KR20210096036A - Food composition containing phycocyanobilin and its derivative derived from spirulina - Google Patents

Abstract

A food composition according to one aspect of the present invention comprises Phycocyanobilin derived from Spirulina as an active ingredient. Accordingly, the present invention exhibits drastic effects of treating skin inflammation disease, having ultraviolet ray resistance, alleviating skin wrinkles, and improving skin whitening.

Description

FOOD COMPOSITION CONTAINING PHYCOCYANOBILIN AND ITS DERIVATIVE DERIVED FROM SPIRULINA comprising phycocyanoblin derived from spirulina and derivatives thereof as active ingredients

The present invention relates to a food composition, and more particularly, to a food composition comprising phycocyanoblin derived from spirulina as an active ingredient.

Spirulina is the oldest algae on Earth and is a multicellular organism with a thin cell wall. Although spirulina was used in ancient Africa and Mexico, it was only recently introduced to modern industrial society.

The color of Spirulina is emitted by phycocyanin (blue) of chlorophyll (green) in the cell, and blue-green algae are also called blue-green algae. Spirulina was given its scientific name in 1827 by the German ornithologist Turpin.

Spirulina means spiral in Latin, and is mainly grown in saline lakes in the tropics.

Spirulina is a high-protein food containing more protein (69.5%) than Chlorella (50%), which is famous for being high in protein, and contains all essential amino acids in a balanced way. It is easy to see how much protein spirulina has compared to soybean (soybean), which is a food with high protein content, which is 39%, beef is 20%, and eggs are about 12%. In addition, spirulina is multicellular and has no cell wall, so the digestibility and absorption rate (95.1%) is high, whereas chlorella is single-celled and has a thick cell membrane, so the digestibility rate (73.6%) is low.

Spirulina's pigment component contains yellow carotenoids, green chlorophyll, and blue phycocyanin. Phycocyanin is a bile pigment contained in spirulina, which is not present in chlorella. Like animal bile pigments, phycocyanin helps digestion of fat and exhibits anti-inflammatory and antioxidant properties. It also contains a large amount of antioxidants such as tocopherol and beta-carotene.

Accordingly, in Prior Art Documents 1 to 4, a fermented beverage with excellent antioxidant activity using spirulina, a cancer cell growth inhibitor using a spirulina extract in Prior Art Document 2, and an obesity treatment composition using spirulina extract as an active ingredient in Prior Art Document 3 This, Prior Art Document 4 discloses a composition for improving liver function containing spirulina.

However, prior art documents 1 and 4 are merely used by pulverizing spirulina, and prior art documents 2 and 3 contain a limitation in that they are compositions using phycocyanin.

That is, when using simply crushed spirulina, this is because the targeting effect on target cells is remarkably reduced, and phycocyanin contained in spirulina is excellent in physiological activity, but it is a very unstable substance.

That is, phycocyanin is structurally destroyed due to a strong interaction that occurs depending on factors such as light and temperature after extraction, and the concentration of phycocyanin contained in the extract is eventually reduced. Therefore, it is necessary to derive a substance having a more stable structure of phycocyanin and introduce food including the same.

- Prior Art Document 1: Korean Patent No. 10-1286214 (2013.7.15) - Prior Art Document 2: Korean Patent Publication No. 10-2017-004862 (2017.5.18) - Prior Art Document 3: Korean Patent No. 10-1771695 (2017.8.29) - Prior Art Document 4: Korean Patent Publication No. 10-2001-0111557 (2001.12.19)

The present invention is to solve the problems of the prior art, and an object of the present invention is to provide a food composition containing phycocyanobilin from spirulina.

In addition, the present invention provides a more stable structure in order to overcome the shortcomings that structural destruction of phycocyanin occurs due to strong interaction that occurs depending on factors such as light and temperature, leading to a decrease in the concentration of phycocyanin contained in the extract. To provide a food composition containing phycocyanobilin as an active ingredient.

The food composition according to one aspect of the present invention includes phycocyanobilin derived from spirulina as an active ingredient.

In this case, the phycocyanobilin can be prepared by centrifuging the spirulina aqueous solution, adding TCA to the separated supernatant, stirring, and then concentrating and freeze-drying the reaction product to which MeOH is added to the precipitate.

In addition, the spirulina may include at least one selected from the group consisting of spirulina platensis, spirulina maxima, and combinations thereof.

On the other hand, the phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and may be used for enhancing the antioxidant effect.

In addition, the phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and may be used for anticancer or immune enhancement.

