KR101813200B1 - MANUFACTURING METHOD OF BLACK GARLIC TABLET COMPRISING HIGH-CONTENT S-allylcysteine INCLUDING PINE NEEDLE - Google Patents

Abstract

The present invention relates to a method for producing a black gum tablet containing a high purity S-aryl cysteine with pine needle added thereto, and more particularly, to a method for producing a black gum tablet containing pine needle powder and a high content S- The present invention relates to a method for producing a black garlic tablet containing a high purity S-aryl cysteine added with pine needle which can increase consumers' ingenuity and purchase preference through a black garlic tablet selected according to an optimum blending ratio of black garlic powder.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for preparing a black garlic tablet containing a high purity S-aryl cysteine and a pine needle-added S-allyl cysteine.

The present invention relates to a method for producing a black gum tablet containing a high purity S-aryl cysteine with pine needle added thereto, and more particularly, to a method for producing a black gum tablet containing pine needle powder and a high content S- The present invention relates to a method for producing a black garlic tablet containing a high purity S-aryl cysteine added with pine needle which can increase consumers' ingenuity and purchase preference through a black garlic tablet selected according to an optimum blending ratio of black garlic powder.

While the average life expectancy of humans has increased dramatically due to modern medicine, civilization and improvement of living standards, many factors inside and outside the human body caused by environmental pollution such as air and water quality, various stresses, . In Korea, dietary patterns of people are changing from vegetable and carbohydrate-based diet to high-fat foods such as meat and processed foods. In addition, cardiovascular diseases such as hyperlipidemia are serious health problems , And the 2008 National Health and Nutrition Examination Survey showed that the prevalence of chronic diseases such as obesity, diabetes, hypercholesterolemia and hypertriglyceridemia was continuously increasing. The increase of such chronic diseases was statistically significant Of the patients.

In particular, cardiovascular diseases caused by hematologic disorders such as arteriosclerosis and stroke are leading causes of death worldwide in the United States, Europe, and Asia. In Korea, The number of cases of cardiovascular disease in the stomach and third in the cerebrovascular disease, and the number of deaths due to vascular diseases was the same as that of the first cancer.

This is due to the recent rapid economic growth and changes in the family structure, which have led to changes in the eating habits and lifestyle of modern people and increased intake of fat and processed foods. As a result, hypercholesterolemia, hypertension, diabetes, metabolic syndrome And the incidence of vascular diseases due to hematological disorders is increasing. Therefore, there is a urgent need to develop health functional food for improving blood circulation.

In addition, the range of functional foods includes foods that have harmful substances neutralization, detoxification excretion, blood pressure, blood sugar and cholesterol lowering, prevention of obesity, dieting effect, and further prevention of bio-defense, immunity, disease prevention and treatment, It is gradually expanding to the role of regulation of the living body.

One of the biggest changes in the living body caused by aging is that the blood vessels become hardened and cause blood flow disorders. The aging of the blood vessels starts with the damage of the endothelial cells, and when the endothelial cells are damaged, LDL is oxidized to form macrophage to form foam cells. When they accumulate in the lower endothelial cell layer, atheroma is formed and further solidified into fat. Calcium accumulation then leads to calcification and arteriosclerosis.

Endothelial cells also inhibit vasodilatation and platelet aggregation by inhibiting NO and PGI2. When endothelial cells are damaged, they cause vasoconstriction and platelet aggregation. In other words, if blood flow is impeded by three factors, arteriosclerosis due to hyperlipidemia, thrombosis due to platelet aggregation, and vasoconstriction, hypoxia is induced in each tissue.

In the normal blood vessels, the homeostasis is maintained by balancing the activation reaction with the hemostatic mechanism, but the homeostasis is maintained. However, blood vessel damage, high fat dietary intake, and lack of exercise activate the blood coagulation process, resulting in abnormal blood coagulation It also causes thrombosis.

The blood clot attaches to the wall of the blood vessel and makes the flow of the blood flow not smooth. When the blood clot moves along the blood stream and blocks the blood vessel of the heart, lung, and brain, .

In order to treat such diseases, anticoagulants and aspirin are prescribed to be taken for a long period of time. However, these medicines have problems such as side effects of bleeding and gastrointestinal disorders.

The formation of thrombosis, which is a major cause of vascular disease, is closely related to dietary and environmental factors since the endothelium begins to be damaged by hyperlipemia, obesity, hypertension, diabetes, smoking, Improvement of blood circulation through ingestion of health functional foods before treatment with drugs due to hematologic disorder is very important for prevention of chronic diseases.

Pinus densiflora sieb, et zucc is an evergreen tree belonging to the Pinaceae family. It has been used as a wild plant in all parts of the country such as leaves, pine cones, pollen, rosin and bark .

In particular, pine leaves that can be easily harvested have been edible for longevity and health due to their pharmacological effects as well as their antioxidant, disinfectant, and fragrance-enhancing effects. In pineapple or folk remedies, pine needles can be used for atherosclerosis, hypertension, diabetes, neuralgia, arthritis It was used as a medicinal plant effective for prevention of aging-related diseases.

The active ingredients of pine leaves are known as chlorophyll, carotene, dietary fiber, terpenoids, phenolic compounds, tannin and alkaloids compounds.

Pine needles have unique pharmacological effects such as antimicrobial, anti-cancer, hypotensive, promoting hormone secretion, antioxidant and cardiovascular disease prevention. They are used in aromatheraphy and contain 16 essential amino acids It is effective for the growth of the body, the vitality enhancement, the blood flow and the hormone secretion are active, and it has an excellent effect on anti-aging, hyperlipidemia and cholesterol lowering.

Garlic (Allium sativum L) is a scaly stem vegetable belonging to Lilliiaceae (Allium). It is a representative food for enhancing the taste and health of food. The growing area and the production volume steadily increased from 1985 to 1998, , Which is about 1 trillion won in production, occupies its position as a major crop after rice in terms of income.

Garlic has been widely used as spice and folk medicine since ancient times due to its distinctive odor and unique taste. It has been known to have effects such as cholesterol lowering, platelet aggregation inhibition, blood pressure strengthening, antioxidant and the like. Thus, it is known that high blood pressure, arteriosclerosis, stroke, Prevention and treatment of anti-thrombotic diseases, immunological diseases, brain aging, arthritis, and chronic diseases such as cataracts, and to improve delay and vigor and circulatory system.

