KR20200044391A - An anti-inflammatory composition comprising an Gynostemma pentaphyllum distillate and hydrogen as an active ingredient - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a Gynostemma pentaphyllum distillate and hydrogen water as active components. A composition containing a Gynostemma pentaphyllum distillate and hydrogen water manufactured by a method of the present invention can remove intrinsic fragrance, smell, and taste of Gynostemma pentaphyllum, so anyone can take the composition. In addition, the composition consists of a distillate, thereby having a quick absorption rate and causes no side effects when taken.

Description

An anti-inflammatory composition comprising an Gynostemma pentaphyllum distillate and hydrogen as an active ingredient}

The present invention relates to an anti-inflammatory composition comprising an extra-distillate and hydrogen water as active ingredients.

According to ancient medical literature such as Donguibogam and Bonchogangmok, various efficacy of traditional natural herbal medicines that have been passed down since ancient times have been reported, and in recent years, their efficacy has been identified one by one through scientific clinical experiments on these herbal medicines. Along with this, there are more and more cases where Chinese medicine is taken and treated, from incurable diseases to incurable diseases.

As a method of extracting natural herbal ingredients that are effective for our human body, conventionally, the Tangje method has been used in the past. Traditionally, the Tangje method was used to put herbal medicines into a water bath made of earthenware or porcelain with water for a long time and then defrost the contents. It was a method of squeezing with a cloth.

However, when using this method, there was a problem that a significant portion of the ingredients of the medicine were vaporized and escaped to the outside, so that a significant portion of the active ingredient was lost and the surrounding environment was contaminated due to the peculiar smell generated during the operation. In particular, there was a lot of problems to popularize because there was a reluctance to drink with the bitter taste, smell, etc. peculiar to greasy and herbal medicines coming from herbal medicines.

Recently, a herbal medicine in the form of a clear and transparent distillate, in which the original flavors, scents, and odors of the original herbal medicines were removed by vaporizing the extracts of the herbal medicines to produce vapor and cooling them, then extracting them.

However, this type of distilled Chinese medicine is only manufactured through a simple distillation process. In fact, there is no medicinal ingredient in distilled liquid or it is extremely incomplete, so it is difficult to be used as a Chinese medicine because it has only a psychological effect and has almost no medicinal effect. there was.

Accordingly, the present inventors made it easy to take by reducing the intrinsic aroma and odor of the original herbal medicine by improving the distillation method using doldol as a herbal medicine material, and prepared a distillate solution in which the original efficacy of the herbal medicine was preserved, and added anti-inflammatory efficacy by adding hydrogen water thereto. This excellent pharmaceutical composition was completed.

Republic of Korea Registered Patent No. 0524659 Republic of Korea Registered Patent No. 0562460 Republic of Korea Patent Publication No. 2014-0137549

An object of the present invention is to provide an anti-inflammatory composition comprising an extra-distillate and hydrogen water as active ingredients.

The present invention provides an anti-inflammatory composition comprising an extra-distillate and hydrogen water as active ingredients.

Specifically, the stone-like distillate may be prepared according to a method for preparing a crude drug distillate composed of the following steps.

(1) preparing an aqueous solution by placing the herbal medicine and water in a water heater and heating;

(2) preparing the distillate by cooling the water vapor generated in the step of discharging;

(3) mixing the prepared distillate into the supernatant again;

(4) repeating steps (2) and (3) to circulate the distillate; And

(5) Extracting the final distillate after circulation.

The known method for preparing a distillate of a herbal medicine was a method of directly extracting a distillate by liquefying water vapor generated by heating the herbal medicine together with water. However, the present inventors have confirmed that the distillate prepared by such a conventional method has a significant drop in intrinsic efficacy of the herbal medicine. Accordingly, the present inventors, unlike the conventional method, reduce the intrinsic aroma and odor of the distillate by repeating the process of mixing and heating the distillate obtained by cooling the water vapor generated by heating the herbal medicines and water again in the bath liquid It was possible to purify and increase the content of the desired functional ingredient.

The content of the herbal medicine and water used to prepare the distillate is an amount that can be prepared once using a hot water heater, and 20000 ml to 30000 ml of water can be used for 1 kg to 5 kg herbal medicine, and the amount of water can be adjusted according to the amount of the herbal medicine. You can.

In the above method, the circulation time in the step of circulating the distillate in step (4) may be 1 to 12 hours, and preferably 7 to 8 hours. When the circulation time is less than 1 hour, sufficient functional components may not be extracted, and when the circulation time is increased by 15 hours, the functional components may be removed from the distillate again.

In the above method, the time for extracting the distillate in the step (5) may be 1 to 5 hours, preferably 3 hours.

In addition, the method for preparing a distillate of the present invention may further include injecting hydrogen gas into the final extracted distillate. Hydrogen is known to have an excellent anti-inflammatory effect, and it was confirmed that by adding hydrogen gas at a certain concentration to the distillate of the present invention, the taste and smell of the distillate were purified. In one embodiment of the present invention, the concentration of the hydrogen gas to be added can be adjusted according to the content of the herbal medicine, preferably 500 to 1000bbp concentration of hydrogen gas can be added.