In addition, the phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and may be used for suppressing obesity.

In addition, the phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and may be used for improving liver function.

The present invention produces a food composition containing phycocyanobilin, a stable substance, as an active ingredient, thereby exhibiting remarkable effects in antioxidant effect, anti-cancer or immunity enhancement, obesity suppression and liver function improvement.

In addition, the present invention provides an advanced effect of being able to obtain a composition that is safe to heat and light and has excellent cosmetic activity through primary and secondary processing of spirulina simple extracts.

1 is a graph measuring the high growth of T cells and B cells of a composition containing phycocyanobilin (PCB).
2 is a result of measuring the weight of the experimental animal according to the dose and time of the composition containing phycocyanobilin (PCB).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them with reference to the accompanying drawings. Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

Terms including an ordinal number, such as first, second, etc., may be used to describe various elements, but the elements are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another.

For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. and/or includes a combination of a plurality of related listed items or any of a plurality of related listed items.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, an embodiment of the present invention will be described.

<Example 1>

from spirulina Phycocyanobilin Method for preparing the contained composition

To prepare a composition containing phycocyanobilin (PCB) from spirulina used in the experiment, put 50 g of spirulina powder in 1.5 L of water and stir for 10 minutes. Thereafter, it is placed in 6 250 mL centrifuge bottles and centrifuged at 8000 RPM, 4° C. for 1 hour. After centrifugation, the obtained supernatant is transferred to a 2L flask, and then 15 g of TCA is added.

After stirring for 1 hour under dark conditions (covered with aluminum foil), centrifuge at 8000 RPM, 4°C for 10 minutes, and collect the precipitate. After that, put this precipitate into a 500 mL Erlenmeyer flask, add 25 mL of pure methanol (MeOH), stir slowly, and then add pure methanol again until the final total volume reaches 250 mL.

After centrifugation at 8000 RPM, 4 °C for 10 min, the supernatant is discarded. Repeat the above steps four times or until the MeOH wash is colorless and then store in foil at -20°C. Thereafter, the pellet obtained in the extraction flask and 1 L of MeOH are added and reacted at 60° C. for 12 hours. Thereafter, the final reaction product obtained was concentrated and lyophilized to obtain a powder containing PCB, which was then used in the experiment.

<Experimental Example 1>

from spirulina Phycocyanobilin Measurement of PCB content of the contained composition

The PCB content of the powder obtained in the above process was measured. For the analysis of the concentration of PCB from the PCB powder obtained in Example 1, 10 mL of distilled water was added to 10 mg of powder and mixed, and the test solution was contrasted with distilled water to measure the absorbance at 1 cm of the liquid layer and a wavelength of 680 nm.

- Analysis and calculation

PCB content (mM) = E680nm/37.9 × dilution factor

PCB content analysis result PCB content (mM) Sample 0.004

As shown in [Table 1], in the method for producing phycocyanobilin according to this example, it was confirmed that the content of phycocyanobilin was about 0.004 mM.

<Experimental Example 2>

from spirulina Phycocyanobilin ( Phycocyanobilin ) Measurement of antioxidant activity of a composition containing (PCB)

The antioxidant activity of the composition obtained in the above process was measured. Antioxidant activity was measured for each process by measuring the reducing power and ROS content measurement experiment.

Antioxidant activity analysis result for each process division treatment concentration Reducing power (O.D.) DPPH (%) Sample 0.01 mg/mL 0.216 8.36 0.05 mg/mL 0.734 12.77 0.1 mg/mL 1.002 20.93

Based on the results of [Table 2] above, it was confirmed that the composition of the present invention exhibited a high reducing power value. In addition, it was confirmed that the composition of the present invention exhibited high DPPH activity in a concentration-dependent manner even in DPPH activity, thereby confirming that the composition of the present invention increased antioxidant activity.

At this time, the maximum concentration in the experiment is 0.01% (0.1mg/ml), which is a very low concentration, and in the case of natural products, the antioxidant activity is increased up to 0.1% (1mg/mL) higher than this, and thereafter it is reduced for toxicity or other reasons. Since there are many cases, a separate upper concentration limit is not limited.

<Experimental Example 3>

from spirulina Phycocyanobilin Measurement of cytotoxicity of the contained composition

The cytotoxicity of the composition containing Phycocyanobilin (PCB) from the spirulina was measured. For the measurement of cytotoxicity, MTT reagent was used. CCD-986sk cells were used as the cells used, and for the cytotoxicity measurement method, the cells were cultured in a 96-well plate at a concentration of 3.0 × 105 for 24 hours, and then each sample was treated by concentration and cultured for another 24 hours.