Allicin, an active ingredient of garlic, is a volatile fragrance component of garlic, which is produced by allinase when garlic tissue is destroyed. The glycosidic glycoside is hydrolyzed to produce diallyl sulfide, an essential oil with a characteristic odor, and disulfide, dithiin, and ajoene, and water-soluble sulfur compounds such as S-allylcysteine (SAC) and S-allylmercaptocysteine.

Most of the physiological activity of garlic is contained in organic sulfur compounds. The content of sulfur compounds in garlic is 1.1-3.5%, which is about 4 times that of onion or broccoli. About 90% .

In many studies, it has been reported that cholesterol, triglyceride, and blood LDL are reduced in raw garlic or raw garlic extract, and blood circulation is improved by the effect of inhibiting hyperlipidemia and platelet aggregation inhibition such as increasing HDL. In recent years, Diallyl and oligosulfides in garlic oil have been reported to inhibit the growth of Helicobacter pylori. It has been reported that diallyl disulfides have improved functionality as the number of sulfur increases.

Garlic is recognized as a high-grade raw material in the Food and Drug Administration's revision bill in January 2015, which can help improve cholesterol in the blood. Because it has the functionality, it can aggressively market health functional foods using garlic, The functional foods market is expected to grow sharply.

Traditionally, a method of removing irritating odor by using a bake-or-boil recipe has been used as a method of facilitating garlic ingestion. When the garlic is aged for a certain period of time while maintaining proper humidity in a high temperature storage state, And enzymes to cause a browning reaction so that the inside of the garlic has a deep blackish brown color and the sweetness is increased and the hot taste is decreased while the incense and viscosity are increased to change the chewiness. S-allylcysteine (SAC) and S-ally melcaptocytein (SAMC), which are soluble components, increase in total phenol and flavonoid, and melanoidins are produced by the non-enzymatic browning reaction of garlic sugar and amino acid. SAC, which is a representative water-soluble sulfur compound of black garlic, is known to be effective against antioxidant, cancer cell proliferation inhibition, cognitive function improvement, liver protection, etc. Was reported.

However, garlic is consumed mainly in the form of raw garlic in Korea, and the processed products are simple and the processing rate is also very low. Also, due to the opening of the agricultural products market, the increase of import of garlic in China and the imbalance of production and consumption, Loss was caused. In addition, when garlic was used as a raw material for food materials or by simple processing (such as dry powder), difficulty in ingesting garlic with a specific odor, irritating taste and strong aroma occurred. However, most of the black garlic is commercialized in the form of whole garlic or garlic, which retains its own form of garlic. Such black garlic is not only for long-term preservation and distribution, but also has its own form of garlic, And the sales of the products are insufficient.

In order to secure safety of raw materials, there is a possibility of contamination with microorganisms and occurrence of malformation due to moisture absorption of raw materials, which may cause problems in manufacturing process and distribution safety. Therefore, production of high-content SAC, removal of irritating odor, Process.

As a result of analyzing the market structure according to formulation, 39.0% of liquid / beverage type, 57.7% of pill type, 41.5% of liquid type / beverage type and 59.9% of pill Type in 2012, It is intended to manufacture as a pill type which can increase consumers' convenience and purchase preference.

Korean Patent No. 10-0794383 discloses a method of extracting an active ingredient containing a physiologically active ingredient from garlic and using it as a main ingredient and adding various nutrients such as top mushroom, citric acid, xylitol, glucose, magnesium stearate, octacosanol and vitamin C Containing garlic tablet and a process for producing the same.

Korean Patent No. 10-1394527 discloses a citrus pulverized powder further coated with a composition containing a black garlic extract and coated on a saccharide seed in the form of powder or granules, Type citron tea and a tablet.

However, in the case of the above-mentioned prior art, there is an advantage of increasing the ease of ingestion by the consumer with the tablet formulation, but since the antioxidant, antioxidant, anti-aging and anti-aging properties of pine needles and black garlic are not expressed well, The effect was insufficient.

The object of the present invention is to provide a black garlic tablet, which is selected according to the optimum blending ratio of black garlic powder having high pine needle powder and high S-aryl cysteine content, which is excellent in consumer's ingenuity and purchasing preference, And a method for producing the same. Therefore, it is an object of the present invention to provide a poultice product containing pine needles, crystalline cellulose, seaweed calcium powder P having a calcium content of 32% or more, whey calcium having a calcium content of 25% or more, carboxymethylcellulose calcium, magnesium stearate, HPMC The present invention also provides a method for producing a black garlic tablet containing a high content of S-arylcysteine, which is a health functional food using a material of a glycerin fatty acid ester mixture as a raw material and which is improved in nutrition, storage stability, .

According to one aspect of the present invention, there is provided a method for producing a fermented garlic, comprising: (a) a step of removing a root portion of garlic and fermenting and aging the garlic; (Step 2) of removing moisture from the black garlic obtained by fermentation and aging; A step of cutting and smoothing the black garment from which water has been removed, and then performing a primary smoothing and drying (step 3); Grinding and drying the first gross and dried black garlic to secondaryize and dry (fourth step); Pulverizing and drying the secondarily germinated and dried black garlic to prepare a black garlic powder (fifth step); Mixing the pine needle powder and the black garlic powder together (step 6); And a step of drying and pressure-molding the mixture (step 7). The present invention also provides a method for producing a black garlic tablet containing a high purity S-aryl cysteine added with pine needle.

In the method of preparing a black garlic tablet containing a high-content S-aryl cysteine with the pine needle of the present invention, the black garlic is preferably put into a white rice crate with mugwort and aged in a heat aging period for 595 to 605 hours.

In the method for producing a black garlic tablet containing a high-content S-aryl cysteine to which the pine needle is added according to the present invention, it is preferable that the aging includes two stages of fermentation by high-temperature hot air treatment and low-temperature hot air treatment.

In the method for preparing a black gum tablet containing the high-content S-aryl cysteine with pine needle of the present invention, it is preferable that the primary gelation and drying are performed at 75 to 85 ° C for 1.5 to 2.5 hours.

