KR20210058741A - Health functional food for ameliorating stress comprising extract of black rice aleurone - Google Patents

Abstract

The present invention relates to a health functional food for ameliorating stress comprising an extract of black rice aleurone, wherein the black rice aleurone extract suppresses weight loss and immune function decline due to the stress, and has excellent effects of regulating the secretion of IL-6, TNF-α, MMP-2, MMP-3, MMP-7, MMP-13, and lipocalin-2 increased due to the stress, and thus can be usefully used as a health functional food for stress relief.

Description

{Health functional food for ameliorating stress comprising extract of black rice aleurone}

The present invention relates to a health functional food for improving stress, and specifically relates to a health functional food for improving stress including black rice whistle extract.

In the modern society, an increasing number of people complain of anxiety disorders and mental disorders caused by various stresses. In general, our living body produces several reactions against stress, which leads to changes in the endocrine system and autonomic nervous system. In particular, persistent or chronic stress is accompanied by psychological changes such as depression, anxiety, and anger, and the pathogenesis of various diseases such as mental disorders, digestive disorders such as central nervous system, immune system disorders, nervous gastric ulcers, and cardiovascular disorders such as angina. It is closely related.

Chronic psychological stress consequently causes depression.In Western society, drugs related to depression are among the drugs with the highest prescription rate among prescription drugs, and the World Health Organization (WHO) reports that by 2020, depression in the U.S. and Western Europe. It is estimated to be the disease with the highest prevalence. In Korea, as urbanization accelerates, the gap between the rich and the poor and feelings of alienation are deepening, and the incidence of depression is rapidly increasing. Currently, Korea is one of the countries with the highest suicide rate among OECD countries.

Specifically, depression (or severe depressive disorder) is a serious mental disorder that accompanies physical and cognitive dysfunction along with subjective emotions such as sadness and lethargy. The most important symptom of depression is the loss of interest and enjoyment for universal stimuli. When depression occurs, daily life becomes difficult, resulting in huge social and economic losses, and death as 60 to 70% of the causes of suicide are depression. It is also a disease that leads to. Therefore, although there is a need for and development of antidepressants, some antidepressants have severe side effects or may not meet expectations.

It usually takes about 2 weeks to improve symptoms such as improvement of depression by administration of antidepressants, and there are many patients who do not respond well to antidepressants, or recurrence of depression, and are unable to take drugs due to severe side effects. Specifically, tricyclic antidepressants such as imipramine and desipramine have severe side effects such as hypotension and heart dysfunction, and are the most widely used selective drugs such as fluoxetine or sertraline. Serotonin absorption inhibitors cause nausea, gastrointestinal bleeding or sexual dysfunction. Therefore, the development of new drugs that are more effective and have fewer side effects than existing antidepressants has been continuously attempted, but the development costs are enormous, so pharmaceutical companies cannot easily develop them.

Therefore, it is necessary to develop a health functional food that can prevent the development of stress stimulation into mental disorders without the problems that appear in the antidepressants. Natural products contain various unknown ingredients and have the advantage of being relatively safe. , It has a very high potential for stress improvement. In this regard, referring to the specific prior art, grapefruit oil; And there is a patent (KR 10-2010-0106062 A) on a fragrance extract containing bergamot oil, which is said to have stress relief and soothing effects, and can be used in various fields such as beauty and cosmetics. However, the stress referred to in the above patent provides an instant refreshing sensation and does not substantially alleviate mental stress.

On the other hand, black rice ( Oryza sativa , black rice) is a type of primitive rice, mainly produced in China, and has a unique color and aroma as a type of colored rice. It has a water-soluble pigment called anthocyanin, which is known to have antioxidant, anticancer and anti-ulcer effects, so it has a black color, and black rice contains four times more anthocyanins than black beans. It contains more than 5 times that of ordinary rice. Therefore, black rice is known to be good for preventing heart disease, stroke, adult diseases, and cancer as well as effectively neutralizing free radicals in the body that cause aging and various diseases.

