KR102618848B1 - Composition containing the hydrolysate of Laminaria by-product extracts for immuno-stimulating activity - Google Patents

Abstract

The present invention relates to an immune-enhancing composition containing a hydrolyzate of kelp by-product extract and a method for producing the same.

Description

Composition for immune enhancement containing the hydrolyzate of kelp by-product extract {Composition containing the hydrolysate of Laminaria by-product extracts for immuno-stimulating activity}

The present invention relates to a composition for enhancing immunity containing a hydrolyzate of kelp by-product extract as an active ingredient and a method for producing the same.

The immune system can be divided into natural resistance, non-specific immune system, and specific immune system. Natural resistance (first line of defense) refers to anatomical and physiological elements that block all invaders, including microorganisms, regardless of their type, and non-specific immunity (second line of defense) refers to phagocytes that eliminate invaders that break through natural resistance and enter the body. The dual-specific immune system is a highly developed immune system with memory and the ability to distinguish between self and non-self (Richard A. Goldsby, et al., KUBY Immunology, 2000).

Macrophages have many lysosomes, which contain acid hydrolase and peroxidase. In addition, it has the property of strongly adhering to glass and plastic surfaces and actively phagocytoses microorganisms and tumor cells.

Macrophages play a major role in the immune response, and immunostimulation is one of the important therapeutic strategies to strengthen the body's defense mechanism against various diseases caused by decreased immune function, such as cancer and inflammatory diseases. Phagocytosis, the main role of macrophages, is to absorb microorganisms and other pyrogenic particles and also secretes a number of cytotoxic molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12. It stimulates immune responses by secreting cytotoxic and inflammatory substances such as cytokines, nitric oxide (NO), and reactive oxygen species (ROS) (Wolf et al., 1994; Lee and Hong) , 2011; Murray and Wynn, 2011).

As such, macrophages play a very important role in the immune response, so they contribute to strengthening immunity through activation and strengthening of the function of these macrophages, and to the prevention, improvement and treatment of various diseases caused by decreased immunity. Development of effective ingredients is required.

The present invention was devised to solve the problems of the prior art,

The purpose is to provide a composition containing as an active ingredient an ingredient that can effectively improve immune function without side effects, and a method for producing the composition.

The present invention,

1) Adding a solvent to the kelp by-product, refluxing at 80 to 100°C, and extracting;

2) Concentrating and freeze-drying the extract prepared in step 1) above; and

3) Preparation of an immune-boosting composition containing a hydrolyzate of a kelp by-product extract as an active ingredient, comprising the step of adding 0.1 to 1 part by weight of an enzyme and hydrolyzing 100 parts by weight of the sample freeze-dried in step 2) above. Provides a method.

In addition, the present invention provides a composition for enhancing immunity comprising a hydrolyzate of kelp by-product extract as an active ingredient.

The composition containing the hydrolyzate of the kelp by-product extract of the present invention as an active ingredient shows an effect of increasing the production of secretion ability of immune enhancing factors such as increased NO production within macrophage Raw 264.7 cells, showing an activity enhancing effect within immune cells. This can provide an immune-boosting effect without side effects.

This immune-boosting effect is excellent, so it can be usefully used in the prevention, suppression, and treatment of hypoimmune disease. Through this, medicines and health functional foods with immune-boosting effects can be provided, and just by consuming these foods, etc. It can provide an immune boosting effect.

Additionally, by using kelp by-products that were discarded in the past as raw materials, pollution of environments such as the sea can be minimized.

Figure 1 is a graph showing the results of an immune enhancement activity test (NO production) in macrophages (RAW 264.7).
Figure 2 is a photograph to explain the edible part, tail, and roots of kelp.
Figure 3 is a graph showing the results of a cell viability experiment in which cytotoxicity was assessed for macrophages (RAW 264.7) through the MTT assay.

