KR102041615B1 - A method of using propolis mixed with other origins and a composition for health functional foods containing propolis - Google Patents

Abstract

The present invention relates to a mixed propolis composition comprising a mixture of two or more propolis extracts selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis, mixed propolis mixed with propolis of various origins By preparing the intake of the daily intake as compared to the intake of each propolis alone, there is an effect that can consume more of the various active ingredients contained in each propolis.

Description

A method of using propolis mixed with other origins and a composition for health functional foods containing propolis}

The present invention relates to a method of using propolis to enhance the efficacy and to a composition for health functional foods using the propolis as an active ingredient, and more particularly, to a mixture of two or more kinds of propolis extracts of different origin.

Propolis is a natural waxy mixture made from honey bees mixed with their saliva to protect the plant's growth and wounds.

Bees use propolis in the gaps of the honeycomb to protect themselves from germs and viruses, and to keep them suitable for spawning, growing, ripening and storing honey by preventing invasion by enemies such as wasps and mice. Especially when the queen bee spawns, it is used to disinfect the spawning place and keep it clean.

Propolis, which is used as a folk remedy in several countries, including South America, is a health functional food recently used in Korea for the treatment and prevention of various diseases. Flavonoids and terpenoids, which are components of propolis, Much research and attention has been drawn to the various pharmacological actions due to the antioxidant activity of). In particular, the therapeutic effects on skin, mucous membranes and oral infections are due to various substances identified and identified in propolis, and at least 150 terpenes, caffeic acid esters, flavonoids and amino acids. Phenylpropane derivatives such as these are known to be involved. These components exhibit antibacterial and antifungal effects as well as antiseptics, indicating that they can be used in various areas.

Ethanol and hot water extract of propolis are used as a herbal medicine for various purposes. Propolis and its phenolic constituents include antiproliferative effects (Guarini et al., 1992), anti-inflammatory effects (Dobrowolski et al., 1991), antibacterial effects (Eley 1999; Grange et al., 1990) in human tumor cells; Steinberg et al., 1996), antiviral effect (Serkedjieva et al., 1992), immunomodulatory effect (Dimov et al., 1992), antioxidant effect (Krol et al., 1990; Scheller et al., 1990) and It is known to exhibit various pharmacological actions such as antiprotozoal effect (Starzyk et al., 1977). It has also been reported that ethanol extracts of propolis have anti-inflammatory effects that significantly improve acute and chronic inflammation and pain (Park et al., 1996; Park and Khang, 1999). These propolis ethanol extracts have excellent anti-inflammatory effects and no adverse effects on the gastrointestinal tract, which is a side effect of non-steroidal anti-inflammatory drugs (NSAIDs), and flavonoids, which are known to exert strong antioxidant activity, It contains a large amount of phenolic acid.

These propolis differ in the active ingredient, color, and taste depending on the type of plant (species) and distribution inhabited by region, because the composition varies depending on the species of worker bees collecting tree species or resins.

Propolis colors vary from green, red, yellow, black, etc., depending on the color of the active ingredients are also different. That is, the active ingredient of green propolis is chlorophyll, the active ingredient of red propolis is anthocyanin, the active ingredient of yellow propolis is flavone, flavonol, and the active ingredient of black propolis is flavonoid (anthanthanthin). In addition, the propolis has a difference in the active ingredient by taste, the active ingredient of sweet propolis is carcon, the active ingredient of bitter propolis is catechin and isoflavones, the active ingredient of spicy propolis is peroleic acid.

Currently, Propolis is produced in New Zealand, Brazil, China, UK, etc., and only products using a single propolis produced in each country are produced.

Republic of Korea Patent No. 10-0420498 Republic of Korea Patent No. 10-1176521 Republic of Korea Patent No. 10-1323178 Republic of Korea Patent No. 10-1418366 Republic of Korea Patent Publication No. 10-2004-0024057 Republic of Korea Patent Registration No. 10-0897778 Republic of Korea Patent Registration No. 10-0980254 Republic of Korea Patent Registration No. 10-1348643 Republic of Korea Patent Registration No. 10-0864006

The present invention is to use a combination of propolis of various origins in the difference in the active ingredient by using a variety of active ingredients when eating a daily intake to increase the efficacy of propolis as a health functional food using a method of using propolis And it aims to provide the composition for health functional foods which uses this mixed propolis as an active ingredient.

In order to achieve the above object, the present invention,

In order to enhance the efficacy of propolis as a dietary supplement, two or more origins selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis are mixed and used as extracts of different propolis. Provides methods of using polis.

The efficacy of the propolis includes any of antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy.

Preferably, the extract of propolis is a water soluble extract that has received an ethanol extract of propolis.

The mixed use of the extract of propolis is preferably a mixture of extracts of Argentine propolis, Chinese propolis and Korean propolis. More preferably, the extracts of Argentine propolis, Chinese propolis and Korean propolis are mixed and used in a weight ratio of 30 to 40:25 to 35:30 to 40.

In another embodiment of the present invention, the mixed use of the extract of propolis is preferably a mixed use of extracts of Argentine propolis, Chinese propolis and Brazilian propolis. More preferably, extracts of Argentine propolis, Chinese propolis and Brazilian propolis are used in a weight ratio of 35 to 45:25 to 35:25 to 35.

In the present invention,

In the composition for health functional foods containing propolis as an active ingredient, in order to increase the efficacy of the propolis, two or more origins selected from propolis of Argentina, Chinese propolis, Brazilian propolis and Korean propolis are selected from different propolis. It provides a health functional food composition comprising a mixed extract.

In the present invention,

In the manufacturing method of health functional food which uses propolis as an active ingredient,

In order to enhance the efficacy of propolis, propolis-containing health function, characterized in that the mixed use of extracts of different propolis from two or more origins selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis Provides a method of preparing food.

In the present invention, "health functional food" is meant to include all of the commonly used health food and functional food, including health functional food under the health functional food law.

In the present invention, by using a mixture of propolis of various origin, when taking the daily intake of propolis, it is possible to ingest more effective ingredients than the intake of each propolis alone by propolis as a health functional food It can increase the efficacy of.

According to the invention, compositions for dietary supplements comprising mixed extracts of propolis of different origin may exhibit more excellent effects, particularly in antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy.