In one specific embodiment of the present invention, an extracellular distillate may be prepared according to the following steps by using extracellular as a medicinal herb:

(1) 3kg stone and 20,000 to 25,000ml water into a water heater and heating to prepare a water solution;

(2) preparing the distillate by cooling the water vapor generated in the step of discharging;

(3) mixing the prepared distillate into the supernatant again;

(4) repeating steps (2) and (3) to circulate the distillate for 8 hours;

(5) extracting the distillate for 3 hours after circulation is over; And

(6) Injecting hydrogen gas into the extracted distillate.

It was confirmed that the stone-like distillate prepared by the above method was soft with little smell and aroma and little herb flavor, high polyphenol and flavonoid content, and excellent anti-inflammatory effect.

The composition containing the stone-like distillate and hydrogen prepared in the method of the present invention can remove the unique odor, smell, and taste of the stone-like, so anyone can take it regardless of age or gender, and it is composed of distillate. It has the advantage of quick absorption rate and no side effects.

Figure 1 shows the results of measuring the total polyphenol content of the primary distillate sample.
Figure 2 shows the results of measuring the total polyphenol content of the secondary distillate sample.
Figure 3 shows the results of measuring the total flavonoid content of the secondary distillate sample.
Figure 4 shows the results of measuring the DPPH scavenging activity of the secondary distillate samples 4-A and 4-B.
Figure 5 shows the results of measuring the cell viability of the extracellular distillate.
6 shows the result of measuring the NO concentration for each concentration of the distillate out of stone.
7 shows the results of measuring the concentration of COX-2 by concentration of the distillate out of stone.
Figure 8 shows the results of measuring the concentration of PEG ₂ by the concentration of the extra-distillate.
9 shows the result of measuring the concentration of TNF-α according to the concentration of the distillate outside the stone.
Figure 10 shows the results of measuring the cell viability to confirm the cytotoxicity for human immune cells TK6 by concentration of the extracellular distillate.
Figure 11 shows the results of measuring the change in weight of the rat by orally administering the extra-distilled hydrogen distillate used in Sprague-Dawley rats.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation and evaluation of extracellular distillate

The present inventors prepared the optimum distillate by variously controlling the circulation time, extraction time and the concentration of hydrogen added for the purpose of producing the most optimal state of distillate using doldol as a crude drug. The specific manufacturing process is as follows.

1. Preparation and sensory evaluation of primary distillate samples

Distillate was extracted by adding 1kg of stone outpost and 22,000 ~ 25,000ml of water to the water heater and liquefying the water vapor generated during water heating at 106 ℃ using a cooler. The extraction of distillate involves extracting after distilling the circulating distillate by repeating the process of directly extracting the distillate liquefied with water vapor and mixing the distillate for a certain period of time with a circulator and then mixing the mixture with the liquid. In order to find out the difference, the distillate was extracted with varying cycle times of 0, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours). . In addition, the extraction time (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours) was extracted to examine the difference in the distillate according to the extraction time. In order to examine the difference in the distillate according to the hydrogen concentration, the hydrogen concentration filling time was measured to measure the hydrogen concentration over time (1 min, 2 min, 3 min, 4 min, 5 min in 500 ml distillate). Filling with hydrogen gas).

As a result, the sensory evaluation of the distillate according to the circulation time resulted in a strong aroma when not circulated or circulated for a short period of time, while having a herb flavor, while increasing the circulation time, the aroma was reduced and the taste was also eliminated. It was confirmed that the rejection was reduced, and the color of the distillate was darkened (Tables 1 to 5). Particularly, when it was cycled for 8 hours, it showed the darkest color, and when it was cycled for more than 8 hours, it was confirmed that the color became pale again. Through this, it was confirmed that the content of the components contained in the distillate was affected by the circulation time (Table 6).

In addition, as a result of performing sensory evaluation of the distillate according to the extraction time, as the extraction time increased, the aroma was reduced and the taste was also eliminated, reducing the rejection. In addition, it was confirmed that the color was the darkest when extracted for 4 hours, and then gradually faded. Through this, it was confirmed that the components included in the distillate were also affected by the extraction time (Tables 7 to 9).

As a result of sensory evaluation of the distillate according to the concentration of hydrogen, it was confirmed that the taste became softer as the hydrogen concentration increased (Table 10).

2. Preparation and sensory evaluation of secondary distillate samples

Through the preparation of the primary sample, it was confirmed that the circulation time, extraction time, and hydrogen concentration influenced the evaluation of the components and sensory properties of the distillate. In order to prepare a secondary sample.

In the second sample, 3 kg of stone outpost and 24,000 to 25,000 ml of water were added to the water heater to liquefy the water vapor generated during water heating at 106 degrees using a cooler to extract the distillate. The extraction of distillate involves extracting after distilling the circulating distillate by repeating the process of directly extracting the distillate liquefied with water vapor and mixing the distillate for a certain period of time with a circulator and then mixing the mixture with the liquid. In order to see the difference, the distillate was extracted while varying the circulation time (5 hours, 6 hours, 7 hours, 8 hours). In addition, the extraction time (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours) was extracted to examine the difference in the distillate according to the extraction time. In order to examine the difference in the distillate according to the hydrogen concentration, the hydrogen concentration filling time was measured to measure the hydrogen concentration over time (the hydrogen dissolved concentration in the 500 ml distillate was 617 ppb, 817 ppb, 933 ppb).

