KR102402445B1 - A composition for enhancing immunity comprising broccoli leaves - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity comprising broccoli leaves treated with a high voltage pulsed electric field as an active ingredient. The composition of the present invention is very useful for the development of a food composition or pharmaceutical composition for enhancing immunity. In addition, the composition of the present invention will be able to contribute to improving the productivity of farmers and increasing added value in that the leaves, which are by-products discarded during broccoli cultivation, are utilized.

Description

Composition for enhancing immunity comprising broccoli leaves as an active ingredient

The present invention relates to a health functional food composition or pharmaceutical composition for enhancing immunity using broccoli leaves as an active ingredient.

The present invention also relates to a method for preparing broccoli leaves for enhancing immunity.

The immune response can be divided into an innate immune response, which is an initial immune response, and an acquired immune response, which is a late immune response. In the initial immune response, the host is protected by suppressing the pathogen by the activation of macrophages and natural killer cells (NK cells), and in this case, the macrophages phagocytose the pathogen and the activation marker TNF-α (tumor necrosis factor- α) is produced and secreted, and NK cells produce and secrete a lot of perforin, an activation marker, to kill pathogen-infected cells. Then, cytotoxic T lymphocytes, helper T lymphocytes, and B lymphocytes involved in adaptive immunity are activated to kill infected cells or protect the host by making antibodies. Cytotoxic T lymphocytes, like NK cells, produce and secrete a lot of perforin to kill pathogen-infected cells, and B lymphocytes protect the host by making antibodies dependent or independent of helper T lymphocytes. Inflammatory cytokines represented by COX-2 (cyclooxygenase-2), IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α) are substances that mediate the immune response and are particularly deeply involved in the initial immune response. It is known that there is In addition, NO (nitric oxide) as a radical is produced from L-arginine by NOS (nitric oxide synthases) enzyme group, and acts as an important secondary transporter of cells. Inducible NOS (iNOS) generates a large amount of NO during inflammation and generates reactive radicals such as peroxynitrite to exhibit anticancer, antibacterial and antiparasitic effects. NO produced by activated macrophages has been identified as a major effective molecule involved in the destruction of tumor cells, and moreover, it non-specifically defends the host, macrophage-mediated killing, microorganisms and tumors in vitro and in vivo. It has been reported that NO is involved in cell proliferation inhibition. Therefore, it is very important to develop an effective immunomodulatory factor capable of activating macrophages in defense against bacterial or environmental pathogens. In many studies so far, immune enhancing activities of natural compounds or extracts including flavonoids and polysaccharides have been reported.

The present inventors have completed the present invention by confirming that the immune enhancing effect appears when high voltage pulse electric field treatment is performed on broccoli leaves, which are by-products remaining after taking an edible part of broccoli while researching on a material effective for enhancing immunity. The broccoli leaf is suitable as a raw material of the present invention because the economical use value is low and the price is low.

KR 10-1729913 B

An object of the present invention is to provide a method for preparing broccoli leaves for immune enhancement by high voltage pulse electric field treatment, and a health functional food composition or pharmaceutical composition comprising the broccoli leaves prepared therefrom.

In order to achieve the above object, the present invention provides a health functional food composition for enhancing immunity comprising broccoli leaves treated with high voltage pulsed electric field (PEF) as an active ingredient.

According to an embodiment of the present invention, the broccoli leaf may be at least one selected from the group consisting of raw material, dried product, pulverized product, and extract.

According to an embodiment of the present invention, the high voltage pulse electric field treatment is performed under the conditions of an electric field of 0.1 to 30 kV/cm, a frequency of 1 to 200 Hz, an energy of 1 to 20 kJ, and a treatment time of 0.1 to 150 seconds in a liquid medium containing broccoli leaves. may be performed 1 to 100 times.

According to an embodiment of the present invention, the high voltage pulse electric field treatment may be performed at 1.5 to 2.5 kV/cm and 3 to 10 kJ of energy.

According to an embodiment of the present invention, the liquid medium may be water, alcohol, or a mixture thereof.

According to an embodiment of the present invention, the composition may be prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverages.

The present invention also provides a pharmaceutical composition for enhancing immunity comprising broccoli leaves treated with high voltage pulsed electric field (PEF) as an active ingredient.

In addition, the present invention is a high voltage pulse electric field treatment 1 to 100 times in a liquid medium containing broccoli leaves under the conditions of an electric field of 0.1 to 30 kV / cm, a frequency of 1 to 200 Hz, an energy of 1 to 20 kJ, and a treatment time of 0.1 seconds to 150 seconds It provides a method for producing broccoli leaves for immune enhancement, including the step of performing a.

The composition comprising broccoli leaves treated with a high voltage pulsed electric field of the present invention as an active ingredient is very useful in the development of a food composition or pharmaceutical composition for enhancing immunity. In addition, the composition of the present invention will be able to contribute to improving the productivity of farmers and increasing added value in that the leaves, which are by-products discarded during the cultivation of broccoli, are utilized.