1A to 1C are chromatograms of an active ingredient of propolis using LC / MS / MS, FIG. 1A is Chrysin, FIG. 1B is Galangin, and FIG. 1C is pinozembrine ( Pinocembrin) chromatogram.
Figure 2a to 2h is a chromatogram analysis of the active ingredient of water-soluble propolis, Figure 2a is Cinnamic acid (Cinnamic acid), Figure 2b is caffeic acid (Caffeic acid), Figure 2c is acecetin (Acacetin), Figure 2d CAPE, Figure 2e is a kaempferol, Figure 2f is a gallic acid (Gallic acid), Figure 2g Chrysin (Chrysin), Figure 2h is a chromatogram of ferulic acid (Ferulic acid).
Figure 3 is a graph showing the effect of propolis samples of various origin on cell viability.
4 is a graph showing the effect of propolis samples of various origin on the production of NO.
5 is a graph showing the effect of mixed propolis on cell viability.
6 is a graph showing the effect of mixed propolis on the production of NO.
Figure 7 is a graph showing the effect of the mixed propolis inhibit cytokine related genes.
8 is a schematic of the therapeutic effect test of mixed propolis against alcoholic gastritis.
Figure 9 shows the results of measuring the liver weight in the therapeutic effect test of mixed propolis for alcoholic gastritis.
Figure 10 shows the result of measuring the spleen weight in the therapeutic effect test of mixed propolis against alcoholic gastritis.
Figure 11 shows the result of measuring the weight of the kidney in the therapeutic effect test of mixed propolis for alcoholic gastritis.
Figure 12 shows the results of histopathological observations in the therapeutic effect test of mixed propolis against alcoholic gastritis.
Figure 13 shows the results confirming the macroscopic damage of the gastric mucosa in the therapeutic effect test of mixed propolis for alcoholic gastritis.
Figure 14 shows the results of the therapeutic effect test of mixed propolis against alcohol gastritis, A shows the result of measuring the area of gastric damage, B shows the rate of gastric injury inhibition.
Figure 15 shows the results of measuring the oxidative damage of gastric tissue in the therapeutic effect test of mixed propolis against alcoholic gastritis, A is the result of the measurement of MDA, B is the result of the measurement of GSH.
Figure 16 shows the results of confirming the expression of inflammatory cytokines in the therapeutic effect test of mixed propolis against alcoholic gastritis, A is the result of measurement of IL-6, B is the result of measurement of IL-1β, C is TNF It is a measurement result of -α.
Figure 17 is a schematic of the therapeutic effect test of mixed propolis against other alcoholic gastritis.
Figure 18 shows the results of measuring the weight in the therapeutic effect test of mixed propolis for alcoholic gastritis.
Figure 19 shows the results of measuring the long-term weight in the therapeutic effect test of mixed propolis against alcoholic gastritis.
20 shows the results of histopathological observations in the therapeutic effect test of mixed propolis against alcoholic gastritis.
Figure 21 shows the results confirming the macroscopic damage of the gastric mucosa in the therapeutic effect test of mixed propolis against alcoholic gastritis.
Figure 22 shows the results of the therapeutic effect test of mixed propolis for alcohol gastritis, A shows the results of measuring the area of gastric damage, B shows the rate of gastric injury inhibition.
Figure 23 shows the results of measuring the oxidative damage of gastric tissue in the therapeutic effect test of the mixed propolis complex composition for alcoholic gastritis, A is the result of the measurement of MDA, B is the result of the measurement of GSH.
Figure 24 shows the results confirming the anti-Helicobacter pylori effect of the propolis samples by country of origin.
FIG. 25 shows the activity of Helicobacter pylori of mixed propolis and natural extract.
Figure 26 shows the ability to inhibit urease activity of various propolis.
Figure 27 shows the results of measuring the ALT and AST in serum to confirm the safety of the mixed propolis in the anti-Helicobacter pylori animal experiments.
Figure 28 shows the histological examination results in anti-Helicobacter pylori animal experiments.
Figure 29 shows the results of visual observation of the gastric tissue of the experimental animals in the anti-Helicobacter pylori animal experiment.
Figure 30 shows the results of microscopic observation of the gastrointestinal animal in the anti-Helicobacter pylori animal experiment.
Figure 31 shows the results of confirming the gastric adhesion of Helicobacter pylori in the anti-Helicobacter pylori animal experiment.
32 is a schematic of an anti-Helicobacter pylori animal experiment.
Figure 33 shows the results of measuring the liver weight in the anti-Helicobacter pylori animal experiment.
Figure 34 shows the result of measuring the spleen weight in the anti-Helicobacter pylori animal experiment.
Figure 35 shows the results of measuring the height of the kidney in anti-Helicobacter pylori animal experiments.
Figure 36 shows the results of histopathological observation of the gastric tissue of the experimental animals in the anti-Helicobacter pylori animal experiment.
Figure 37 shows the results of confirming the gastric adhesion of Helicobacter pylori in the anti-Helicobacter pylori animal experiment.
Figure 38 shows the results of confirming the oxidative damage of gastric tissue in the anti-Helicobacter pylori animal experiment, A is the result of confirming the MDA, B is GSH.
Figure 39 shows the results of confirming the expression of inflammatory cytokines in anti-Helicobacter pylori animal experiment, A is the result of confirming the expression of IL-6, B is TNF-α, C is IL-1β.

The use method of propolis provided by the present invention,

In order to enhance the efficacy of propolis as a dietary supplement, two or more origins selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis are mixed and used.

The efficacy of the propolis includes all of the efficacy of the general propolis, and in particular, any one of the antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy.

The extract of the propolis is not particularly limited, but is preferably an ethanol extract of the propolis. More preferably, they are water soluble extracts which have ethanol extract of propolis.

In the preferred embodiment of the present invention, the mixed use of the extract of propolis is to use the mixed extract of Argentine propolis, Chinese propolis and Korean propolis. At this time, more preferably, extracts of Argentine propolis, Chinese propolis and Korean propolis are used in a weight ratio of 30 to 40:25 to 35:30 to 40.

In another preferred embodiment of the present invention, the mixed use of the extract of the propolis is to use a mixture of extracts of Argentine propolis, Chinese propolis and Brazilian propolis. At this time, more preferably, extracts of Argentine propolis, Chinese propolis and Brazilian propolis are used in a weight ratio of 35 to 45:25 to 35:25 to 35.

Health functional food composition comprising a propolis as an active ingredient provided in the present invention, in order to increase the efficacy of the propolis, at least two origins selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis Mixed extracts of different propolis.

In a preferred embodiment of the present invention, the mixed extract includes extracts of Argentine propolis, Chinese propolis and Korean propolis. More preferably, the extract of Argentine propolis, Chinese propolis and Korean propolis is included in the weight ratio of 30-40: 25-35: 30-40.

In another preferred embodiment of the present invention, the mixed extract includes extracts of Argentine propolis, Chinese propolis and Brazilian propolis. More preferably, the extract of Argentine propolis, Chinese propolis and Brazilian propolis is included in a weight ratio of 35 to 45:25 to 35:25 to 35.

The description of the efficacy and extract of the propolis to be improved is the same as described in the above method of using propolis.

The method for producing a health functional food comprising the propolis provided by the present invention as an active ingredient includes at least two selected from Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis in order to increase the efficacy of the propolis. Use a mixture of extracts of different propolis origin.

Mixing and using the extract of propolis, the efficacy of the propolis to improve and the description of the extract is the same as described in the above method of using propolis.

The composition of propolis varies depending on the type of surrounding plants and the time of collection, and the color, flavor, and functional characteristics of the propolis are also different depending on the collection area. In the present invention, while using the propolis of a variety of origin through the confirmation of the characteristics according to each origin, the effectiveness is kept constant.

The mixed use method of propolis of different origin according to the present invention can be applied to the use of propolis for dietary supplements. According to the present invention, it is possible to further improve the efficacy of propolis, and in particular, an improved effect can be obtained in terms of antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy.

"Health functional food" as defined in the present invention means a food prepared and processed using raw materials or ingredients having a useful functionality to the human body, "functional" is to control nutrients or physiology for the structure and function of the human body Ingestion is intended for the purpose of obtaining a beneficial effect on health uses such as a pharmaceutical action. The "health functional food" of the present invention includes all of the commonly used health food and functional food, including health functional food under the health functional food law. The health functional food may generally have one of tablets, capsules, powders, granules, liquids, and pills, but is not limited thereto, and may have a general food form.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. The following Examples and Experimental Examples illustrate the present invention, but the scope of the present invention is not limited thereto.

< Experimental Example  1>

Analysis of Propolis by Country of Origin

The composition of propolis varies depending on the type of surrounding plants and the time of collection, and the color, flavor, and functional characteristics of the propolis are also different depending on the collection area. Propolis raw materials with anti-inflammatory effects were selected by examining the active ingredient content by country of origin (Korean, Chinese, Argentine, Brazilian, etc.) using propolis currently on the market.

Preparation of Propolis Extract by Country of Origin

50 rpm each of natural propolis from Korea, China, Argentina, and Brazil, 500 rpm using two kinds of extraction solvents: 95% fermented alcohol 100% solution, mixed solution of 95% fermented alcohol 70% and purified water 25% -SS200) for 6 hours, extracted with stirring at room temperature and left for 24 hours, the extracted propolis was filtered using filter paper (Advantec No. 2).

Analysis of active ingredients

1. Determination of total flavonoid content

Propolis is a dietary supplement that is a dietary supplement and contains total flavonoids based on the Nutraceutical Code.

(1) How to measure

Total flavonoid content was determined according to dietary supplement. This is a method of extracting a flavonoid-based material from the sample using ethanol and analyzing it using a spectrophotometer, which is quantified by measuring the absorbance at 415 nm, which is the maximum absorption wavelength of the flavonoid.

