mainly in construction materials, furniture, packaging materials, plywood, pulp, charcoal, etc., and its seed production is also excellent, so that pine trees are classified as trees with high economic value (Lee et al ., 2013; ). Korean pine nut, a seed of Pinus koraiensis, is one of the main consumption nuts of Korea and is known as a local specialty of Gapyeong, the main producing area. Pine nuts have unique flavors and aromas, and they are used as food additives or ingredients such as oil, porridge, noodles, noodles, tofu, moon tea, makkolli, jelly, cold noodles,

Pine nuts are composed of 64.2% of lipids, 18.6% of protein, 5.5% of moisture, 4.3% of carbohydrates and 1.5% of ash. It contains various minerals such as calcium, magnesium, potassium, vitamin B1 and vitamin E, Nuts (Park et al , 2005). Or a high-calorie foods with the highest energy in a much higher lipid content as seen in comparison with other nuts, such as general nut (43.5%) consumed by, almond (43.2%), peanut (49.7%) (Chung et al, 2013 ). In particular, about 80% of the lipid constituting more than half of the components of pine nut is composed of 48.6% of linoleic acid, 22.8% of oleic acid and 14.4% of arachidonic acid, which are unsaturated fatty acids.

Recently, studies using components of pine nut have been reported. Typically, lipid components of pine nut have been reported to inhibit weight gain by increasing lipid metabolism and greatly increasing IL-1β (Park et al , 2013) It has been reported that increased expression of AMP-activated protein kinase (AMPK) and oxidative metabolism-related proteins may inhibit skeletal muscle lipid accumulation and reduce brown fat tissue to improve obesity and its associated metabolic diseases Le et al , 2012). Or antioxidant activity of the jatgireum extracted with carbon dioxide pressure of the high-temperature (Chen et al, 2010), decreased appetite, due to the increase of the CCK (cholecystokinin) (hughes et al , 2008), lipid accumulation in the liver inhibits (Ferramosca et al, 2008) Were studied and reported as the physiological activity of nuts oil.

Despite the various functions and functions of pine nuts and pine nuts, the residue left after squeezing process is mostly discarded because there is no report on nutrients and useful physiological activities. Currently, studies on the development of foodstuffs of pine nuts coated with pine nuts have been reported, but there have been no reports or studies on the components and useful physiological activities of pine nuts (pine nuts) Patent No. 10-0679676).

As described above, despite the pharmacological and food-related functions and effects of pine nut, pine nut oil is used for edible purposes, while the research on the pine nut which is expected to have a pharmacological effect is very small at home and abroad.

Therefore, the inventors of the present invention have invented a composition for enhancing immunity by using a pine nut which was not industrially valuable in a specific method, and completed the present invention.

Accordingly, an object of the present invention is to provide a composition for enhancing immunity, which comprises an extract of Fowl Fungus as an active ingredient.

Yet another object of the present invention is to provide a pharmaceutical composition, a food composition, a health functional food, and a feed additive which comprise a snort extract as an active ingredient.

It is yet another object of the present invention to provide a method for producing the nutshell fraction effective for immunity enhancement.

In order to accomplish the above object, the present invention provides a composition for enhancing immunity, which comprises a nut shell extract as an active ingredient.

According to another preferred embodiment of the present invention, the composition for enhancing immunity may comprise 0.01 to 200 μg / ml of a nasal suppressant extract.

According to another preferred embodiment of the present invention, the bark extract is extracted with at least one solvent selected from the group consisting of lower alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, trichloroacetic acid, diethyl ether, water and hexane .

According to another preferred embodiment of the present invention, the bark extract may be a bark fraction obtained by fractionating the nut shell extract.

According to another preferred embodiment of the present invention, the nutrient extract is selected from the group consisting of uronic acid, galacturonic acid, And may include one or more selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose, and rhamnose. .

The present invention also relates to a process for producing a pine nuts, comprising: (a) removing an oil by adding an organic solvent to the pine nuts; (b) hot water extraction of the nuts from which the oil has been removed in the step (a); (c) removing the protein from the hot-water extract of the nuts obtained in the step (b); (d) treating the extract obtained in step (c) with an alcohol solvent and then dialyzing with distilled water to obtain a crude shoot extract (PNE); And (e) purifying the crude nuts extract (PNE) obtained in the step (d) to obtain a fraction (PNE-P1) of a nutshell.

In order to accomplish still another object of the present invention, the present invention provides a pharmaceutical composition for enhancing immunity comprising an extract of Fowl Fungus as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a food composition for enhancing immunity, comprising the snort extract as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a health functional food for immunity enhancement comprising a nut shell extract as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a feed additive for immunity enhancement comprising a nut shell extract as an active ingredient.

Accordingly, the present invention provides a composition for enhancing immunity, which comprises an extract of Fowl Fungus as an active ingredient. The snort extract of the present invention is excellent in immunostimulating effect because it amplifies the amount of NO, cytokine, and chemokine that induce an immune response without mass cytotoxicity against macrophages.