Aleurone is also a term for the aleurone layer, which is a cell layer containing a large amount of aleurone. In addition to storing nutrients, the algal layer serves to secrete enzymes such as amylase and supply them to the embryo, and contains enzymes related to proteolysis. The algae layer can be found in the outermost layer of seed endosperm such as Crested family and sedum plants, and has a high content of protein, fat, and vitamins.

Since there was no prior art for a health functional food for stress improvement including black rice whistle extract, therefore, the present inventors were studying to find an extract for stress improvement from natural substances, the black rice whistle extract prevented weight loss due to stress. The present invention was completed by confirming that there is an effect of inhibiting and regulating the secretion of cytokines, MMPs, and lipocalins.

An object of the present invention is to provide a health functional food for improving stress.

Another object of the present invention is to provide a pharmaceutical composition for improving stress.

To achieve the above object,

One aspect of the present invention provides a health functional food for improving stress containing black rice whistle extract as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for improving stress containing black rice whistle extract as an active ingredient.

The black rice whistle extract of the present invention inhibits weight loss and immune function decline due to stress, and increases IL-6, TNF-α, MMP-2, MMP-3, MMP-7, MMP-13, lipocalin due to stress. It has an excellent effect of controlling the secretion of -2, so it can be usefully used as a health functional food for stress improvement.

1 is a graph measuring the weight of rats of each group prepared in an experimental animal model after breeding for 12 weeks.
FIG. 2 is a graph showing the weight of the spleen extracted by sacrificing the rats of each group prepared in the experimental animal model after 12 weeks of breeding.
3 is a graph showing the weight of the thymus gland extracted by sacrifice of rats of each group prepared in an experimental animal model after 12 weeks of rearing.
4 is a graph showing serum IL-6 levels in blood by analyzing serum obtained from rats of each group prepared in an experimental animal model after 12 weeks of breeding.
FIG. 5 is a graph showing serum TNF-α levels in blood by analyzing serum obtained from rats of each group prepared in an experimental animal model after breeding for 12 weeks.
6 is a graph showing serum MMP-2 levels in blood by analyzing serum obtained from rats of each group prepared in an experimental animal model after 12 weeks of breeding.
FIG. 7 is a graph in which serum MMP-3 levels in blood were measured by analyzing serum obtained from rats of each group prepared in an experimental animal model after breeding for 12 weeks.
FIG. 8 is a graph in which serum MMP-7 levels in blood were measured by analyzing serum obtained from rats of each group prepared in an experimental animal model after breeding for 12 weeks.
9 is a graph showing serum MMP-13 levels in blood by analyzing serum obtained from rats of each group prepared in an experimental animal model after 12 weeks of rearing.
FIG. 10 is a graph showing serum lipocalin-2 levels in blood by analyzing serum obtained from rats of each group prepared in an experimental animal model after breeding for 12 weeks.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a health functional food for improving stress comprising a black rice whistle extract.

The black rice flour extract may be prepared in the form of a liquid or powder.

The black rice flour extract may be prepared by a manufacturing method comprising the step of preparing an extract by adding an extraction solvent to the black rice flour.

The black rice flour extract may be prepared, including one or more of filtration, concentration under reduced pressure, and drying, if necessary.

The black rice flour extract may be prepared in the form of a powder by adding an extraction solvent to the black rice flour to prepare an extract, followed by filtration, concentration under reduced pressure, and lyophilization.

In the above manufacturing method, black rice can be used without limitation, such as grown or commercially available.

In addition, the extraction solvent may be water, _{a lower alcohol of C 1} to C ₃ , or a mixed solvent thereof, and specifically, the _{lower alcohol of C 1} to C ₃ may be ethanol. Further, the extraction solvent may be ethanol or an aqueous ethanol solution, and the aqueous ethanol solution is 20 to 90% (v/v), 30 to 80% (v/v), 20 to 80% (v/v), 30 to 70% (v/v) or 40 to 60% (v/v).