1 is a graph showing radical scavenging activity according to an increase in the concentration of a broccoli leaf extract (hereinafter, referred to as 'BLE') according to an embodiment of the present invention.
Figure 2 is a graph showing the effect of BLE on cell viability after treatment of RAW 264.7 cells (Figure 2a), HepG2 cells (Figure 2b) and 3T3-L1 cells (Figure 2c) with various concentrations of BLE for 24 hours; to be.
3 is a graph showing the effect of BLE on NO production when the RAW 264.7 cells treated with various concentrations of BLE were treated again with LPS to induce inflammation.
4 is a graph showing the effect of BLE on the amount of TNF-α cytokine production when the RAW 264.7 cells treated with various concentrations of BLE were again treated with LPS to induce inflammation.
5 is a graph showing the effect of BLE on the amount of IL-6 cytokine production when the RAW 264.7 cells treated with various concentrations of BLE were treated again with LPS to induce inflammation.
6 is a graph showing the effect of BLE on IL-1β cytokine production when the RAW 264.7 cells treated with various concentrations of BLE were treated with LPS again to induce inflammation.
7 is a view showing the effect on the expression of COX-2 and iNOS when the RAW 264.7 cells treated with various concentrations of BLE were treated again with LPS to induce inflammation. Specifically, Figure 7a shows the results of analysis of iNOS and COX-2 expression levels in RAW 264.7 cells treated with various concentrations of BLE by Western blotting, and Figure 7b is RAW 264.7 cells treated with various concentrations of BLE. It is a graph showing a relatively comparative comparison of iNOS expression in , and FIG. 7c is a graph showing relative comparison of COX-2 expression in RAW 264.7 cells treated with various concentrations of BLE.
8 shows the results of analysis of the mobility pattern of total RNA isolated from RAW 264.7 cells treated with BLE through agarose gel electrophoresis and the RNA peak pattern through an electropherogram.
9 is a result of analyzing the RIN value of total RNA isolated from RAW 264.7 cells treated with the BLE sample (1250 μg/ml) of the present invention.
10 is a gene ontology analysis result using RAW 264.7 cells treated with LPS.
11 is a gene ontology analysis result using RAW 264.7 cells treated with LPS and BLE.

Hereinafter, the present invention will be described in detail.

According to one embodiment of the present invention, the present invention provides a health functional food composition for enhancing immunity comprising broccoli leaves treated with high voltage pulsed electric field (PEF) as an active ingredient.

Only the bud part of broccoli is used for food, and the leaves and stems of broccoli, which account for 70% of all other broccoli, are thrown away. The broccoli leaves and stems not only have a poor texture compared to the bud portion, but also have a low content and efficacy of useful ingredients, so their utility is very low.

Accordingly, the present inventors have completed the present invention while researching to increase the utilization of broccoli by-products, discovering that when broccoli leaves are subjected to high voltage pulse electric field treatment, an immune enhancing effect similar to that of broccoli flowers appears.

In the present invention, the broccoli leaves treated with the high voltage pulse electric field are placed in a liquid medium containing broccoli leaves under the conditions of an electric field of 0.1 to 30 kV/cm, a frequency of 1 to 200 Hz, an energy of 1 to 20 kJ, and a treatment time of 0.1 seconds to 150 seconds. It may be manufactured by performing high voltage pulse electric field treatment to 100 times.

The high voltage pulse electric field treatment is preferably performed at 1.5 to 2.5 kV/cm and 3 to 10 kJ of energy.

The broccoli leaves immersed in the liquid medium before the high voltage pulse electric field treatment may be raw materials that have not been pulverized or ground or cut products thereof.

The liquid medium is preferably water, ethanol, or a mixture thereof. Organic acids such as acetic acid, citric acid, and lactic acid or inorganic salts such as salts may be further added to the liquid medium.

In addition, the broccoli leaves after the high voltage pulse electric field treatment may be at least one selected from the group consisting of a raw material, a dried product, a pulverized product, and an extract. That is, it may be the raw material of broccoli leaves that have been subjected to high voltage pulse electric field treatment, or may have been dried after high voltage pulse electric field treatment, pulverized, dried after pulverization, pulverized, dried or extracted. The extract may be extracted using a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, preferably water, methanol, ethanol, or a solvent of butanol, more preferably may be an extract extracted under reflux cooling using 60 to 80% (v/v) methanol as a solvent, or a hot water extract extracted by heating at 90 to 100° C. for 1 to 3 hours using water as a solvent. In addition, the extract shall include any one of an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a crude product or a purified product.

The high-voltage pulsed electric field-treated broccoli leaf or a health functional food composition comprising the same as an active ingredient has an immune enhancing effect.