In the method for preparing a black garlic tablet containing the high-content S-aryl cysteine with pine needle of the present invention, the secondary gelation and drying are preferably performed at 75 to 85 ° C for 1.5 to 2.5 hours.

In the method of manufacturing a black gum tablet containing the high-content S-aryl cysteine with the pine needle of the present invention, it is preferable that the size of the powder of the black gum is in the range of 16 to 18 mesh.

In the method for preparing a black garlic tablet containing a high purity S-aryl cysteine with pine needle of the present invention, the mixture is preferably mixed at a ratio of 50 to 53% by weight of the black garlic powder and 18 to 21% by weight of the pine needle powder .

In the method for preparing a black gum tablet containing the high purity S-aryl cysteine with pine needle of the present invention, the amount of the mixture is preferably 68 to 74% by weight of the total tablet weight.

In the method for producing a black garlic tablet containing the high-content S-aryl cysteine with the pine needle of the present invention, it is preferable that the pressure molding is performed at a pressure of 100 to 300 kg / cm 2 for 60 to 120 seconds.

A method of manufacturing a tablet that black garlic and the pine needle of this invention containing an added amount S- aryl cysteine, the black garlic tablet cellulose (M101) 16.7% by weight of calcium less than 32% calcium seaweed powder P (AQUA knife ^® using available) also known as a 5% by weight of calcium of 25% or more whey calcium (or milk calcium) 3% by weight, carboxymethylcellulose calcium 2% by weight, magnesium stearate 1% by weight, HPMC 0.8% by weight and a glycerol fatty acid ester mixture (US Bassett ^® - Liquid phase usable) 0.1% by weight of pine needle-containing high purity S-aryl cysteine.

Hereinafter, a method for producing a black garlic tablet containing a high-content S-aryl cysteine to which pine needle is added is provided.

It is preferable to remove the root portion of the garlic in a state where the first portion produced in the southern sea is selected and the stem portion is left to be about 1 to 2 cm.

The high-temperature hot air treatment preferably maintains a temperature of 50 to 95 ° C and a humidity of 35 to 85% in a heat aging machine and conducts the hot air treatment while raising and lowering the low temperature and humidity, high temperature and humidity for 120 to 288 hours . At this time, since the change of the temperature will affect the composition and quality of the garlic, careful production is required, and the most suitable temperature is 82-88 ° C. High temperature hot air treatment is very important factor in reducing or eliminating the bitter smell of heartburn or garlic when raw garlic is consumed. In this case, garlic has various organic sulfur compounds and exhibits antioxidant activity, and the color of unfleshy fresh green is changed to reddish brown.

The low-temperature hot air treatment is carried out at a temperature of 30 to 49 ° C for 36 to 90 hours in a heat aging machine. The intrinsic effective ingredient such as alicin of garlic is changed into an organic sulfur compound, and the color of garlic is changed from reddish brown to dark brown When ingested, the sugar content of garlic will appear.

In this case, it is desirable to control the temperature of the automatic thermostat by time-series computerized programming and input management.

In the present invention, the black garlic obtained by fermentation and maturation is weighed, and the maximum amount of water is removed through primary drying. The resultant is cut into a size of 5 to 7 mm, and subjected to a primary smoothing process at 78 to 82 ° C for 1.5 to 2.5 hours It is preferable to undergo a drying process at a temperature of 58 to 62 DEG C or less. Through this process, the starch and carbohydrates of black garlic are converted into simple sugars, which increases the total equivalent weight and removes adhesion and customization. After the dried product is cut into a size of 2 to 4 mm, and then subjected to a secondary gelatinization process and drying process, it is preferable that the black garlic powder is pulverized to a size of 0.8 to 1.2 mm and dried so that the moisture content is within 2.8 to 3.2%.

In the process of producing the black garlic powder, it can be confirmed that the black garlic which contains a large amount of simple sugars is not powdered due to the problem that the internal moisture can not escape to the outside due to the coating phenomenon only by freeze drying. On the basis of this, it was possible to obtain a powder form which was subjected to a glazing process at a high temperature and a low temperature. However, at the high temperature, all of the SACs were destroyed during the drying process. Therefore, it is desirable to obtain the black garlic powder which is free from stickiness and irritability through the process of converting the starch into glucose by a process of gypsum at 50 to 60 ° C.

In the food industry, a tablet is usually made of granules by adding the excipients, binders, disintegrants, lubricants and the like as they are, and molding the granules by using a compressor. The tablet is accurate in capacity, convenient to carry and store, Suitable. It is known that the addition of high molecular materials such as zeolite or gum arabic as a binder causes a difference in the disintegration depending on the type and amount of the constituents, but it is also known that there is a considerable difference depending on the molding pressure and time. When granulated, the fluidity and compression ratio are improved, so that the powder is granulated and then subjected to a tableting process. As a method of making a tablet, first, an excipient is added to a raw material to form wet granules and dry granules, and second, there is a direct tablet method without a granulating step.

According to the present invention, a black garlic tablet selected according to the optimum blending ratio of black garlic powder having high pine needle powder and S-aryl cysteine content which are excellent in anti-aging effect and blood circulation improving effect, And a high content of S-arylcysteine added with pine needle which is easy to carry, can be provided.

