KR20140097718A - A health food composition for preventing inflammatory comprising the extracts or fractions of Amaranth as effective component - Google Patents

Abstract

The present invention relates to a health-promotional food composition which an anti-inflammatory effect containing an extract or a fraction of amaranth (Mastixia arborea C.B.Clarke) as an active ingredient. In particular, the extract or the fraction of amaranth exhibit an NO eliminating activity and an effect of inhibiting iNOS generation depending on the density, and thus has an excellent anti-inflammatory effect. Therefore, the extract or the fraction of amaranth can be advantageously used for active ingredients of health-promotional food, a pharmaceutical composition, and an antiaging cosmetic composition. Moreover, the composition of the present invention: has an effect of inhibiting generation of melanin; contains excellent inorganic nutrients such as iron and calcium; includes sulfur-containing amino acids, squalene, and tocopherol, plants of the legume family lack; and thus can be used for various applications such as growth-promotional food, nutrient supplements, cosmetic materials, and pharmaceutical compositions.

Description

[0001] The present invention relates to a health food composition comprising an amaranth extract or a fraction thereof as an active ingredient,

The present invention relates to an anti-inflammatory composition comprising an extract of Mastixia arborea CBClarke or a fraction thereof as an active ingredient. More specifically, the extract of Amaranth or the fraction thereof according to the present invention has NO-scavenging activity and iNOS production inhibitory effect And has an excellent anti-inflammatory effect, it can be effectively used as an active ingredient, a pharmaceutical composition and a cosmetic composition for preventing aging of health food.

Amaranth is a biennial-like grains belonging to the family Amaranthus and is a biplot plant, not a monocotyledon such as corn, oats, rice, and wheat. It was the main food source along with corn and peas in the Inca era of Peru and the Aztec era of Mexico. The size is slightly smaller than the size of a grain of sand, and it is very small in size. The shape of a seed encircles the endosperm like a ring. It has a protein and fat accumulation in an annular pear, and starch It is compassion. The length of the ears is usually 20 to 60cm, and the color of the seed coat is yellow, light flesh, purple. It is usually planted from late April to mid May, and the flowering period usually varies from late June to mid-August depending on the variety of flowering period. The plant length varies depending on the soil fertility, but small varieties are about 120 cm. It is about 250cm.

The present inventors measured the inhibition of NO production and iNOS and cytokine inhibitory activity involved in inflammation by using the extract of Amaranth cultivated in Jeju Island and found that the amaranth seeds And purple outpost showed excellent NO inhibitory effect, inhibition of iNOS protein expression, and cytokine inhibitory effects such as inflammation-related TNF-α and IL-6. In addition, the present inventors completed the present invention by examining the nutritional components of the amaranth, and confirming the superior nutritional components compared to the amaranth grown in the other lipid.

Recently, society is rapidly changing, and various factors such as the advancement of women into society are overflowing with fast food foods for busy modern people. As a result, consumers are routinely exposed to diets that do not have sufficient nutritional components, and various unbalanced body developments and diseases arise, resulting in social problems.

Nitric oxide (NO), one of the substances that induce human diseases and aging, is an air pollutant. It is one of the many chemicals contained in tobacco smoke, which is a very unstable gas with toxicity. At high concentration, It is one of the free radicals. Nitric oxide synthase (NOS) is classified into three types of isoenzymes, Type I, II, and III, depending on their physicochemical properties. In other words, Type I (neuronal NOS, nNOS) and Type Ⅲ (endothelial NOS, eNOS) are classified as constitutive NOS because they are continuously present in the cells. In some cells, lipopolysaccharide (LPS), cytokines and bacteria There is an inducible NOS (iNOS), Type II, which is expressed only when exposed to specific stimuli such as toxins. This NOS converts NO to L-arginine into L-citrulline.