FIG. 1 is a schematic view showing the extraction process of crude extract (PNE) through hot water extraction from a pine nut and ethanol precipitation according to an embodiment of the present invention.
FIG. 2 is a schematic diagram (A) of a crude extract (PNE) obtained from a pine nuts obtained by purifying a fraction (PNE-P1) of a nutshell through a DEAE-cellulose column and a graph (B) in which sugar was detected by a phenol-sulfuric acid method.
FIG. 3 shows the result of qualitative and quantitative analysis of neutral sugars by HPLC (High Performance Liquid Chromatography) using HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection). Specifically, Is the result of neutrophil measurement of PNE, and C is the graph of neutrophil measurement result of PNE-P1. Peak 1 represents rhamnose, peak 2 represents arabinose, peak 3 represents galactose, peak 4 represents glucose, and peak 5 represents xylose.
FIG. 4 is a graph showing qualitative and quantitative analysis of uronic acid by HPLC using HPAEC-PAD method, wherein A is a graph of standard uronic acid and B is a graph of uronic acid analysis of PNE-P1. Peak 1 represents galacturonic acid and peak 2 represents glucuronic acid.
5 is a graph showing molecular weight measurement results of PNE and PNE-P1 using HPLC.
6 is a graph showing the cytotoxicity of PNE-P1 to macrophages measured by MTT assay.
FIG. 7 is a graph showing the results of changes in nitrogen oxide production of macrophages treated with PNE-P1 at different concentrations. FIG.
FIG. 8 is a graph (A) and a graph (B) showing the results of measuring the expression amount of iNOS in macrophages treated with PNE-P1 at different concentrations by PCR.
FIG. 9 shows the results of measurement of cytokine production of macrophages treated with PNE-P1 at different concentrations. A is a graph showing the amount of production of TNF-α, and B is a graph showing the amount of production of IL-6.
FIG. 10 shows the results of measurement of chemokine production of macrophages treated with PNE-P1 at different concentrations. A is a graph showing a change in the amount of MIP-1α produced, and B is a graph showing a change in the amount of RANTES produced.
Figure 11 shows that PNE-P1 inhibits various types of cancer cells (human colorectal cancer cells (HCT 116), mouse skin cancer cells (B16F10). Human prostatic cancer cells (PC-3), human liver cancer cells (HepG2)] and normal cells [monkey kidney epidermal cells (Vero)].
12 is a graph comparing the inhibitory effects of PNE and PNE-P1 on human liver cancer cell proliferation.
FIG. 13 is a microscope photograph of cells comparing PNE and PNE-P1 induced human liver cancer cell shape change (cell shape reduction).
FIG. 14 is a graph showing data on changes in the activation of caspase and PARP proteins in liver cancer cells by PNE-P1.
FIG. 15 is a graph showing the breakdown of mitochondrial membrane potential in liver cancer cells by PNE-P1. FIG.
FIG. 16 is a graph showing changes in the expression level of Bax protein in liver cancer cells by PNE-P1.

Hereinafter, the present invention will be described in more detail.

As described above, despite the pharmacological and food-related functions and effects of pine nut, the noodle oil is used for edible purposes, whereas the pine nut is used only for the purpose of firewood or mulching. Was very small at home and abroad.

Thus, Applicants invented a composition for enhancing immunity by using a punch which was not industrially valuable in a specific way. The snort extract of the present invention is excellent in immunostimulating effect because it amplifies the amount of NO, cytokine, and chemokine that induce an immune response without mass cytotoxicity against macrophages.

The present invention provides a composition for enhancing immunity, which comprises a nut shell extract as an active ingredient.

, Pine nut, of the present invention is pine (Pinus koraiensis ), and Pinus koraiensis (Pinus koraiensis ) is an alpine plant that is a coniferous tree of Pinus koraiensis (Pinus koraiensis ). The 'pine nut' of the present invention means 'pine nut'. In the present invention, 'pine nut' refers to the debris left over from the pine nuts by kneading nuts.

The composition for enhancing immunity of the present invention may comprise 0.01 to 200 μg / ml of the nut shell extract. According to one embodiment of the present invention, low cytotoxicity was observed in the nutshell extract at a concentration of 10-200 μg / ml (see FIG. 6). In addition, it was confirmed that the effect was enhanced in a concentration-dependent manner in the immunity enhancing effect (Figs. 7 to 10).

More specifically, as a result of measurement of nitrogen oxide production, nitrogen oxide was produced in a concentration-dependent manner when PNE-P1 was treated with macrophages, and nitrogen oxide was about 14.5 times higher than that of the control not treated at the highest concentration of 200 μg / ml Respectively. Thus, it was confirmed that PNE-P1 stimulates NO secretion in macrophages and induces immune functions such as tumor necrosis and viral death (see FIG. 7).

In addition, the expression level of iNOS in macrophages following treatment with PNE-P1 was increased by 2.3-fold when compared with the control without any treatment at 200 μg / ml. Thus, it was confirmed that PNE-P1 increased the amount of iNOS expression of macrophages to induce NO production and enhance immunity (see FIG. 8).

In addition, TNF-α and IL-6 levels at 100 μg / ml were significantly increased by 14.6-fold and 84.1-fold, respectively, when compared with the control without any treatment Respectively. In particular, IL-6 is produced more in PNE-P1 than LPS (lipopolysaccharide), which is used as a typical immune response stimulating substance, at 0.1 μg / ml as a control, suggesting that PNE- To induce the differentiation and activity of lymphocytes to promote an adaptive immune response (see FIG. 9).

Finally, when the concentration of PNE-P1 was treated in a concentration-dependent manner, the amount of chemokine production increased exponentially and the amount of RANTES and MIP-1α at 100 μg / ml was about 25 times and 26 times higher than that of the control without any treatment Respectively. PNE-P1 treatment significantly increased the production of chemokines than the LPS control, leading to excellent immune cell recruitment.

 More preferably, the preferred concentration range of the present invention may be 5-100 μg / ml, more preferably 10-75 μg / ml. If less than 0.01 μg / ml of bovine skin extract is present, effective immune enhancement is difficult to achieve, and more than 200 μg / ml may result in cytotoxicity.

The snort extract of the present invention may be extracted with at least one solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, trichloroacetic acid, diethyl ether, water and hexane. Preferably one or more solvents selected from the group consisting of hexane, water, trichloroacetic acid, diethyl ester and ethanol.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.