The health functional food composition can be prepared in various forms according to conventional methods known in the art, for example, powder, granules, pills, tablets, capsules, candy, syrup and beverages in any one formulation selected from can be According to the present invention, the content of the broccoli leaf treated with the high voltage pulse electric field in the health functional food composition may be contained in an amount of 0.1 to 100% by weight based on the total weight of the composition, preferably 0.2 to 80% by weight, more preferably may be contained in an amount of 0.3 to 60% by weight. When the content of the broccoli leaves is less than the above range, there is a problem in that it is difficult to expect the effect of the broccoli leaves.

When the composition of the present invention is used as a health drink, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotenstrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. Although the proportion of these additives is not very important, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

The present invention also provides a pharmaceutical composition for enhancing immunity comprising the high-voltage pulsed electric field (PEF)-treated broccoli leaf as an active ingredient.

The pharmaceutical composition for enhancing immunity of the present invention may include 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the high voltage pulsed electric field (PEF)-treated broccoli leaves based on the total weight of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, and a mixture of methylhydroxybenzo.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents water and liquid paraffin, various excipients such as wetting agents, suspensions, emulsions, freeze-dried formulations, and preservatives etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witabsol, Macrogol, Tween 61, cacao butter, lausinji, glycerogelatin, etc. may be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the high voltage pulsed electric field (PEF)-treated broccoli leaves of the present invention are preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention includes the steps of placing broccoli leaves in a batch or continuous processing vessel containing a liquid medium; and 1 to 100 high voltage pulse electric field treatments in the liquid medium under the conditions of an electric field of 0.1 to 30 kV / cm, a frequency of 1 to 200 Hz, an energy of 1 to 20 kJ, and a treatment time of 0.1 seconds to 150 seconds. It provides a method for producing broccoli leaves for immune enhancement, including the step;

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited by the following examples.

Example

Broccoli leaves were purchased and used at Jeju Techno Park. The high voltage pulse electric field treatment device used the model name ELCRACK HVP5 of German Institute of Food Technology (Germany).

embodiment (BLE)

100 g of fresh broccoli leaf raw material itself is placed in a batch treatment container filled with water in a high voltage pulse electric field treatment device, and one pulse electric field is applied under the treatment conditions of an electric field of 2.1 kV/cm, a frequency of 200 Hz, an energy of 7 kJ, and a treatment time of 3 seconds. (PEF) treated.

Then, the broccoli leaves treated with the high voltage pulse electric field were crushed, 2 L of purified water was added, and then extracted with heating at 95° C. for 2 hours. The extract was filtered, concentrated and dried to prepare a high-voltage pulsed electric field-treated broccoli leaf extract.

Comparative Example 1 (broccoli stem)

Except in the same manner as in Example, a broccoli stem extract was prepared using a broccoli stem instead of a broccoli leaf.

Comparative Example 2 (broccoli flower)

Except in the same manner as in Example, a broccoli flower extract was prepared using broccoli flowers instead of broccoli leaves.

Comparative Example 3 (broccoli leaf + PEF untreated)

A broccoli leaf extract was prepared in the same manner as in Example, but without high voltage pulse electric field treatment.

Comparative Example 4 (broccoli stem + PEF untreated)

A broccoli stem extract was prepared in the same manner as in Comparative Example 1, but without high voltage pulse electric field treatment.

Comparative Example 5 (broccoli flower + PEF untreated)

A broccoli flower extract was prepared in the same manner as in Comparative Example 2, but without high voltage pulse electric field treatment.

Experimental example

Experimental Example 1: Analysis of glucoraphane and sulforaphane content

The content of sulforaphane contained in the leaves and stems of broccoli before or after high voltage pulsed electric field (PEF) treatment was analyzed as follows, and the results are shown in Table 1. At this time, the experimental group (broccoli leaves, stems and flowers) subjected to high voltage pulse electric field treatment was treated in the same manner as in Example except for the raw material to prepare samples, and the experimental group not subjected to high voltage pulse electric field treatment (broccoli leaves, stems and flowers) were treated in the same manner as in Example except that raw materials and high voltage pulse electric field treatment were not performed to prepare samples.

A standard solution was prepared with a glucoraphane standard and a sulforaphane standard (90%, Sigma) and analyzed by UPLC-MS/MS (manufacturer Waters) to prepare a standard quantitation curve, and 1 g of the concentrated powder of the samples was dissolved in 1 ml of methanol. was analyzed by UPLC-MS/MS for glucoraphane content and sulforaphane content, and is shown in Table 1 below. The UPLC system is an Acquity UPLC system (Waters corporation, milford, MA), a binary solvent manager, and a sample manager. The MS system is Waters Quattro Premier XE Tandem MS (Micromass UK limited), and the Software is Masslynx V4.1. .

content
(mg/100g) broccoli leaves Broccoli Stems before PEF
(Comparative Example 3) after PEF
(Example) before PEF
(Comparative Example 4) after PEF
(Comparative Example 1) glucoraphane 247.1 129.1 581.2 389.3 sulforaphane 16.8 39.9 3.57 18.8

Referring to Table 1, the sulforaphane content of the broccoli leaf extract (Example) treated with the high voltage pulse electric field was significantly increased.