1 is a graph showing S-aryl cysteine content of a black garlic powder according to the present invention.
2 is a graph showing the DPPH radical scavenging activity of the high-t content S-aryl cysteine-containing black gum according to the present invention.
3 is a graph showing TRAP activity of the high-content S-aryl cysteine-containing black gum according to the present invention.
4 is a graph showing the ORAC activity of the high-content S-arylcysteine-containing black gum according to the present invention.
FIG. 5 is a graph showing the intracellular ROS scavenging activity of the high-content S-arylcysteine-containing black glands according to the present invention.
FIG. 6 is a graph showing anti-genetic toxicity of the high-content S-aryl cysteine-containing black garlic according to the present invention.
FIGS. 7 and 8 are graphs showing gene expression of anti-aging genes sirtuin 1 and 6 by treatment with high-content S-arylcysteine-containing black germ in 3Te-L1 senescing cells according to the present invention.
9 is a graph showing the NO production ability of the high-content S-arylcysteine-containing black gum according to the present invention.
10 is a graph showing the ACE inhibitory activity of the high-content S-arylcysteine-containing black garlic according to the present invention.
11 is a graph showing the platelet coagulating ability of the high-content S-aryl cysteine-containing black gum according to the present invention.
12 and 13 are graphs showing reaction surface curves of total polyphenol contents and ORAC activity according to mixing conditions of the high content S-aryl cysteine black garlic powder and pine needle powder according to the present invention.
Figures 14 and 15 show the total polyphenols and total flavonoid contents of garlic powder (G), high S-aryl cysteine garlic powder (AG), high S-aryl cysteine garlic powder and pine needle blended powder (AG + P) Graph.
16 and 17 show DPPH radical scavenging activity and DPPH IC ₅₀ of garlic powder (G), high content S-arylcysteine black garlic powder (AG), high content S-arylcysteine black garlic and pine needle mixed powder (AG + Graph.
18 is a graph showing TRAP activity of garlic powder (G), a high content of S-arylcysteine black garlic powder (AG), and a high content of S-arylcysteine black garlic and pine needle blended powder (AG + P).
19 is a graph showing ORAC activity of garlic powder (G), high-content S-arylcysteine black garlic powder (AG), and high-content S-arylcysteine black garlic and pine needle mixed powder (AG + P).
20 is a graph showing the intracellular ROS scavenging activity of garlic powder (G), high S-arylcysteine black garlic powder (AG), and high-content S-arylcysteine black garlic and pine needle mixed powder (AG + P).
FIG. 21 is a graph showing the antimutagenic activity of garlic powder (G), high-content S-arylcysteine black garlic powder (AG) and high-content S-arylcysteine black garlic and pine needle mixed powder (AG + P).
22 and 23 show the expression of anti-aging genes sirtuin 1 and 6 of garlic powder (G), high-content S-arylcysteine black garlic powder (AG), high content S-arylcysteine black garlic and pine needle blended powder (AG + Fig.
24 is a graph showing the NO production ability of garlic powder (G), a high content of S-arylcysteine black garlic powder (AG), and a high content of S-arylcysteine black garlic and pine needle blended powder (AG + P).
25 is a graph showing the ACE inhibition performance of garlic powder (G), high-content S-arylcysteine black garlic powder (AG) and high-content S-arylcysteine black garlic and pine needle blended powder (AG + P).
26 is a graph showing platelet coagulation ability of garlic powder (G), a high content of S-arylcysteine black garlic powder (AG), and a high content of S-arylcysteine black garlic and pine needle blended powder (AG + P).
FIG. 27 is a graph showing the effect of a high content of S-arylcysteine black garlic and pine needle powder on the platelet aggregation ability of a cholesterol-induced animal model.
28 is a photograph showing a black garnet tablet containing a high content of S-aryl cysteine added with pine needle produced according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. S-aryl cysteine content analysis

The powder obtained from the three conditions used for the production of high-content S-aryl cysteine-containing black garlic powder was used for the analysis. The sample was dried with dextrin (Case 1), hot-hot air-dried powder (Case 2), and gelled powder (Case 3), and the finished sample was collected and added to 100 mL of distilled water in an amount of 30 g Of black garlic was soaked and sonicated for 1 hour. This process was applied to the analysis three times.

HPLC conditions for S-aryl cysteine content analysis Analysis condition

column
GL Sciences Inc. C ₁₈
Interstil ODS-2
5 mm, 150 mm * 4.6 mm


Mobile phase
5% B solvent
A - 0.1% Trifluoroacetic acid in water
B - Acetonitrile

Temperature
38 ° C


Calculation of detection amount
SAC content (mg / kg) = C * V / S
C: Concentration (μg / mL) of SAC obtained from the calibration curve
V is the final volume (mL) of the test solution, S is the sample volume (g)

Experimental Example 1 Analysis of S-aryl cysteine content

In order to prepare high-content S-arylcysteine-containing black garlic powder, the content of S-arylcysteine was analyzed by using the powder obtained through the preparation process (see FIG. 1). Case 1 was prepared by mixing 60 brix concentrated black garlic juice with 10% dextrin, followed by hot air drying, and case 2 was dried at 90 ° C for 12 hours. Finally, case 3 is the result of the SAC content of the powder produced by the hydrolysis process. As a result, it was found that SAC content was not found in the case of powder dried for a long time at high temperature, and SAC was contained in the concentrated sample, but the level was 0.8 mg / g, which was 1/7 Respectively. Therefore, it was concluded that Case 3 is suitable for producing high-content S-arylcysteine-containing powder.

Example 2. Production of high-content S-aryl cysteine-containing black garlic extract

To 5 g of powdered, high-content S-aryl cysteine white garlic, 100 mL of ethanol was added and the mixture was extracted at room temperature for 72 hours. The extract was filtered under reduced pressure through a filter paper (Whatman No. 1), and then washed with a rotary vacuum concentrator (EYELA N-1000, Tokyo Rikakikai Co., Tokyo, Japan) at 37 ° C. The concentrate was dissolved in DMSO at a concentration of 50 mg / mL and stored at 4 ° C and diluted to an appropriate concentration.

Example 3. Antioxidant efficacy of high-content S-aryl cysteine-containing black garlic

3-1. Total polyphenol content measurement

Phenol content was determined by colorimetry according to the Folin-Denis method. Take 1 mL of the constantly diluted S-aryl cysteine extract, dilute it with 1 mL of distilled water, add 2 mL of 1 N Folin-Ciocalteu's phenol reagent, leave at room temperature for 3 minutes, add 2 mL of 10% Na2CO3 solution, And allowed to stand at room temperature for 1 hour. After 1 hour, the mixture was centrifuged at 13,400 g for 5 minutes. The supernatant (200 μL) was taken and absorbance was measured at 690 nm using an ELISA reader (Sunrise, Tecan Co. Ltd., Grodig, Austria) The total phenol content was expressed in units of 100 mg of garlic acid equivalents (GAE).

3-2. Total flavonoid content measurement

Flavonoid content was determined by colorimetry according to the Davis method. Take 0.1 mL of the constantly diluted S-aryl cysteine extract, add 1 mL of diethyl glycol, and allow to stand at room temperature for 5 minutes. Add 0.1 mL of 1 N NaOH solution, leave the mixture at room temperature for 30 minutes, Absorbance was measured at 420 nm using UV / VIS spectrometer- (UV 1601, shimadzu, Kyoto, Japan). Calibration curves were prepared using quercetin as a standard, and the total flavonoid content was expressed in units of mg quercetin equivalents (GAE) / 100 g.