Stimulated iNOS produces a large amount of NO over a long period of time, and the produced NO is characterized by the activity of guanylyl cyclase and cytotoxicity to surrounding tissues. NO is very small, but reactive and electrically neutral, so it diffuses straight from the synthesis site to spread in all directions, promoting inflammatory reactions such as vascular permeability and edema, promoting the biosynthesis of inflammatory mediators, JH, "Inhibition of nitric oxide synthesis by 8-epi-xanthatin in activated RAW 264.7 cell", Yakhak, Hoeji . , 1998 , 42 : 5.). Many research reports on NO have been shown to act as mediators, neurotransmitters or regulators as messenger molecules of various physiological biochemical phenomena in living organisms (Kuo, PC and Schroeder, RA "The cmerging multifaceted roles of nitric oxide. Ann Surg ", 1995 , 221 (3): 220-235.). Although normal NO formation plays an important role in killing bacteria or eliminating tumors, excessive NO formation due to pathological causes causes damage to normal cells, causing inflammation, and damages tissue, gene mutation, and nerve damage The cause (Weisz, A., C Icatiello, I., and Esumi, H., "Regulation of the mouse inducible type nitric oxide synthase gene promoter by interferon- gamma, bacterial lipopolysaccharide and NG-monomethyl-L- arginine", Biochem J., 1996 , 316 : 209).

When inflammation occurs, cells involved in inflammatory reactions such as lymphocytes, macrophages, endothelial cells, and fibroblasts are involved When these cells are exposed to stimulants such as lipopolysaccharide (LPS), interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α), the expression of iNOS is increased, .

Activation of AP-1 transcription factors through the activation of c-Jun by inflammatory mediators and cytokines during inflammatory reactions promotes the expression of matrix metalloproteases (MMPs) that break down mucopolysaccharides and binding fibers in the dermis, (Burger D, Rezzonico R, Li JM, Modoux C, Pierce RA, Welgus HG, et al., &Quot; Imbalance between interstitial collagenase and tissue inhibitor of metalloproteinases (TIMPs) 1 in synoviocytes and fibroblasts upon direct contact with stimulated T lymphocytes: involvement of membrane-associated cytokines ", Arthritis Rheum ., 1998 , 41 : 1748-59.).

Minerals are an essential element in controlling body composition and some body functions. In particular, calcium constitutes bones and nerve cells, and iron constitutes the hemoglobin of red blood cells, forming the bones and blood of the human body, which is an important mineral component for human growth and development from childhood to old age.

Calcium is the most abundant element in the body. The total amount of calcium in the body is about 2% of body weight. Most of calcium is calcium phosphate, which is a component of bone and teeth. It is necessary for blood coagulation. Calcium is an essential ingredient throughout life, especially in growing, pregnancy, and lactation, and is an important component of growth, osteoporosis and arthritis (Lee Sung Hyun, Hwangbo Youngsook, Kim Ji Yeon, Lee Yun Sook, , Korean Nutrition Society, 30 (5): 499-505, 1997)

Iron is a trace element that is involved in oxygen transport as a component of hemoglobin. Therefore, it is a component which is easily deficient if there is a high demand for hemorrhagic disease, menstrual onset period, pregnancy, childbirth, and growing period, (4): 371-384, 2007). In recent years, in order to prevent and treat various diseases such as inflammatory diseases, various foods such as functional foods, functional cosmetics and therapeutic agents Researches using natural materials that are not artificial materials are being actively carried out. The present inventors have measured the anti-inflammatory effect of amaranth which is excellent in the content of minerals such as iron and calcium, has an excellent protein content as compared with other grains, and contains excellent components such as squalene and tocopherol. Respectively.

The object of the present invention is to prevent the various diseases caused by inflammatory diseases through development of safer and more effective natural substances through the use of harmless and environmentally friendly materials to the human body and to develop products containing healthy nutrients, And to provide excellent nutritional formula to children and the elderly. Another object of the present invention is also the first to disclose its anti-inflammatory and whitening effects in Amaranth for the first time.

To achieve the above object, the present invention provides an extract of Amaranth which has an NO inhibitory effect, an iNOS and COX-2 protein inhibitory effect, and an inhibitory effect on cytokine expression by extracting amaranth with ethanol and other organic solvents. Also, the present invention provides a solvent fraction obtained by extracting the above ground part and seedling part of amaranth with 70% ethanol, and then extracting again with an organic solvent such as an organic solvent such as hexane, dichloromethane, ethyl acetate, butanol and the like. As the extraction solvent of the present invention, ethanol, hexane, dichloromethane, ethyl acetate, dimethylformamide (DMF), tetrahydrofuran (THF), chloroform, diethyl ether and the like can be used.

In the present invention, the amaranth seeds and the seedling are dipped in 70% ethanol and repeatedly extracted and concentrated to obtain an ethanol extract, which is then suspended in 20% ethanol and an organic solvent such as hexane, dichloromethane, ethyl acetate and butanol is added successively And a corresponding solvent-extracted fraction is prepared.