Experimental Example 2: Evaluation of antioxidant efficacy ( in vitro )

The antioxidant effect of broccoli leaf extract according to an embodiment of the present invention was measured by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) to scavenging free radicals.

First, the sample of Example (BLE; broccoli leaf extract) was dissolved in DMSO and diluted by concentration (0, 500, 1,000, 2,000, 2,500 μg/mL).

Then, 50 μL of the sample was added to 950 μL of a 0.1 mM DPPH solution dissolved in ethanol, and after reacting at room temperature for 30 minutes, absorbance was measured at 517 nm using an absorption spectrophotometer. Then, using the measured absorbance, DPPH radical scavenging activity was obtained according to the following formula, and the results are shown in FIG. 1 .

Antioxidant activity (%) = { 1 - (absorbance of sample added group / absorbance of non-sample group) } x 100

Referring to FIG. 1 , the radical scavenging activity of the sample of the present invention was increased by 20%, 40%, and 43% at concentrations of 1,000 μg/mL, 2,000 μg/mL and 2,500 μg/mL, respectively. Through the above results, it can be confirmed that the BLE of the present invention increases the DPPH radical scavenging activity in a concentration-dependent manner and exhibits excellent antioxidant efficacy.

Experimental Example 3: Cytotoxicity measurement by MTT assay

The cytotoxicity of each concentration of BLE to RAW 264.7 cells, HepG2 cells and 3T3-L1 cells was confirmed by MTT assay.

To do this, first, RAW 264.7 cells, HepG2 cells, and 3T3-L1 cells were equally aliquoted in a 96 well plate at 5×10 ⁵ cells/well, and then cultured at 37°C, 5% CO ₂ in an incubator for 24 hours, and then the culture medium was removed. did

After diluting BLE by concentration (0, 1, 10, 100, 1500, 2500, 5000 μg/mL) and adding it to the medium, 37° C., 5% CO ₂ incubator (CLM-170B-85, ESCO Co., Korea) for 24 hours. EZ-Cytox (DoGen Bio Co., Ltd., Korea) was added to each well by 10% of the medium and reacted for 3 hours at 37°C, 5% CO ₂ in an incubator. Absorbance was measured at 450 nm with an ELISA reader. shown in

Figure 2 is a graph showing the effect of BLE on cell viability after treatment of RAW 264.7 cells (Figure 2a), HepG2 cells (Figure 2b) and 3T3-L1 cells (Figure 2c) with various concentrations of BLE for 24 hours; to be.

Referring to FIG. 2 , for BLE, cytotoxicity at a concentration of 1 to 1,500 μg/mL in RAW 264.7 cells, 1 to 1,250 μg/mL in HepG2 cells, and 1 to 2,500 μg/mL in 3T3-L1 cells did not show any change in

Experimental Example 4: Nitric oxide (NO) production inhibitory activity

It was intended to confirm the NO production inhibitory activity of BLE.

To this end, first, RAW 264.7 cells were equally aliquoted in a 96-well plate at 1×10 ⁶ cells/well, and then cultured at 37° C., 5% CO ₂ in an incubator for 24 hours, and then the culture medium was removed. BLE was diluted and added to the medium by concentration (0, 0 (+LPS), 100, 500, 1000, 1250 μg/mL), and then incubated at 37° C., 5% CO ₂ in an incubator for 1 hour, and LPS (lipopolysacchride) , 1 μg/mL) was added to all wells except for the control and stimulated. After 24 hours, after reacting with griess reagent for 10 minutes, absorbance was measured at 550 nm with an ELISA reader, and the amount of NO production was estimated therefrom and shown in FIG.

3 is a graph showing the effect of BLE on NO production when the RAW 264.7 cells treated with various concentrations of BLE were treated again with LPS to induce inflammation.

Referring to FIG. 3 , the amount of NO production in RAW 264.7 cells induced by LPS significantly increased, whereas in the experimental group treated with BLE, the amount of NO production at a concentration of 1000 μg/mL was significantly reduced by about 77% compared to the control group. did For reference, at a concentration of 1250 μg/mL, 50% NO production inhibitory effect was exhibited. GRN and SFN used as positive controls, when treated at a concentration of 5 μM in LPS-stimulated RAW 264.7 cells, showed an effect of reducing NO production by 32% and 79%.

That is, it was specifically confirmed that BLE reduced NO production in a concentration-dependent manner when inflammation was induced.