Most preferably, the nutshell extract is prepared by removing oil with hexane, subjecting it to hot extraction, removing the protein with trichloroacetic acid, removing trichloroacetic acid with diethyl ether and washing with 80 to 100% It may be a crude extract from which ethanol is removed after reacting with an alcohol.

The nut shell extract of the present invention may be the nut shell fraction obtained by fractionating the nut shell extract. The snort extract may be a bovine fraction obtained by subjecting the crude extract thus prepared to purification and fractionation using chromatography, followed by dialysis, as described in Example 1-1.

The nutmeg extract of the present invention is uronic acid, galacturonic acid. And may include one or more selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose, and rhamnose. .

According to one embodiment of the present invention, arabinose, glucose, xylose, galactose, and laminose were found to be 5490.90, 5773.93, 4225.09, 1344.84, and 580.91 pmoles per 10 μg of crude nutshell extract (PNE) And the major sugar components were arabinose, glucose and xylose. In addition, the purified polysaccharide (PNE-P1) was found to have 6676.25, 5443.22, 2984.78, 1668.58 and 756.83 pmoles of arabinose, glucose, xylose, galactose and rhamnose per 10 μg, It was confirmed to be xylose (see Fig. 3).

As a result of uronic acid analysis, it was confirmed that galacturonic acid and glucuronic acid were present at 2436.82 and 34.04 pmole per 10 μg of PNE, respectively, and galacturonic acid was found to be the main uronic acid. Galacturonic acid and glucuronic acid were detected per 10 μg of PNE-P1 3351.24, and 54.62 pmole, and it was also confirmed that the main uronic acid was galacturonic acid (see FIG. 4).

Thus, the nail bitter extract of the present invention comprises 4490 to 6490 pmoles of arabinose, 4773 to 6773 pmoles of glucose, 3225 to 5225 pmoles of xylose, 344 to 2344 pmoles of galactose, 0.001 to 1580 pmoles of gallinose, Rhamnose, 1436 to 3436 pmoles of galacturonic acid, and 0.001 to 500 pmoles of glucuronic acid. In addition, the nasal cut fractions of the present invention may comprise fractions of arabinose, from 4476 to 6443 pmoles of glucose, from 1984 to 3984 pmoles of xylose, from 668 to 2668 pmoles of galactose, from 0.001 to 1756 pmoles of lambda Os, 2351 to 4351 pmoles of galacturonic acid, and 0.001 to 500 pmoles of glucuronic acid.

The present invention also relates to a process for producing a pine nuts, comprising: (a) removing an oil by adding an organic solvent to the pine nuts; (b) hot water extraction of the nuts from which the oil has been removed in the step (a); (c) removing the protein from the hot-water extract of the nuts obtained in the step (b); (d) treating the extract obtained in step (c) with an alcohol solvent and then dialyzing with distilled water to obtain a crude shoot extract (PNE); And (e) purifying the crude nuts extract (PNE) obtained in the step (d) to obtain a fraction (PNE-P1) of a nutshell.

The organic solvent in step (a) is not particularly limited as long as it is a solvent capable of removing an oil component, but hexane may be preferably used.

The step (b) of extracting hot water may be carried out at a temperature of 100 ° C for 2 to 5 hours while stirring. If the extract is extracted for less than 2 hours, the active ingredient exhibiting the immunopotentiating effect may not be sufficiently extracted, and if it is extracted for more than 5 hours, the active ingredient may be denatured or lost.

The step (c) is not limited to the use of a solvent capable of removing proteins. In an embodiment of the present invention, the filtrate is dissolved in distilled water, and then the solution is reacted with 10% trichloroacetic acid at room temperature for 24 hours It can be done.

The alcohol to be treated in the step (d) may be an alcohol having 1 to 4 carbon atoms, but is not limited thereto, preferably ethanol or methanol, and most preferably 90 to 100% ethanol. The alcohol treatment in step (d) may be carried out at 4 ° C for 12 hours to 36 hours.

The purification of step (e) may be purified by a conventional purification method, and preferably purified by chromatography. More preferably, anion exchange column chromatography can be used.

The present invention provides a pharmaceutical composition for enhancing immunity, which comprises an extract of the snake root as an active ingredient.

The composition of the present invention may be a pharmaceutical composition. The pharmaceutical composition may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, , A binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredient (snort extract) for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the snort extract of the present invention may be contained in the composition at a concentration of 0.01 μg / ml to 200 μg / ml, and may be included in an amount of 0.1 to 99% by weight based on the total weight of the composition.

In addition, the pharmaceutical composition of the present invention can be used as an immunity enhancer, an adjuvant additive capable of improving the efficacy of a vaccine, an adjuvant for improving therapeutic efficacy, etc.

The present invention provides a food composition for enhancing immunity, which comprises an extract of the snake root as an active ingredient.

The food composition may contain, as an additional component, various flavors or natural carbohydrates, as well as conventional food compositions, in addition to containing the snort extract as an active ingredient.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

The present invention provides a health functional food for immunity enhancement comprising a nut shell extract as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of enhancing immunity.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture of the nutrient extract of the present invention, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives in a usual manner, Alternatively, the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule form of the health functional food can be prepared by filling a normal hard capsule with a mixture of the nut shell extract, which is an effective ingredient of the present invention, with an additive such as an excipient, and the soft capsule is prepared by adding the nut shell extract to additives such as excipients And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the nut shell extract, the active ingredient of the present invention, excipient, binder, disintegrant, etc. by a conventionally known method, and if necessary, Or the surface may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of the nutrient extract, the active ingredient of the present invention, excipient, binder, disintegrant and the like into a granule by a known method, and if necessary, adding a flavoring agent, ≪ / RTI >

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

In addition, the present invention provides a feed additive for immunity enhancement comprising the snort extract as an active ingredient.

The composition for the animal feed additive may be liquid, powdery or granular.