After injecting 50 µL of MTT reagent at a concentration of 200 µg/mL in the dark, incubating for 3 hours, washed twice with PBS buffer, 200 µL of DMSO was injected into each well, and absorbance was measured at 570 nm after 20 minutes. The method of calculating the cell viability was calculated as O.D value of the sample-added group / O.D value of the sample-free group x 100%.

Cytotoxicity analysis results by concentration division treatment concentration Cytotoxicity (%) Sample 0.025 mg/mL 5.63 0.05 mg/mL 7.70 0.075 mg/mL 8.55 0.1 mg/mL 10.63

The cytotoxicity results of the composition containing Phycocyanobilin (PCB) from Spirulina are shown in [Table 3]. As a result of the measurement, it was confirmed that cytotoxicity was increased in a concentration-dependent manner in a composition containing phycocyanobilin (PCB) from spirulina.

At this time, the maximum concentration in this experiment is 0.01% (0.1mg/ml), which is a very low concentration, and in the case of natural products, usually 1% (= 10mg/mL) or more, most of them show cytotoxicity, so maximum cytotoxicity is allowed. The concentration is preferably 5% or less.

<Experimental Example 4>

from spirulina Phycocyanobilin Measurement of NO production of the contained composition

In order to confirm the immune activity of the composition containing phycocyanobilin (PCB) from the spirulina, RAW 264.7 cells of macrophages from mice were cultured to measure NO production. For the culture and differentiation medium, 10% Fetal bovine serum was added to Dulbecco's modified Eagle's medium (DMEM) high glucose and cultured in an incubator at 5% CO2 and 37° C. ml at a density of 96 well plates and cultured for 24 hours.

After 24 hours, the composition containing phycocyanobilin (PCB) from Spirulina was divided into LPS-treated and untreated groups, and 200 ul was added and cultured for another 24 hours. After 24 hours, as much as 50 ul of the supernatant of the cultured sample was transferred to a new 96 well plate, 50 ul of Griess reagent was added, and the absorbance was measured at 540 nm wavelength after leaving at room temperature for 20 minutes. Thus, the amount of NO production was quantified.

Measurement result of NO production by concentration division treatment concentration NO production (uM) Control - 0 LPS - 30.13 Sample 0.025 mg/mL 1.76 0.05 mg/mL 2.62 0.075 mg/mL 2.34 0.1 mg/mL 2.21 Sample + LPS 0.025 mg/mL 13.50 0.05 mg/mL 15.69 0.075 mg/mL 17.98 0.1 mg/mL 17.51

As a result, as shown in [Table 4], as a result of confirming the NO production amount of each extract, it was confirmed that the NO production amount of the composition containing phycocyanobilin (PCB) from spirulina decreased.

That is, in this experiment, when only a toxic substance called LPS was added, a high value of about 30 uM appeared, and it was confirmed that the amount of NO was significantly reduced when this toxic substance and PCB were added together.

At this time, it is remarkable that the treatment concentration in the sample + LPS was found to have a critical significance according to the upper and lower limits of the treatment concentration because the most desirable reduction in the amount of NO production was confirmed between 0.02 and 0.07 mg/mL.

<Experimental Example 5>

from spirulina Phycocyanobilin ( Phycocyanobilin ) (PCB)-containing composition T cell and B cell viability measurement

To confirm the immune activity of the composition containing phycocyanobilin (PCB) from the spirulina, the growth degree of immune cells T cells and B cells was checked, and each cell was Roswell Park Memorial Institue (RPMI) 1640 media and 10% Fetal bovine serum, the cultured cells were put in a 6-well plate at an amount of 2.5 × 105 cells/ml and cultured for 24 hours. After 24 hours, a sample of the composition containing phycocyanobilin (PCB) was treated from spirulina, and the growth degree of each cell was measured by checking the number of cells treated with the extract for 7 days using a hematocytometer.

As confirmed in Figure 1, it was possible to confirm the high growth degree of T cells and B cells.