In addition, the amount of NO produced when the extracts of Examples (BLE) and Comparative Examples 1 to 5 were treated at a concentration of 1,250 μg/mL, respectively, in the same manner as above were measured and shown in Table 2 below.

division Example Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 NO production
(% of control) 23.3 22.6 21.8 80.1 76.4 62.7

Looking at Table 2, it can be seen that the NO production of the experimental group ingesting the BLE (Example) of the present invention is reduced to a degree similar to that of broccoli flowers.

From these results, it can be seen that the BLE of the present invention has the effect of increasing immunity by reducing the amount of NO production.

Experimental Example 5: TNF-α, IL-6 and IL-1β cytokine production inhibitory activity

It was attempted to confirm the TNF-α, IL-6 and IL-1β cytokine production inhibitory activity of BLE.

To this end, first, RAW 264.7 cells were equally aliquoted in a 96-well plate at 1×10 ⁶ cells/well, and then cultured at 37° C., 5% CO ₂ in an incubator for 24 hours, and then the culture medium was removed. BLE was diluted and added to the medium by concentration (0, 0 (+LPS), 100, 500, 1000, 1250 μg/mL), and then incubated at 37 ° C., 5% CO ₂ in an incubator for 1 hour, LPS (lipopolysacchride) , 1 μg/mL) was added to all wells except for the control and stimulated. After 24 hours, the secretion of cytokines in the cell culture was measured using ELISA kits (Koma biotech., Korea), and the results are shown in FIGS. 4 to 6 .

4 is a graph showing the effect of BLE on TNF-α cytokine production when the RAW 264.7 cells treated with various concentrations of BLE were treated with LPS again to induce inflammation, and FIG. 5 is treated with BLE of various concentrations. When one RAW 264.7 cell was treated with LPS again to induce inflammation, it is a graph showing the effect of BLE on the amount of IL-6 cytokine production, Figure 6 is RAW 264.7 cells treated with BLE of various concentrations treated with LPS again This is a graph showing the effect of BLE on IL-1β cytokine production when inflammation is induced.

4 to 6, the LPS-induced inflammation Raw 264.7 cells significantly increased the content of TNF-α, IL-6 and IL-1β compared to the control group, whereas in the case of the BLE-treated experimental group, the inflammatory cytokine TNF -α, IL-6 and IL-1β production was decreased in a concentration-dependent manner.

Specifically, in the case of the BLE-treated experimental group, TNF-α production was 14%, 25%, 31%, 68 at concentrations of 100 μg/mL, 500 μg/mL, 1,000 μg/mL and 1,250 μg/mL compared to the control group, respectively. % inhibition, IL-6 production was inhibited by 24%, 35%, 44%, and 65%, respectively, and IL-1β production was inhibited by 19%, 28%, 34%, and 39%, respectively.

That is, it was specifically confirmed that BLE reduced the production of inflammatory cytokines (TNF-α, IL-6 and IL-1β) in a concentration-dependent manner.

In addition, the amount of inflammatory cytokines (TNF-α, IL-6 and IL-1β) produced when each of the extracts of Examples (BLE) and Comparative Examples 1 to 5 was treated at a concentration of 1,000 μg/mL in the same manner as this was measured Thus, it is shown in Table 3 below.

division Example Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 TNF-α production
(% of control) 32.4 36.3 30.6 94.2 91.2 89.4 IL-6 production
(% of control) 36.2 41.6 34.4 96.8 93.1 92.3 IL-1β production
(% of control) 61.7 66.4 60.5 92.7 88.5 85.6

Looking at Table 3, it can be seen that the production of inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the experimental group ingesting the BLE (Example) of the present invention is reduced to a degree similar to that of broccoli flowers.

From these results, it can be seen that the BLE diet of the present invention has the effect of increasing immunity by reducing the amount of inflammatory cytokines (TNF-α, IL-6 and IL-1β) production.

Experimental Example 6: COX-2 and INOS protein expression inhibitory activity

We tried to confirm the relationship between BLE and COX-2 and iNOS protein expression.

To this end, RAW 264.7 cells were first washed with PBS, lysed with RIPA buffer, and then centrifuged at 4° C. at 12,000 rpm for 10 minutes. The supernatant obtained by the centrifugation was quantified by Bradford assay and separated by electrolinkage in SDS-PAGE (Tris-Glycine-PAG, non-SDS Precast Gel (10%)). The separated protein was transferred to a nitrocellulose membrane, and the membrane was blocked with a blocking buffer (5% skim milk in TBST (Tris-buffered saline with Tween 20)) to inhibit non-specific binding of the antibody and washed with TBST.

Thereafter, the primary antibody (COX-2, iNOS) was diluted and reacted at 4 °C overnight, then washed 3 times at 10 minute intervals using TBST, and the membrane was diluted 45 with each secondary antibody polymerized with HRP. reacted for a minute. COX-2, iNOS bands and quantification were measured using Chemi-luminescence Bioimaging Instrument (NeoScience Co., Ltd., Korea).