As a result, the sensory evaluation of the distillate according to the circulation time, as a result of increasing the circulation time, the scent decreased and the feeling of rejection decreased, and it was confirmed that the color of the distillate became darker (Tables 11 and 12). Particularly, when it was cycled for 8 hours, it showed the darkest color, and when it was cycled for more than 8 hours, it was confirmed that the color became pale again. Through this, it was confirmed that the content of the components contained in the distillate was affected by the circulation time (Table 6).

In addition, as a result of performing sensory evaluation of the distillate according to the extraction time, there was little difference in taste, aroma and color as the extraction time increased, and only the amount of extraction increased (Table 13). Considering the economic evaluation according to the amount of extraction and the difference in the pharmacology according to the concentration of the extract, it was determined that the extraction time is preferably 3 hours because the medicinal properties of the extract at a dark concentration are superior.

As a result of performing sensory evaluation of the distillate according to the concentration of hydrogen in the secondary sample, it was confirmed that the taste of the distillate became softer as the hydrogen concentration increased, and the taste was softest at the concentration of 900 ppb (Table 14).

Example 2. Analysis of the components of the extracellular distillate sample

Based on the results in Example 1, the component analysis of the distillate samples prepared under various conditions was confirmed by requesting the Korea Institute of Functional Food. In addition to stone, it was commissioned to analyze the content of ginseng saponins rb-1, rg1-, and rg-3, which are known to be abundant. Preparation of the distillate sample commissioned for analysis was performed as follows, and specific compositions and conditions are shown in Table 15 below.

A-1: 3 kg of stone and 25,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 16 hours using a circulation device that pushes it back to the water heater. When the circulation of 16 hours was over, the circulator was terminated and the sample made by collecting the distillate for 3 hours was designated as A-1.

A-2: 3 kg of stone and 25,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate, and the distillate is continuously circulated to the water heater for 12 hours using a circulation device that pushes it back to the water heater. When the circulation of 12 hours was over, the circulator was terminated and the sample made by collecting the distillate for 3 hours was designated as A-2.

A-3: 3 kg of stone and 24,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 8 hours by using a circulator that pushes it back to the water heater. When the 8-hour cycle was over, the circulator was terminated and the sample made by collecting the distillate for 3 hours was named A-3.

A-4: After adding 3 kg of stone and 24,000 ml of water to the water heater, the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate, and the distillate is continuously circulated to the water heater for 6 hours using a circulation device that pushes it back to the water heater. After 6 hours of circulation, the circulator was terminated and the sample made by collecting the distillate for 3 hours was designated as A-4.

B-1: 3 kg of stone and 24,000 ml of water were added to the water heater, and the final heating temperature was set to 106 degrees to start the water heater. When water vapor began to be generated and the distillate was started to be extracted from the time the cooler started to operate, the circulator was turned off and the distillate was collected immediately, and a sample in which the distillate was collected for a total of 5 hours was designated as B-1.

B-2: 3 kg of stone and 24,000 ml of water were added to the water heater, and the final heating temperature was set to 106 degrees to start the water heater. When water vapor began to be generated and the distillate was started to be extracted from the time the cooler started to operate, the circulator was turned off and the distillate was collected immediately, and a sample in which the distillate was collected for a total of 3 hours was designated as B-2.

B-3: 3 kg of stone and 25,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 16 hours using a circulation device that pushes it back to the water heater. When the circulation of 16 hours was over, the circulator was terminated and the sample made by collecting the distillate for 1 hour was named B-3.

B-4: 3 kg of stone and 25,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 16 hours using a circulation device that pushes it back to the water heater. After 16 hours of circulation, the sample was made by collecting the distillate for 1 hour from 1 hour to 2 hours at the time when the circulator was terminated and the distillate was started to be collected.

B-5: 3 kg of stone and 25,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 16 hours using a circulation device that pushes it back to the water heater. After 16 hours of circulation, the sample was made by collecting the distillate for 1 hour from 2 hours to 3 hours at the time when the circulator was terminated and the distillate was started to be collected.

B-6 (Sample for confirming the reproducibility of the A-3 sample): 3 kg of stone and 24,000 ml of water were added to the water heater, and the final heating temperature was set to 106 ° C to start the water heater. The water vapor starts to be generated and the distillate starts to be extracted from the point when the cooler starts to operate. The distillate was continuously circulated to the water heater for 8 hours by using a circulator that pushes it back to the water heater. When the 8-hour cycle was over, the circulator was terminated and the sample made by collecting the distillate for 3 hours was designated as B-6.

Table 16 shows the results of the component analysis.

