KR101722448B1

KR101722448B1 - Food composition with the fruit extract of Phyllanthus emblica Linn. and the leaf extract of Psidium guajava for the improvement of immunity

Info

Publication number: KR101722448B1
Application number: KR1020160122921A
Authority: KR
Inventors: 김지훈; 심태진; 장영선; 민서영
Original assignee: 주식회사 프롬바이오
Priority date: 2016-09-26
Filing date: 2016-09-26
Publication date: 2017-04-03
Also published as: WO2018056497A1

Abstract

The present invention relates to a food composition for immunity enhancement containing a complex composed of an Amaranthaceae fruit extract and a Guava leaf extract. The present invention relates to a food composition for enhancing macrophage activity, an increase in NK cell activity, an increase in IL-2 expression and an increase in IL- And has an immunity-enhancing effect, and can help prevent and treat diseases caused by decreased immunity.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a food composition for enhancing immunity, which comprises an extract of Amara fruit and a guava leaf extract. and the leaf extract of Psidium guajava for the improvement of immunity}

The present invention relates to a food composition for enhancing immunity, and more particularly, to a food composition for enhancing immunity containing Amaranth Fruit extract and Guava leaf extract.

'Immunity' refers to a state in which life forms a strong resistance to a particular pathogen or toxin. Immunity defends itself from damage by external stimuli or invasion of hospital microorganisms, but it can also damage your own tissues, such as inflammatory reactions.

The function of regulating the immune function to regenerate to normal or to reduce the range of change is classified into immune function suppression or immune function enhancement. Modification of the immune response is meant to inhibit an undesirably increased immune response, such as allergic reactions caused by adverse reactions to foreign substances, reactions to self-antigens or modified self-antigens.

Immunity is largely divided into congenital immunity, inherited from birth, and adaptive immunity, inherited from birth.

Congenital immunity refers to a natural immune response that directly attacks and destroys cells to be attacked even when they are not exposed to specific antigens. They respond nonspecifically to antigens and have no special memory effect. Congenital immune systems include skin and mucous tissue that block the entry of antigens, strong acidic stomach, and complement in blood. Phagocytes (acidic leukocytes, neutrophils, monocytes) and NK cells, one of the lymphocytes, attack the invading microorganisms, which are the cellular defenses of the innate immune system. Mononuclear cells grow into macrophages when they reach the wound area. They play the most important role before the defense with NK cells. In fact, most of the infections are defended by innate immunity.

Adaptive immunity is also called acquisition immunity. It can remember about the first invaded antigen, reacts specifically when invading again, and can effectively remove the antigen, thereby reinforcing innate immunity. Adaptive immunity is divided into humoral immunity involving B lymphocytes and cellular immunity involving T lymphocytes. Humoral immunity is the ability of B lymphocytes to differentiate after recognizing the antigen and secrete the antibody, which mainly removes infected bacteria. Cellular immunity recognizes antigens from T lymphocytes derived from the thymus and secretes lymphocytes or kills directly infected cells.

As a busy modern society, children, examinees, and elderly people suffer from chronic fatigue and impaired immunity regardless of age. Stress, overwork, lack of sleep, excessive drinking, unbalanced diet, exposure to various environmental influences are known to be the main causes of decreased immunity. Because of these problems, the natural healing function of our body gradually weakens, and the natural healing function of our body is gradually weakened, and the natural healing function of our body is gradually weakened to become such as atopic dermatitis, allergic rhinitis, urticaria, hypersensitivity pneumonia, otitis media, weak constitution, Various diseases or symptoms are induced.

With the prolonged epidemic of MERS in 2015, there were 186 confirmed patients nationwide, of which 38 died. The World Health Organization (WHO) classifies patients with diabetes, renal failure, chronic lung disease, and immunosuppression as high-risk patients with mers infection. It is known that the most effective way to prevent mans infection without vaccines and medicines is to increase their natural resilience, or immunity.