The inventors of the present invention have found that a mixed substance extracted and fractioned by the above process exhibits activity such as anti-inflammatory effect, and amino acid and inorganic components are identified by a food or drink method in order to analyze nutritional components, and squalene and tocopherol Respectively.

The anti-inflammatory health supplement composition containing the amaranth extract of the present invention or the solvent fraction of the amaranth extract as an active ingredient may be provided in a form such as a porridge, a wire, a cereal, a drink, and the like. The amaranth extract or the amaranth solvent It is preferred that the fraction contains 0.01 to 10% by weight of the total weight of the composition. When it is 0.01% by weight or less, it is difficult to expect the inflammatory effect of the present invention, and when it is contained by 10% by weight or more, there is a problem in formulation.

The amaranth extract of the present invention has an excellent anti-inflammatory effect. Especially, the amaranth outermost portion has excellent efficacy as an ethanol extract, and can be used as a food in a variety of seeds and outpost portions. Thus, it has a safe and effective anti- It can be widely used as a food composition.

The amaranth extract obtained according to the present invention can be widely applied not only to foods but also to cosmetics and pharmaceuticals.

FIG. 1 shows that the inhibitory effect of genes involved in NO production in the ethyl acetate layer isolated from the seeds using Western blot analysis showed that iNOS was significantly suppressed and COX-2 was also inhibited in a concentration-dependent manner there was.
FIG. 2 shows that the inhibition of iNOS and COX-2 protein expression of the ethanol extract of purple amaranth upper layer was remarkably inhibited by both proteins in a concentration-dependent manner.
Figure 3 shows the amaranth extraction method.
Fig. 4 shows the NO inhibitory effect and cytotoxicity
Figure 5 shows the NO inhibitory effect and cytotoxicity
Figure 6 shows the NO inhibitory effect and cytotoxicity of the amaranth seed ethyl acetate layer
Figure 7 shows the effect of TNF-alpha inhibition
A: purple amaranth 70% EtOH extraction (100 ug / mL),
B: Purple Amaranth 70% EtOH Extraction (200 ug / mL).
C: purple amaranth 70% EtOH extraction (400 ug / mL).
D: Amaranth mixture 70% EtOH extraction (100 ug / mL),
E: Amaranth mixture 70% EtOH extraction (200 ug / mL),
F: Amaranth mixture 70% EtOH extraction (400 ug / mL)
FIG. 8 shows the effect of the extract of the above-ground Amaranth extract on the IL-6 inhibitory effect
A: purple amaranth 70% EtOH extraction (100 ug / mL),
B: Purple Amaranth 70% EtOH Extraction (200 ug / mL).
C: purple amaranth 70% EtOH extraction (400 ug / mL).
D: Amaranth mixture 70% EtOH extraction (100 ug / mL),
E: Amaranth mixture 70% EtOH extraction (200 ug / mL),
F: Amaranth mixture 70% EtOH extraction (400 ug / mL)
Figure 9 shows the effect of the amaranth seed ethyl acetate layer on the TNF-
10 shows the PGE2 inhibitory effect of the amaranth purple pre-plant ethanol extract
11 shows the PGE2 inhibitory effect of? Acetate extract on amaranth seeds
Figure 12 shows the protein content of amaranth leaves and seeds
* A: Raw seeds (grinding)
B: Roasted seeds
C: Raw Seeds (do not grind)
D: leaf
Figure 13 shows the iron content of amaranth leaves and seeds
14 shows the calcium content of amaranth leaves and seeds
15 shows the comparison of the protein content of amaranth and other grains
Figure 16 compares the iron content of amaranth and other grains
17 shows the comparison of the calcium content of amaranth and other grains
18 shows the squalene content of amaranth seeds
19 shows the squalene content of raw amaranth hexane extract
20 shows the squalene content of roasted amaranth hexane extract
Figure 21 shows the alpha-tocopherol content of the amaranth seeds
22 is a graph showing the change in the? -Tocopherol content
23 is a graph showing the effect of the roasted amaranth hexane extract on? -Tocopherol
Figure 24 shows the < RTI ID = 0.0 > β-tocopherol content

The present invention provides amaranth ethanol extract having anti-inflammatory activity by repeatedly extracting amaranth surface portion and seed portion by immersing in 70% ethanol, respectively. In addition, the ethanol extract of amaranth is further extracted with an organic solvent such as hexane, dichloromethane, ethyl acetate, butanol, etc., to provide an extract fraction having anti-inflammatory effect.