The composition for animal feed additive of the present invention is a composition containing an organic acid such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, a phosphate such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin ), Natural antioxidants such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid and phytic acid.

Animal feed additive containing the composition of the present invention and feeds containing the composition of the present invention may be supplemented with various adjuvants such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, living microbial agents, For example, crushed or shredded wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; It is preferable to mix all of the dry ingredients comprising the sugar and the milk product such as the various powdered milk and the whey powder and the dry additive and then mix the liquid ingredient with the ingredient to be liquid after heating, In addition to the main components such as fat and vegetable fat, can be used together with materials such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The composition for the animal feed additive can be administered alone to the animal in combination with other feed additives in the edible carrier.

In addition, the composition for animal feed additives can be easily administered as top dressing or they can be mixed directly with animal feed, or separately from feed, in separate oral formulations, by injection or percutaneously, or in combination with other ingredients. Typically, a single daily dose or a divided daily dose can be used as is well known in the art.

When the composition for animal feed additives is administered separately from an animal feed, the dosage form of the composition, as is well known in the art, can be prepared in an immediate release or sustained release formulation in combination with these composition-toxic pharmaceutically acceptable edible carriers have. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, it may be in the form of a soft gelatin capsule, or a syrup or liquid suspension, emulsion or solution. In addition, the dosage form may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoting agents and the like.

In addition, animal feeds in which the extract of the present invention is included as an animal feed additive can be any protein-containing organic fructose commonly used to meet animal dietary needs. These protein-containing flours usually consist mainly of corn, soy flour or corn / soy flour mix.

The animal feed additive may be added to the animal feed by immersion, spraying or mixing.

The present invention is applicable to a number of animal diets including mammals, poultry, and fish. More specifically, the diets include, but are not limited to, commercially important mammals such as pigs, cows, sheep, goats, laboratory animals (rats, mice, hamsters and gerbils), fur- Zoo animals (eg, monkeys and tailless monkeys), as well as livestock (eg, cats and dogs). Common commercially important poultry include chicken, turkey, duck, goose, pheasant and quail. Commercial breeding fish such as trout can also be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Extraction and purification of crude extract derived from pine nut

(1) Extraction of crude extract derived from pine nut

Pinus noodles of Gapyeong country of origin, Gyeonggi-do koraiensis) foil was separated and the filtrate and solids with a filter paper (Whatman # 4) After stirring for 3 minutes homogenizer by the addition of hexane (Hexane) of the 10-fold volume to remove the remaining oil on 100 g. This process was repeated three times, and the solids were dried and sufficiently dissolved in distilled water having a capacity of 40 times, followed by hot water extraction with stirring at 100 ° C for 3 hours. The filtrate was recovered through a filter paper, dried and dissolved in distilled water at a concentration of 0.5% to remove proteins, and then reacted with 10% trichloroacetic acid (TCA) at room temperature for 24 hours. The supernatant was recovered by centrifugation (10,000 rpm, 30 minutes) to remove precipitated proteins, and the mixture was stirred with diethyl ether of the same volume, and the TCA was removed while neutralizing the pH by layer separation. Then, 3 times volume of 95% ethanol was added and reacted at 4 ° C for 24 hours, followed by centrifugation (10,000 rpm, 30 minutes) to recover the precipitate. After removing all of the ethanol remaining in the precipitate using a vacuum concentrator, the polymeric polysaccharide was obtained by dialysis (MWCO 1,000) for 3 days in distilled water and lyophilized. About 1.4 g of pine nut derived crude extract (PNE) was obtained and showed a yield of about 1.4% (see FIG. 1).

(2) Purification of crude extract derived from pine nut

Anion exchange column chromatography was used to purify the polysaccharide from the crude nuts extract according to Example 1. 10 mg of pine nuts extract was dissolved in 1 ml of distilled water, and the solution was poured into DEAE-cellulose (3 × 22 cm). After thoroughly draining the distilled water, all impurities unbound to the column were removed, 1 M) was added to elute the polysaccharide bound to the column. In each eluted fraction, the sugar was detected using the phenol-sulfuric acid method. The sugar-detected fractions were collected and dialyzed (MWCO 1,000) for 3 days in distilled water and lyophilized. Approximately 6.2 mg of pine nut fraction (PNE-P1) was obtained and showed a yield of about 0.8% (see FIG. 2).

Chemical composition analysis of pine nut derived crude extract (PNE) and purified fraction (PNE-P1)

The total sugar content of PNE and PNE-P1 was measured at 490 nm using phenol-sulfuric acid method using glucose as a standard, and uronic acid analysis was performed using carbazole assay (glucuronic acid) And measured at 550 nm. Protein analysis was performed at 595 nm using a bradford assay using bovine serum albumin as a standard.

As a result of chemical composition analysis, as shown in Table 1, the total sugar content of PNE was about 31.11%, the uronic acid content was 25.65%, and no protein was detected. The total sugar content of PNE-P1 was about 45.80%, the uronic acid content was 32.53%, and no protein was detected.

sample Neutral sugar (mg) Uronic acid (mg) Protein (mg) Fowl barley extract (PNE) 1 g 31.11 ± 0.45 25.65 ± 0.01 Non-detection FROZEN FOOT Fraction (PEN-P1) 1 g 45.80 0.60 32.53 + - 0.01 Non-detection

Neutral sugar determination and quantitative analysis of pine nut derived crude extract (PNE) and purified fraction (PNE-P1)

To neutralize and qualitatively analyze PNE and PNE-P1, 1% solution was dissolved in distilled water. Trifluoroacetic acid (TFA) was added to give a final concentration of 2 M and reacted at 100 ° C for 4 hours Acid hydrolysis was performed and the sample was dried using Speed Vac. The dried samples were dissolved in 100 μl of distilled water and neutral sugars were identified by HPAEC-PAD using Bio-LC (LC 20 Chromatography Enclosure, Dionex, Co., USA). Standard neutral sugars include fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, and fructose, purchased from Sigma (USA) (xylose) was used. The column was eluted with CarboPac PA1 (4 × 250 mm, Dionex, Thermo Scientific, USA) and the concentration was adjusted to 0.7 ml / min with distilled water and 200 mM NaOH. Qualitative analysis compares the retention time (min) values of each peak and quantitative analysis of peak areas using Chromeleon software (Thermo Scientific, USA).