In the A-3 and B-6 samples, the content of ginsenoid and saponin was the highest. In addition, the sample was prepared under the condition that the distillate was circulated for 8 hours and the distillate was extracted for 3 hours, and the color was as dark as possible. Through this, it was confirmed that the distillate components and contents were also changed according to the distillate production conditions and methods.

Example 3. Antioxidant effect and stability evaluation of extra-stone distillate

3-1. Sample preparation

According to the conditions shown in Tables 17 and 18, primary and secondary extracellular distillate samples prepared by the method of Example 1 were used.

For comparison with the distillate, the sample of the organic solvent dolce extract is crushed by washing and drying dolce with distilled water, followed by lyophilization (PVTFI 10A, Ilshin Lab, Korea), and pulverizing the powdered sample at -80 ℃. Stored in a freezer for use. Each sample was extracted with 100% methanol (MeOH) and 70% ethanol (EtOH) as an extraction solvent, with an extraction solvent of 5 times the building weight (g / mL, W / V) for the active ingredient. 100% MeOH and 70% EtOH were extracted for 72 hours at 150 rpm and 25 ° C using a shaking incubator. After the extraction time, the extracellular sample was centrifuged at 1500 rpm for 15 minutes, and suspended components were removed using a 0.45 μm filtration filter.

3-2 Measurement of total polyphenol content

Polyphenol compounds are generic to flavonoids, anthocyanins, tannins, catechins, isoflavones, lignans, resveratrols, etc., and are widely distributed in plant systems, especially in fruits and leafy vegetables. The resonant structure of a number of hydroxyl groups (-OH) and phenol rings present in polyphenols has the property of easily binding to various compounds, contributing to lipid oxidation, free radical removal, etc., thereby pharmacological effects such as antioxidant, anti-cancer, and anti-inflammatory Is known to be excellent. Accordingly, pharmacological effects such as anti-inflammatory effects can be predicted by measuring the content of the polyphenol compound contained in the stone distillate.

The total polyphenol content of the sample was measured by modifying the Folin-Denis method. To the sample prepared by the above method, 50% Folin-Ciocalteu reagent, 95% EtOH, and DW were added. After 5 minutes, 5% sodium carbonate solution was added, mixed, and shaded for 1 hour to develop color. Then, absorbance was measured at 725 nm using a microplate reader (Spectra MR, Dynex, VA, US), and a gallic acid standard solution was used. It was quantified by the calibration curve obtained.

As a result, as a result of measuring the content of polyphenols contained in the primary stone-extra distillate sample, 4-A and 4-B were 13.1 and 12.4 mg gallic acid equivalence (GAE) / L, respectively. As shown, the cycle time was estimated as a factor, but overall, it was observed that the total polyphenol content of all samples was low (FIG. 1). This shows that the total polyphenol content of the secondary distillate sample was 4-A and 4-B, respectively, showing 322 and 334 mg gallic acid equivalence (GAE) / L, respectively, which was 6-7 times higher than other samples. It is estimated that the high content of the circulation time increased condition was a major factor, and it was determined that the distillate extraction time and the hydrogen concentration were independent (FIG. 2).

3-3 Total Flavonoid Content Measurement

Flavonoid is a polyphenolic component. It is a generic term for phenolic compounds with yellow or pale yellow color as the basic skeleton of C6-C3-C6.It is contained in almost all parts such as leaves, flowers, fruits, stems and roots of vegetables and plants. In addition, it is abundantly contained in nature, such as grains and fruits. Flavonoids, like polyphenols, are known to have high antioxidant capacity by effectively removing reactive oxygen species, and have been reported to act on various physiological activities. Specifically, after adding 5% sodium nitrite solution and DW per 30 μL of sample and adding 10% aluminum chloride solution after 6 minutes, incubate again at room temperature for 6 minutes. After adding 4% NaOH solution, the total volume was adjusted to DW, mixed well, and then reacted at room temperature for 15 minutes. After the reaction time was over, absorbance was measured at 510 nm using a microplate reader and quantified by a calibration curve obtained using a rutin standard solution.

As a result, the flavonoid content of the secondary sample of the extracellular distillate was 43.5 times higher than other samples with 203.5 and 206.6 mg rutin equivalence (RE) / L in 4-A and 4-B as shown in FIG. It was shown. The reason why the total flavonoid content is measured to be remarkably high was estimated to be an important factor in increasing the circulation time as in the result of total polyphenol content.

3-4. cell Survival rate  Measure

The CCK-8 assay is easier to test than the existing MTT, MTS, and XTT assays, and is known as a technique capable of producing accurate results. The test method is as follows. The test was performed using CCK-8 (Dojindo Molecular Technologies, Inc., Lot No. KL709).

① Each cell suspension was prepared and cells were counted using a hemocytometer.

② Cells suspended in the culture medium were dispensed into 2 × 104 cells / well in a 96-well plate and cultured in a 37 ° C. CO2 incubator for 24 hours.

③ After removing the existing medium, the sample was treated and cultured for 24 hours.

④ After adding 10 μl CCK-8 solution based on 100 μl of cell culture medium per well, incubated for 2 hours in a CO2 incubator, absorbance at 450 nm was measured using a Microplate reader (Molecular devices, SoftMax Pro5, USA). Cell viability was calculated according to the following equation based on the measured OD value.