The present invention provides an amaranth ethyl acetate fraction having an excellent anti-inflammatory effect by fractionating the upper part of amaranth and the seedling part with 70% ethanol and fractionating the concentrated ethanol extract with an organic solvent.

The present invention provides a composition having anti-inflammatory activity using the above-described amaranth ethanol extract as an active ingredient. Specifically, the extract can be used as a composition for preventing inflammation and inhibiting the production of inflammation, as well as for an anti-inflammatory food, an aromatic product, and a medicine.

The above extract of Amaranth showed excellent NO inhibitory effect and inhibitory effect on iNOS and COX-2 protein in ethanol crude extract, and IL-6 and PEG2 also showed a dose-dependent inhibitory effect on inflammation. The anti - inflammatory effect of seed extract was best measured by the ethyl acetate fraction in the organic solvent fraction. In order to confirm the inhibitory effect of iNOS and COX-2 protein expression, Western blotting assay showed that iNOS and COX-2 protein expression was suppressed in a concentration-dependent manner in the ethyl acetate layer. In particular, Respectively.

In addition, in the present invention, it was confirmed that the nutritional content of amaranth grown in Jeju Island was superior to other regions, and the content of calcium and iron was higher than that of other regions, and the protein content was also confirmed to be relatively high. Especially, the content of squalene and tocopherol was significantly higher than that of other areas, indicating that Jeju amaranth can be used in various foods, cosmetics and pharmaceuticals.

≪ Example 1: Amaranth  From above Of the solvent fraction  Manufacturing>

Amaranth plantlets and seedlings used in this experiment were grown and harvested from Jeju Island. The amaranth outpost was experimented with amaranth mixture of three colors and purple amaranth outpost. The seedlings were pulverized using a pulverizer.

Amaranth outposts and seedlings cultivated on Aewol-eup berry cultivated in Jeju Island were picked and finely crushed. After finely crushing, the outpastes were extracted with 3L 3 times with 80% ethanol and concentrated under reduced pressure to obtain 15 g of ethanol extract. The seeds were extracted with 5% ethanol with 80% ethanol and concentrated under reduced pressure to obtain 12 g of ethanol crude extract. 12 g of the dried crude extract was suspended in 1 L of water, and 1 L of hexane was added to separate the hexane layer. This was repeated twice to obtain a hexane fraction layer. To the remaining water layer was added 1 L of dichloromethane to separate the dichloromethane layer, and this was repeated twice to obtain a dichloromethane fraction layer. To the remaining water layer was added 1 L of ethyl acetate, and the ethyl acetate layer was separated. This was repeated twice to obtain an ethyl acetate fraction layer. Finally, 1 L of butanol was added twice to the water layer to separate into a butanol layer and a water layer.

To investigate the anti-inflammatory effect of the extract of Amaranth, the anti-inflammatory effect of each fraction obtained in Example 1 was investigated through NO inhibitory effect and RAW 264.7 cells.

Experimental Example 1: NO  And Cell survival rate ( LDH ) Measurement>

Raw264.7 cells were plated at 1.8 × 10 ⁵ cells / mL in a 24-well plate using a medium containing 10% FBS. After 18 hours, LPS (1 ug / mL) was included in the medium containing 10% FBS The extracts were treated with 100, 200, and 400 ug / mL of the extracts in the medium containing or without aminobutyric acid and fractionated with n-hexane, MC, EA, BuOH and H ₂ O An amaranth seed fraction was treated with 200 ug / ml or fractionated (25, 50, 100, 200 ug / ml) and cultured at 37 ° C and 5% CO ₂ for 24 hours. The amount of NO produced from Raw264.7 cells was measured using a Griess reagent system (Promega, Madison, WI, USA) as the form of NO ₂ present in the cell culture. The supernatant was dispensed into 96-well plates, and the Griess reagent was added and reacted at the wavelength of 540 nm using a microplate reader (BIO-RAD, Hercules, CA, USA).

In order to measure the cell viability, 50ul of the upper liquid of the same medium in which the nitrite was measured was dispensed into each well of a 96-well plate, and 50ul of the LDH reagent was dispensed and left for 30 minutes. After 30 minutes, stop the reaction by adding 50 μl of stop solution, and measure with a microplate reader (BIO-RAD, Hercules, CA, USA) at a wavelength of 490 nm.