So far, a number of vaccine immunity enhancers have been under development for the purpose of improving immunity, but there are only 5 to 6 immunopotentiators actually licensed. Alum adjuvant, the most widely used immunological enhancer, has reported side effects such as relatively low immune enhancement efficiency and autoimmune response. Due to the safety problem of the immunostimulant, the use of relatively safe health functional foods, especially health functional foods using natural products, has been pointed out as a more preferable method, and researches thereof have been actively conducted.

Korean Patent Laid-Open Publication No. 10-2015-0132616 (published on November 25, 2015) discloses a technique for an immunostimulating composition comprising an apiose extract or an apiose fermented extract as an active ingredient. Korean Patent Laid-Open Publication No. 10-2014-0133689 (published on Apr. 20, 2014) describes a technique for an immunity enhancing composition containing a complex extract containing herringbone. Korean Patent No. 10-1555025 (Registered on Feb. 21, 2015) describes a technique for producing a composition for improving immunity, which comprises a staff serums extract. Korean Patent No. 10-1339706 (Registered on Dec. 31, 2013) describes a technique for an immune enhancing complex composition containing red ginseng inclusion compound, immunoconcentrate and lactic acid bacterium as an active ingredient.

An object of the present invention is to develop and provide a food composition derived from a natural product and having an excellent immunity enhancement.

The present invention provides a food composition for enhancing immunity, which comprises an Amaranthaceae fruit extract and a Guava leaf extract.

The inventors of the present invention have investigated new food materials that exhibit immunity enhancing effects from natural substances having no toxicity and side effects, and as a result, they confirmed that synergy is exerted when a combination of Amara fruit extract and Guava leaf extract is used.

When combined with Amaranthus fructus extract and Guava leaf extract, increased macrophage activity, increased NK cell activity, increased IL-2 expression, and increased IL-12 expression were observed, respectively, It was confirmed that it exerts the effect.

Phyllanthus emblica Linn.) is a major pole and a deciduous arboreal tree, commonly known as Indian gooseberry. It is widely distributed in tropical and sub-tropical countries such as China, India, Indonesia and Taiwan. It is known to contain vitamin C, minerals, amino acids, tannins, filrambic acid, rutin, glucuronide, embolic and phenolic compounds. (Zhang YJ et al., J Nat Prod, 63: 1507-1510, 2000). Cancer contains 20 times more vitamin C than orange, and it is very stable in the heat, so even if it is exposed to high temperatures for a long time, vitamins are hardly destroyed like freshly harvested from trees. The antioxidant, antitumor and antiinflammatory activity of Amaranthus fructus extract is known to be influenced by high content of vitamin C and polyphenol components (Scartezzini P et al. , J. Ethnopharmacology, 104: 113-118, 2006).

Guadava ( Psidium guajava ) is a cultivar plant originating in the Americas tropical region and widely spread to the subtropical region, and is the main food of tropical and subtropical countries and used as a private drug such as anti-inflammatory, anticonvulsant, diarrheal, dysentery, gastrointestinal and respiratory disorders come. Guava is known to be rich in nutrients such as dietary fiber, folic acid, vitamin A, and vitamin C. Guava leaves contain polyphenols such as (+) - galocatechin and leucocyanin, and copper, selenium and zinc It is known that the minerals are abundant, especially zinc rich in 2% or more (Seshadri TR et al., Phytochemistry, 4: 989-992, 1965).

On the other hand, in the food composition for immunostaining according to the present invention, the above Amara fruit extract or Guava leaf extract is preferably extracted with water or a lower alcohol having 1 to 4 carbon atoms or a mixture thereof as an extraction solvent. As the extraction method, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction can be used.

On the other hand, in the food composition for immunostaining according to the present invention, the Amaranth Fruit extract or Guava leaf extract contains the concentrate obtained by extraction, filtration and concentration under reduced pressure or vacuum concentration, and the concentrate obtained by lyophilization or hot air drying It may be in powder form.

In the present invention, it is preferable that the Amaranth fruit extract and Guava leaf extract are mixed in an amount of 0.5 to 1.5 parts by weight based on 1 part by weight of the Amaranthaceae fruit extract. This is because the synergy effect can be maximized in the mixing ratio. More preferably, 1 part by weight of Amaranthaceae fruit extract is mixed with 1 part by weight of guava leaf extract.