The test substance was a colorless liquid substance with no viscosity, and the cell viability was measured by setting 8, 3.2, 1.28, and 0.51% by setting 20% (v / v) to the highest concentration and setting the azeotrope to 2.5.

As a result, cell viability was measured by treating test substances at concentrations of 0.51, 1.28, 3.20, 8.00, and 20.00% using CCK-8 for the preliminary test for setting the concentration of the sample solution. It showed abnormal cell viability (Table 20 and FIG. 5).

SUMMARY OF CELL VIABILITY STUDY: NT18-00009 Viability
(%) GROUP G1:
Control G2:
Distilled Hydrogen Distillate
(0.51%) G3:
Distilled Hydrogen Distillate
(1.28%) 24h 100 ± 1.11 (3) 119.90 ± 0.80 (3) 120.27 ± 0.51 (3) Viability
(%) GROUP G4:
Distilled Hydrogen Distillate
(3.20%) G5:
Distilled Hydrogen Distillate
(8.00%) G6:
Distilled Hydrogen Distillate
(20.0%) 24h 120.13 ± 0.81 (3) 120.97 ± 1.20 (3) 122.17 ± 0.47 (3)

_{Mean ± S.D  (Number of wells)}

3-5 Evaluation of anti-inflammatory effects

In the primary and secondary distillate samples, the secondary distillate sample 4-B having a high polyphenol and flavonoid content was selected as a final distillate candidate, and reproducibility and anti-inflammatory activity of the prepared distillate efficacy were measured.

In order to measure the anti-inflammatory activity, LPS was treated with macrophages (Raw 264.7) to induce inflammation, and the test substances were treated with concentrations of 3.2 3.0, 8.0 and 20.0%, followed by Nitric oxide, Cyclooxygenase-2, Prostaglandin E2 and inflammatory cytokine. The results of measuring are as follows.

1) NO (Nitric oxide) measurement

① The cell culture solution was separated 24 hours after the test substance treatment and used as a test solution.

② The amount of NO generated was measured in the form of Nitrite (NO2-) present in the cell culture using Griess reagent. To quantify the amount of NO generated, a standard calibration curve was prepared using Nitrite standards and the concentration was calculated. Did.

③ The standard solution of 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000 μM was prepared by diluting the Nitrite standard material with 1 mM solution using a culture solution and then stepwise diluting it.

④ 100 μl of test solution and 50 μl of substrate solution (Sulfanilamide in the reaction buffer) are treated in each well for reaction at room temperature for 10 minutes, and then 50 μl of Naphthylethylenediamine in the stabilizer buffer is treated in each well for 10 minutes at room temperature. To react. After the reaction, the absorbance was measured at 540 nm to calculate the concentration of NO.

As a result, in the test groups of 3.2, 8.0, and 20.0% of the test substance, it was statistically significantly reduced (p <0.01) compared to the LPS-induced control group (Table 21 and FIG. 6).

SUMMARY OF NITRIC OXIDE LEVEL STUDY: NT18-00009 NO
(μM) GROUP G1:
Control G2:
1 μg / ml LPS G3:
1 μg / ml LPS
+3.2% extrahydrogen hydrogen distillate 24h 8.53 ± 0.71 (3) 28.36 ^## ± 0.14 (3) 26.67 ^** ± 1.10 (3) NO
(μM) GROUP G4:
1 μg / ml LPS
+8.0% Hydrogen Distilled Water G5:
1 μg / ml LPS
+20.0% Distilled Hydrogen Distillate 24h 19.90 ^** ± 0.87 (3) 11.75 ^** ± 0.36 (3)

_{Mean ± S.D  (Number of wells)}

_{** indicates significant differences among G2∼G5 at p <0.01 according to one-way ANOVA}

_{## indicates significant differences between G1 and G2 at p <0.01 according to the Student's  t-test}

2) COX2  ( Cyclooxygenase -2) Measurement

① After 24 hours of incubation, the cells were collected and then treated with Cell Extraction Buffer to crush the cells and centrifuged at 48,000 ° C. and 18,000 g for 20 minutes to use the supernatant as a sample solution.

② The amount of COX2 was measured using a cell lysate using the Mouse COX2 ELISA kit (abcam, Lot No. GR3182641-1), and a standard in the Mouse COX2 ELISA kit was used to quantify the amount of COX-2 generated. A standard calibration curve was prepared and the concentration was calculated.

③ Add 1 ml of Cell Extraction Buffer to the standard material in the Mouse COX2 ELISA kit to make a 15 ng / ml solution, then dilute it step by step to standardize 0, 0.23, 0.47, 0.94, 1.88, 3.75, 7.5, 15 ng / ml The solution was prepared.

④ 50 μl of test solution and standard solution and 50 μl of Antibody Cocktail were added to each well and reacted for 1 hour in a plate shaker.

⑤ After the reaction for 1 hour, the contents were removed and washed 3 times with 350 µl of Wash Buffer.