As a result of neutral sugar analysis, there were 5490.90, 5773.93, 4225.09, 1344.84 and 580.91 pmole of arabinose, glucose, xylose, galactose and rhamnose per 10 μg of crude extract (PNE), and the main sugar components were arabinose, Glucose, and xylose. The purified fraction (PNE-P1) contained arabinose, glucose, xylose, galactose and rhamnose at 10 μg, respectively, at 6676.25, 5443.22, 2984.78, 1668.58 and 756.83 pmole, and the main sugar components were arabinose and xylose (See Fig. 3).

Qualitative and quantitative analysis of uronic acid in crude extract (PNE) and purified fraction (PNE-P1)

To qualitatively and quantitatively analyze PNE and PNE-P1, 1% solution was dissolved in distilled water. Trifluoroacetic acid (TFA) was added to the resulting solution to a final concentration of 2 M and reacted at 100 ° C for 4 hours Acid hydrolysis was performed and the sample was dried using Speed Vac. The dried sample was dissolved in 100 μl of distilled water and uronic acid was confirmed by HPAEC-PAD method. As standard uronic acid, galacturonic acid and glucuronic acid purchased from Sigma (USA) were used. The column was CarboPac PA1 (4 × 250 mm, Dionex, Thermo Scientific, USA) and the concentration gradient was 600 mM sodium acetate (100 mM NaOH) and 100 mM NaOH at a rate of 0.5 ml / min Lt; / RTI > Qualitative analysis compares the retention time (min) values of each peak and quantitative analysis of peak areas using Chromeleon software (Thermo Scientific, USA).

As a result of uronic acid analysis, it was confirmed that galacturonic acid and glucuronic acid were present at 2436.82 and 34.04 pmole per 10 μg of PNE, respectively, and galacturonic acid was found to be the main uronic acid. Galacturonic acid and glucuronic acid were detected per 10 μg of PNE-P1 3351.24, and 54.62 pmole, and it was also confirmed that the main uronic acid was galacturonic acid (see FIG. 4).

Molecular weight measurement of pine nut derived crude extract (PNE) and purified fraction (PNE-P1)

Molecular weight measurements of PNE and PNE-P1 in powder form were determined using HPLC. The HPLC used for the measurement of the molecular weight was the product of Waters Alliance HPLC 2695. The column used was the SB-showdex 804 HQ column, and the mobile phase was the third distilled water. Pullulan and blue dextran (P-112 kDa, P-200 kDa, P-366 kDa, P-805 kDa and B-2000 kDa) were used as reference materials. The molecular weights of PNE-P1 were 1394 and 705 kDa in the order of PNE-1 and PNE-2. The molecular weights of PNE-P1 were 1274, 640 and 307 kDa in the order of PNE-P1-1, PNE- (See Fig. 5).

Immune enhancement effect of purified fraction (PNE-P1) derived from pine nut

(1) Measurement of cell viability of macrophages with PNE-P1 stimulation

MTT (methylthiazolyldiphenyl tetrazolium bromide) assay was performed to evaluate the cytotoxic ability of PNE-P1 in mouse-derived macrophages (Raw 264.7). (Welgene, Korea) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (10,000 Units / μg Penicillin, 10,000 μg / μl Streptomycin, Hyclone, USA) Cells were cultured in 96-well plates at a density of 1 × 10 ⁴ cells / ml at 36 ° C and 5% CO ₂ for 24 hours. Then, PNE-P1 was added to each concentration (PNE-P1 10, 25, 50 , 100, 200 μg / ml) and cultured for 24 hours in the same conditions as above. After removing the culture medium, MTT reagent (3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide, Roch, Germany) was added to phosphate buffered saline (PBS, %, Mixed with the culture solution at a ratio of 1: 5, and then added to each well in an amount of 120 μl, followed by culturing for 4 hours. After removing all of the MTT solution, 200 μl of dimethyl sulfoxide (DMSO) was dissolved in the purple water-insoluble formazan glass and the absorbance at 570 nm was measured with a microplate reader (Molecular devices, UK) Respectively.

The cell viability was measured by MTT assay. As the concentration of PNE-P1 increased, the cell viability was increased and decreased. There was no significant difference when compared with the control group. Thus, the toxicity of PNE-P1 to cells It was found to promote cell growth at a low or rather low concentration (see FIG. 6).

(2) Measurement of nitrogen oxide (Nitrite) production

Nitrogen oxide production of PNE-P1 was measured by Griess assay. Raw 264.7 cells were cultured in 6-well plates at a density of 1.25 × 10 ⁵ cells / ml for 24 hours at 37 ° C and 5% CO ₂ , and then PNE-P1 (10, 25, 50, / ml) and cultured for 24 hours under the above conditions. After that, the culture solution of each concentration was mixed 1: 1 with Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% N- (1-Naphthyl) ethylene diamine dihydrochloride) and absorbance was measured at 540 nm with a microplate reader.

As a result of measurement of nitrogen oxides production, nitrogen oxide was produced in a concentration dependent manner when PNE-P1 was treated with macrophages, and nitrogen oxide was increased by about 14.5 times at 200 μg / ml at the highest concentration compared with the control without the sample. Thus, it was confirmed that PNE-P1 stimulates NO secretion in macrophages and induces immune functions such as tumor necrosis and viral death (see FIG. 7).