7 is a view showing the effect on the expression of COX-2 and iNOS when the RAW 264.7 cells treated with various concentrations of BLE were treated again with LPS to induce inflammation. Specifically, FIG. 7a shows the results of analyzing the iNOS and COX-2 expression levels in RAW 264.7 cells treated with various concentrations of BLE by Western blotting, and FIG. 7b is RAW 264.7 cells treated with various concentrations of BLE. It is a graph showing a relatively comparative comparison of iNOS expression in , and FIG. 7c is a graph showing relative comparison of COX-2 expression in RAW 264.7 cells treated with various concentrations of BLE.

Referring to FIG. 7 , the expression of COX-2 and iNOS protein in RAW 264.7 cells induced by LPS significantly increased, whereas in the experimental group treated with BLE, the expression of COX-2 and iNOS protein was decreased in a concentration-dependent manner.

That is, it was specifically confirmed that BLE reduced the expression levels of COX-2 and iNOS proteins in a concentration-dependent manner when inflammation was induced.

In addition, the expression levels of COX-2 and iNOS proteins when the extracts of Examples (BLE) and Comparative Examples 1 to 5 were treated at a concentration of 1000 μg/mL, respectively, were measured in the same manner as shown in Table 4 below.

division Example Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 COX-2 expression level 0.41 0.46 0.37 1.73 1.81 1.54 iNOS expression level 0.37 0.51 0.31 1.24 1.26 1.21

Looking at Table 4, it can be seen that the expression levels of COX-2 and iNOS proteins in the experimental group ingesting the BLE (Example) of the present invention are reduced to a degree similar to that of broccoli flowers.

From these results, it can be seen that the BLE of the present invention has the effect of increasing immunity by reducing the expression levels of COX-2 and iNOS proteins.

Experimental Example 7: Analysis of increase or decrease of gene expression by BLE

7-1. Isolation of total RNA from mouse macrophage RAW 264.7 cells

First, RAW 264.7 cells were aliquoted at 1×10 ⁶ cells/well in a 96 well plate and cultured in an incubator at 37° C. and 5% CO ₂ condition.

When the cells were about 80% adhered (after about 1 day), they were washed with 1×PBS, and BLE and LPS were injected into the wells (experimental group: BLE (0 μg/mL), BLE (0 μg/mL) )+LPS, BLE (100 μg/mL)+LPS, BLE (1,250 μg/mL)+LPS).

After 1 day, the cells in each well were dissolved in 500 µL of Trizol to isolate total RNA, and then stored at -80 °C until analysis.

7-2. Total RNA quality check (QC)

The QC (quality check; OD _260/280 & OD _260/230 ) of the isolated total RNA was analyzed and shown in Table 5 below.

No. sample Nano-drop (ND 2000) Bioanalyzer (nano) result Content (ng/μl) Total (μg) OD _260/280 OD _260/230 Ratio(28s/18s) RIN One (-)control 7327.1 146.5 2.10 2.22 1.9 10.0 PASS 2 (+) control 5027.2 100.5 1.95 2.00 1.7 9.8 PASS 3 100 μg/ml
BLE 4530.9 90.6 2.00 2.09 1.8 9.9 PASS 4 1250 μg/ml
BLE 4762.5 95.3 1.99 2.08 1.7 10.0 PASS

< QC standard >

·PASS: A sample that has passed all of the following criteria.

- Content > 100 ng/μL (Total over 500 ng)

- OD _260/280 > 1.8 , OD _260/230 > 1.6

- For eukaryotes, Ratio(28s/18s) > 1.0, RIN value > 7.0

·Check: It can be used for experiments, but proceed to the next step after consulting with the customer.

- Content : 100 ng/μL ~ 0.5 ng/μL (Total 5 ng or more)

- OD _260/280 1.2 ~ 1.8, OD _260/230 > 0.5 ~ 1.5

·Fail: A sample that cannot be used for testing.

7-3. RNA Mobility Analysis by Agarose Gel Electrophoresis (Migration pattern)

RAW 264.7 cells treated with BLE and LPS (Experimental group: BLE (0 μg/mL), BLE (0 μg/mL)+LPS, BLE (100 μg/mL)+LPS, BLE (1,250 μg/mL)+ LPS) was put into a 0.8% agarose gel (loading), electrophoresis was performed at 100 V, and the analysis results are shown in FIG. 8 .

8 shows the results of analysis of the mobility pattern of total RNA isolated from RAW 264.7 cells treated with BLE through agarose gel electrophoresis and the RNA peak pattern through an electropherogram.

In the case of RNA isolated through this experiment, the appropriate quality that can be applied to the analysis experiment was secured, and the reliability of the analysis result was secured through this.

7-4. Rin (RNA Integrity Number)

The quality of total RNA isolated from RAW 264.7 cells treated with the BLE sample of the present invention (1250 μg/ml) was measured using an Agilent 2100 Bioanalyzer, and the results are shown in FIG. 9 .