0.1-0.5 g (mL) of propolis extract was weighed, 20 mL of 90% ethanol was added, dissolved and separated (3,000 rpm, 10 minutes), and the supernatant was collected. The residue was extracted three times with 8 mL of 80% ethanol, and the whole extract was extracted. After the addition, 80% ethanol was used to make 50 ml of the total amount and was used as a test solution. 0.5 ml of this test solution was taken into a test tube, 1.5 ml of ethanol, 0.1 ml of 10% aluminum nitrate solution, 0.1 ml of 1M potassium acetate solution, and 2.8 ml of water were added and stirred sufficiently. After standing for 40 minutes at room temperature, the absorbance was measured at 415 nm using a UV / VIS spectrophotometer (Spectrophotometer, Varian, Inc. USA) using water as a control solution using a 10 mm cell. The total flavonoid content was calculated by Equation 1 below using the absorbance difference minus the absorbance of adding 0.1 ml of water instead of the aluminum nitrate solution during the above operation separately from the absorbance of the sample.

(2) Measurement result

The extraction yield and total flavonoid content of the propolis by country of origin are shown in Table 1 below.

origin Solvent (1: 3 weight ratio w: w) Extract amount (g) Total Flavonoid Content (%) China 95% fermented alcohol 162 1.60 70% fermented alcohol 132 1.35 Argentina 95% fermented alcohol 130 1.20 70% fermented alcohol 125 0.935 Korea 95% fermented alcohol 128 0.84 70% fermented alcohol 120 0.74 Brazil 95% fermented alcohol 123 0.9 70% fermented alcohol 116 0.93 Brazil
95% fermented alcohol 120 0.72 70% fermented alcohol 118 0.83

In the results of Table 1, even if extracted under the same conditions, it can be seen that the content of the active ingredient extracted according to the extraction solvent, depending on the origin of the propolis.

2. LC Of MS Of MS Active ingredient using Chrysin , Galangin , Pinocembrin Quantitative analysis

In Table 1, using a 95% fermented alcohol extract having a high extraction rate of the yield and the active ingredient, the glycerin (Chrysin), galangin (Galangin) and pinosembrine (indicators) having an anti-helicobacter pylori effect reported in the literature ( Pinocembrin) was analyzed by LC / MS / MS to confirm the content.

Acetonitrile (HPLC grade, Merck) and formic acid (Sigma Aldrich) were used as mobile phase reagents. Finnigan Surveyor MS pump plus and Autosampler and Finnigan TSQ Quantum Discovery were used. The analysis conditions of LC / MS / MS are shown in Table 2 below, and the optimization conditions of the compounds are shown in Table 3 below.

Analysis system Analytical System LC-MS / MS Mass Spectrometry TSQ Quantum Discovery MAX (Thermo) HPLC Pump Surveyor MS Pump Plus (Finnigan) Autosampler Surveyor Autosampler Plus (Finnigan) software Xcalibur 2.1.0 column Sunniest C18 (2.0 x 100mm, 3um) Analysis condition Mobile Phase ACN: DW (0.1% formic acid) = 65:35 (v / v) Flow Rate 0.250 / min Injection Volume 5 MRM (MS / MS) Condition (Voice) Crysin (m / z 253.0 to m / z 143.02),
Galangin (m / z 255.0 to m / z 213.05),
Pinosembrine (m / z 269.0 to m / z 169.05), IS (5-NIO, m / z 321.0 to m / z 291.12) Source / Gas / Temperature Parameters (Optimized) Ion Source ESI (HESI, Negative) Ion Spray Voltage (V) 3500 Sheath Gas Pressure 35 Aux Gas Pressure 20 CID Gas Pressure 1.5 Vaporizer Temp.,) 125 Capillary Temp. 315 Tube Lens Offset 105

Compound optimization Krisin (m / z 253.0 to m / z 143.02) Collision Energy 32 Galangin (m / z 255.0 to m / z 213.05) Collision energy 23 Pinosembrine (m / z 269.0 to m / z 169.05) Collision energy 28 IS (5-NIO) (m / z 321.0 to m / z 291.12) Collision energy 21

Chrysine, gallangin and pinosembrine contents of the propolis according to the country of origin of the analysis are shown in Table 4 below, and chromatograms of the respective samples are shown in FIGS. 1A to 1C.

Krisin (ppm) Galangin (ppm) Pinosembrine (ppm) Made in China (Hyesong) 29.7 38.5 15.7 Made in Korea 47.1 12.3 27.0 Brazil (Green) 0.2 0.2 1.6 Brazil 0.1 0.1 0.7 Argentina 102.3 26.1 36.4

3. Analysis of active ingredient content of ethanol extract propolis and water soluble propolis

Ethanol extract propolis has a problem that does not easily dissolve in water and sticky to the mouth or stick to the cup when ingested so that it is not easily removed. In order to solve this problem, the present invention prepares and uses a water-soluble propolis that is easily soluble in water without ethanol.

The water-soluble propolis used in the present invention is prepared by extracting ethanol and concentrating to remove ethanol and then solubilizing with purified water. In order to confirm the difference between the effective ingredient of the water-soluble propolis and ethanol extract propolis of the present invention, the quantitative test of 15 components known as the active ingredient through LC / MS / MS was carried out as follows.

Acetonitrile (Acetonitrile, HPLC grade, Merck) was used as the mobile phase reagent, and Finnigan Surveyor MS pump plus and Autosampler and Finnigan TSQ Quantum Discovery were used as equipment. The analysis conditions of LC / MS / MS are shown in Table 5 below. As a standard solution, a standard calibration curve solution of 1, 0.1 ppm was prepared using a standard product (Sigma), and a calibration curve was prepared.

Memantine column Capcell PAK MGII (3um, 2.0 × 100mm) Mobile phase Acetonitrile / H2O (65:35, v / v) Flow rate 300 / min Injection volume 5 Mass condition
(Mass Condition) XCALIBUR 2.0 sofrware
Spray voltage:-Negative 3.5 kV
Positive 5.0 kV
Transfer capillary temperature: 315, 295
Sheath gas pressure: 35, Aux gas pressure: 20
Evaporator Temperature: 125, 50

As a result, the processing of the ethanol-extracted propolis with water-soluble propolis did not significantly reduce the content of the active ingredient, but also concentrated through the solubilization process to increase the content of some active ingredients (data not shown). .

Cinnamic acid, caffeic acid, acecetin, CAPE, kaempferol, gallic acid, chrysin, ferullic acid The quantitative analysis results for are shown in Figures 2a to 2h.

< Experimental Example  2>

Cytotoxicity of Propolis Samples by Origin

In order to determine the effect (toxicity) of propolis samples of different origins on the growth of macrophages, propolis extracts from Korean, Chinese, Brazilian, and Argentinian cells were treated with RAW 264.7 cells, a macrophage line, and the cells were analyzed by MTT assay. Toxicity was confirmed. The experiment was repeated three times and the results are expressed as mean ± SD of three experiments. The results are shown in FIG.

Domestic and Argentine propolis showed toxicity above 50 µg / ml. Brazilian propolis showed little toxicity up to 100 μg / ml and Chinese propolis showed little toxicity up to 50 μg / ml but weakly at 100 μg / ml.

< Experimental Example  3>

Of propolis samples by country of origin NO  Confirmation of production inhibitory activity

To determine the effect of propolis samples by origin on NO production, raw 264.7 cells were stimulated with LPS and treated with propolis samples by concentration for 24 hours. The experiment was repeated three times and the results are expressed as mean ± SD of three experiments. The results are shown in FIG.

As can be seen from the results of Figure 4, domestic and Argentine propolis showed NO production inhibitory activity, but at a concentration of 50 µg / ml or more was considered to be inhibitory activity due to cytotoxicity (see Figure 3). In the case of propolis from China and Brazil, it was confirmed that the inhibitory activity was dependent on the concentration.

< Example  1>

Selection of mixing ratio of mixed propolis

Since the types and contents of the active ingredients contained in propolis vary depending on the surrounding environment, the contents of the active ingredients contained in the extract of propolis are analyzed by LC / MS / MS based on the results of analysis by LC / MS / MS. After mixing the propolis alcohol extract, the total flavonoid content of the aqueous solution obtained through solubilization was measured.