Here, the extraction method may be immersion, shaking extraction, Soxhlet extraction, or reflux extraction. At this time, the extraction temperature is preferably 10 to 70 °C, more preferably 30 to 50 °C, but is not limited thereto. At this time, the extraction time is 10 to 96 hours; 20 to 82 hours; 30 to 70 hours; Alternatively, it may be 40 to 56 hours, but is not limited thereto. The extraction may be repeated 1 to 5 times. In addition, the extraction solvent is preferably extracted by adding 2 to 20 times the weight of the black rice flour used for extraction, more preferably 5 to 15 times for extraction, and 8 to 12 times for extraction. Although preferred, it is not limited thereto. The content of the active ingredient in the black rice flour extract may be insignificant because extraction is not sufficiently performed within a range outside the conditions of the extraction time or the number of extractions. Alternatively, the extraction yield may no longer increase, and thus the efficiency of the extraction operation may decrease.

On the other hand, the filtration may use a gravity filter, a vacuum filter, a pressurized filter, a compressed filter, a centrifugal filter, or a membrane separation filter, but is not limited thereto, and the vacuum concentration may be performed using a rotary vacuum concentrator, a vacuum vacuum concentrator, or a vacuum rotary evaporator. However, the drying is not limited thereto, and the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and specifically, may be freeze drying, but is not limited thereto.

In addition, the extract of the present invention includes not only the extract by the above-described extraction solvent, but also an extract obtained through another conventional extraction method, or an extract that has undergone purification and fermentation processes. It includes not only the extract by the decompression by carbon dioxide, the extraction by the supercritical extraction method by high temperature, the extraction by the extraction method using ultrasonic waves, but also the fractions such as the fraction using the difference in solvent polarity for the crude extract. Separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography, or extracts obtained by fermentation products using natural or various microorganisms are also included in the extract of the present invention.

The black rice flour extract can suppress weight loss due to stress.

In a specific embodiment of the present invention, the weight of the rat decreases due to the pain stress caused by performing a knee incision, but it was found that the stress was suppressed and the weight loss was suppressed when the black rice flour extract was fed to the stress-induced rat (Experimental Example 1 and Fig. 1).

The black rice whistle extract can suppress the decrease in immune function due to stress. In a specific embodiment of the present invention, the weight of the spleen and thymus, which are immune tissues of the rat, is reduced due to the pain stress caused by performing a knee incision, but the stress is suppressed and the weight of the spleen and thymus is reduced when the black rice flour extract is applied to the stress-induced rat. Was found to be suppressed (see Experimental Example 2, FIGS. 2 and 3).

The black rice whistle extract may reduce cytokines increased due to stress. Here, the cytokine may be one or more selected from the group consisting of IL-6 and TNF-α.

The black rice nutrient extract may reduce matrix metallopeptidase (MMP) increased due to stress. Here, the MMP may be at least one selected from the group consisting of MMP-2, MMP-3, MMP-7, and MMP-13.

The black rice whistle extract may reduce lipocalin increased due to stress. Here, the lipocalin may be lipocalin-2.

In a specific embodiment of the present invention, levels of IL-6, TNF-α, MMP-2, MMP-3, MMP-7, MMP-13 and lipocalin-2 in the blood of rats due to pain stress induced by performing a knee incision However, when black rice wheat extract was fed to stress-induced rats, stress was suppressed and blood IL-6, TNF-α, MMP-2, MMP-3, MMP-7, MMP-13, and lipocalin-2 levels decreased. (See Experimental Examples 3 to 5 and FIGS. 4 to 10).

From this, the black rice flour extract of the present invention has the effect of inhibiting weight loss and immune tissue weight reduction due to stress induction, and blood IL-6, TNF-α, MMP-2, MMP-3, MMP-7 increased by stress induction. , MMP-13 and lipocalin-2, since it has the effect of reducing, it can be seen that it can be usefully used as a health functional food for stress improvement.

"Health functional food" as used herein refers to a food manufactured using nutrients that are liable to be deficient in daily meals or raw materials or ingredients having useful functions for the human body, and refers to foods that help maintain the health of the human body, but is not limited thereto. It is not used in a sense that includes all health foods in the usual sense.

The form and type of the health functional food is not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional food may contain various flavoring agents, sweetening agents, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include taumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.