While researching natural ingredients effective in preventing, improving, or treating diseases caused by decreased immune function, such as AIDS caused by damage to the immune system and cancer caused by decreased immune function, the present inventor discovered kelp ( Laminaria japonica ) by-product extract. The present invention was completed after confirming that the hydrolyzate could perform such a function.

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

The present invention relates to a composition for enhancing immunity containing the hydrolyzate of kelp by-product extract as an active ingredient.

Kelp ( Laminaria japonica ) belongs to brown algae. It is about 2~4m long, 20~30cm wide, and has a yellow-brown or black-brown band shape. Kelp is rich in minerals involved in biological metabolism, such as calcium, potassium, iodine, and zinc, and contains a lot of vitamin C.

Kelp can be largely divided into edible parts, tails, and roots (Figure 1). The edible parts are consumed for food, but kelp by-products such as tails and roots are discarded in the sea because they have no possibility of being utilized.

In the present invention, kelp by-products refer to the tail and roots of kelp excluding the edible part, and more preferably, kelp tails (Figure 2).

The immune-enhancing composition may be a hydrolyzate of a kelp by-product extract itself, may contain the hydrolyzate as the main ingredient as an active ingredient, and may further include a generally acceptable carrier or one or more additives.

The hydrolyzate can be produced through hydrolysis using an enzyme, and the type of the enzyme is not particularly limited, but an example may include Termamyl SC.

In addition, the present invention

1) Adding a solvent to the kelp by-product, refluxing at 80 to 100°C, and extracting;

2) Concentrating and freeze-drying the extract prepared in step 1) above; and

3) A method for producing the composition of the present invention is provided, which includes the step of adding 0.1 to 1 part by weight of an enzyme to 100 parts by weight of the sample freeze-dried in step 2) and subjecting it to a hydrolysis reaction.

The kelp by-product in step 1) above can be immersed in fresh water for about 10 minutes to remove impurities and salts, desalted, and dried before use. It can also be used by pulverizing it.

In step 1), it is preferable to use 100 to 2,000 parts by weight of solvent for 10 parts by weight of kelp by-product, and it is more preferable to use 300 to 1,000 parts by weight in terms of preventing waste of solvent and excellent extraction efficiency of useful components. can do.

The type of solvent is not particularly limited, but one or more types selected from water, C1 to C4 alcohol, and hexane can be used. Using water as a polar solvent can more effectively extract components useful for enhancing immunity. It may be desirable.

In addition, extraction is preferably performed at 80 to 100°C, preferably 90 to 100°C, for 2 to 8 hours, more preferably 2 to 4 hours. If the extraction time is too long or the temperature is high, additional unuseful components may be extracted and there is a risk of deterioration or destruction of useful components. On the other hand, if the extraction time is too short and the temperature is low, a problem may occur in which useful components are not sufficiently extracted.

The extract prepared in this way can also be filtered and used using filter paper.

The concentration in step 2) above can be concentrated at about 50 ℃ and 50 psi using a rotary concentrator as an example of the present invention, but is not limited to this, and can also be manufactured in powder form by freeze-drying at about -80 ℃. possible.

The enzyme used in the hydrolysis reaction in step 3) is not particularly limited, but Termamyl SC can be used as an example.

The hydrolysis reaction in step 3) is preferably carried out at a temperature of 40 to 65 ℃, pH 4.5 to pH 8.5, more preferably 50 to 60 ℃, pH 6.5 to pH 8.5 to optimize the hydrolytic activity of the enzyme. , it is not necessarily limited to this.

Before or after the hydrolysis reaction in step 3), it is possible to further include reacting at about 90°C for about 15 minutes to deactivate the endogenous enzyme or hydrolytic enzyme.

After the reaction to deactivate the hydrolytic enzyme, the supernatant is recovered through reduced pressure filtration and centrifugation and freeze-dried to produce a hydrolyzate of the kelp by-product extract.

Accordingly, the present invention can provide a composition for enhancing immunity containing the hydrolyzate of the kelp by-product extract prepared using the above production method as an active ingredient.