Propolis alcohol extract mixture was based on a mixture of Argentinean and Chinese propolis extracts, and Korean and Brazilian propolis extracts were added, respectively.

1. Mix of Argentinean, Chinese and Korean Propolis

Argentinean, Chinese and Korean propolis alcohol extracts were mixed in the weight ratio of Table 6 below, and the total flavonoid content of the aqueous solution obtained through the solubilization process was measured.

Ethanol Extract Propolis Blend Ratio Total Flavonoid Content
(%) Argentina Made in China Made in Korea 35 30 35 0.80 35 35 30 0.75 30 35 35 0.71 20 40 40 0.72 40 20 40 0.71 40 40 20 0.75 30 30 40 0.71 30 40 30 0.72 40 30 30 0.75

As a result, the total flavonoid content was 0.8% in the aqueous solution obtained by mixing the propolis alcohol extract in the ratio of Argentine: Chinese: Domestic = 35:30:35, which was higher than that of the Propolis extract mixed in different proportions. .

2. Mix of Argentinean, Chinese and Brazilian Propolis

Argentinean, Chinese and Brazilian propolis alcohol extracts were mixed in the weight ratio of Table 7 below, and the total flavonoid content of the aqueous solution obtained through the solubilization process in the same manner as in Table 6 was measured.

Ethanol Extract Propolis Blend Ratio Total Flavonoid Content
(%) Argentina Made in China Brazil 35 35 30 0.85 35 30 35 0.84 30 35 35 0.84 20 40 40 0.80 40 20 40 0.75 40 40 20 0.77 30 30 40 0.68 30 40 30 0.71 40 30 30 0.99

Argentine; Chinese: Brazilian = 40:30:30 The total flavonoid content of the aqueous solution containing the mixed mixture of propolis extracts was 0.99%, which was the highest in Korea. The total flavonoid content was measured higher than the aqueous solution. The aqueous solution of propolis prepared with brazilian mixed alcohol extract was detected even after accommodating the spicy taste of Brazilian propolis, but the degree of spicy was weak compared to the aqueous solution prepared with brazilian propolis alone.

< Experimental Example  4>

Propolis and by country of origin Mixed-process  Active ingredient

Argentine propolis, Chinese propolis, Korean propolis, and the active ingredient content of the mixed propolis mixed with them are shown in Table 8 below.

The mixed propolis samples were mixed propolis mixed with Argentine propolis, Chinese propolis and Korean propolis in a weight ratio of 35:30:35.

ingredient Argentina Made in China Made in Korea mix Flavonone Acecetin 185.5 51.5 154.6 243.6 Krisin 4,505.9 2,284.0 3,062.1 5,036.5 Flavonoids Galangin 5,390.0 1,791.4 4,485.7 5,730.9 Quercetin 21.0 6.8 21.2 26.8 Camper Roll 583.9 126.0 360.0 282.5 Routine 3.9 3.4 9.4 6.1 Flavonone Hesperetin 10.8 13.5 35.3 38.0 Pinosembrine 3,491.2 935.2 1,745.1 1,763.6 Phenolic
compound Galsan 20.9 11.1 33.7 23.5 Ferulic acid 234.2 103.5 268.3 258.0 Caffeic Acid 1,898.8 18.8 1,413.6 1,723.7 CAPE 855.2 18.7 639.6 827.3 Sinnamsan 6,988.9 14,911.3 2,508.5 Kumaric acid 1,417.7 69.7 1,427.0 1180.2

As can be seen in the results of Table 8, by mixing the propolis of different origin can be ingested a larger amount of the unique active ingredients of each propolis together.

< Experimental Example  5>

Rise of Mixed Propolis ( synergy Check the effect

As a sample for confirming the synergistic effect of the mixed propolis, mixed propolis was prepared by mixing extracts of Argentine propolis, Chinese propolis and Brazilian propolis in a weight ratio of 40:30:30.

1. Check cytotoxicity

In order to confirm the effects (toxicity) of the mixed propolis samples on the growth of macrophages, the mixed propolis samples were treated with Raw 264.7 cells, which are macrophages, and cell viability was confirmed by MTT assay 24 hours later.

The experiment was repeated three times and the results are expressed as mean ± SD of three experiments. The results are shown in FIG.

As can be seen from the results in FIG. 5, the samples did not all show toxicity.

2. NO  Confirmation of production inhibitory activity

In order to confirm the NO production inhibitory activity of the mixed propolis samples, the mixed propolis samples were treated in LPS-treated RAW 264.7 cells by concentration and the amount of NO produced was measured. The experiment was repeated three times and the results are expressed as mean ± SD of three experiments. The results are shown in FIG.

3. Confirmation of cytokine related gene inhibitory effect

In order to confirm the cytokine-related gene inhibitory effect of the mixed propolis samples, the concentration of the mixed propolis samples in RAW 264.7 cells treated with 50% ethanol extract of each concentration for 24 hours with or without 1 μg / ml LPS MRNA levels of TNF-α, IL-1β and IL-6, which are proinflammatory cytokines produced by LPS stimulation, were measured by quantitative RT-PCR. The experiment was repeated three times and the results are expressed as mean ± SD of three experiments. The results are shown in FIG.

< Experimental Example  6>

Therapeutic Effect Test of Mixed Propolis against Alcoholic Gastritis

The following tests were performed to evaluate the therapeutic effect of mixed propolis in alcohol-induced gastric injury model.

Test Methods

(1) test animals

Sprague-Dawley rats were used as test animals, and were used by Orient Bio Co., Ltd. (Seongnam, Gyeonggi-do).

Body weights ranged from 150-180 g and male rats. Only healthy animals were used for testing with quarantine and purifying periods for one week after acquisition. Starting dose ranged from 180 to 200 g and a total of 82 animals were used for testing. Breeding was carried out at a temperature of 20 ± 2 ℃, humidity 40-60%, 12 hours night / day cycle.

(2) induction of alcoholic gastritis

After fasting the rats for 24 hours, 5 ml / kg of absolute alcohol (absolute alcohol) was administered by gavage, and the test substances were administered after 1 hour and 12 hours, respectively. Autopsy was performed 24 hours after alcohol administration to compare gastritis lesions. A schematic diagram of the experimental design is shown in FIG. 8.

(3) Test group  Configuration

The test group consisted of 4 groups (G1 ~ G4), each of which used 5 or 7 animals. The configuration of the test group is shown in Table 9 below.

Test group Damage
Gastric injury process The number of animals G1 Normal control - Saline 5 G2 Alcohol group ethanol Saline 10 G3 Positive control group ethanol Omeprazole 10 G4 Experimental group ethanol Mixed Propolis 10

(4) Dosage and Method

The dosages and methods of alcohol, control drug (positive control; omeprazole) and test drug are shown in Table 10 below.

process Dosage Dosing method Number of doses ethanol 5 / kg (anhydrous alcohol) Gastrointestinal Nutrition Law One Omeprazole 20 mg / kg / day 2 Mixed Propolis 175 mg / kg / day 2

(5) Statistical processing

For statistical analysis, one-way ANOVA was performed using GraphPad PRISM program, and post-mortem analysis was performed using Turkey test to verify the difference between groups. Statistical significance was determined as p <0.5.

Mortality and clinical symptoms

The clinical symptoms and death / rare animal occurrence were observed once per day during the study period. Records were kept only for subjects with clinical symptoms (not recording if normal), and weekends and holidays were replaced by one morning observation. However, at least twice a day was observed on the day of administration.

As a result, no special clinical symptoms were observed in all animals during the period of acclimatization, and there were no dead animals on the test day. The results are shown in Table 11 below.