NO inhibitory effect and cytotoxicity of ground amaranth division Treatment concentration (ug / mL) NO inhibition% LDH release%
A mixture of three kinds of amaranth
100 ug / mL 13.14 -3.85 200 ug / mL 16.02 -7.41 400 ug / mL 38.47 -6.19
Purple amaranth
100 ug / mL 25.45 -10.42 200 ug / mL 39.59 -15.55 400 ug / mL 67.16 -21.47

As shown in Table 1, in the case of Amaranth crude extract,

Respectively. Especially, it was possible to confirm a good NO inhibitory effect in a concentration - dependent manner in the absence of cytotoxicity of the purple single extract from the mixture of three kinds of the above - ground parts.

NO inhibitory effect and cytotoxicity of ground amaranth division Treatment concentration (ug / mL) NO inhibition% LDH release% Crude crude


100 ug / mL

17.37 -26.25 n-hexane 25.71 -6.885 CH ₂ Cl ₂ 22.87 -3.302 EtOAc 45.00 -6.393 BuOH -4.11 -1,268 H ₂ O -4.38 -3.067

As shown in Table 2, it was confirmed that the best inhibitory effect of NO on the ethyl acetate layer was obtained when the seeds were seeded.

Experimental Example 2: Cytokine  ( TNF -a, IL -6) inhibition experiment

Raw 264.7 cells were cultured to cell density of 1.8 x 10 ⁵ cell / mL. RAW 264.7 cells were stimulated with LPS (1 ug / mL) and the extracts were treated at various concentrations and cultured in a 37 ° C, 10% CO ₂ incubator for 24 hours. Twenty-four hours later, cell supernatants were obtained and cytokine expression was measured by ELISA. ELISA was performed by purchasing a mouse ELISA kit for IL-6, IL-1b and TNF- from BD pharmingen (San Diego, CA, USA).

Experimental Example 3: Western blot Through iNOS  And COX -2 inhibitory effect experiment>

Raw 264.7 cells were seeded in a 60 mm dish at a density of 1.2 × 10 ⁶ cells / ml. After 18 hours, the culture medium containing 10% FBS or without LPS (1 μg / ml) 50, 100, 200, and 400 ug / ml, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, remove the culture medium, wash with PBS 2-3 times, add 100 μl of lysis buffer, and lysis at 4 ° C for 30 minutes. After 30 minutes, the cells were centrifuged at 15,000 rpm for 15 minutes. Protein concentration was determined by standardizing BSA (Bovine serum albumin) using Protein Assay Kit (Bio-Rad, USA). Total cell lysate was separated by size from 4-12% Bis-Tris Gel 1.0 mm X 15 well and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA) for 120 minutes at 200 mA. The membrane was blocked for 3 hours at room temperature in TTBS (TBS-buffered saline + 0.1% Tween 20) containing 5% skim milk. After the reaction, each of the antibodies (iNOS 1: 2000, COX-2 1: 2000, and β-actin 1: 2000) to be measured was added and reacted at room temperature for 2 hours or 3 hours and then at 4 ° C for 24 hours. After washing with TTBS and DW for 3 times and then reacting with secondary antibody (1: 5000) for 3 hours at room temperature, the cells were washed with TTBS and DW for 4 to 5 times, and the ECL substrate (Amersham Biosciences, Piscataway, Nj, USA) After 1 to 3 minutes of reaction, the film was exposed to X-ray film.

Example 2: amaranth From seedling HPLC  Analytical sample preparation>

Amaranth seeds used in this analysis were harvested from Jeju Island.

Raw and popping samples were used for each seed, and popping samples were used for roasting at high temperatures.

extraction

○ 70% EtOH Extraction: 500 g of the plant sample in the Raw state and 120 g of the plant sample in the popped state were extracted in 6 L of 5 L and 1.2 L of 70% ethanol, respectively. It was filtered, concentrated using a vacuum concentrator, lyophilized and then used.

○ Hexane extraction

Squalene analysis: Five grams of each of the two milled samples were added to 40 ml of hexane and extracted with stirring at room temperature for 24 hours.

- Tocopherol analysis: Twenty grams of each of the two milled samples were mixed with 1 L of hexane, and the mixture was sonicated three times for 30 minutes each.