<Experimental Example 6>

from spirulina Phycocyanobilin Measurement of changes in body weight of experimental animals of the contained composition

In order to confirm the anti-obesity activity of the composition containing phycocyanobilin (PCB) from the spirulina, the weight change was confirmed by ingesting the experimental animals by concentration. Specifically, according to the dietary composition of the laboratory mice described in Table 1 below, the laboratory mice (C57BL/6N, male, 5 weeks old) were given a basic diet (solid feed, Cargill Agripurina, Gunsan, Korea / free drinking water) for one week. Ingestion) and after adaptation to the environment, 10 numbers were placed so that each group had a similar average body weight. In the control diet, 10 kcal was ingested, the high-fat diet was 60 kcal, and the high-fat diet was ingested at 20 mg/kg of anthocyanin cyanidin-3-O-galactoside to measure obesity suppression activity. Water was provided ad libitum, and body weight was measured at weekly intervals.

Dietary Table of Experimental Rats Control diet (10 kcal % fat) High fat diet (60 kcal % fat) g kcal g kcal Casein, 80 Mesh 200 800 200 800 L-Cystine 3 12 3 12 corn starch 315 1,260 0 0 Maltodextrin 10 35 140 125 500 Sucrose 350 1,400 68.8 275 Cellulose 50 0 50 0 Soybean oil 25 225 25 225 Lard 20 180 245 2,205 Mineral mix 10 0 10 0 Dicalcium phosphate 13 0 13 0 Calcium carbonate 5.5 0 5.5 0 Potassium citrate, 1H ₂ O 16.5 0 16.5 0 Vitamin mix 10 40 10 40 Choline bitartrate 2 0 2 0

As a result of checking the body weight as shown in FIG. 2, compared with the general high-fat diet, the same body weight was shown at the first week 0, but the composition administration group containing phycocyanobilin (PCB) from the spirulina from the first week It was confirmed that the weight decreased compared to the high-fat diet.

<Experimental Example 7>

from spirulina Phycocyanobilin Blood and liver in rats of the contained composition within the organization Triglyceride measurement

In order to confirm the fat-suppressing activity of the composition containing phycocyanobilin (PCB) from the spirulina, the following experiment was performed. In the case of blood triglyceride analysis, triglyceride was measured using a blood biochemistry analyzer (KoneLab 20, Thermo Fisher Scientific, Waltham, MA, USA) for serum isolated from the blood collected on the day of autopsy from mice.

For triglyceride analysis of liver tissue, a mixed solution of chloroform : methanol = 2:1 (v/v) was added to 0.5 g of liver, homogenized using a homogenizer, and stirred to extract lipids, and lipids were extracted at 3,000 rpm for 10 minutes. After centrifugation, the lower layer containing lipids was taken and dried. To measure the triglyceride content by weighing, 3 ml of chloroform was added to dissolve the lipids. Using an enzymatic quantitative measurement kit, it is presented in the kit manual. Triglyceride was measured by referring to the method described above.

Triglyceride content in liver and serum diet test substance triglycerides
(mg/g liver) triglycerides
(g/L Serum) contrasting diet - 4.11 ± 0.33 0.927 ± 0.027 high fat diet - 8.31 ± 0.27 1.336 ± 0.034 high fat diet Sample
(100 mg/kg) 5.90 ± 0.73 0.706 ± 0.086

As confirmed in [Table 6], while triglycerides increased in the liver and serum in the high-fat diet group, the composition containing phycocyanobilin (PCB) from Spirulina at 100 mg/kg administration group, Both serum showed the effect of reducing the triglyceride content.

<Experimental Example 8>

from spirulina Phycocyanobilin ( Phycocyanobilin ) (PCB)-containing composition to improve liver function and measure hangover relieving activity

In order to confirm the liver function improvement and hangover relieving activity of the composition containing phycocyanobilin (PCB) from the spirulina, the following experiment was performed. The blood alcohol concentration of the composition was measured as follows. Six SD rats weighing around 200 g received from Orient Co., Ltd. (Gyeonggi-do, Korea) were set up as one group, and they were used in the experiment after being acclimatized in the laboratory for one week.

A composition containing phycocyanobilin (PCB) from Spirulina at a concentration of 400 mg/kg was mixed with 0.1% CMC and then orally administered. Alcohol was administered to each experimental group at 3 g/kg of ethanol (50%, v/v) diluted with physiological saline. The experimental group to which neither sample nor ethanol was administered was set as a control group, and the experiment was conducted by dividing it into a group administered with only ethanol and a group administered with a composition containing phycocyanobilin (PCB) from spirulina.

Immediately after administering ethanol to each experimental group, 1, 2, and 4 hours later, blood was collected from the orbital vein, centrifuged at 4°C at 3000 rpm for 10 minutes, and serum was separated using an ethanol analysis kit (Sigma, USA). The amount of alcohol remaining in me was measured.