Test group
process The number of animals Our company
The number of animals Mortality rate
(%) Clinical
observe G1 Normal control Saline / Saline 5 - - N G2 Alcohol group Ethanol / saline 7 - - N G3 Positive control group Ethanol / omeprazole 7 - - N G4 Experimental group Ethanol / Mixed Propolis 7 - - N * N: looks normal

2. Weight measurement

Upon receipt, body weights were measured once per week during the acclimation period, during group separation and during the test period. In particular, the weight measurement during the test period was measured using the same scale on the same day and the same time zone (before and after 4 pm) to minimize the error in each measurement.

In the alcohol group (G2 ~ G4) it was confirmed that the weight is reduced, and compared to G1, G2, G3 and G4 group was statistically significant weight loss. The results are shown in Table 12 below (data is presented as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 respectively when compared to G1).

Test group
process Weight (g) Initial (before ethanol administration) Final (ethanol
After administration) % Increase G1 Normal control Saline / Saline 319.8 ± 10.5 322.4 ± 9.6 100.9 ± 0.5 G2 Alcohol group Ethanol / saline 297.8 ± 4.3 278.3 ± 3.2 93.5 ± 1.0 ^* G3 Positive control group Ethanol / omeprazole 316.4 ± 19.1 290.7 ± 24.1 91.5 ± 3.8 ^** G4 Experimental group Ethanol / Mixed Propolis 291.5 ± 2.5 273.6 ± 2.8 93.8 ± 0.6 ^*

3. Autopsy and organ weight calculation

After 24 hours of alcohol administration, the abdominal aorta and the abdominal vein were cut open and bleeding after being opened under CO ₂ anesthesia. The absolute organ weight of each organ (liver, spleen, kidney) was measured after visual observation of abnormality of internal organs. The relative organ weights for fasted body weights before necropsy were calculated.

(One) Liver weight

As a result of calculating the relative organ weight for fasting body weight, liver weight was increased after alcohol administration, and liver weight was significantly increased in G2, G3 and G4 groups compared with G1. However, no significant difference was observed between G3 and G4 compared to G2. The results are shown in FIG. 9 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 respectively when compared to G1).

(2) Spleen weight

As a result of calculating the relative organ weight of the spleen to the fasted body weight, the weight of the spleen increased after alcohol administration, but there was no statistically significant difference. The results are shown in FIG. 10 (data shown as mean ± SEM).

(3) weight

As a result of calculating the relative organ weight of the kidney relative to the fasted body weight, the weight of the kidney was increased after alcohol administration, and the weight of the kidney was significantly increased in the G2, G3, and G4 groups compared to the G1 group. However, no significant difference was observed between G3 and G4 compared to G2. The results are shown in FIG. 11 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 respectively when compared to G1).

4. Histopathological Analysis

After euthanasia, the individual stomach was removed and a part of it was fixed in 10% buffered neutral formalin. The fixed tissue was cut to a certain thickness, and then paraffin embedded through a general tissue treatment process to produce 4-5 μm tissue sections, followed by hematoxylin & eosin staining (H & E stain). Histopathological changes (inflammation of inflammatory cells, structural destruction of epithelial cells, ulcers and bleeding) were observed.

H & E staining of the stomach tissue was confirmed to determine the extent of tissue damage. In G1, no abnormalities were observed in all layers including gastric mucosa, but G2 showed necrosis and hemorrhage with the loss of mucosa and some wall cells (parietal). cells and pepsinogen-secreting cells (chief cells) have been identified. Infiltration of inflammatory cells, such as macrophages and neutrophils, was observed in the mucosa and submucosa. However, gastric mucosal damage was reduced in G3 and G4 groups. The results are shown in FIG.

5. Damage  Inhibition rate measurement

The extracted stomach was incised along the Taiwan part, and the damage length (mm) and the damage area (mm ² ) generated were measured under a microscope and calculated according to Equation 2 below. It was judged to be effective if significant effects were demonstrated over the control and positive controls under the 5% significance level (p <0.05).

(1) gross damage to the gastric mucosa

Alcohol directly stimulates the gastric mucosa and induces swelling in the submucosal muscle layer, causing a temporary ischemic state, causing necrosis of cells due to oxidative damage, and stimulating the gastric mucosa directly, causing bleeding and ulceration. In order to evaluate the therapeutic effect of the propolis complex composition on alcohol-induced gastric mucosal damage, the lesions were visually examined after autopsy, and G1 showed no gross abnormalities. In G4), thick bleeding was observed on the gastric mucosa. However, these bleeding sites have been shown to decrease in G3 and G4 compared to G2. The results are shown in FIG.

(2) Damage  area

As a result of measuring the length of injury and the area of injury (mm ² ) of the gastric mucosa, the relative area with respect to the area of the gastric mucosa was compared. Injuries (20.7 ± 1.7%) were observed, and gastric mucosal injuries decreased significantly in both G3 and G4 groups compared to G2. The results are shown in A of FIG. 14 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

(3) Damage  Suppression rate

Compared with G2 (alcohol group; 0.0 ± 0.0%), the inhibition rate of gastric mucosa was significantly increased in both G3 and G4 groups. The results are shown in FIG. 14B (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared with G1; ^† and ^†† , respectively, P <0.05 when compared with G2). and P <0.01).

6. In the stomach tissue Oxidative  Damage measurement

(1) lipid peroxidation

Lipids and oxides are caused by damage to tissues by the removal of hydrogen atoms from phospholipid cell membranes in tissues by oxidative stresses such as ROS and free radicals. MDA, a lipid peroxidation product of liver tissue, was measured as an indicator of gastric damage.

A portion of the stomach tissue was homogenized using a homogenizer by adding four-fold 150 mM KCl. The homogenized tissue was centrifuged at 4,000 rpm for 30 minutes, the supernatant was taken to measure glutathione (GSH), and the remainder was centrifuged again at 20,000 rpm for 1 hour, and then the supernatant was taken to measure malondialdehyde (MDA). . TBARS assay kit (Cell Biolabs, USA) was used to measure the absorbance at 586nm and MDA was calculated.

Compared with G1 (normal group; 1.2 ± 0.6μmol / mg protein) group, G2 (alcohol-administered group; 11.7 ± 1.8μmol / mg protein) significantly increased the MDA of gastric tissue, compared with G2 at G3 and G4. MDA levels were significantly reduced. The results are shown in A of FIG. 15 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

(2) GSH

The glutathione analysis kit (Cell Biolabs, USA) was used to measure the absorbance at 405 nm and GSH concentration was calculated.

GSH is one of the antioxidants in vivo that protects against oxidative stress damage caused by free radicals and peroxides. In this experiment, GSH of the gastric tissues was significantly decreased in G2 (alcohol-administered group; 17.0 ± 2.4μmol / mg protein) compared to G1 (normal group; 1.2 ± 0.6μmol / mg protein). However, no significant difference was observed between G3 and G4 compared to G2, and the results are shown in B of FIG. 15 (data are shown as mean ± SEM. ^* And ^** are P <0.05 respectively when compared to G1. and P <0.01; ^† and ^†† , P <0.05 and P <0.01, respectively, compared to G2).

7. Inflammatory cytokines measured in gastric tissue

(1) gun RNA Separation and cDNA  synthesis

Dissolve 1 ml of Trizol Lysis buffer in each of the tissues and homogenize it. 200 μl of chloroform was dispensed and inverted for 20 seconds. After centrifugation at 13,200 rpm for 15 minutes, the supernatant was transferred to a tube containing 500 μl of isopropanol. After centrifugation again at 13,000 rpm for 10 minutes, the supernatant was removed, and 1 ml of 70% ethanol was dispensed into each tube, followed by centrifugation at 13,000 rpm for 5 minutes, and then the supernatant was removed and dried at room temperature. 40 μl of D.W. containing DEPC was dissolved to dissolve RNA, and then 1 μl of the RNA solution was taken to measure absorbance at 260/280 nm using a NanoDrop instrument to calculate the total RNA concentration of the solution. CDNA was synthesized using the extracted RNA and RT DriyMIX cDNA synthesis kit.