In addition to the above-described additional ingredients, the health functional food of the present invention includes nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pexane and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , Glycerin, alcohol, and the like may be further included. These ingredients can be used independently or in combination. The ratio of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the extract of the present invention.

The black rice whistle extract of the present invention may be added to food as it is or used with other foods or food ingredients. At this time, the content of the added active ingredient may be determined according to the purpose. In general, the content of the health functional food may be 0.01 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be below the above range, and if there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another aspect of the present invention provides a pharmaceutical composition for improving stress containing black rice whistle extract as an active ingredient.

The description of the black rice whistle extract is the same as the description of the black rice whistle extract in the health functional food.

The pharmaceutical composition for improving stress containing the black rice flour extract of the present invention as an active ingredient contains 0.1 to 99.9% by weight of the extract of the present invention as an active ingredient based on the total weight of the composition for administration, and is pharmaceutically acceptable. Carriers, excipients or diluents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and is, for example, selected from the group consisting of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol and ethanol. Any one of these components or one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to form an injectable formulation such as an aqueous solution, a suspension, an emulsion, a powder, a tablet, a capsule, a pill, a granule, or an injection solution. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art.

In addition, the pharmaceutical composition for improving stress containing the black rice whistle extract of the present invention as an active ingredient can be administered orally or parenterally, and when administered parenterally, a subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection injection method is used. It is desirable to choose. In order to formulate a formulation for parenteral administration, the composition is mixed with the composition in water to prepare a suspension, and it is formulated in a unit dosage form of an ampoule or vial.

The pharmaceutical composition for improving stress containing the black rice whistle extract of the present invention as an active ingredient is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

Hereinafter, the present invention will be described in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

<Example 1> Preparation of black rice flour extract

The aleurone layer of black rice (Jindo Gumjeong Rice Farming Corporation, Jindo) ( Oryza sativa , black rice) was pulverized and extracted for 48 hours at 40°C with 50% (v/v) alcohol 10 times the weight thereof. . The extract was filtered through a filter paper, concentrated, lyophilized, and powdered, which was used in the following experiment.

<Experiment preparation>

1. Preparation of experimental animal model

In order to confirm the stress improvement effect of the black rice whistle extract, a stress-induced rat model was prepared. The normal rat model was Sprague-Dawley (SD) male white rat, and the stress-induced rat model had pain stress by performing an anterior cruciate ligament transaction on the rat. During the breeding period of normal rats and stress-induced rats, general solid feed (central laboratory animal, Seoul, Korea) and water were freely ingested, and the temperature of the breeding environment was 23±2℃ and the humidity was 50±10%. The contrast period was adjusted in units of 12 hours.

The black rice whistle extract prepared in Example 1 was dissolved in a solvent (Phosphate Buffer Saline, PBS), and the black rice whistle 1 group was fed with 150 mg of black rice whistle extract per 1 kg of rat body weight, and the black rice whistle 2 group was stressed. 300 mg of black rice flour extract per 1 kg of rat body weight was fed, and the control group was fed only the same amount of PBS to stress rats. That is, the experiment was conducted by dividing into a normal group, a control group, a black rice flour group 1, and a black rice flour group 2 groups.

This was bred for a total of 12 weeks, and after the breeding was completed, the body weight was measured _{, anesthetized with CO 2} , and blood was collected. The collected blood was placed in a centrifugal test tube and centrifuged for 15 minutes at 2000 rpm and 4°C to obtain serum. The collected rats were _{anesthetized with CO 2} and euthanized, and the spleen and thymus were excised. The weight was measured using.

<Experimental Example 1> Analysis of the effect of inhibiting weight loss by inducing stress

In order to confirm whether the black rice flour extract according to the present invention has a stress suppressing effect, an experiment was performed to analyze the effect of suppressing weight loss due to stress induction by the following method, and the results are shown in FIG. 1.

For the normal group, the control group, the black rice flour group 1 and the black rice flour group 2 prepared as in the experimental animal model, the body weight was measured every week for 12 weeks, and the body weight at the 12th week at the end of the experiment period is shown in FIG. I got it. The weight of the rat was measured and recorded in a state where the numerical value was stabilized on a scale capable of measuring to one decimal place.