The two extracts were filtered, concentrated using a vacuum concentrator, and lyophilized before use.

Sample preparation

The 70% EtOH extract was prepared by adding ACN solvent to the lyophilized extract at a concentration of 100 mg / ml and sonication so that the mixture could be mixed well. The syringe was filtered using a syringe filter of 0.45 μm and prepared in a vial.

Hexane extract was prepared by adding ACN solvent to the lyophilized extract at a concentration of 10 mg / ml and sonication so as to be mixed thoroughly. The extract was filtered and prepared in a vial using a 0.45 μm syringe filter.

Preparation of standard samples

Squalene was dissolved in ACN solvent and prepared at concentrations of 0.1, 0.5, and 1 mg / ml.

α-Tocopherol was dissolved in ACN solvent and prepared at concentrations of 0.05, 0.1, 0.5 and 1 mg / ml.

β-Tocopherol was dissolved in ACN solvent and prepared at concentrations of 0.1, 0.5 and 1 mg / ml.

Experimental Example 4: Squalene  And Tocopherol Flow analysis>

Squalene analysis

The wavelength of the PDA detector for squalene analysis was 208 nm. The ACN solvent used as the mobile phase was divided into solvent A and solvent B, and a ratio of 50:50 was used. The flow rate was 0.4 ml / min and each run for 10 minutes.

Tocopherol Flow analysis

The wavelength of the PDA detector for tocophenol analysis was 284 nm. The ACN solvent used as the mobile phase was divided into solvent A and solvent B, and a ratio of 50:50 was used. The flow rate was 0.4 ml / min and each run for 10 minutes.

Squalene and tocopherol content of amaranth seeds Squalene α-Tocopherol β-Tocopherol unit Ministry of Agriculture and Forestry Research Report 3 species
(mean) Raw 2915 9.3 1.23 ppm Germinated 2282 7.63 One ppm
Jeju Compilation A 160 310 100 ppm B 80 - - ppm  A ' 22800 320 60 ppm  B ' 21800 - 50 ppm

* A: Raw seeds (70% EtOH extraction)

  B: Roasted seeds (70% EtOH extraction)

  A ': Raw seeds (Hexans extraction)

  B ': Roasted seeds (Hexans extraction)

As shown in Table 3, amaranths grown in Jeju harvesting showed higher content of squalene and tocopherol than the amaranth grown in the Gyeonggi province, which was presented in the previous report of the Ministry of Agriculture and Forestry (high-nutrient grain resource amaranth application of active ingredients and processing product development, 2002) It was able to confirm that it was remarkably high.

≪ Example 3: Preparation of amino acid analysis solution >

Protein-constituting amino acids

○ Test solution for quantification of amino acids except cystine and tryptophan

A sample containing about 10 mg of protein is precisely weighed into a hydrolysis test tube. Add about 1,000 times, that is, 10 mL of 6 N hydrochloric acid containing 0.05% (w / v) 2-mercaptoethanol (C ₂ H ₆ SO) to the amount of protein.

After freezing with dry ice ethanol, install it in the degasser, melt and freeze repeatedly several times and thoroughly degas. With respect to the rods, hydrolyze in a constant temperature furnace at 110 ° C ± 1 ° C for 22-24 hours. After completion of the hydrolysis, the shaft tube is cut, and immediately decompressed, and concentrated and dried at 40 ° C to remove hydrochloric acid. To remove the hydrochloric acid as much as possible, water is added to the residue, and it is concentrated and dried again. Prepare a test solution with a certain amount of 0.2 N sodium citrate buffer (pH 2.2) or 0.02 N hydrochloric acid solution. If there is a precipitate, filter using a membrane filter.

○ Test solution for cystine determination

10 mL of protein and 10 mL of a solution of formic acid, which had been cooled to 0 ° C beforehand, were precisely weighed and stored at 0 ° C for 4 hours when the protein was soluble and at 0 ° C overnight if insoluble. After left standing, air is sucked in and a small amount of water is added to freeze-dry. Transfer the residue to a hydrolysis test tube using 10 mL of 6 N hydrochloric acid containing 0.05% (v / v) 2-mercaptoethanol. After freezing, it is mounted on a degasser, thoroughly degassed, and then disassembled at 110 ± 1 ° C for 18 hours. In the following method, (1) Prepare the test solution according to the test solution for quantification of amino acids other than cystine and tryptophan.