In the present invention, the Amaranth Fruit extract and Guava leaf extract are preferably contained in an amount of 0.01 to 50% by weight based on the whole food composition. If the added amount is less than 0.01% by weight, the effect of enhancing the immune function is insignificant. If the added amount is more than 50% by weight, the effect on the usage amount is insignificant and not economical.

The food composition for immunostaining according to the present invention has excellent immunosuppressive effect by exhibiting excellent macrophage activity, increased NK cell activity, increased IL-2 expression and IL-12 expression, Can help. Further, the present invention is advantageous in that it is not toxic, has no side effects, and is made of a natural extract which is harmless to the human body, so that there is no sense of rejection to consumers.

FIG. 1 is a graph showing the results of experiments to confirm the effect of the complex of the present invention (Amaranth Fruit extract and Guava leaf extract) on macrophage activation.
Fig. 2 is a graph showing the effect of the complex of the present invention (Amara fruit extract and Guava leaf extract) on NK cell activation.
FIG. 3 is a graph showing the results of an experiment to confirm the effect of the complex of the present invention (Amaranthaceae fruit extract and Guava leaf extract) on the expression of IL-2 cytokine.
FIG. 4 is a graph showing the results of tests on the effect of the complex of the present invention (Amaranthaceae extract and Guava leaf extract) on IL-12 cytokine expression.

Hereinafter, the structure of the present invention will be described more specifically with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Manufacturing example One: Amla Preparation of fruit extract]

1 kg of Amaranth Fruit was dried and then ground, and 300 g of the powder was placed in a bowl and 3 L of water was added thereto. The mixture was repeatedly extracted three times at 60 ° C for 6 hours to obtain Amara Fruit Extract. Thereafter, the mixture was filtered under reduced pressure through a filter paper (whatman no. 1, England), concentrated under reduced pressure at 60 ° C in a vacuum rotary condenser, and spray-dried to prepare an amaranthin extract powder to be used in the present invention.

[ Manufacturing example 2: Guava Preparation of leaf extract]

After drying 1 kg of guava leaves, 300 g of the powder obtained by grinding was put in a collecting bottle, and 3 L of water was added, followed by hot water extraction at 90 ° C to obtain a guava leaf extract. Thereafter, the mixture was concentrated under reduced pressure at 60 ° C by a vacuum rotary condenser, and then spray-dried to prepare guava leaf extract powder to be used in the present invention.

[ Example 1: Preparation of complex of the present invention]

Composite was prepared from the Amla fruit extract powder and Guava leaf extract powder prepared in Preparation Examples 1 to 2 at the weight ratio shown in Table 1 below.

Amla fruit extract powder
(weight%) Guava leaf extract powder
(weight%) Example 1 50 50

[ Experimental Example 1: Determination of effect of complex of the present invention on macrophage activation]

In this experimental example The effect of the single extract prepared in each of Preparation Examples 1 and 2 and the complex of the present invention prepared in Example 1 on macrophage activation was examined.

Macrophages are distributed in all tissues of the body and are involved in the digestive phagocytosis of foreign substances, bacteria, viruses, body waste and the like, and maintenance of the bioimmune function of transmitting antigens to antigen-specific lymphocytes. Macrophages are known to produce cytokines such as tumor necrosis factor (TNF-α), IL-6, and IL-12, and to degrade digested foreign substances.

Three days before autopsy, 2 mL of thioglycollate medium is intraperitoneally injected into 7-week-old Balb / c mice. On the day of the autopsy, 8 mL of DMEM was placed in the abdominal cavity and massaged. The recovered periplasmic cells were seeded on a 96-well plate at 1 × 10 ⁵ cells / well, and then cultured overnight at 37 ° C. under 5% CO ₂ The cells were cultured.