3-3. Measurement of DPPH radical scavenging ability

The DPPH radical scavenging activity was determined by adding 20 μL of S-aryl cysteine extract to 80 μL of 0.2 mM DPPH ethanol solution, mixing for 10 seconds, and incubating at room temperature for 10 minutes. The ELISA reader (Sunrise, Tecan Co. Ltd., Grodig, Austria) The absorbance at 492 nm was measured.

3-4. Total antioxidant capacity (TRAP) measurement

Total antioxidant activity was determined by absorbance of the ABTS radical cation formed by the interaction of ferryl myoglobin radical species generated by activation of 150 μM ABTS [2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate) and 2.5 μM metmyoglobin with 75 μM H2O2 The reaction mixture containing S-aryl cysteine extracts at different concentrations was incubated for 6 min at 30 ° C, and analyzed by UV / VIS spectrophotometer (UV 1601, Shimazu, Kyoto, Japan) at 740 nm The absorbance was measured. TRAP concentrations were calculated using the calibration curve of Trolox (6-hydroxy-2,5,7,8-tetramethy lchroman-2-carbonyl acid) and expressed as TEAC (Trolox equivalent antioxidant capacity, mM).

3-5. Oxygen radical absorbance capacity (ORAC) value measurement

Peroxyl radical scavenging capacity (ORAC _ROO ) assay was used to measure antioxidative activity through peroxyl radical scavenging ability. The AUCH for peroxyl radical formation was treated at a final reaction concentration of 20 nM in the S-aryl cysteine extract by concentration, fluorescein, a fluorescence standard solution, was treated to a final reaction concentration of 40 nM, As a control standard. The fluorescence reduction rate, which indicates the decrease of peroxyl radical, was measured for 2 hours every 2 minutes at excitation wavelength 485 nm and emission wavelength 535 nm using GENios fluorescence plate reader (Tecan Trading AG, Salzburg, Austria). The ORAC value was calculated as 1 μM trolox equivalents (TE) by calculating the net area under the curve where the fluorescence of each sample decreases.

3-6. Cellular antioxidant capacity assay (CAC assay)

The CAC assay for measuring the antioxidant activity of S-aryl cysteine extract in living cells using HepG2 cells was determined by modifying the DCFH-DA assay. In a 96-well plate, 5 × 10 4 cells / mL of HepG2 were added to DMEM medium and incubated in a 5% CO2 incubator at 37 ° C with 10% fetal bovine serum. After 24 hours, the medium was removed and 200 μL of fluorescence-stable Hank's balanced salt solution (HBSS) was added to each well. After treatment with S-aryl cysteine extract at different concentrations, the cells were cultured for 30 minutes. After incubation, the cells were washed with HBSS, and 2 μL of 80 mM AAPH was added to 200 μL of HBSS to induce peroxyl radical formation and cultured for 30 minutes. After incubation for 30 min with addition of 40 mM DCFH-DA with a fluorescent probe, the fluorescence was detected at 485 nm and 535 nm using a GENios fluorescence plate reader (Tecan Trading AG, Salzburg, Austria) The degree of occurrence was measured. Negative controls were treated with DCFH-DA only and positive controls with AAPH and DCFH-DA. The relative fluorescence values of each sample and AAPH were compared with the fluorescence value of the control group as 100%.

3-7. Measurement of Antigen Genotoxic Activity (DNA Damage Protection)

1) Leukocyte cell separation in blood

After collecting whole blood from healthy adult men, 200 μL of fresh whole blood and 800 μL of phosphate buffered saline (PBS) were mixed and carefully shaken in 200 μL of Histophaque-1077 (Sigma Chemical Co., St Louis, MO, USA) After centrifugation at 1,450 rpm for 5 minutes, leukocytes were separated and used for analysis.

2) Treatment of sample and induction of oxidative stress

S-aryl cysteine extracts were treated with 1-50 μg / mL of the extracted white blood cells at 37 ° C for 30 min. After the reaction, the leukocytes were washed with phosphate buffered saline (PBS) , 200 μM H2O2 was treated at 4 ° C for 5 minutes and then washed with PBS. Positive control was treated with 200 μM H2O2. The leukocytes were stained with ethidium bromide (20 μg / mL) and analyzed by fluorescence microscope (LEICA DMLB, Wetzlar, Germany) using a CCD camera (Nikon, Tokyo, Japan) Each nucleus image was analyzed by comet image analyzing system (Komet version 5.0, Kinetic Imaging, Liverpool, UK).

Experimental Example 3. Antioxidant efficacy of high-content S-aryl cysteine-containing black garlic

3-1. Total polyphenol content and flavonoid content

Total phenol content measurement is a widely used method for antioxidant research. Major polyphenol compounds present in nature are reported to have physiological activity including radical scavenging ability and strong antioxidant ability. The total polyphenol content of the high-t content S-aryl-cysteine-containing black garlic was 1481.5 ± 6.9 mg GAE / 100 g and the total flavonoid content was 248.6 ± 0.4 mg QE / 100 g. This study was carried out to investigate the antioxidant activity of black garlic by using reaction surface analysis and to establish the extraction conditions for the optimum antioxidant activity of Korean garlic (Korean Journal of Food Preservation and Distribution, 19: 577 ~ 585 (2012)). The total polyphenol content of the high-content S-arylcysteine-containing black garlic produced by the manufacturing process developed in this study was about 26 times higher I could confirm that it was high.

Total polyphenols and total flavonoid content of high-content S-aryl cysteine-containing black garlic
High-content S-aryl cysteine-containing black garlic

Total polyphenol (mg GAE / 100 g)

1481.5 ± 6.9
Total flavonoid (mg QE / 100 g)

248.6 ± 0.4

3-2. Measurement of DPPH radical scavenging ability

DPPH assay is a fast and simple method to measure the antioxidant activity of plants, food extracts or single compounds. DPPH is reduced by accepting electrons of hydrogen radicals at a very rapid rate when encountered with antioxidants, -diphenyl-1-picrylhydrazine, and has a characteristic of dark purple color. The DPPH radical scavenging ability of the high-titer S-arylcysteine-containing black garlic was increased in a concentration-dependent manner and showed 20 to 85% DPPH radical scavenging ability at 1 to 10 mg / mL (see FIG. 2).