The composition of the present invention is effective in preventing, improving, and treating diseases caused by decreased immune function, and such diseases include infectious diseases such as colds, inflammatory diseases, allergic diseases such as atopy, AIDS, and cancer. However, it is not limited to this, and all diseases caused by decreased immune function known to those skilled in the art are included.

The immune-enhancing composition according to the present invention may be a hydrolyzate of the kelp by-product extract itself, or may include one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans or mammals. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl. Examples include cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives are added. It can be included as .

That is, the present invention also provides a pharmaceutical composition for enhancing immunity containing the hydrolyzate of kelp by-product extract as an active ingredient.

The pharmaceutical composition can be prepared as injections, liquids, pills, tablets, capsules, powders and suspensions according to conventional methods in the pharmaceutical field to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be formulated into preparations. The dosage form may be in the form of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder, and is preferably in tablet, capsule, or liquid form.

The pharmaceutical composition according to the present invention can be administered through several routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, and the dosage of the active ingredient varies depending on the route of administration, the patient's age, gender, weight, and severity of the patient. It can be appropriately selected depending on the factors. However, for a desirable effect, the hydrolyzate of the kelp by-product extract of the present invention is preferably administered at 0.0001 to 20 mg/kg per day, preferably at 0.001 to 10 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. Additionally, the composition of the present invention can be administered in combination with known compounds that have an effect on enhancing immunity.

Therefore, the present invention can provide a drug containing an immune-boosting composition containing a hydrolyzate of kelp by-product extract as an active ingredient.

The composition according to the present invention is a natural product, not a chemically synthesized compound, and is a composition containing hydrolyzate of kelp by-product extract as an active ingredient. It has no side effects from the drug, so it can be used safely even when taken for a long period of time, and is very harmful to the body. It has the characteristic of being stable.

Accordingly, the composition of the present invention can be used as a composition for health functional foods for the production of health functional foods as well as pharmaceutical compositions for the purpose of enhancing immunity as described above.

The composition for health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components can be used independently or in combination.

Therefore, the present invention provides a composition for immune-boosting health functional food containing the hydrolyzate of kelp by-product extract as an active ingredient, and can be used as a health functional food, such as a main ingredient, secondary ingredient, food additive, functional food or beverage. It can be used easily.

Foods to which the immune-boosting health functional food composition according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, etc., which can be manufactured by conventional manufacturing methods. You can.

In addition, the above-mentioned health functional food refers to a food group or food composition that has added added value to the food by using physical, biochemical, biotechnological methods, etc. to function and express the function of the food for a specific purpose, such as regulation of biological defense rhythm, disease prevention, and other functions. It may be a food that has been designed and processed to sufficiently express body regulatory functions related to recovery, etc. to the living body. The health functional food may contain food supplements that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of health functional foods.

In the food containing the composition for health functional food of the present invention, the amount of fermented product of the kelp extract according to the present invention may be 0.001% by weight to 15% by weight of the total weight of the food, preferably 0.1% by weight to 0.1% by weight. It may be included at 10% by weight, and in the case of beverages, it may be included at a ratio of 1 mg to 100 mg, preferably 1 mg to 20 mg, based on 100 ml, but long-term intake for the purpose of health and hygiene or health control. In the case of, it may be below the above range, and since the active ingredient is derived from a natural product and there is no problem in terms of safety, it can be used in an amount above the above range and is not necessarily limited to the above range.

Hereinafter, the present invention will be described in more detail using examples and comparative examples. However, the following examples are for illustrating the present invention, and the present invention is not limited by the following examples and may be modified and changed in various ways. The scope of the present invention will be determined by the technical spirit of the claims described later.