Blood alcohol concentration measurement result Sample time (h) 0 One 2 4 Control 0.004 0.004 0.004 0.004 Ethanol only 0.004 0.156 0.144 0.129 PCB composition 0.004 0.119 0.088 0.071

In addition, for the measurement of alcoholase activity in the liver homogenate, immediately after blood collection of mice administered with a composition containing Phycocyanobilin (PCB) from Spirulina, the liver was removed and mixed with 0.25 M sucrose 4 It was homogenized using a homogenizer at ℃. Then, after centrifugation at 600 × g for 15 minutes, the liver cytosol was separated by ultra-high speed centrifugation at 15,000 × g for 20 minutes and then at 130,000 × g for 45 minutes. Enzyme activity of ADH and ALDH present in the obtained cytosol was measured.

To measure ADH activity, 0.1 mL of the liver homogenate was mixed with 0.2 mL ethanol, 0.02 mL of 0.5M semicarbazide, and 2 mL of a reaction solution containing 0.1 M NAD, reacted at 37°C, and absorbance was measured at 340 nm for 10 minutes.

In addition, for ALDH activity, 0.1 mL of liver homogenate is added to 0.9 mL of 50 mM sodium pyrophosphate buffer (pH 8.5) to make a total of 1.0 mL, 1 mM NAD, 2 mM pyrazol, and 10 mM acetaldehyde are added, and 340 nm of 340 nm in a cycle of 10 minutes at 37°C. Absorbance was measured continuously.

Measurement result of alcoholase activity in liver homogenate Sample ADH activity (%) ALDH activity (%) Control 24.3 28.8 PCB composition 37.4 45.6

In addition, for measuring the enzyme activity of Glutamic oxaloacetic transaminase (GOT) and Glutamic pyruvic transaminase (GPT) in serum, the GOT and GPT activity in serum of the composition containing Phycocyanobilin (PCB) from Spirulina were measured as follows. was carried out as ICR mice (weight 30 g ± 1.5 g) purchased from Orient Co., Ltd. (Gyeonggi-do, Korea) were used, and 6 mice were used as group 1, and Phycocyanobilin (PCB) from CCl4 and Spirulina. The composition containing this was administered.

2 hours before the start of the experiment, the normal group and the CCl4 group were administered 0.9% physiolohical saline, and the experimental group was administered 1mg of a composition containing phycocyanobilin (PCB) from spirulina. Then, at the start of the experiment, CCl4 was administered once at 0.3 mL/kg, and after 24 hours, a composition containing 0.9% physiolohical saline and spirulina was administered, respectively, and then 48 hours passed. Blood was collected and used as samples for GOT and GPT activity. After that, it was measured using a kit for measuring GOT and GPT activity (Yeongdong Pharmaceutical (Seoul, Korea)).

Result of enzyme activity measurement of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in serum Sample GOT (IU/L) GPT (IU/L) 1 Day 2 Day 1 Day 2 Day Normal 156.7 159.3 44.1 43.8 Control 357.9 302.0 99.2 93.6 PCB composition 291.1 265.5 70.3 62.7

As confirmed in [Table 7] to [Table 9], it was confirmed that the composition containing phycocyanobilin (PCB) from spirulina was excellent in liver function improvement and hangover relieving function in all of the administration groups.

As described above, preferred embodiments of the present invention have been disclosed in the present specification and drawings, and although specific terms are used, these are only used in a general sense to easily explain the technical contents of the present invention and to help the understanding of the present invention. , it is not intended to limit the scope of the present invention. It will be apparent to those of ordinary skill in the art to which the present invention pertains that other modifications based on the technical spirit of the present invention can be implemented in addition to the embodiments disclosed herein.

Claims (7)

A food composition comprising phycocyanobilin derived from spirulina as an active ingredient.
According to claim 1,
The spirulina is a food composition comprising at least one selected from spirulina platensis or spirulina maxima.
According to claim 1,
The phycocyanobilin is a food composition, characterized in that it is prepared by centrifuging the spirulina aqueous solution, adding TCA to the separated supernatant, stirring, and then concentrating and freeze-drying the reaction product added with MeOH to the precipitate.
According to claim 1,
The phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and is used for enhancing the antioxidant effect.
According to claim 1,
The phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and is used for anticancer or immune enhancement.
According to claim 1,
The phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and is used for obesity suppression.
According to claim 1,
The phycocyanobilin is contained in an amount of 0.0001 to 20% by weight based on the total weight of the food composition, and is used for improving liver function.

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