(2) real time PCR

Real-time PCR was performed to investigate gene expression of IL-1β, IL-6 and TNF-α. Primer sequences used in the experiments are shown in Table 13 below.

gene primer Sequence (5 '→ 3') IL-1β Forward AGG ACC CAA GCA CCT TCT TT Reverse AGA CAG CAC GAG GCA TTT T IL-6 Forward TAG TCC TTC CTA CCC CAA CT Reverse TTG GTC CTT AGC CAC TCC TT TNF-α Forward GTC TGT GCC TCA GCC TCT TC Reverse CCC ATT TGG GAA CTT CrC CT GAPDH Forward ACA GCA ACA GGG TGG TGG AC Reverse TTT GAG GGT GCA GCG AAC TT-

(3) Expression of Inflammatory Cytokines

① IL Expression of -6

IL-6 levels were measured in gastric tissues to evaluate the effects of alcohol administration on the gastric inflammatory response and propolis complex composition. The expression of IL-6 was significantly increased in G2 compared to G1, and the expression of IL-6 was significantly decreased in G3 compared to G2. The results are shown in A of FIG. 16 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

② IL Expression of -1β

The expression of IL-1β was significantly increased in G2 compared to G1, and a decrease in the expression of IL-1β was observed in G3 and G4 compared to G2. The results are shown in B of FIG. 16 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

③ TNF -α

The expression of TNF-α was significantly increased in G2 compared to G1, and a significant decrease in TNF-α expression was observed in both G3 and G4 groups compared to G2. The results are shown in C of FIG. 16 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

8. Comprehensive review of therapeutic effects of mixed propolis against alcoholic gastritis

To investigate the therapeutic effect of mixed propolis on the damaged gastric mucosa after alcohol administration, the results of gross findings, histopathologic findings, oxidative stress analysis and expression of proinflammatory cytokines were compared. The results are shown in Table 14 below.

Test group process Microscopic features Antioxidant
stress Anti-inflammatory Stomach ulcer
Area ^A Inhibition rate ^B MDA ^C GSH ^D IL-6 ^E IL-1β TNF-α ^G G1 Normal control Saline / Saline - - - - - - - G2 Alcohol group Ethanol / saline - - - - - - - G3 Positive control group Ethanol / omeprazole + + ++ + +++ +++ ++ G4 Experimental group Ethanol / Mixed Propolis ++ ++ + ++ ++ ++ +++ A: ulcer area> 10 mm ² ; +, Ulcer area = 10-7 mm ² ; ++, ulcer area <7 mm ² ; +++.
B: inhibition rate <50%; +, Inhibition = 50-70%; ++, inhibition rate <70%; +++.
C: MDA> 5 uM / mg protein; +, MDA = 3-5 uM / mg protein; ++, MDA <3 uM / mg protein; +++.
D: GSH <20 uM / mg protein; +, GSH = 20-50 uM / mg protein; ++, GSH> 50 uM / mg protein; +++.
E: IL-6> 5 fold; +, IL-6 = 2-5 folds; ++, IL-6 <2 times; +++.
F: IL-1β> 50 fold; +, IL-1β = 20-50 fold; ++, IL-1β <20 fold; +++.
G: TNF-α> 0.5 fold; +, TNF-α = 0.2-0.5 fold; ++, TNF-α <0.2 fold; +++.

As shown in Table 14, the mixed propolis was confirmed to exhibit anti-ulcer, antioxidant and anti-inflammatory effects.

< Experimental Example  7>

Therapeutic Effect Test of Mixed Propolis against Alcoholic Gastritis

The following tests were performed to evaluate the therapeutic effect of mixed propolis in alcohol-induced gastric injury model.

Test Methods

(1) test animals

Sprague-Dawley rats were used as test animals, and were used by Orient Bio Co., Ltd. (Seongnam-si, Gyeonggi-do).

Body weights ranged from 150-180 g and male rats. Only healthy animals were used for testing with quarantine and purifying periods for one week after acquisition. Starting dose ranged from 180 to 200 g and a total of 75 animals were used for testing. Breeding was carried out at a temperature of 20 ± 2 ℃, humidity 40-60%, 12 hours night / day cycle.

(2) induction of alcoholic gastritis

After fasting the rats for 24 hours, 5 ml / kg of absolute alcohol (absolute alcohol) was administered by gavage, and the test substances were administered after 1 hour and 12 hours, respectively. Autopsy was performed 24 hours after alcohol administration to compare gastritis lesions. A schematic of the experimental design is shown in FIG. 17.

(3) Test group  Configuration

The test group consisted of 4 groups (G1 ~ G4), each of which used 5 or 7 animals. The configuration of the test group is shown in Table 15 below.

Test group Gastric injury process The number of animals G1 Normal control - Saline 5 G2 Alcohol group ethanol Saline 10 G3 Positive control group ethanol Omeprazole 10 G4 Experimental group ethanol Mixed Propolis 10

(4) Dosage and Method

The dosages and methods of alcohol, control drug (positive control; omeprazole) and test drug are shown in Table 16 below.

process Dosage Dosing method Number of doses ethanol 5 / kg (anhydrous alcohol) Gastrointestinal Nutrition Law One Omeprazole 20 mg / kg / day 7 Mixed Propolis 175 mg / kg / day 7

(5) Statistical processing

For statistical analysis, one-way ANOVA was performed using the GraphPad PRISM program, and post-mortem analysis was performed using the Turkish test to verify the differences between the groups. Statistical significance was determined as p <0.5.

1. Observation of death and clinical symptoms

The clinical symptoms and death / rare animal occurrence were observed once per day during the study period. Records were kept only for subjects with clinical symptoms (not recording if normal), and weekends and holidays were replaced by one morning observation. However, at least twice a day was observed on the day of administration.

No specific clinical symptoms were observed in all animals during the acclimatization period. In addition, no animals died until the autopsy was performed after administration of the test substance (7 days) and alcohol.

2. Weight measurement

Upon receipt, body weights were measured once per week during the acclimation period, during group separation and during the test period. In particular, the weight measurement during the test period was measured using the same scale on the same day and the same time zone (before and after 4 pm) to minimize the error in each measurement.

No significant weight differences were observed in all groups (G2-G4) compared to G1 during the weekly administration of the test substance. The results are shown in FIG.

3. Autopsy and long-term weight measurement

After 24 hours of alcohol administration, the abdominal aorta and the abdominal vein were cut open and bleeding after being opened under CO ₂ anesthesia. The absolute organ weight of each organ (liver, spleen, kidney) was measured after visual observation of abnormality of internal organs. The relative organ weights for fasted body weights before necropsy were calculated.

As a result of calculating the relative organ weight of the liver for fasting body weight, there was no statistically significant difference in all groups (G2 ~ G4) compared to G1. There was also no significant difference in the relative organ weights of the spleen and kidney relative to the fasted body weight. The results are shown in FIG. 19 (data shown as mean ± SEM).

4. Histopathological Analysis

After euthanasia, the individual stomach was removed and a part of it was fixed in 10% buffered neutral formalin. The fixed tissue was cut to a certain thickness, and then paraffin embedded through a general tissue treatment process to produce 4-5 μm tissue sections, followed by hematoxylin & eosin staining (H & E stain). Histopathological changes (inflammation of inflammatory cells, structural destruction of epithelial cells, ulcers and bleeding) were observed.

H & E staining of the stomach tissue was confirmed to determine the extent of tissue damage. In G1, no abnormalities were observed in all layers including gastric mucosa, but G2 showed necrosis and bleeding with the loss of gastric mucosa. Invasion of inflammatory cells such as macrophages and neutrophils was observed in the mucosa and submucosa. However, damage to the gastric mucosa was reduced in both G3 and G4. The result is shown in FIG.

5. Damage  Inhibition rate measurement

The extracted stomach was cut along the taiwan section, and the damage length (mm) and damage area (mm ² ) generated were measured under a microscope and calculated according to Equation 2 above. It was judged to be effective if significant effects were demonstrated over the control and positive controls under the 5% significance level (p <0.05).

(1) gross damage to the gastric mucosa

In order to evaluate the therapeutic effect of the propolis complex composition on alcohol-induced gastric mucosal damage, the lesions were visually examined after autopsy, and G1 showed no gross abnormalities. In G4), thick bleeding was observed on the gastric mucosa. However, compared to G2, this bleeding site was found to decrease in G3. The results are shown in FIG.