Compared to the normal group, the control group lost 8.4% in weight. In addition, the black rice flour group 1 had a weight of 0.4% higher than that of the control group, and the black rice flour group 2 had a body weight of 7% higher than that of the control group (see FIG. 1).

The above results indicate that the weight of the rat is reduced due to the pain stress caused by performing a knee incision, but the stress is suppressed and the weight loss is suppressed when the black rice flour extract is fed to the stress-induced rat. From this, it can be seen that the black rice flour extract of the present invention has an effect of suppressing weight loss due to stress induction, and thus can be usefully used as a health functional food for improving stress.

<Experimental Example 2> Analysis of the inhibitory effect on reducing immune tissue weight by inducing stress

In order to confirm whether the black rice whistle extract according to the present invention has a stress suppression effect, an experiment was performed to analyze the suppression effect of reducing immune tissue weight by stress induction by the following method, and the results are shown in FIGS. 2 and 3 . When stress is induced, the function of the spleen or thymus, which is an immune tissue, decreases and an abnormality occurs in immunity, so the following experiment was performed.

According to the preparation of the experimental animal model, the normal group, the control group, the black rice flour group 1, and the black rice flour group 2 were reared, and after 12 weeks of breeding was completed, the body was euthanized, and the immune organs of the spleen and thymus were extracted, and the weight was weighed with a chemical balance. It was measured.

Compared to the normal group, the control group had a 13% reduction in spleen weight. In addition, the black rice flour group 1 had a 10% higher spleen weight compared to the control group, and the black rice flour group 2 had a 12% higher body weight than the control group (see FIG. 2).

In addition, compared to the normal group, the control group decreased the thymus weight by 23%. In addition, the black rice flour group 1 had a thymus weight higher than that of the control group by 2%, and the black rice flour group 2 had a thymus weight higher than the control group by 19% (see FIG. 3).

The above results show that the weight of the spleen and thymus of the rat is reduced due to pain stress caused by performing a knee incision, but the weight of the spleen and thymus is suppressed when the stress is suppressed when the black rice whistle extract is fed to the stress-induced rat. . From this, it can be seen that the black rice flour extract of the present invention has an inhibitory effect on reducing the weight of immune tissues caused by stress induction, and thus can be usefully used as a health functional food for improving stress.

<Experimental Example 3> Analysis of the modulatory effect of IL-6 and TNF-α in blood increased by stress induction

In order to check whether the black rice whistle extract according to the present invention has a stress inhibitory effect, the modulating effect of IL-6 (Interleukin-6) and TNF-α (Tumor necrosis factor-α) in blood, which is increased by stress induction by the following method, An experiment to analyze was performed, and the results are shown in FIGS. 4 and 5. When stress is induced, cytokines IL-6 and TNF-α are secreted in the body, so they are well known as representative blood stress indicators.

According to the preparation of the experimental animal model, the normal group, the control group, the black rice flour group 1 and the black rice flour group 2 were reared, blood was collected after 12 weeks of breeding, and the collected blood was centrifuged to obtain serum. It was taken as a sample.

Quantitative analysis was performed using a mouse enzyme-linked immnunosorbent assay (ELISA) kit. The sample was placed on a plate coated with a monoclonal antibody, and then reacted at room temperature for 150 minutes in a 400 rpm rotary incubator, and then washed three or more times with the provided washing buffer. Then, polyclonal antibody was added to attach IL-6 (abcam, ab119548) to the plate, reacted at room temperature for 1 hour at 400 rpm, washed three or more times with the provided washing buffer, and then added the conjugate solution of IL-6. It was reacted for 30 minutes at room temperature at 400 rpm. The reacted plate was washed three or more times with a washing buffer and color was developed at room temperature with a substrate solution, and then a stop solution was added to stop the color reaction, and the absorbance was measured at 450 nm. In addition, an experiment for TNF-α (abcam, ab46070) was performed in the same way.