○ Test solution for quantitation of tryptophan

A sample containing 10 mg of protein is precisely weighed into a hydrolysis test tube, 100 mg of soluble starch is added, and then 3 mL of 4.2 N sodium hydroxide solution is added. After freezing, it is mounted on a degasser, thoroughly degassed, sealed, and hydrolyzed at 135 ± 1 ° C for 22 hours. After hydrolysis, cut off the capillary tube, neutralize with 6 N hydrochloric acid and make a test solution with 0.2 N sodium citrate buffer (pH 4.25). If the solution is cloudy, centrifuge at 30,000 to 40,000 rpm for 30 minutes to take the supernatant.

Free amino acid

○ Extraction by water

It is applied to solid or paste-like specimens.

Approximately 1 g of a homogeneous sample is precisely weighed, and about ten times the amount of water is added. The mixture is heated in a boiling water bath to coagulate and then filtered to take the water layer. The residue is washed with a small amount of water two to three times, and the washing liquid is combined with the above-mentioned water. If there is fat, remove with ether.

The water layer is concentrated and dried under reduced pressure, and the residue is dissolved in 0.2 N sodium citrate buffer (pH 2.2) or 0.02 N hydrochloric acid to obtain a test solution. When insoluble substances are present, they are filtered using a membrane filter.

○ trichloroacetic acid method

Apply to liquid specimens.

1.0 to 3.0 mL of the specimen is placed in a test tube, and 16% trichloroacetic acid solution is added to the same volume and shaken for 15 minutes. Centrifuge at 3,000 rpm for 15 minutes and use the supernatant as the test solution.

Experimental Example 5: Amino acid analysis >

Amino Acid Analyzer Condition

Check the presence of buffers, color reagents, temperature of the column oven, set the automatic buffer switching mechanism, constant flow pump, and flow rate according to predetermined conditions and perform measurement.

Qualitative examination

For qualitative analysis, the standard material and the test solution are analyzed under the same conditions and the components with the same retention time are compared and identified.

Quantitative test

For quantitative analysis, prepare a calibration curve from the peak area and content of the amino acid standard of the previously obtained concentration, and calculate the amino acid content in the test solution at the peak area obtained from the test solution.

Experimental Example 6: Calcium analysis [

In the potassium permanganate capacity method, a hydroxide is added to a solution containing Ca, precipitated as calcium hydroxide CaC ₂ O ₄ H ₂ O which is very hardly soluble in water, the precipitate is dissolved in H ₂ SO ₄ , and the aqueous solution in the solution is dissolved in KMnO ₄ solution .

Inorganic components 40 mL (corresponding to 1 to 12 mg of Ca) of the test solution obtained in the preparation of the test solution is placed in a 200 mL beaker, and a few drops of methyl red solution and 10 mL of ammonium hydroxide solution are added. It melts. Cover the beaker with a watch glass and continue heating slowly. When the color of the methyl red solution changes from red to orange-yellow (pH of about 5.6), stop heating and allow to cool. Leave it at room temperature for 2 hours or more (overnight when precipitation is little). The amount of liquid at this time is about 20 ~ 30 mL. Precipitate is precipitated, calcium carbonate precipitate is filtered by suction with a glass filter (15AG-4), and 30-40 mL of ammonia water for washing is divided into several times and the precipitate and filter paper are washed well. After washing, the beaker used for the precipitation is placed under the front glass filter, and the glass rod used for filtration is put into a glass filter and the sulfuric acid solution (1 → 25) preheated to 70 ° C or more is injected into the glass filter. Stir with a glass rod, allow to stand for a while, dissolve calcium hydroxide and inhale. After the next inhalation, add about 5-7 mL of warm sulfuric acid solution (1 → 25) to the glass filter, stir with glass rod, and wash the inside wall and inhale. This operation is repeated twice. The beaker is removed from the filtration apparatus and heated to 65 to 80 ° C and titrated with 0.02 N potassium permanganate solution. Perform the same procedure for the blank test.

a: consumption of 0.02 N potassium permanganate solution for blank test

b: consumption of 0.02 N potassium permanganate solution to the sample solution

F: 0.02 N Potency of potassium permanganate solution

V: Dilution of test solution

S: Amount of sample (g)

Experimental Example 6: Analysis of iron powder [

Ortho-phenanthroline (C ₁₂ H _{8n 2} ) is a red complex [(C ₁₂ H _{8n 2} ) ₃ Fe ²⁺ ] reacting quantitatively with Fe ²⁺ 1 atom and O-phenanthroline 3 molecules in the sample solution under a certain range of pH. . Since the red concentration is proportional to the Fe concentration in the range of Fe 100 to 200 γ and the pH range of 2.9 to 9.0, the Fe content in the sample can be determined by colorimetry.