(2) Damage  area

The damage length and area of injury (mm ² ) of the gastric mucosa were measured and the relative area of the entire gastric mucosa was compared. The most damage was observed in G2 compared to G1, and this bleeding in G3 compared to G2. The site was significantly reduced. The results are shown in A of FIG. 22 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

(3) Damage  Suppression rate

Compared with G2, the rate of gastric injury inhibition was significantly increased in G3. The results are shown in B of FIG. 22 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

6. In the stomach tissue Oxidative  Damage measurement

(1) Fraction and Lipid Peroxidation of Gastric Tissues

MDA, a lipid peroxidation product of liver tissue, was measured as an indicator of gastric damage.

A portion of the stomach tissue was homogenized using a homogenizer by adding four-fold 150 mM KCl. The homogenized tissue was centrifuged at 4,000 rpm for 30 minutes, the supernatant was taken to measure glutathione (GSH), and the remainder was centrifuged again at 20,000 rpm for 1 hour, and then the supernatant was taken to measure malondialdehyde (MDA). . TBARS assay kit (Cell Biolabs, USA) was used to measure the absorbance at 586nm and MDA was calculated.

MDA of gastric tissue increased in G2 (alcohol-administered group; 55.3 ± 12.3μmol / mg protein) compared to G1 (normal group; 20.0 ± 2.9μmol / mg protein) group, and MDA levels in both G3 and G4 compared to G2 Significantly decreased. The results are shown in A of FIG. 23 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2. and P <0.01).

(2) GSH

The glutathione analysis kit (Cell Biolabs, USA) was used to measure the absorbance at 405 nm and GSH concentration was calculated.

Compared to the G1 (normal group; 1.2 ± 0.6 μmol / mg protein) group, G2 (alcohol-administered group; 17.0 ± 2.4 μmol / mg protein) of GSH decreased significantly but no significant difference was observed. There was also no statistical difference between G3 and G4 compared to G2. The results are shown in FIG. 23B (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 when compared to G2). and P <0.01).

7. Comprehensive review of therapeutic effects of mixed propolis against alcoholic gastritis

In order to investigate the therapeutic effect of mixed propolis on damaged gastric mucosa after alcohol administration, gross findings, histopathologic findings, and oxidative stress analysis were comprehensively reviewed. The results are shown in Table 17 below.

Test group process Microscopic features Antioxidant stress Stomach ulcer
Area ^A Inhibition rate ^B MDA ^C GSH ^D G1 Normal control Saline / Saline - - - - G2 Alcohol group Ethanol / saline - - - - G3 Positive control group Ethanol / omeprazole +++ +++ + +++ G4 Experimental group Ethanol / Mixed Propolis + ++ ++ ++ A: ulcer area> 10 mm ² ; +, Ulcer area = 10-8 mm ² ; ++, ulcer area <8 mm ² ; +++.
B: inhibition rate <30%; +, Inhibition = 30-50%; ++, inhibition rate <50%; +++.
C: MDA> 30 uM / mg protein; +, MDA = 15-30 uM / mg protein; ++, MDA <15 uM / mg protein; +++.
D: GSH <10 uM / mg protein; +, GSH = 10-30 uM / mg protein; ++, GSH> 30 uM / mg protein; +++.

As shown in Table 17, the mixed propolis showed anti-ulcer and antioxidant effects.

< Experimental Example  8>

Anti-helicobacter Pylori  Check active

Test Methods

(1) Preparation of Propolis Sample

As propolis samples, 50% ethanol extracts of Korean (D), Brazilian (B), Chinese (C) and Argentine (A) propolis were used.

(2) Helicobacter Pylori  Cultivation of Strains

Experiments, we used the Helicobacter pylori (Helicobacter pylori) four kinds, to the types of Helicobacter pylori strains used in this experiment are shown in Table 18. 10% New Born Calf serum was added to Brucella broth (BD, USA) as a liquid medium for the pre-culture and main culture of Helicobacter pylori, and Brucella broth (BD) was used as a solid medium. , USA) was used to add 10% newborn calf serum and bacto-agar (BD, USA). Culture conditions were incubated at 37 ℃ in a 10% CO ₂ incubator (incubator).

number Strain Related characteristics source One Helicobacter pylori ATCC 26695 Sequenced clinical isolate
(cagA +, vacA +, reference strain) Gyeongsang National University
College of Medicine 2 Helicobacter pylori SS1 Murine passaged isolate
(cagA +, vacA +) 3 Helicobacter pylori 52 4 Helicobacter pylori CH150

1.Propolis Samples by Origin Anti-helicobacter Pylori  Check the effect

Antimicrobial activity against Helicobacter pylori of propolis samples of each origin was measured by the paper disc agar diffusion method. In other words, each strain cultured on a plate medium was incubated with platinum and cultured in 10 ml of liquid medium for 18-24 hours to activate, and then inoculated with 0.1 ml of the bacterial solution in 10 ml of the liquid medium to be incubated for 3 to 6 hours. After the main culture, the inoculation was carried out so that the number of bacteria per plate medium was about 1 × 10 ⁷ cells and evenly spread with a sterile swab. The sterilized filter paper disc (filter paper disc, 8mm, Whatman, Japan) was placed on a solid plate medium, and the sample was absorbed by concentration to 0.5 mg / disc and incubated at 37 ° C. for 72 hours. The diameter of the clear zone (mm) was measured. The results are shown in Table 19 and FIG. 24.

Although the results were slightly different for each strain, on average, propolis A, C, D, and B showed the highest effect. In particular, the effects of A and C propolis samples were better.

Dish No. sample Concentration (/ disc) Clear zone size (mm) ATCC 26695 Propolis A 500 17.17 B 15.83 C 17.17 D 15.17 SS1 Propolis A 500 23.67 B 16.5 C 22.5 D 21 CH150 Propolis A 500 18.67 B 13.67 C 18.67 D 17.17 52 Propolis A 500 18.67 B 13.67 C 15.33 D 17.5

2. Mixed Propolis Anti-helicobacter Pylori  Check the effect

Minimum inhibitory concentration analysis MIC assay )

The anti-helicobacter pylori effect of mixed propolis was confirmed by MIC (minimum inhibitory concentrations) analysis. MIC is a unit used to express the antimicrobial activity or developmental inhibition of antibiotics, and refers to the minimum concentration (mg / l) of the target substance having developmental inhibition against a specific microorganism. Above this concentration value, the microorganism does not develop and shows a constant number.

The mixed propolis used as a sample was a mixture of Argentine propolis, Chinese propolis and Brazilian propolis in a weight ratio of 40:30:30. Licorice extract and olive leaf extract were used as samples for comparison.

In order to measure the minimum inhibitory concentration for Helicobacter pylori, each sample was serially diluted with medium to contain 1 mg / ml concentration, and the cells were inoculated at an OD600 = 0.1 concentration and incubated for 16 hours under aerobic conditions. After the reaction, the solution was diluted to 10 ⁻³ with PBS, loaded on a plate, and incubated for 3 days in an aerobic condition, and the number of colonies formed was counted.

Alternatively, after the sample was treated, the same amount of urea broth was added, the reaction was performed at 37 ° C. in a 10% CO ₂ incubator, and the number of viable cells was indirectly measured by a 560 nm spectrophotometer.

The results for the mixed propolis samples are shown in Table 20 below, and the results for each sample are shown in FIG. 25.

Dish No. Sample concentration (/ disc) Clear zone size (mm) ATCC 26695 500 17.17 SS1 500 23.67 CH150 500 18.67 52 500 18.67

As can be seen from the results in Table 20 and FIG. 25, mixed propolis showed high anti-Helicobacter efficacy.