3-3. Total antioxidant capacity (TRAP) measurement

TRAP assay was used to measure the antioxidant abilities of 2,2-azinobis- (3-ethylbenzothiazoline-6-sulphonate) cations (ABTS +). The TRAP activity of high-titer S- (See FIG. 3).

3-4. Oxygen radical absorbance capacity (ORAC) value measurement

The ORAC assay measures the antioxidative activity of peroxy radicals derived from oxidants by reacting with fluorescent probes to form nonfluorescent products. The antioxidant activity is measured by evaluating the amount and the rate of reduction of non - fluorescent products over time. The ORAC activity of the high-titer S-arylcysteine-containing black gum was increased in a concentration-dependent manner, and the ORAC activity was increased by about 4.8 times as compared to 1 mM trolox at a concentration of 50 μg / mL (see FIG.

3-5. Cellular antioxidant capacity assay (CAC assay)

Active oxygen (ROS) is a reducing metabolite of oxygen that is formed from the inside by normal metabolic processes in the cell or enzymatic action in the cytoplasm, or is formed by various external factors. Cells are equipped with an antioxidant defense mechanism to counter the accumulation and damage of ROS. Exposure to oxidative stress when ROS production exceeds the antioxidant capacity of the cell. The level of ROS production in HepG2 cells after 8 mM AAPH administration was significantly decreased in a concentration-dependent manner by the high content of high-content S-arylcysteine-containing black germ (see FIG. 4).

3-6. Measurement of Antigen Genotoxic Activity (DNA Damage Protection)

DNA damage protection by oxidative stress The comet assay, well known as a single-cell gel electrophoresis (SCGE) assay, has been used as a useful indicator to directly detect the degree of DNA damage in the cell upon oxidative stress induced by reactive oxygen species . The effect of inhibiting DNA damage by 200 μM H ₂ O ₂ in high-titer S-aryl cysteine-containing black garlic is shown in Fig. DNA damage was significantly increased in 200 μM H ₂ O ₂ treatment induced oxidative stress in healthy adult white blood cells, but the damage was significantly reduced in the high-titer S-aryl cysteine-containing black garlic treatment. The cell viability of the leukocyte was measured and the cell viability was measured. As a result, 90% or more of the cell viability was not observed, and cytotoxicity due to treatment with high-S amount of S-arylcysteine was not observed (see FIG.

Example 4. Evaluation of antioxidant efficacy of high-content S-arylcysteine-containing black garlic

4-1. Measurement of anti-aging gene (Sirtuin) expression

SIRT1 (sirtuin-1) and SIRT6 (sirtuin-1) were treated with 3-D3-L1 adipocytes with high fat S-arylcysteine concentration for 24 h at a concentration of 100 μg / sirtuin-6) was analyzed.

Primer sequence for PCR analysis Gene Primer sequence
SIRT1
forward: 5'-GAC TTC CCA GAC CTC CCA GA-3 '
reverse: 5'-CTG CAA CCT GCT CCA AGG TA-3 '

SIRT6
forward: 5'-AAC CTG CAA CCC ACA AAACA-3 '
reverse: 5'-CAT GCA CTG CAC CAT TGA GA-3
'
β-actin
forward: 5'-GAT TAC TGC TCT GGC TCC TA-3 '
reverse: 5'-ATC GTA CTC CTG CTT GCT-3
'

Experimental Example 4. The anti-aging efficacy evaluation result of the high content S-aryl cysteine-containing black garlic

4-1. Measurement of anti-aging gene (Sirtuin) expression

In the human body, the sirtuin 1 gene is known to deacetylate histone and non-histone proteins and thus has anti-inflammatory and anti-aging properties. It has been reported that the method of activating sirtuin 1 can be activated by antioxidant components. It has been reported that Sirtuin 6 can reduce the life span in the case of a functional error and increase the life span if the function is restored. In the present study, when the 3T3-L1 preadipocytes were differentiated into adipocytes, the high-content S-arylcysteine-containing black garlic was treated for 24 hours at 14th day. As a result, the sirtuin 1 and sirtuin 6 were significantly increased by about 1.9 times and about 4 times, respectively (see FIGS. 7 and 8).

Example 5. Evaluation of anti-aging effect and blood circulation improvement effect of high-content S-aryl cysteine-containing black garlic

5-1. Measurement of nitric oxide (NO) inhibitory activity; Blood vessel relaxation index

Raw 264.7 macrophages were cultured in DMEM medium at 37 ° C in a 5% CO 2 incubator maintained at 95% humidity with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin. After incubation for 24 h at 6 × 10 ⁵ cells / well in a 24-well plate, S-arylcysteine extract and 1 μg / mL LPS were treated at the same time for 24 h. The amount of nitric oxide produced from Raw264.7 macrophage was measured and NO2- present in the cell culture was measured using a griess reagent. 100 μL of the cell culture supernatant and 100 μL of the griess reagent were mixed and reacted for 15 minutes. The absorbance was measured at 540 nm using a microplate reader. The amount of NO ₂ ^- was quantified using a standard curve of NaNO ₂ .

5-2. Angiotensin converting enzyme (ACE) inhibitory activity; Antihypertensive index

Rabbit lung acetone powder (Sigma) was suspended in 10 times 50 mM sodium borate buffer (pH 8.3) and homogenized at 4 ° C to extract the enzyme. This was centrifuged at 40,000 g for 40 minutes and the supernatant was used as an ACE. Hippuryl-L-histidyl-L-leucine (HHL) was used as a substrate for the enzyme reaction. 50 mM sodium borate buffer (pH 8.3), 300 mM NaCl and 0-10 mU of enzyme solution were added to the reaction solution at 37 ° C for 30 minutes without adding the substrate, and 0.25 mL of 1 N HCl was added to the reaction solution. Add 1.5 mL of ethyl acetate to this solution, extract hippuric acid, centrifuge at 3,000 rpm for 10 minutes, transfer 1.0 mL of ethyl acetate layer to a new test tube, evaporate at 100 ° C for 30 minutes, add 50 mM sodium borate buffer 8.3) was added to dissolve hippuric acid and absorbance was measured at 228 nm using a spectrophotometer.