9 is a result of analyzing the RIN value of total RNA isolated from RAW 264.7 cells treated with the BLE sample (1250 μg/ml) of the present invention.

Referring to FIG. 9, since the RIN value for the BLE sample of the present invention is 10.0, it can be seen that the sample is of high quality.

For reference, the RIN value presented by the Agilent 2100 Bioanalyzer is a statistical value based on migration and peak pattern, the ratio of 28s/18s ribosomal RNA, and has a range of 1 to 10, and is a quality indicator of eukaryotic total RNA samples. is being used The recommended value for DNA microarray analysis requiring high quality samples is 7.0 or higher.

7-5. RNA Sequencing

Analysis method

RNA from the experimental group administered with BLE and LPS to mice (BLE (0 μg/mL), BLE (0 μg/mL)+LPS, BLE (100 μg/mL)+LPS, BLE (1,250 μg/mL)+LPS) was isolated, and the difference in gene expression of each group was compared from the RNA. Table 6 shows the RNA sequencing analysis method of the present invention.

division The details RNA sample Measurement of total RNA quality using Agilent's 2100 Bioanalyzer System Library construction Made with Lexogen's Quant-Seq library Prep kit Sequencing Platform Illumina NextSeq500 Library layout Illumina SE75 Organism Mouse Build mm10 (UCSC) alignment Transcriptome Transcript Model UCSC mm10 Qualtiry Trimming >average Q20 Normalization Method Quantile normalization between samples

Analysis

(1) Gene Ontology

Inflammation and immune response-related genes regulated by LPS stimulation and BLE treatment were identified through gene ontology (GO).

10 and Table 7 are results of gene ontology analysis using RAW 264.7 cells treated with LPS.

division Total Gene Number 23282 % of Total 100 Up significant 2314 % UP significant 9.9 Down significant 2579 % of Down significant 11.1 Total Significant 4893 % of Total Significant 21.0

11 and Table 8 are results of gene ontology analysis using RAW 264.7 cells treated with LPS and BLE.

division Total Gene Number 23282 % of Total 100 Up significant 2539 % UP significant 10.9 Down significant 2482 % of Down significant 10.6 Total Significant 5021 % of Total Significant 21.5

(2) cDNA microarray

Table 9 shows 25 genes that were up-regulated more than twice by LPS stimulation and then recovered to the control level by BLE treatment.

gene LPS/Negative control BLE+LPS/Negative control Explanation Fasl 3.180 1.014 Fas ligand (TNF superfamily, member 6) Prdm1 3.227 1.026 PR domain containing 1, with ZNF domain Nlrp1b 2.118 1.268 NLR family, pyrin domain containing 1B Mpo 2.119 1.014 myeloperoxidase Hist1h2ba 2.100 1.007 histone cluster 1, H2ba Peli2 2.119 1.014 pellino 2 Mx1 3.296 1.049 MX dynamin-like GTPase 1 CD6 2.097 1.253 CD6 antigen Nmi 2.868 1.314 N-myc (and STAT) interactor Mecom 2.119 1.014 MDS1 and EVI1 complex locus Il12a 2.100 1.007 Inteleukin 12 a Cdh17 2.100 1.007 Cadherin 17 Prkcz 3.180 1.014 Protein kinase C Oas1e 2.100 1.007 2'-5' oligoadenylate synthetase 1E Crcp 2.044 1.637 calcitonin gene-related peptide-receptor Gstk1 2.119 1.014 glutathione S-transferase kappa 1 March 8 2.310 1.355 membrane-associated ring finger Bcl3 4.225 1.541 B cell leukemia/lymphoma 3 Chid1 2.129 1.377 chitinase domain containing 1 Cx3d1 2.136 1.020 chemokine (C-X3-C motif) ligand 1 cd3d 2.100 1.007 CD3 antigen Gcnt3 2.100 1.007 glucosaminyl (N-acetyl) transferase 3 Tdgf1 2.100 1.007 teratocarcinoma-derived growth factor 1 Cybb 2.349 1.064 cytochrome b-245, beta polypeptide

In addition, the 27 genes that were down-regulated to less than 0.5 times by LPS stimulation and recovered to the control level by BLE treatment are shown in Table 10 below.