< Manufacturing example >

Manufacturing example 1. Sample preparation

1) Preparation of kelp tail hot water extract

① Pretreatment of samples

Kelp ( Laminaria japonica ) used in this study was purchased from Wando, Jeollanam-do. The edible parts and roots of the kelp were removed, and only the tail was used. To remove impurities and salt from the kelp tail, desalination was performed by immersing it in 100 times more fresh water than the dried material for 10 minutes. After desalting, hot air drying (45-50°C), the dried kelp tails were pulverized using a grinder, and those with a standard sieve of 30 mesh or less were used as samples for extraction.

② Production of kelp tail hot water extract

The hot water extract of kelp tail was prepared by adding 600 ml of distilled water to 10 g of the pulverized sample above and extracting under reflux at 100°C for 3 hours. The extract was filtered through 25 ㎛ filter paper and then evaporated through a rotary evaporator. It was concentrated at 50°C and 50 psi, and then freeze-dried at -80°C. The powder form obtained by freeze-drying was used in a control experiment (Comparative Example 1) and enzymatic hydrolysis.

< Example > Kelp tail of hot water extract hydrolyzate manufacturing

① Deactivation of endogenous enzymes

To the freeze-dried sample prepared in Preparation Example 1, 60 times (by weight) of tertiary distilled water was added, and the sample was placed in a heating mantle at 90° C. for 15 minutes to deactivate the endogenous enzyme. Afterwards, it was cooled to 4°C, and the optimal pH for the enzyme reaction was adjusted to 1 M-NaOH.

② Hydrolysis using enzymes

The corresponding hydrolytic enzyme was added at 0.5% of the sample weight in a water vessel with controlled reaction conditions and reacted for 3 hours while stirring at 420 rpm.

The enzyme used for hydrolysis was Termamyl SC, a product purchased from Novozymes.

After 3 hours of reaction, reaction was performed at 90°C for 15 minutes to deactivate the hydrolytic enzyme. This was filtered under reduced pressure with Whatman No.2, the filtrate was centrifuged at 6,000 rpm for 30 min, the supernatant was recovered, and freeze-dried at -80°C to prepare the hydrolyzate of Example 1.

Reaction temperature and pH conditions for each enzyme are listed in Table 1 below.

Enzyme name Reaction temperature (℃) reaction pH Example 1 Termamyl S.C. 60 6.5~8.5

< Experiment example >

Experiment example 1. Evaluation of cytotoxicity on macrophages (RAW 264.7)

To confirm the toxic safety of kelp tail hot water extract and hydrolyzate to macrophages (RAW 264.7), cytotoxicity was assessed using a colorimetric method using the MTT assay.

In the cytotoxicity test method, each cell was seeded at 1X 10 ⁴ cells/well in a 96-well plate and cultured for 24 hours in a 37°C, 5% CO ₂ incubator.

The cultured cells were treated with kelp tail hot water extract and hydrolyzate at various concentrations (1, 50, 100 ug/ml) and cultured in a 5% CO ₂ incubator at 37°C for 24 hours. After 24 hours of incubation, the cell culture medium was removed, and medium containing MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] at 1 mg/ml was added to each well. Treatment was performed at a time of ㎖, and cultured for an additional 4 hours in an incubator at 37°C and 5% CO _2. After removing the medium, tetrazolium bromide salt was removed, and 200 ㎕ of DMSO was dispensed into each well. Formazan crystals were dissolved, and cell viability was confirmed by measuring absorbance at a wavelength of 570 nm in a microplate reader.

As a result, it was confirmed that the hydrolyzate of kelp tail hot water extract did not exhibit cytotoxicity up to a concentration of 100 ug/ml (Figure 3). Therefore, up to 100 ug/ml was judged to be a safe range for toxicity, and the subsequent immune enhancement functional evaluation (NO assay) was conducted within the concentration range of 100 ug/ml.

Experiment example 2. Immune-boosting functionality evaluation (Nitric oxide assay)

Nitric oxide (NO) assay was performed to evaluate the immune-enhancing functionality of kelp tail extract and hydrolyzate in macrophages (RAW 264.7).