(3) Expression of inducible nitric oxide synthase (iNOS)

The degree of iNOS expression of PNE-P1 was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Raw 264.7 cells were cultured in a 6-well plate at a density of 1.25 × 10 ⁵ cells / ml for 24 hours at 36 ° C and 5% CO _2. PNE-P1 (10, 25, 50, / ml) and cultured for 24 hours under the above conditions. After removing the culture medium, each well was washed with phosphate-buffered saline, and 1 ml of trizol (Invitrogen, USA) was added thereto. The cells were lysed at room temperature for 30 minutes and then 200 μl of chloroform (Sigma, USA) Followed by centrifugation (13,000 rpm, 15 min). The supernatant containing RNA was collected and RNA was extracted using a total RNA extraction kit (Intron Biotechnology, Korea). 1 μg of the cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems, USA), and 1 μl of cDNA and 10 μl of forward primer (10 pmol / L, 5'-CTGCAGCACTTGGATCAGGAACCTG) were added to the maxime PCR premix kit (Intron Biotechnology, (Denaturation at 94 ° C for 30 seconds; annealing at 55 ° C for 30 seconds; extension (SEQ ID NO: 1) and reverse primer (10 pmol / L; 5'-GGGAGTAGCCTGGAACCTGA- 72 ° C, 40 seconds; 30 cycles). PCR products were developed by electrophoresis in 2% agarose gel containing ethidium bromide (EtBr; ethidium bromide, Sigma, USA) according to their molecular weight and identified by banding with image station 4000MM (Kodak, USA). Expression levels were compared using imageJ (NIH, USA) program.

The expression level of iNOS in macrophages following treatment with PNE-P1 was increased by 2.3-fold when compared with the control group without any treatment at 200 μg / ml. Thus, it was confirmed that PNE-P1 increased the amount of iNOS expression of macrophages to induce NO production and enhance immunity (see FIG. 8).

(4) Measurement of cytokine production

The amount of cytokine production of macrophages with stimulation of PNE-P1 was measured. Cytokines refer to all substances secreted by cells, and they play an important role in immune and inflammatory responses including innate immunity, antigen presentation, differentiation of bone marrow cells, cell recruitment and activation, and cell adhesion molecule expression.

Raw 264.7 cells were cultured in 12-well plates at a density of 6 × 10 ⁴ cells / ml for 24 hours at 37 ° C and 5% CO ₂ , and then PNE-P1 (0.1, 1, 5, 50, 100 μg / ml) and cultured for 24 hours under the above conditions. After that, the amount of cytokine production was examined using an ELISA kit (mouse TNF alpha platinum ELISA, Affymetrix, USA; mouse IL-6 duoset ELISA, R & D Systems, USA).

The concentration of TNF-α and IL-6 at 100 μg / ml increased 14.6-fold and 84.1-fold, respectively, compared with the control group without any treatment. In particular, IL-6 is produced more in PNE-P1 than LPS (lipopolysaccharide), which is used as a typical immune response stimulating substance, at 0.1 μg / ml as a control, suggesting that PNE- To induce the differentiation and activity of lymphocytes to promote an adaptive immune response (see FIG. 9).

(5) Measurement of chemokine production

The amount of chemokine production of macrophages with stimulation of PNE-P1 was measured. Chemokines are small chemical molecules that induce chemotaxis (properties that affect the direction of movement in response to certain chemicals) in neutrophils, monocytes, lymphocytes, eosinophils, fibroblasts, and keratinocytes. Chemokines secreted by pro-inflammatory cytokines stimulate the lymphocyte, neutrophil and monocyte to the site of infection and initiate an immune response to treat the inflammation.

Raw 264.7 cells were cultured in 12-well plates at a density of 6 × 10 ⁴ cells / ml for 24 hours at 37 ° C and 5% CO ₂ , and then PNE-P1 (0.1, 1, 5, 50, 100 μg / ml) and cultured for 24 hours under the above conditions. After that, the culture medium was collected and the amount of chemokine produced was determined using an ELISA kit (murine RANTES ELISA kit, Peprotech, USA; murine MIP-1 alpha ELISA kit, Peprotech, USA).

When PNE-P1 was treated at different concentrations, the amount of chemokine production increased exponentially. At 100 μg / ml, the amounts of RANTES and MIP-1α were increased about 25-fold and 26-fold, respectively, compared to the control group without any treatment. PNE-P1 treatment significantly increased the production of chemokines than the LPS control, leading to excellent immune cell recruitment.

Inhibitory effect of PNE-P1 on growth of cancer cells

(1) Measurement of inhibitory effect of PNE-P1 on growth of cancer cells according to cell type

The inhibitory effect of PNE-P1 on cancer cell growth was tested by MTT assay. Human colorectal cancer cells (HCT 116), mouse skin cancer cells (B16F10). Human prostate cancer cells (PC-3), human hepatoma cells (HepG 2), monkey 37 ℃ for 24 hours in a 96-well plate for kidney epithelial cells (Vero) at a density of ^{1 × 10 5 cells / ml,} 5% CO ₂ < / RTI > The cells were treated with PNE-P1 at concentrations of 1, 10, 30, and 50 μg / ml, respectively. After incubation for 72 hours at 37 ° C and 5% CO ₂ , 20 μl of 5 mg / ml MTT reagent was added and reacted for 4 hours. After the reaction, 200 μl of dimethylsulfoxide was added to dissolve the formed formazan and the absorbance was measured at 570 nm using a microplate reader.