The experiment was carried out in the same manner as in Example 1, except that the control group (control), the preparation group 1 (70 μg / mL of Amara Fruit extract), the preparation group 2 (70 μg / mL of Guaba leaf extract) And extract (70 μg / mL)). After the sample was treated for each group, the cells were cultured for 2 hours. After that, the cells were washed twice with serum-free medium, 100 μL of fixation solution was added, incubated for 5 minutes at room temperature, and washed twice with 1 × PBS. After adding 100 μL of 1 × blocking reagent, shaking at room temperature for 60 minutes, washing 3 times with 1 × PBS, adding 100 μL of 1 × permeabilization solution, incubating at room temperature for 5 minutes And then washed. After adding 100 μL of 1 × detection solution, the plate was shaken at room temperature for 60 minutes, washed 3 times with 1 × PBS, and 50 μL of detection buffer was added to each well, followed by shaking at room temperature for 10 minutes. Finally, 100 μL of the substrate was added, reacted for 15 minutes, and absorbance was measured at 405 nm.

As shown in FIG. 1, the effect of increasing the macrophage activity about 4-fold higher than that of the normal control group in the Example 1 group was significantly confirmed. In addition, it was confirmed that the effect of increasing the macrophage activity was better in the complex of Example 1 than the single extract (Preparation Example 1, Preparation Example 2).

[ Experimental Example 2: The complex of the present invention NK Identification of effect on cell activation]

In this experiment, a method of measuring LDH released from YK-1 cells (natural killer cell sensitive cell line) destroyed by NK cells was used in order to examine the effect of the complex ingestion of the present invention on NK cell activation.

For isolation of NK cells, the spleens isolated from the mice were washed with RPMI 1640 (10% FBS, 2 mmol / L glutamine, 100 mg / L penicillin-streptomycin) I made a suspension. After that, NK cells were isolated from the spleen cell suspension, which was hemolyzed with erythrocyte lysis buffer using 'mouse NK cell enrichment kit (Stem Cell Technologies, Inc., Vancouver, BC, Canada)'.

NK cells were cultured in RPMI 1640 (10% FBS, 1% penicillin) at 37 ° C and 5% CO ₂ . The number of YAC-1 cells per well was adjusted to 1 × 10 ³ cells / 100 μL per well in a 96-well plate and the number of NK cells was adjusted so that the effect non-cell / target cell ratio (NK cell / YAC- . The experiment was carried out in the same manner as in Example 1, except that the control group (control), the preparation group 1 (70 μg / mL of Amara Fruit extract), the preparation group 2 (70 μg / mL of Guaba leaf extract) Extracts (70 μg / mL). The samples were treated with the samples at 37 ° C and 5% CO ₂ for 4 hours. After 4 hours of cell culture, the cells were centrifuged for 4 minutes to collect 50 μL of the supernatant from which the LDH was liberated, transferred to a 96-well plate, and 50 μL of the recovered substrate mixture was added to each well. After that, the absorbance was measured at 490 nm.

For the spontaneous LDH measurement, add only the culture medium, and add 10 μL of the lysis solution 45 minutes before the supernatant was collected in the maximum LDH well to determine the maximum value of the LDH released from the YAC-1 cells. To allow complete dissolution of the cells. The cytotoxicity percentage of NK cells (% of cytotoxicity) was calculated as LDH liberated from each culture.

As a result, the cytotoxic activity of NK cells increased about 1.3 times higher than that of the control group in Example 1. In addition, it was confirmed that the effect of increasing the cytotoxic activity of NK cells in the complex of Example 1 was better than that of the single extract (Preparation Example 1, Production Example 2) (Fig. 2).

[ Experimental Example 3: Evaluation of the effect of the complex of the present invention on cytokine expression]

In this Example, the effect of the complex of the present invention on the cytokines IL-2 and IL-12 expression in spleen cells was examined.

IL-2 is a cytokine involved in immune regulation and is known as T-cell growth factor (TCGF). It acts to proliferate T-cells and acts on T-cells to secrete interferon-γ, and has activity to stimulate B-cells. It also activates immune cells such as NK cells and lymphokine-activated killer cells.

IL-12 is a cytokine that is involved in the immune regulation of activated macrophages and dendritic cells. It activates NK cells and promotes the differentiation and proliferation of assisted T1 lymphocytes, thus initiating a cell mediated immune response. .