5-3. Platelet aggregation ability and aggregation inhibition ability; Blood circulation improvement index

1) Rat Platelet Preparation

Prepared rats were anesthetized with ethyl ether, and 1 mL of 0.38% sodium citrate as an anticoagulant was added to the syringe and 9 mL was collected from the abdominal artery. The blood was centrifuged at 1000 rpm for 10 minutes The supernatant was re-separated at 3000 rpm for 5 minutes. The precipitated platelets were suspended in platelet-free plasma and used as platelet rich plasma (PRP). Platelet counts were measured using a cell counter (Hema-vet HV950FS, Drew scientific, USA) and diluted with plasma to adjust platelets to 1 × 10 ⁸ platelet / mL.

2) Platelet aggregation ability measurement

The inhibition of platelet aggregation in rats (SD rats) was measured by turbidity measurement using Aggregometer (Chrono-Log Co., Ltd., Havertown, Pa. USA). PRP was incubated at 37 ° C for 2 minutes, and then 500 μl of platelet-poor plasma (PPP) and 50 μl of Platelet Rich Plasma (PRP) were added to quartz, and the stirrer bar was added to each well. The samples were incubated for 2 minutes with 500, 200 and 100 ㎍ / mL of ADP (10 μM), and the platelet aggregation reaction was measured for 5 minutes and then the flocculation activity was calculated. The sample was not treated, and the control was performed.

Experimental Example 5 Inhibition of blood vessel aging and blood circulation improvement effect of high-content S-aryl cysteine-containing black garlic

5-1. Measurement of nitric oxide (NO) inhibitory activity; Blood vessel relaxation index

In order to measure the inhibitory activity of NO, which is an inflammatory mediator, Law 264.7 cells were stimulated with LPS, and the NO production ability of the high-titer S-arylcysteine-containing black garlic extract was decreased in a concentration-dependent manner Reference). The IC ₅₀ value, which is 50% inhibition of NO produced, was 204.4 μg / mL.

5-2. Angiotensin converting enzyme (ACE) inhibitory activity; Antihypertensive index

ACE is an enzyme that converts Angiotensin II from Angiotensin I, which is a cause of blood pressure rise, and inhibiting ACE activity is widely used as an index to suppress blood pressure rise. The results of ACE inhibition of high-t-content S-arylcysteine-containing black garlic were shown (see FIG. 10). ACE inhibition was increased in a concentration dependent manner, and ACE inhibition was about 74% at a concentration of 10 mg / mL.

5-3. Platelet aggregation ability and aggregation inhibition ability; Blood circulation improvement index

When blood vessels are damaged, platelets are activated in response to the stimulation of various agonists such as collagen, thrombin, and ADP, resulting in adhesion, secretion, and aggregation, thereby damaging blood vessels After hemorrhage occurs, blood clots in the bloodstream or tissue, resulting in thrombosis. Although platelets play a crucial role in preventing blood loss in response to vascular injury, they cause acute atherosclerotic thrombosis, which can be an early cause of cardiovascular disease. When S-arylcysteine-containing black gum containing high amounts of S-arylcysteine was reacted with platelets, the ADP-treated group was significantly reduced at a concentration of 500 μg / mL compared to the group without the addition of the sample (see FIG. 11).

Example 6. Performance Analysis of Black Garbage Tablet

The results of the comparison of SAC contents of conventional black garlic powder products and the antioxidative, anti-aging, anti-aging effects and blood circulation improvement effects of the final 'pine needle-added high-content S-aryl cysteine-containing black garlic tablet' Respectively. To evaluate platelet coagulability, male Sprague - Dawley 5 - week - old male rats were adapted to solid - fed diet for one week and were divided into four groups of 8 rats per each treatment group for 6 weeks. In the experimental group, normal diet (ND), 1% high cholesterol diet (HC), 1% high cholesterol + 3% black garlic intake (HC + G), 1% high cholesterol + 3% And pine needle mixed powder intake group (HC + GPA). The diets were prepared on the basis of AIN-93. Dietary composition was given below, and water and diet were supplied without restriction. During the rearing period, the temperature of the feeding chamber was 20 ° C, the humidity was 55% 08: 00 ~ 20: 00). This animal study was conducted under the approval of KUIAC-1502 (Kyungnam National University). After sacrifice at 6 weeks, whole blood was used for platelet coagulation assay.

Ingredient ND HC HC + AG HC + AG + P    Casein 15 15 14.30322 13.8541222    Cornstarch 61.07 59.57 57.75428 58.1987135    Sucrose 10 10 10 10    Soybean Oil 4 4 3.91393 3.883067    Cellulose 5 5 5 5    Mineral mix 3.5 3.5 3.5 3.5    Vitamin mix One One One One    L-Cystein 0.18 0.18 0.18 0.18    Choline Bitartrate 0.25 0.25 0.25 0.25    Cholesterol 0 One One One    Cholic acid 0 0.5 0.5 0.5 Treatment    Pine needle - - 0.83    Aged garlic - - 3 2.16 Total 100 100 100.4 100.3

Comparison of Optimal Mixing Conditions, ORAC Activity and Total Polyphenol Contents and Observed Values of S-Arylcysteine Black Garlic Powder and Pine Needle Powder with High Content pine needles
(g) Black garlic
(g) ORAC (uM TE) TP (mg GAE / 100 g) Predicted value Actual value Predicted value Actual value Optimal Mixing Condition 1.97 5.08 2.0 6.4 1798.7 2017.8

Experimental Example 6. Performance analysis of black garbage tablet

The total polyphenol content of garlic powder (G), high content S-arylcysteine black garlic powder (AG) and high content S-arylcysteine black garlic and pine needle blend powder (AG + P) (AG + P) and increased by 36% when compared to the high content of S-arylcysteine black garlic powder (AG) (see FIGS. 14 and 15). The total flavonoid content was also determined by adding garlic powder (G) (58.3 mg QE / 100 g), high content S-arylcysteine black garlic powder (AG) (248.6 mg QE / 100 g) (AG + P) (524.2 mg QE / 100 g) was significantly higher than that of S-arylcysteine black garlic powder (AG) Respectively.