The cell viability of human hepatoma cells (HepG2) was decreased in a dose-dependent manner in various cell groups treated with PNE-P1 at the concentrations of 1, 10, 30 and 50 μg / ml, Showed a cell growth inhibitory effect of about 77%, and no cell viability reduction effect was observed in normal cells (monkey kidney epidermal cells) (see FIG. 11).

(2) Inhibitory effect of PNE and PNE-P1 on the growth of cancer cells in human liver cancer cells

In order to compare the inhibitory effect of PNE-P1 on liver cancer cell growth with that of crude extract, the inhibitory effect of PNE and PNE-P1 on liver cancer cell growth was examined by MTT assay. Human hepatoma cells (HepG2) were cultured in 96-well plates at a density of 1 × 10 ⁵ cells / ml for 24 hours at 37 ° C and 5% CO ₂ . PNE and PNE-P1 were treated at concentrations of 1, 10, 30, and 50 μg / ml, respectively. After incubation for 72 hours at 37 ° C and 5% CO ₂ , 20 μl of MTT (3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide) And the mixture was allowed to react for 4 hours. After the reaction, 200 μl of dimethylsulfoxide was added to dissolve the formed formazan and the absorbance was measured at 570 nm using a microplate reader.

As a result, it was confirmed that the survival rate of the cell group treated with PNE-P1 at the concentrations of 1, 10, 30, and 50 μg / ml was much higher than that of the cells treated with PNE at the respective concentrations, At 50 μg / ml, PNE-P1 showed about 77% inhibition of cell growth and PNE did not show any cell growth inhibitory effect (see FIG. 12).

(3) Measurement of change of liver cancer cell shape by PNE and PNE-P1

In order to compare the inhibitory effect of PNE-P1 with that of PNE, the inhibitory effect of PNE and PNE-P1 on liver cancer cell growth was evaluated by observing cell shape change.

The process of cell death can be roughly divided into two ways: apoptosis and necrosis. Among them, apoptosis (programmed cell death) is an important mechanism that maintains the overall physical health by causing the abnormal cells, damaged cells and aged cells to kill themselves by suicide. In particular, one of the characteristics of cancer cells is that cell death is suppressed. Specifically, there is an increasing tendency to treat cancer by inducing apoptosis only in cancer cells. The process of cell death begins with the collapse of the cell, which then creates a gap between adjacent cells, activates apoptosis-specific proteins, and progresses as the DNA, the genetic material, is cleaved regularly. Therefore, in order to determine whether PNE-P1 inhibits the growth of cancer cells through apoptosis, the morphology of PNE and PNE-P1 treated cells was observed using a phase contrast microscope.

To investigate the effect of PNE and PNE-P1 on cell shape changes, HepG2 cells were cultured on a 6-well cell culture plate for 24 hours, and the concentrations of PNE and PNE-P1 (0, 20, 50 μg / ml) and further cultured for 48 hours.

As a result, no significant changes in cell shape were observed in the group treated with PNE (20 and 50 μg / ml) compared to the control group, but the group treated with PNE-P1 (50 μg / ml) The change was observed. Thus, it was confirmed that PNE-P1 induces apoptosis by shrinking cells differently from PNE (see Fig. 14).

Studies on the hepatocyte apoptosis mechanism of PNE-P1

In order to investigate the mechanism of the death of liver cancer cells in PNE-P1, cell death mechanism by cysteinyl-aspartate-specific protease and PARP [Poly (ADP-ribose) polymerase] Respectively.

Human hepatoma cells (HepG2) were cultured on a 6-well plate for 24 hours. Cells were treated with polysaccharides at concentrations of 1, 10, 30, and 50 μg / ml and cultured for 48 hours. After 48 hours of incubation, the cells were washed with PBS and centrifuged at 1,400 rpm for 5 minutes to obtain cells. The precipitated cells were added with 50 μl of lysis buffer, reacted on ice for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes to obtain proteins. Proteins were quantitated using the Bradford assay, followed by loading dye, denaturing at 95 ° C for 5 min, and electrophoresis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After the electrophoresis, the cells were transferred to a nitrocellulose membrane, and the cells were incubated with primary anti-cleaved caspase 9 antibody, anti-cleaved caspase 3 antibody, anti-cleaved PARP antibody and anti-beta actin antibody (1: 500-1000) milk) were added to the nitrocellulose membrane and reacted at 4 ° C for 24 hours. The HRP (horseradish peroxidase) -conjugated secondary antibody was diluted 1: 2000 in 5% non-fat milk milk and reacted at room temperature for 2 hours. After washing three times with TBST in the same manner as described above, ECL (Enchanced Chemiluminescence) substrate solution was reacted on the membrane for 1 minute and developed on X-ray film.

Caspase, a key protein in apoptosis, is known to induce cell shape changes, DNA fragmentation and PARP activation by cleavage of the protein. When the amount of cleaved caspase 9 and 3 was examined using PNE-P1, the expression level of PNE-P1 increased as the concentration of PNE-P1 was increased. The expression level of PARP [poly (ADP-ribose) polymerase], which is a characteristic index of apoptosis and nuclear enzyme, increased as the concentration of PNE-P1 treated with cells increased. (Fig. 15)

Investigation of mitochondrial dysfunction in hepatocellular carcinoma cells by PNM-P1 fraction

 Mitochondrial dysfunction is caused by loss of mitochondrial membrane potential, which is known to release cytochrome C into the cytoplasm and induce apoptosis by forming apoptosome 9 and apoptosome.