Compared to the normal group, the control group had a 25% increase in blood IL-6 (pg/mL) levels. In addition, the level of IL-6 (pg/mL) in the blood in the black rice flour group 1 decreased by 8% compared to the control group, and the level of IL-6 (pg/mL) in the blood in the black rice flour group 2 decreased by 11% compared to the control group (Fig. 4).

In addition, compared to the normal group, the control group had a 31.1% increase in blood TNF-α (pg/mL) levels. In addition, the level of TNF-α (pg/mL) in blood in the black rice flour group 1 decreased by 7% compared to the control group, and the level of TNF-α (pg/mL) in the blood in the black rice flour group 2 decreased by 24% compared to the control group (Fig. 5).

The above results show that the level of IL-6 and TNF-α in the blood of rats increased due to pain stress caused by performing a knee incision, but when black rice wheat extract was fed to the stress-induced rats, stress was suppressed and thus blood IL-6 and TNF- It indicates a decrease in the α level. From this, it can be seen that the black rice whistle extract of the present invention has a modulating effect of IL-6 and TNF-α in blood, which is increased by inducing stress, and thus can be usefully used as a health functional food for improving stress.

<Experimental Example 4> Analysis of the modulating effect of MMP in blood increased by inducing stress

In order to confirm whether the black rice whistle extract according to the present invention has a stress suppressing effect, an experiment was performed to analyze the modulating effect of matrix metallopeptidase (MMP) in blood, which is increased by stress induction, by the following method, and the results are shown in Figs. It is shown in 9. Since it is well known that the MMP level in the body increases when stress is induced, experiments for MMP-2, MMP-3, MMP-7, and MMP-13 in blood were performed as follows.

According to the preparation of the experimental animal model, the normal group, the control group, the black rice flour group 1 and the black rice flour group 2 were reared, blood was collected after 12 weeks of breeding, and the collected blood was centrifuged to obtain serum.

Dispense 100 μL of rat serum into each well in a 96 well plate of the MMP-7 ELISA kit (abcam, ab213910), react at 37°C for 120 minutes, and dispense 250 μL of wash buffer into each well. Leave to be washed for 1 minute to 2 minutes, and repeat the process of wiping off on a sterile tissue 5 or more times. Apply 100 μL of biotinylated antibody to each well, react at 37°C for 60 minutes, dispense 250 μL of wash buffer, leave to wash for 1 to 2 minutes, repeat the process of wiping off on sterile tissues 5 or more times. After that, it is left to dry for at least 5 minutes.

100 μL of Streptavidin peroxidase was dispensed into each well and reacted at 37° C. for 30 minutes, then 250 μL of streptavidin peroxidase was dispensed into each well and washed for 1 minute to 2 minutes, and shaken off with a sterile tissue. 100 μL of 3,3',5,5'-tetramethylbenzidine substrate was dispensed into each well, left at 37°C for 20 minutes, and 100 μL of stopping solution was dispensed. After confirming the stopping reaction changing from blue to yellow, the absorbance (optical density) of each well was measured at 450 nm in a microwell plate reader (Molecular Devices, Sunnyvale, CA, USA) within 30 minutes, and each concentration group for the control group The percentage of absorbance was calculated and evaluated as the relative activity of MMP-7. In addition, the quantitative experiments of MMP-2, MMP-3, and MMP-13 were performed in the same manner as this except for using the ELISA kit corresponding to each MMP.

Compared to the normal group, the control group had a 3.4% increase in blood MMP-2 (ng/mL) levels. In addition, the level of MMP-2 (ng/mL) in the blood in the black rice flour group 1 decreased by 8% compared to the control group, and the MMP-2 (ng/mL) level in the blood in the black rice flour group 2 decreased by 6% (see FIG. 6). .

In addition, compared to the normal group, the control group had a 42% increase in blood MMP-3 (ng/mL) levels. In addition, the level of MMP-3 (ng/mL) in the blood in the black rice flour group 1 decreased by 31% compared to the control group, and the MMP-3 (ng/mL) level in the blood in the black rice flour group 2 decreased by 20% (see FIG. 7). .