Pipette 10 mL of the test solution from the preparation of the test solution into a 25 mL volumetric flask and take the same 10 mL into a test tube (or small Erlenmeyer flask).

The latter is used as a control solution for pH control. Add 4 drops of bromophenol blue TS to the test tube, pipette 1 mL of the hydroquinone solution and 2 mL of the phenanthroline solution to the 25 mL volumetric flask . The sodium citrate solution was either burette or 5 mL When the pH of the solution becomes 3.5, drop the solution at the point where the color changes from yellow to yellow green when the indicator solution is added. Record the number of mL of sodium citrate solution consumed until this point.

Add the sodium citrate solution of this volume to the measuring flask, dilute to 25 mL of water with water, and allow to stand at a temperature of 20 ° C or more for 1 hour or more. It is safe to leave overnight. Once fully developed, the solution does not fade for at least 48 hours.

The absorbance of the coloring liquid is measured at a wavelength of 510 nm in a spectrophotometer and in a photoelectric colorimeter using a green filter to obtain a non-absorbance coefficient. This is compared with the calibration curve prepared in advance to obtain the amount of iron.

The calibration curve is made as follows. That is, iron standard solution is diluted with water exactly 10 times. 1, 2, 5, 10, 15, and 20 mL each into a 25 mL volumetric flask and test tube or small Erlenmeyer flask. Add 10 mL of water to make approximately 10 mL. Similarly to the above-mentioned coloring operation, color development is performed in each of the measuring flasks at a pH of 3.5, absorbance is measured, and a concentration absorption curve is prepared.

Iron (mg / 100 g) = x V x 100

             A: Amount of iron (mg) determined from the extinction curve

             S: Sample weight (g)

V: Dilution of test solution

Analysis of calcium, iron and protein content of amaranth

Item Domestic research report
Jeju Compilation

unit Ministry of Agriculture and Forestry
Research Report 2002 Gyeonggi-do Northwest  Mixed varieties (Mix) Raw Roasting Popping A B C D protein 15 14.4 12.3 16.3 16.2 17.6 17.9 20.8 g /
100g iron 7.5 7.4 8 7.6 46.7 33.4 15.5 1.7 mg /
100g calcium 185 154.8 509.6 492.3 155.6 153.7 319 3544 mg /
100g

* A: Raw seeds (grinding)

 B: Roasted seeds

 C: Raw Seeds (do not grind)

 D: leaf

In Table 4, it can be seen that the amaranth juice from Jeju has excellent protein contents as a whole, and iron and calcium particularly have superior contents than the amounts analyzed in the other lipids.

Comparing the components of other grains and amaranth Item Protein
(g / 100 g) Ca
(mg / 100g) Fe
(mg / 100g) brown rice 7.2 41 2.1 Wheat 12 7 3.2 Barley 10.6 43 5.4 Oats 13 55 4.6
Jeju Compilation A 16.2 155.6 46.7 B 17.6 153.7 66.4 C 17.9 319 15.5 D 20.8 3544 1.7

In Table 5, it was confirmed that protein, calcium and iron were superior to those of other grains in Jeju amaranth.

Claims (6)

A health supplement composition containing as an active ingredient an amaranth ground and seedling extract or a solvent fraction of Amaranth ground and seedling extract The health supplement product composition according to claim 1, which comprises 0.01 to 10% by weight of the total weight of the composition, the amaranth ground part and seedling extract or the amaranth ground part and the solvent fraction of the seedling extract. An anti-inflammatory cosmetic composition containing as an active ingredient an amaranth ground and seedling extract or a solvent fraction of Amaranth ground and seedling extract An anti-inflammatory pharmaceutical composition containing as an active ingredient an amaranth ground part seedling extract or a solvent fraction of an amaranth ground part and seedling extract Anti-inflammatory effect containing the active ingredient of the amaranth ground and seedling extract or the solvent fraction of the amaranth ground and seedling extract Composition of supplements for the amaranth and top seeds

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