(1) Measurement of mitochondrial membrane potential change in cancer cells by PNE-P1

(5,5 ', 6,6'-tetrachloroc-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide) to detect the loss of mitochondrial membrane potential of liver cancer cells (HepG2) by PNE- A potential measurement kit was used. Human liver cancer cells were cultured in a 96-well plate at a density of 1 x 10 ⁵ cells / ml for 24 hours at 37 ° C and 5% CO ₂ . After culturing, cells were treated with PNE-P1 at concentrations of 1, 10, 30, 50 μg / ml and cultured for 48 hours. After incubation, 100 μl of JC-1 was added to each well and incubated at 37 ° C for 30 minutes. Fluorescence intensity was measured at 600 nm excitation and 600 nm fluorescence using a fluorescence spectrophotometer. 485 nm, and emission 535 nm. Red fluorescence is measured in all living cells, and green fluorescence is measured in dead cells by apoptosis.

As shown in FIG. 16, when the PNE-P1 was treated at 1, 10, 30, and 50 μg / ml, mitochondrial membrane potentials were higher than those of the control group 3%, 10%, 14% and 18%, respectively (see Fig. 15). This suggests that the mitochondrial membrane potential of liver cancer cells is disrupted by PNE-P1.

(2) Measurement of the expression level of Bax protein in liver cancer cells by PNE-P1

The Bax protein, which plays an important role in the apoptosis process, causes dysfunction of the mitochondria. Bax protein is present in the cytoplasm, and when stress such as DNA damage is transmitted, it migrates to the outer membrane of the mitochondria, causing permeability and loss of membrane potential. Therefore, we examined the expression level of Bax protein by Western blotting to determine whether the mitochondrial membrane potential loss of hepatocarcinoma cells (HepG2) by PNE-P1 was induced by Bax protein.

Human hepatoma cells (HepG2) were cultured on a 6-well plate for 24 hours, treated with PNE-P1 at 1, 10, 30, and 50 μg / ml for 48 hours. After 48 hours of incubation, the cells were washed with PBS and centrifuged at 1,400 rpm for 5 minutes to obtain cells. The precipitated cells were added to 50 μl of lysis buffer, reacted on ice for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes to obtain proteins. Proteins were quantitated using the Bradford assay, followed by loading dye, denaturing at 95 ° C for 5 minutes, and electrophoresis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After electrophoresis, the cells were transferred to a nitrocellulose membrane, and the primary anti-Bax antibody and anti-β-tubulin antibody (1: 500-1000) were diluted in 5% skim milk at each ratio and placed in a nitrocellulose membrane Followed by reaction at 4 ° C for 24 hours. The HRP (horseradish peroxidase) -conjugated secondary antibody was diluted 1: 2000 in 5% non-fat milk milk and reacted at room temperature for 2 hours. After washing three times with TBST in the same manner as described above, ECL (Enchanced Chemiluminescence) substrate solution was reacted on the membrane for 1 minute and developed on X-ray film.

When the amount of Bax expression was examined using PNE-P1, the expression level of Bax was significantly increased as the concentration of PNE-P1 was increased. Therefore, it was confirmed that mitochondrial membrane potential collapse in cancer cells by PNE-P1 was induced by activation of Bax protein (see FIG. 16).

In addition, PNE-P1, which was obtained by separation and purification by ethanol precipitation after hot water extraction from pine nuts, was a polysaccharide containing mainly arabinose and galactose and galacturonic acid as a main sugar component, and iNOS The expression level is increased to induce NO production, and the amount of cytokine and chemokine production is also increased, thereby having an immune enhancing activity. In addition, the polysaccharide has an anticancer activity against caspase 9, caspase 3, activation of PARP and expression of Bax by inducing the loss of membrane potential of mitochondria in human liver cancer cells (HepG 2) It was confirmed to be a natural physiologically active substance.

The present invention provides a composition for enhancing immunity comprising a nut shell extract by confirming the immunity enhancing effect of a nutshell which is used only as a firewood because of low industrial availability. The nutritional extract of the present invention has high cytotoxic activity against macrophages and has a high immunosuppressive effect because it produces a large amount of NO, cytokine, and chemokine that induce an immune response and thus is highly industrially applicable.

Claims (13)

Uronic acid, galacturonic acid. A nasal patch containing at least one polysaccharide selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose and rhamnose, A composition for enhancing immunity comprising a water fraction of a hot-water extract as an active ingredient
[Claim 2] The composition for immunomodulation according to claim 1, wherein the composition for enhancing immunity comprises the water fraction of the nutshell hot-water extract at a concentration of 0.01 to 100 [mu] g / ml.
delete delete The immunomodulating composition according to claim 1, wherein the water fraction of the nutshell hot-water extract is purified by chromatography and then dialyzed.
delete The method according to claim 1, wherein the water fraction of the nutshell hot-water extract has an effect of increasing the production of nitrogen monoxide (NO); Increased expression of inducible nitric oxide synthase (iNOS); Gene expression enhancement of cytokines TNF-a and IL-6; , And chemokine RANTES (regulated on activation, normal T cell expressed and secreted) and MIP-1α (macrophage inflammatory protein 1-α).
delete Uronic acid, galacturonic acid. A nasal patch containing at least one polysaccharide selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose and rhamnose, A pharmaceutical composition for enhancing immunity comprising a water fraction of a hot-water extract as an active ingredient.
Uronic acid, galacturonic acid. A nasal patch containing at least one polysaccharide selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose and rhamnose, Wherein the water fraction of the hot-water extract is contained as an active ingredient.
Uronic acid, galacturonic acid. A nasal patch containing at least one polysaccharide selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose and rhamnose, A health functional food for immunity enhancement comprising the water fraction of hot water extract as an active ingredient.
12. The method of claim 11, wherein the health functional food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, Wherein the immunogenic enhancer is selected from the group consisting of:
Uronic acid, galacturonic acid. A nasal patch containing at least one polysaccharide selected from the group consisting of glucuronic acid, arabinose, glucose, xylose, galactose and rhamnose, A feed additive for enhancing immunity comprising a water fraction of hot-water extract as an active ingredient.

Images