And, compared to the normal group, the control group had a 7.8% increase in blood MMP-7 (ng/mL) levels. In addition, the level of MMP-7 (ng/mL) in the blood in the black rice flour group 1 decreased by 20% compared to the control group, and the MMP-7 (ng/mL) level in the blood in the black rice flour group 2 decreased by 18% (see FIG. 8). .

Finally, compared to the normal group, the control group had a 40.6% increase in blood MMP-13 (ng/mL) levels. And, compared with the control group, the level of MMP-13 (ng/mL) in the blood in the black rice flour group 1 decreased by 22%, and the level of MMP-13 (ng/mL) in the blood in the black rice flour group 2 decreased by 26% (see FIG. 9). .

The above results show that the levels of MMP-2, MMP-3, MMP-7, and MMP-13 in the blood of the rat are increased due to pain stress caused by performing a knee incision, but the stress is suppressed by feeding black rice flour extract to the stress-induced rat. This indicates that the levels of MMP-2, MMP-3, MMP-7 and MMP-13 in the blood are decreased. From this, it can be seen that the black rice whistle extract of the present invention has a modulating effect of increasing blood MMP by inducing stress, and thus can be usefully used as a health functional food for improving stress.

<Experimental Example 5> Analysis of the modulating effect of lipocalin-2 in blood increased by stress induction

In order to confirm whether the black rice flour extract according to the present invention has a stress suppressing effect, an experiment was performed to analyze the modulating effect of lipocalin-2 in blood, which is increased by stress induction by the following method, and the results are shown in FIG. . Since it is well known that lipocalin-2 is upregulated in the body by stress, the following experiment was performed.

According to the preparation of the experimental animal model, the normal group, the control group, the black rice flour group 1 and the black rice flour group 2 were reared, blood was collected after 12 weeks of breeding, and the collected blood was centrifuged to obtain serum. Thereafter, a quantitative experiment was performed in the same manner as in Experimental Example 4, except that the lipocalin-2 ELISA kit was used.

Compared to the normal group, the control group had a 2.6% increase in the level of lipocalin-2 (ng/mL) in the blood. In addition, the level of lipocalin-2 (ng/mL) in the blood was decreased by 7% in the black rice flour group 1 compared to the control group, and the lipocalin-2 (ng/mL) level in the blood in the black rice flour group 2 decreased by 10% (see FIG. 10). .

The above results indicate that the level of lipocalin-2 in the blood of the rat is increased due to the pain stress induced by performing a knee incision, but the level of lipocalin-2 in the blood is reduced by suppressing the stress when the black rice whistle extract is fed to the stress-induced rat. From this, it can be seen that the black rice whistle extract of the present invention has a modulating effect of lipocalin-2 in blood, which is increased by inducing stress, and thus can be usefully used as a health functional food for stress improvement.

Claims (12)

Health functional food for improving the lowering of immunity due to stress containing black rice whistle extract as an active ingredient.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that extracted with _{water, C 1} to C _{3 lower alcohol or a mixed solvent thereof.}
The method of claim 2,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that it is extracted with ethanol or an aqueous ethanol solution.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that it suppresses weight loss due to stress.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity decrease due to stress, characterized in that it suppresses the decrease in immune function due to stress.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that it reduces the cytokines increased due to stress.
The method of claim 6,
The cytokine is a health functional food for improving immunity reduction due to stress, characterized in that at least one selected from the group consisting of IL-6 and TNF-α.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that to reduce the increased MMP (Matrix metallopeptidase) due to stress.
The method of claim 8,
The MMP is a health functional food for improving immunity reduction due to stress, characterized in that at least one selected from the group consisting of MMP-2, MMP-3, MMP-7 and MMP-13.
The method of claim 1,
The black rice whistle extract is a health functional food for improving immunity reduction due to stress, characterized in that to reduce the lipocalin increased due to stress.
The method of claim 10,
The lipocalin is a health functional food for improving immunity reduction due to stress, characterized in that lipocalin-2.
A pharmaceutical composition for improving the lowering of immunity due to stress, comprising a black rice flour extract as an active ingredient.

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