gene LPS/Negative control BLE+LPS/Negative control Explanation CD28 0.460 0.956 CD28 antigen Inpp5d 0.495 0.885 inositol polyphosphate-5-phosphatase D Sesn1 0.330 0.738 sestrin 1 Haver2 0.283 1.148 hepatitis A virus cellular receptor 2 Pnma1 0.460 0.956 paraneoplastic antigen MA1 Naip6 0.469 0.863 NLR family, apoptosis inhibitory protein 6 Ang 0.457 0.784 angiogenin Trim13 0.455 0.934 tripartite motif-containing 32 Klhl6 0.410 0.971 kelch-like 6 Rab12 0.458 1.013 RAB12, member RAS oncogene family gsn 0.177 0.912 gelsolin ptgs1 0.389 0.861 prostaglandin-endoperoxide synthase 1 F2 0.237 0.974 coagulation factor II McColn2 0.460 0.956 mucolipin 2 Trim32 0.475 1.041 tripartite motif-containing 32 Tlr4 0.188 0.999 toll-like receptor 4 Epha2 0.470 1.220 Eph receptor A2 Tec 0.456 1.255 tec protein tyrosine kinase Clec5a 0.495 0.913 C-type lectin domain family 5, member a Cyp26b1 0.323 1.001 cytochrome P450, family 26, subfamily b Trim30b 0.355 0.738 tripartite motif-containing 30B Tnfsf13b 0.378 0.738 tumor necrosis factor (ligand) sf13b Hmgb2 0.408 0.795 high mobility group box 2 Cxcr5 0.180 0.781 chemokine (C-X-C motif) receptor 5 Smad6 0.201 0.828 SMAD family member 6 Cxcr3 0.228 1.172 chemokine (C-X-C motif) receptor 3

As a result of analyzing the expression of LPS-stimulated RAW 264.7 cell genes, in the LPS-treated experimental group, a total of 25 genes (Mpo, cd6, Il12a, Il3ra2, Cd3d, etc.) 27 genes (Cd28, Tlr4, Cxcr5, Cxcr3, etc.) were down-regulated by 0.5 fold or less, and it was confirmed that their expression level was restored to the control level by BLE treatment.

From the above experimental results, the high voltage pulse electric field-treated broccoli leaves of the present invention are very economical because they utilize the leaves, which are by-products thrown away during broccoli cultivation, and are very useful in that they can achieve the same immune enhancing effect as broccoli flowers.

Formulation example

Formulation Example 1: Preparation of tablets

Example High Voltage Pulsed Electric Field Treated Broccoli Leaf Extract 10 mg

100 mg cornstarch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

Formulation Example 2: Preparation of capsules

Example High Voltage Pulsed Electric Field Treated Broccoli Leaf Extract 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 3: Preparation of powdered health functional food

1,000 mg of high-voltage pulsed electric field-treated broccoli leaf extract of Example

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

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 phosphate monobasic 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health functional food, but the composition is mixed in a preferred embodiment. Then, the granules can be prepared and used in the preparation of a health functional food composition according to a conventional method.

Formulation Example 4: Preparation of beverage

1,000 mg of high-voltage pulsed electric field-treated broccoli leaf extract of Example

1,000 mg citric acid

100 g of oligosaccharides

2 g of plum concentrate

1 g taurine

Add purified water to total 900 mL

After mixing the above ingredients according to the usual health drink manufacturing method, after stirring and heating at 85 ° C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, then refrigerated. It is used to prepare the functional beverage composition of the present invention.

Although the present invention has been described as the above-mentioned preferred embodiment, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also intended that the appended claims cover such modifications and variations as fall within the scope of the present invention.

Claims (8)

High voltage pulsed electric field (PEF) treated broccoli leaves as an active ingredient,
The PEF treatment is to be performed with an electric field of 1.5 to 2.5 kV / cm and an energy of 3 to 10 kJ in a liquid medium containing broccoli leaves,
The PEF-treated broccoli leaves,
It exhibits nitric oxide (NO) production inhibitory activity, TNF-α, IL-6 or IL-1β production inhibitory activity, and COX-2 or iNOS expression inhibitory activity,
39.9 mg/100 g of sulforaphane,
A health functional food composition for immunomodulation and anti-inflammatory, characterized in that the PEF-treated broccoli leaf is reduced to a level of 23.3% compared to the control group not treated with the PEF-treated broccoli leaf. The method of claim 1,
The broccoli leaf is a health functional food composition, characterized in that at least one selected from the group consisting of a raw material, a dried product, a pulverized product, and an extract. delete delete The method of claim 1,
The liquid medium is a health functional food composition, characterized in that water, alcohol, or a mixture thereof. According to claim 1,
The composition is a health functional food composition, characterized in that it is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup and beverages. High voltage pulsed electric field (PEF) treated broccoli leaves as an active ingredient,
The PEF treatment is to be performed with an electric field of 1.5 to 2.5 kV / cm and an energy of 3 to 10 kJ in a liquid medium containing broccoli leaves,
The PEF-treated broccoli leaves,
It exhibits nitric oxide (NO) production inhibitory activity, TNF-α, IL-6 or IL-1β production inhibitory activity, and COX-2 or iNOS expression inhibitory activity,
39.9 mg/100 g of sulforaphane,
A pharmaceutical composition for immunomodulation and anti-inflammatory, characterized in that the PEF-treated broccoli leaf is reduced to a level of 23.3% compared to the control group not treated with the PEF-treated broccoli leaf.
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