The level of NO production was determined by culturing RAW 264.7 cells at ⁵ Cultured for 24 hours. Each sample was treated at different concentrations (1, 50, 100㎍/㎖) and cultured at 37°C, 5% CO ₂ for 1 hour, and then treated with 100 ng/mL of LPS (lipopolysaccharide) as a positive control group and sample alone. Treated and cultured for 18 hours.

In 50 μL of cell culture medium, 50 μL of 5% phosphoric acid containing Griess reagent, 1% sulfanilamide (Sigma chemical Co., St. Louis, MO, USA) and 0.1% naphthylethylenediamine dihydrochloride (Merck KGaA, Darmstadt, Germany) ) After adding 50 μL and reacting for 10 minutes, the absorbance was measured at 540 nm using a microplate reader (Molecular Device, Sunnyvale, CA, USA).

As a result, compared to the positive control group treated with LPS alone, Comparative Example 1 and Example 1 treated with kelp tail hot water extract or fermented product of kelp tail hot water extract increased the amount of NO released, and it was confirmed that the amount of NO released increased in a concentration-dependent manner. (Figure 1).

Specifically, Comparative Example 1 did not show a significant increase in NO emission, but Example 1, which treated the fermented product of kelp tail hot water extract, showed a significant increase in NO emission.

In particular, at a concentration of 100 ug/ml, the kelp tail hot water extract released 7.61 uM nitric oxide, but the hydrolyzate prepared using Termamyl SC enzyme, a glycolytic enzyme, was confirmed to release 9.48 uM nitric oxide. In other words, it was confirmed that the hydrolyzate of kelp tail extract showed an immunity-boosting effect that was about 24.57% higher than when hydrolysis was not performed. In addition, the control group treated only with LPS released 6.37 uM of nitric oxide, confirming that the hydrolyzate using Termamyl SC enzyme showed a 48.82% higher immune-boosting effect.

It was confirmed that the hydrolyzate of the kelp tail extract of the present invention contributes to significantly activating the immune enhancement of macrophages and is significantly effective in enhancing immunity without significant cytotoxicity.

The immune-enhancing composition containing the hydrolyzate of the kelp by-product extract of the present invention as an active ingredient has immune-boosting activity by inducing the production of NO (Nitric Oxide), an immune indicator, in macrophages, which is caused by decreased immune function. It can be useful in the prevention, improvement, and treatment of diseases.

Claims (10)

1) Adding a solvent to the kelp by-product, refluxing at 100°C for 3 hours, and extracting;
2) Concentrating the extract prepared in step 1) at 50°C and then freeze-drying; and
3) Preparation of an immune-boosting composition containing hydrolyzate of kelp by-product extract as an active ingredient, comprising the step of adding 0.1 to 1 part by weight of enzyme and hydrolyzing 100 parts by weight of the sample freeze-dried in step 2) above. in a way,
The enzyme is Termamyl SC,
The hydrolysis reaction in step 3) is carried out at 60°C and pH 6.5 to pH 8.5 for 3 hours,
Before or after the hydrolysis reaction in step 3), further comprising reacting at 90°C for 15 minutes to deactivate the endogenous enzyme or hydrolytic enzyme,
When the composition prepared by the above manufacturing method is treated with macrophages at a concentration of 100 ug/ml, 9.48 uM of nitric oxide is released, and nitric oxide is 24.57% more than the extract prepared in steps 1) and 2) above. A method of producing a composition for enhancing immunity, which is released. delete delete In claim 1,
After the hydrolysis reaction in step 3), the production method further includes recovering the supernatant by centrifugation and freeze-drying it. In claim 1,
A production method, characterized in that the solvent is at least one selected from water, C1 to C4 alcohol, and hexane. In claim 1,
A manufacturing method, characterized in that the kelp by-product extract is extracted using water. In claim 1,
A production method, characterized in that the kelp by-product extract is a kelp by-product hot water extract. delete delete delete