⑥ 100 µl of substrate solution was added to each well, the light was blocked to react for 10 minutes at room temperature, and then 100 µl of Stop Solution was added to stop color development, and absorbance was measured at 450 nm to calculate the concentration of COX-2.

As a result, the test group of 8.0 and 20.0% of the test substance showed a statistically significant decrease (p <0.01) compared to the LPS-induced control group (Table 22 and FIG. 7).

SUMMARY OF COX-2 LEVEL STUDY: NT18-00009 COX2
(ng / ml) GROUP G1:
Control G2:
1 μg / ml LPS G3:
1 μg / ml LPS
+3.2% extrahydrogen hydrogen distillate 24h 1.441 ± 0.088 (3) 3.718 ^## ± 0.174 (3) 3.734 ± 0.087 (3) COX2
(ng / ml) GROUP G4:
1 μg / ml LPS
+8.0% Hydrogen Distilled Water G5:
1 μg / ml LPS
+20.0% Distilled Hydrogen Distillate 24h 3.183 ^** ± 0.156 (3) 1.675 ^** ± 0.099 (3)

_{Mean ± S.D  (Number of wells)}

_{** indicates significant differences among G2∼G5 at p <0.01 according to one-way ANOVA}

_{## indicates significant differences between G1 and G2 at p <0.01 according to the Student's  t-test}

3) PGE ₂  (Prostaglandin E2) Measurement

① The amount of PGE2 was measured using the PGE2 ELISA kit (abcam, Lot No. GR3208950-3) for 24 hours after treatment with the test substance, and the PGE2 ELISA kit was used to quantify the amount of generated PGE2. A standard calibration curve was prepared using my standard substance and the concentration was calculated.

② A standard solution of 0, 39.1, 78.1, 156, 313, 625, 1,250, and 2,500 pg / ml was prepared by diluting the PGE2 ELISA kit step by step.

③ 100 μl of the test solution and standard solution were added to each well, and 50 μl of PGE2 Alkaline Phosphatase Conjugate and 50 μl of PGE2 Antibody were added to each well and reacted for 2 hours in a plate shaker.

④ After the reaction for 2 hours, the contents were removed and washed 3 times with 400 μl of Wash Buffer.

⑤ 200 µl of substrate solution was added to each well, reacted for 45 minutes at room temperature, and 50 µl of Stop Solution was added to stop color development, and absorbance was measured at 405 nm to calculate the concentration of PGE2.

As a result of PGE2 measurement, it decreased in the test materials of 3.00, 6.00 and 12.00% test group compared to the LPS induction control group. Similar PGE ₂ production inhibitory effects were observed in all test groups (Table 23 and FIG. 8).

SUMMARY OF PGE₂ LEVEL STUDY: NT18-00009 PGE₂
(ng / ml) GROUP G1:
Control G2:
1 μg / ml LPS G3:
1 μg / ml LPS
+3.2% extrahydrogen hydrogen distillate 24h 0.559 ± 0.038 (3) 3.597 ^## ± 0.008 (3) 0.983 ^** ± 0.047 (3) PGE₂
(ng / ml) GROUP G4:
1 μg / ml LPS
+8.0% Hydrogen Distilled Water G5:
1 μg / ml LPS
+20.0% Distilled Hydrogen Distillate 24h 0.668 ^** ± 0.020 (3) 0.561 ^** ± 0.026 (3)

_{Mean ± S.D  (Number of wells)}

_{** indicates significant differences among G2∼G5 at p <0.01 according to one-way ANOVA}

_{## indicates significant differences between G1 and G2 at p <0.01 according to the Student's  t-test}

4) TNF -α measurement

① The amount of TNF-α was measured using a mouse TNF-α Quantikine ELISA kit (R & D systems, Lot No. P126681) for the sample solution that separated the cell culture solution 24 hours after the test substance treatment. To quantify, a standard calibration curve was prepared using a standard in the Mouse TNF-α Quantikine ELISA kit and the concentration was calculated.

② Add 1 ml of sterile distilled water to the standard material in the Mouse TNF-α Quantikine ELISA kit to make a 7,000 pg / ml solution, then dilute in steps to 0, 10.9, 21.9, 43.8, 87.5, 175, 350, 700 pg / ml A standard solution of was prepared.

③ 50 μl of Assay Diluent was added to each well, 50 μl of test solution and standard solution were added, shaken, and reacted for 2 hours at room temperature.

④ After the reaction for 2 hours, the contents were removed and washed 5 times with 400 μl of Wash Buffer.

⑤ 100 μl of Mouse TNF-α Conjugate was added to each well and reacted for 1 hour at room temperature.

⑥ After the reaction for 1 hour, the contents were removed and washed 5 times with 400 μl of Wash Buffer.

⑦ 100 µl of substrate solution was added to each well, and the light was blocked to react for 30 minutes at room temperature. Then, 100 µl of Stop Solution was added to stop color development, and absorbance was measured at 450 nm to calculate the concentration of TNF-α.

As a result of TNF-α measurement, the test groups of 8.0 and 20.0% of the test substance decreased compared to the LPS-induced control group. The 3.2% treatment test group did not show a TNF-α production inhibitory effect, and the other concentration groups showed a concentration-dependent TNF-α production inhibitory effect (Table 24 and FIG. 9).