The mice were anesthetized with anesthesia and the mice were anesthetized. The spleens were harvested, washed with PBS, and placed on a 0.45 μm cell strainer. Cells were collected by centrifugation in RPMI 1640 medium containing 10% FBS, incubated with erythrocyte lysis solution for 1 minute, and centrifuged two more times. After cells were stained with trypan blue dye, the number of cells per well was adjusted to 1 x 10 ⁶ cells / 200 μL in a 96-well plate. Cells were cultured for 24 h at 37 ° C and 5% CO ₂ for IL-2 and IL-12. The supernatants were collected and analyzed by ELISA (DuoSet ELISA Kit, R & D Systems, Minneapolis, USA) absorbance at 650 nm was measured with a reader.

As a result of the measurement of IL-2, the effect of increasing the IL-2 expression was 4.6 times higher than that of the normal control group in Example 1 (FIG. 3). In addition, as a result of measuring IL-12, the effect of increasing the expression of IL-12 was confirmed to be about four times higher than that of the normal control group in the Example 1 group (Fig. 4). In addition, it was confirmed from all the results that the complex of Example 1 had a greater effect of increasing the cytokine expression than the single extract (Preparation Example 1, Production Example 2).

On the other hand, excessive secretion of cytokine, an immune substance, can lead to an excessive inflammation, resulting in the DNA of normal cells being transformed into an inflammatory reaction. In order to confirm this, in Experiment 1, Preparation Example 2, and Example 1, the control group A, which caused an inflammatory reaction with mitogen, induces excessive cytokine secretion in normal immune cells, Inflammation. As a result, it was confirmed that the increased cytokine IL-2 and IL-12 secretion did not cause an inflammatory response in all of Production Example 1, Production Example 2 and Example 1.

[Example 2: Preparation of food composition for immunity enhancement]

In this Example, a food composition for enhancing immunity was prepared as follows.

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then the mixture was prepared into powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds and perilla seeds were each steamed and dried by known methods, and then power distribution and pulverization were carried out to prepare powder having a particle size of 60 mesh. Thereafter, 30 wt% of brown rice, 15 wt% of yulmu, 20 wt% of barley, 9 wt% of glutinous rice, 7 wt% of perilla seed, 8 wt% of black soybean, 7 wt% of black sesame seed, 3 wt% 0.5% by weight of manure, and 0.5% by weight of sulfur.

(2) Production of chewing gum

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of a flavor, 2% by weight of water and 0.1% by weight of a complex of the present invention (Example 1).

(3) Candy manufacturing

Candy was prepared by mixing 60 wt% of sugar, 39.8 wt% of starch syrup, 0.1 wt% of flavor and 0.1 wt% of the complex of the present invention (Example 1).

(4) cereal manufacturing

7.51 weight% of the complex of the present invention (Example 1), 40.49 weight% of rice, 24.29 weight% of brown rice, 2.02 weight% of sorghum, 2.02 weight% of barley, 2.02 weight% of glutinous rice, 0.49 weight% of salt, 0.16 weight% , 1.21% by weight of sunflower oil, and 2.04% by weight of water were blended to prepare cereal by a conventional method.

(5) health drink manufacturing

0.0001% by weight of honey, 0.0002% by weight of chitosanic acid amide, 0.0004% by weight of nicotinic acid amide, 0.0001% by weight of sodium riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoacetic acid, 98.7362% (Example 1) were mixed together to prepare a health drink in a conventional manner.

(6) Health supplement manufacturing

50% by weight of the complex of the present invention (Example 1), 0.01% by weight of vitamin B1 hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, To prepare a refillable health supplement food by a conventional method.

Claims

Amaranth Fruit Extract and Guava Leaf Extract,
Wherein the guava leaf extract is mixed in an amount of 1 part by weight based on 1 part by weight of the Amaranthaceae fruit extract.

The method according to claim 1,
The above-mentioned Amaranth Fruit extract or Guava leaf extract may contain,
Wherein the extract is extracted with water or a lower alcohol having 1 to 4 carbon atoms or a mixture thereof as an extraction solvent.

delete

The method according to claim 1,
The above-mentioned Amaranth Fruit extract and Guava leaf extract,
Wherein the food composition comprises 0.01 to 50% by weight of the whole food composition.