The DPPH radical scavenging ability was increased in a concentration-dependent manner in both the garlic powder (G), the high content S-arylcysteine black garlic powder (AG), and the high content S-arylcysteine black garlic and pine needle mixture powder (AG + P) 17). When comparing DPPH IC ₅₀ , which is capable of eliminating 50% DPPH radicals, G and AG were 5.9 mg / mL, but AG + P was 5.4 mg / mL, which was lower than DPPH radical scavenging ability And it was confirmed that it is excellent.

Comparing TRAP activity, TRAP activity was increased in a concentration-dependent manner in all groups of G, AG and AG + P (see FIG. 18). The highest TRAP activity was AG + P (1.1 mM TEAC) and AG (0.9 mM TEAC)> P (0.6 mM TEAC) in the order of 200 μg / mL. Results of ORAC activity are shown (see FIG. 19). ORAC activity increased in all three samples in the order of concentration. In the highest ORAC activity, AG + P (6.4 mM TE)> G (5.8 mM TE)> AG (4.4 mM TE)

ROS scavenging activity of the cells was significantly increased in the AAPH-treated PC compared to the AAPH-untreated NC, and it was confirmed that ROS scavenging ability was decreased in a dose-dependent manner in both G, AG and AG + P treatments compared to PC Reference).

Figure 30 shows the effect of 200 μM H ₂ O ₂ on the DNA damage inhibition of high-titer S-aryl cysteine-containing black garlic and pine needle. DNA damage was significantly increased in 200 μM H ₂ O ₂ treatment induced oxidative stress in healthy adult white blood cells, but the damage was significantly reduced in G, AG and AG + P treatments. Cell viability of the leukocytes was measured. The cell viability of the leukocytes was more than 90%, indicating no cytotoxicity due to G, AG and AG + P treatment. In comparison with the ED ₅₀ for reducing the damage to oxidative stress by H ₂ O ₂ to 50%, AG, 22.66 μg / mL and AG + P were 22.26 μg / mL and 19.20 μg / (See Fig. 21).

3T3-L1 cells were treated with 200 μg / mL of G, AG and AG + P for 24 h after adipocyte differentiation on day 14, and the expression levels of sirtuin 1 and 6, known as senescence-related genes, And it was confirmed that it was significantly increased in the sample treated (refer to FIG. 22 and FIG. 23). In sirtuin 1, AG + P and G were significantly higher than AG, but sirtuin 6 showed significant increase in AG + P compared to other treatments.

In the Law 264.7 cells, NO production was significantly increased in LPS treated group compared with LPS treated group, and NO production was decreased in a concentration dependent manner by treatment with G, AG and AG + P. As a result of comparing the IC ₅₀ of each sample which can inhibit NO production by 50%, it was found that G 201.8 μg / mL, AG 204.4 μg / mL and AG + P 183.7 μg / mL inhibited NO produced by LPS treatment And AG + P was the most excellent (see FIG. 24).

ACE inhibitory activity of each sample increased in a concentration dependent manner. In the case of 10 mg / mL, ACE inhibition was in the order of AG + P (79.1%)> AG (74.4%)> G (66.2%) (See Fig. 25).

In comparison of in vitro platelet coagulability, G and AG decreased significantly at 500 μg / mL when compared with 100% platelet aggregation without sample, whereas AG + P significantly decreased platelet coagulation at 200 μg / mL Respectively.

As a result of the in vivo platelet aggregation (see FIG. 27), the platelet aggregation ability of the high cholesterol diet group was significantly higher than that of the normal diet group, and the platelet aggregation ability of the high cholesterol diet + AG group, high cholesterol diet + AG + P group Was significantly lower than that of the high cholesterol diet group. And the effect was significantly higher in the AG + P group than in the AG (see FIGS. 26 and 27).

Claims (10)

Removing the root portion of the garlic and fermenting it in a fermentation stage (Step 1); (Step 2) of removing water from the black garlic obtained by fermentation and aging; Cutting off the water-removed black garlic, and primary smoothing and drying at 75 to 85 캜 for 1.5 to 2.5 hours (third step); Grinding and drying the first gross and dried black garlic to secondaryize and dry (fourth step); Pulverizing and drying the secondarily germinated and dried black garlic to prepare a black garlic powder (fifth step); Mixing the pine needle powder and the black garlic powder together (step 6); And a step of drying and press-molding the mixture (step 7). The method of producing a black gargal tablet according to claim 1, wherein the pine needle-added S-aryl cysteine is added. The method according to claim 1,
Wherein the black garlic is obtained by aging the white garlic in a heat aging machine for 595 to 605 hours by placing the black garlic together with the mugwort in a white wooden box, and then adding the pine needle-added S-aryl cysteine to the black garlic tablet. 3. The method of claim 2,
Characterized in that the fermentation includes fermentation and fermentation two times by high-temperature hot air treatment and low-temperature hot air treatment, and the high-content S-aryl cysteine added with pine needle. delete The method according to claim 1,
Wherein the secondary gelatinization and drying are carried out at 75 to 85 캜 for 1.5 to 2.5 hours, wherein the pancreas-added high-content S-aryl cysteine is added. The method according to claim 1,
Wherein the black garlic powder is such that the powder particles have a size of 16 to 18 mesh. The method of manufacturing a black garlic tablet according to claim 1, The method according to claim 1,
Wherein the mixture is prepared by mixing 50 to 53% by weight of black garlic powder and 18 to 21% by weight of pine needle powder. 8. The method of claim 7,
Wherein the amount of the mixture is 68 to 74 wt% of the weight of the whole tablet. The method according to claim 1,
Wherein the pressure molding is performed at a pressure of 100 to 300 kg / cm < 2 > for 60 seconds to 120 seconds. The method according to claim 1,
Wherein the step of calcining comprises the steps of: 16.7% by weight of crystalline cellulose, 5% by weight of seaweed calcium powder P of not less than 32% calcium, 3% by weight of whey calcium not less than 25% calcium, 2% by weight of carboxymethylcellulose calcium, 1% by weight of magnesium stearate, And 0.1% by weight of a mixture of glycerin fatty acid ester and a pine needle-added S-aryl cysteine.

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