SUMMARY OF TNF-α LEVEL STUDY: NT18-00009 TNF-α
(ng / ml) GROUP G1:
Control G2:
1 μg / ml LPS G3:
1 μg / ml LPS
+3.2% extrahydrogen hydrogen distillate 24h 8.59 ± 0.14 (3) 35.54 ^## ± 2.28 (3) 34.58 ± 1.01 (3) TNF-α
(ng / ml) GROUP G4:
1 μg / ml LPS
+8.0% Hydrogen Distilled Water G5:
1 μg / ml LPS
+20.0% Distilled Hydrogen Distillate 24h 31.71 ^** ± 0.74 (3) 26.55 ^** ± 0.24 (3)

_{Mean ± S.D  (Number of wells)}

_{** indicates significant differences among G2∼G5 at p <0.01 according to one-way ANOVA}

_{## indicates significant differences between G1 and G2 at p <0.01 according to the Student's  t-test}

3-6. Human immune cell line  Cytotoxicity test using TK6 cells

TK6 (human lymphocytes) used in this experiment was added to RPMI-1640 medium by adding 10% heat-inactivated fetal bovine serum (FBS), 100 units / mL penicillin, 100 μg / mL streptomycin and L-glutamine 37 C. Incubated in a humidified CO2 incubator (5% CO2 / 95% air).

All cells were cultured by passage with 0.25% trypsin-EDTA after washing with PBS to maintain the appropriate number of cells when they proliferated to about 80% of the culture vessel, and the culture medium was changed every 3-4 days.

Cell viability was measured by performing an MTT assay. Specifically, cells were diluted in a culture solution to be 5 × 10 ⁴ cells / well, and 100 μL of the cell suspension was attached to each well of a 96 well plate for 24 hours and then attached. , After adding extra-distillate by concentration, incubated for the treatment time, and then treated with 3- (4,5-dimethylthiazol) -2,5-diphenyl-tetrazolium bromide (MTT, 5 mg / mL) solution in each well for 4 hours. The absorbance was measured at 550 nm using a microplate reader by dissolving 100 μL of formazan. The measurement was made three times, and the relative survival rate of each test substance was obtained by setting the number of control cells to which no test substance was added to 100%.

As a result of performing the experiment under the above conditions to determine whether the extracellular hydrogen distillate exhibits toxicity to normal human immune cells, it was confirmed that the cytotoxicity of the distillate was not induced, and thus it was possible to develop a safer health functional food material (Fig. ).

3-7. Sprague- Dawley Rats  Used Stone  Of hydrogen distillate Single  Oral administration toxicity test

1) Test system

(1) Species and lineage

No specific pathogen (SPF) Sprague-Dawley rats (NSam: SD). For females, those who had never had a baby in the past and are not currently pregnant were used.

(2) Supply and production: Samtaco BIOKOREA Co., Ltd. (105, Seorang-ro, Osan-si, Gyeonggi-do)

(3) Reason for selection of test system

This is because the rats used in this test are widely used in toxicity tests and have accumulated abundant test basic data, which can be used for interpretation and evaluation of test results.

(4) Date of acquisition: May 31, 2018

(5) Gender number and number of animals: 11 male and female, 22 total

(6) Age at the time of acquisition: 7 weeks of age

(7) Weight range at the time of acquisition

① Male: 184.96∼201.38 g

② Female: 165.07∼185.66 g

(8) Quarantine and purification

On the day of acquisition, the microbiological test report and the appearance of the animals were examined (Annex 1). After the acquisition, a period of purification was given for 7 days, and general symptoms were observed during the purification period, and only healthy animals were used for the test.

(9) Age at the start of administration: 8 weeks of age

(10) Weight range at the start of administration

① Male: 203.22∼229.07 g

② Female: 175.74 ~ 195.44 g

(11) Number of animals used: 10 male and female, 20 total

(12) Group separation

The body weight was measured the day before administration and group separation was performed using the excel program to rank the body weight. Only animals within the range of ± 20% of average body weight were used for the test.

(13) Individual identification

For the individual identification, the notation of the tail and the identification of the individual identification card for each breeding box were used. At the entrance of the animal room, an animal room use record sheet is attached with the test number, test title, animal room usage period, name of the person in charge of the test, and the name of the tester.

(14) Treatment of residual animals

It was processed in accordance with the standard work instructions of the test institute.

(15) Compliance with animal testing ethics regulations

The test was conducted with the approval of the animal laboratory ethics committee of this test institution (approval number: IA18-00486).

2) Animal room and breeding environment

(1) Animal room: SPF breeding area room 1

(2) Range of temperature and humidity: temperature 22 ± 3 ℃, relative humidity 50 ± 20% RH

(3) Number of ventilation: 10 ~ 15 times / hr

(4) Contrast cycle: Fluorescent lighting 12 hr (08:00 lights up ~ 20:00 lights off)

(5) Illuminance: 150∼300 Lux

(6) Noise: 60 dB or less

(7) Ammonia concentration: 15 ppm or less

(8) Breeding box and breeding density (KCL / ANI / 010, AQI / 001)

During quarantine, purification, administration and observation periods, no more than 3 animals were housed in a stainless steel mesh box (250W × 350L × 180H mm). The replacement of the breeding box was carried out when the group was separated.

(9) Feed and water

The feed (Teklad Certified Irradiated Global 18% Protein Rodent Diet, Harlan Co. Ltd., USA) was supplied from Dooyeol Biotech (Dooyeol Building, 14, 17-gil, Yangjaecheon-ro, Seocho-gu, Seoul). Water was purified by drinking drinking water and putting it in a polycarbonate water bottle.

For pollutant testing, data supplied from feed producers and data from nationally recognized inspection agencies were referred to (Annex 2, 3).

3) Test method

(1) Method of administration

① Route of administration and reasons for selection

It was selected to examine the toxicity of oral administration and oral administration.

② Method of administration

After administration, the stomach contents were emptied for one night and then forced orally administered using a sonde for oral administration. After about 3-4 hours, the feed was re-supplied and negative water was supplied even during fasting.

③ Frequency and time of administration

It was administered once in the morning on the day of administration.

④ Dosage amount

The dose amount of 10 ml / kg was calculated based on the body weight measured on the day of administration.

(2) Composition of test group

group gender Number of animals
(Mari) Animal number Dosage amount
(Ml / kg) Dosage
(Mg / kg) G1 Male 5 G1-1 to G1-5 10 0 Female 5 G1-11 to G1-15 G2 Male 5 G2-6 to G2-10 10 5,000 Female 5 G2-16 to G2-20

G1: excipient control group, G2: test substance administration group

(3) Reasons for setting the dose

A preliminary test was conducted at a dose of 5,000 mg / kg using one test animal each male and female. As a result, it was judged that no toxic symptoms caused by the test substance were observed. In this test, a limit test was conducted by setting 5,000 mg / kg as a single dose group. In addition, there was an excipient control group that administered only excipients.

(4) Test method and items

① General symptom observation

All animals were observed once a day for general symptoms, and on the day of administration, they were observed every hour until 30 minutes after administration and 4 hours thereafter. General symptoms were observed until 14 days after administration.

② Weight measurement

All test animals were measured at the time of acquisition, group separation, administration day (before administration), and 1, 7, 7 and 14 days after administration (before necropsy).

③ Autopsy

On the 14th day after administration, all surviving animals were anesthetized with CO2 gas, opened, and then bleeded to kill the abdominal aorta and posterior vena cava.

(5) Data evaluation and statistical test

The weights of all test animals were summarized as the mean and standard deviation, and the trends were analyzed using tables and figures. The weight change between the excipient control group and the test substance administration group was checked for significance through the Independent Samples t-Test and the variance was tested. The incidence rate was expressed as a percentage and was judged to be statistically significant when p <0.05.

Statistical analysis was conducted according to the standard work guideline for statistical processing of this laboratory, and the commercially used statistical package SPSS 12.0 K program (SPSS, Chicago, IL, U.S.A.) was used.

As a result, 1) no dead animals and unusual general symptoms were observed during the experiment period, 2) weight measurement result, only normal weight change was observed, and no weight change related to the test substance was observed (FIG. 11), 3) As a result of the autopsy at the end of the experiment, no unusual gross autopsy findings were observed. As a result of the above results, no toxic symptoms were observed by the test substance extra-hydrogen distillate under these test conditions, and the approximate lethal dose was judged to be> 5,000 mg / kg.

Claims (7)

Anti-inflammatory composition comprising an extra-distillate and hydrogen as an active ingredient.
The anti-inflammatory composition according to claim 1, wherein the extra-distillate is prepared according to the following steps:
(1) step of preparing a solid solution by heating the outside of the stone and water into a water heater;
(2) preparing the distillate by cooling the water vapor generated in the step of discharging;
(3) mixing the prepared distillate into the supernatant again;
(4) repeating steps (2) and (3) to circulate the distillate; And
(5) Extracting the final distillate after circulation.
According to claim 2,
Including the step of injecting a hydrogen gas to the final extracted distillate, the inflammation composition.
The method of claim 2 or 3,
The circulation time of the distillate in step (4) is 1 to 12 hours, anti-inflammatory composition.
The method of claim 2 or 3,
The time for extracting the distillate in step (5) is 1 to 5 hours, anti-inflammatory composition.
(1) 3kg stone and 20,000 to 25,000ml water into a water heater and heating to prepare a water solution;
(2) preparing the distillate by cooling the water vapor generated in the step of discharging;
(3) mixing the prepared distillate into the supernatant again;
(4) repeating steps (2) and (3) to circulate the distillate for 8 hours;
(5) extracting the distillate for 3 hours after circulation is over; And
(6) prepared according to the step of injecting hydrogen gas into the extracted distillate,
Anti-inflammatory composition comprising an extracellular distillate and hydrogen.
The method of claim 6,
The concentration of hydrogen gas in step (6) is 900 to 1000 PPB, anti-inflammatory composition.

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