3. Confirmation of urease activity of propolis samples by country of origin

The pathogenic mechanisms of Helicobacter pylori are not yet fully understood, but Helicobacter pylori, which is present in the stomach, has the ability to produce powerful urease, which is used to break down the urea in the stomach. It has been found to survive in the stomach while neutralizing. Urea dehydrogenase activity, the most widely used method to measure the antibacterial activity of Helicobacter pylori, is easy to measure urease activity and Helicobacter pylori Correlation with viable cell counts was also found to be very high, and urease activity was used as an analysis method for overall antibacterial activity.

In order to measure urease activity inhibitory effect, Helicobacter pylori was cultured, only cells were isolated, and then crushed by an ultrasonic crusher (sonicator), ultra-centrifuged for 12,000rpm, 4 ℃, and 30 minutes. Got. 5 mg / ml of the sample and 50 µl of total protein were mixed with 100 µl of urea broth. The change in color intensity of phenol-red according to the decomposition of urea was measured at 560 nm using a spectrophotometer (TECAN, USA).

The urease inhibitory activity of the identified propolis samples by each country of origin is shown in Table 21 and FIG. 26.

Inhibition of urea activity of propolis samples by origin was B, D, A, and C in order. This result is slightly different from the disk diffusion test, because the main substance in propolis affects the activity of urease to show anti-helicobacter pylori activity, but also affects other mechanisms.

A B C D mix (-) Blank 0.144 0.283 0.267 0.440 1.140 reaction 0.222 0.342 0.380 0.513 1.178 R-(-) 0.078 0.058 0.113 0.073 0.038 % Inhibition 54.99 66.34 34.99 57.93 77.98

< Experimental Example  9>

Anti-Helicobacter Pylori Animal Experiment (Preliminary Experiment)

To test the anti-helicobacter effect of mixed propolis in a gastritis model of mice induced with Helicobacter pylori, Helicobacter pylori Gastric adhesion inhibitory effect and gastric protective effect were evaluated as follows.

Test Methods

(1) Test substance and test animal

Propolis complexes were used as test materials, and 5 weeks old female C57BL / 6 mice were used as test animals.

(2) Helicobacter Pylori  Gastritis Model

In order to obtain a gastritis model by Helicobacter pylori, C57BL / 6 mice were prepared with 1 × 10 ⁹ cfu / ml of Helicobacter pylori and inoculated three times a week by oral gavage per mouse three times per week. 3 weeks after the observation. At this time, Helicobacter pylori was used by adapting the mouse to Sydney strain (SS1, cagA + / vacA +), which was sold at Ajou University School of Medicine.

(3) Test group  Configuration and Dose Settings

Test group composition and dose were configured as shown in Table 22 below.

Test group Helicobacter pylori dose Test substance The number of animals G1 Untreated control C57BL / 6 Mouse 3 × 10 ⁸ cfu / mouse - 8 G2 Negative Control 3 × 10 ⁸ cfu / mouse - 8 G3 Positive control group 3 × 10 ⁸ cfu / mouse Omeprazole 8 G4 Experimental group 3 × 10 ⁸ cfu / mouse Mixed Propolis 8

(4) Administration site and administration method

Oral administration was performed, and the individual dose was converted based on body weight immediately before administration.

(5) Number of administration

In a model infecting Helicobacter pylori, test substances were administered once per day for 3 weeks.

(6) Inspection item

① Safety evaluation of propolis complex

② Visual and Microscopic Observation

③ Helicobacter pylori Gastric adhesion ( H.pylori colonization)

1. Safety Evaluation of Mixed Propolis

(1) Mortality and clinical symptoms

No special clinical symptoms were observed in all animals during the trial. There were no dead animals and no dead animals on the test day.

(2) weight measurement

During 4 weeks of breeding, weight gain in each group showed normal growth curve in all groups and there was no statistical difference in weight.

(3) serological tests

ALT and AST in serum were measured to evaluate the safety of the propolis complex, and the results are shown in FIG. 27. There was no significant difference in the ALT and AST levels between the G1 group that received no treatment (untreated control group) and the G2 group that received Helicobacter pylori. In addition, there was no significant difference in the G3 group (positive control group) and G4 group (mixed propolis administration group) compared with the G1 group.

(4) histological examination

Mouse CO ₂ Euthanasia and liver, lung, spleen and kidney were removed and fixed with 10% buffered neutral formalin. The fixed tissue was cut to a certain thickness, and then paraffin-embedded through a general tissue treatment process to produce 4-5 μm tissue sections, followed by the general staining method, Hematoxylin & Eosin stain (H & E stain). Was carried out. Microscopic examination of the stained tissue revealed no specific tissue lesions in all groups. The results are shown in FIG.

2. Visual and Microscopic Observation

(1) visual observation

Three weeks after the administration of Helicobacter pylori and test substance, the mice were sacrificed and the stomach tissues were extracted to observe the morphological changes. The results are shown in FIG. 29. As can be seen in Figure 29, no special tissue lesions were observed in all groups.

(2) microscopic observation

The stomach was removed and part of it was fixed with 10% neutral buffered formalin solution, followed by H & E staining to observe histopathological findings (histopathological changes such as invasion of inflammatory cells and structural destruction of epithelial cells). 30 is shown. Egg in Figure 30 As can be seen, three weeks after Helicobacter pylori infection, mice were observed with a slight infiltration of inflammatory cells in the G2, G3 and G4 groups, but no specific tissue lesions were observed.

3. Helicobacter Pylori Stomach adhesion ( H. pylori colonization )

Helicobacter pylori The mechanism by which infection causes gastrointestinal disease is that Helicobacter pylori penetrates the mucous layer of the stomach and attaches to the gastric mucosa, causing it to proliferate and cause inflammation. The amount of Helicobacter pylori attached to the gastric mucosa was measured to determine how propolis and the propolis complex play a role in the gastric adhesion of these Helicobacter pylori. Helicobacter pylori was quantified by real-time PCR of 16s DNA of Helicobacter pylori after extracting DNA from each mouse. The results are shown in FIG.

As a result, Helicobacter pylori in G3 and G4 groups compared to G2 group administered Helicobacter pylori The number of infections decreased slightly but no significant difference was observed.

4. Comprehensive review of anti-helicobacter potency test of mixed propolis

Helicobacter pylori of mixed propolis in a gastritis model of mice induced with Helicobacter pylori As a result of evaluating gastric adhesion inhibitory effect and gastric protective effect, no significant difference was observed in the group treated with mixed propolis (G4) compared with the group treated with Helicobacter pylori alone (G2) without drug treatment. The effect was similar to that of omeprazole (G3).

< Experimental Example  10>

Anti Helicobacter Pylori Animal Experiment

Helicobacter pylori of mixed propolis in a gastritis model of mice induced with Helicobacter pylori Gastric adhesion inhibitory effect and gastric protective effect were evaluated as follows.

Test Methods

(1) test animals

C57BL mice were used as test animals, and clean mice not infected with Helicobacter were supplied from Korea Research Institute of Bioscience and Biotechnology.

Body weights ranged from 9-14 g and 4 week old male mice. Only healthy animals were used for testing with quarantine and purifying periods for one week after acquisition. It was 5 weeks old at the start of the administration and had a weight range of 10-15 g. A total of 120 animals were used for the test. Breeding was carried out at a temperature of 20 ± 2 ℃, humidity 40-60%, 12 hours night / day cycle.

(2) Test group  Configuration

The test group used a total of 10 animals in each group of 5 groups, and the composition of the test group is shown in Table 23 below.

Test group Helicobacter
Pylori Test substance The number of animals G1 Normal control - Phosphate buffered saline (PBS) 10 G2 HP control + PBS 10 G3 Positive control group + Omeprazole 10 G4 + Omeprazole + amoxicillin + clarithromycin 10 G5 Test group + Mixed Propolis 10

(3) Helicobacter Pylori  Gastritis caused by

Mice were fasted for 24 hours and then Helicobacter pylori was administered by gavage.

Helicobacter pylori was used as a strain of Sydney strain (SS1, cagA + / vacA +) with high infection rate in mice from Ajou University School of Medicine.

Helicobacter pylori was prepared in C57BL / 6 mice at 1 × 10 ⁹ cfu / ml, and 0.2 ml per mouse were inoculated three times every other day by gavage for 8 weeks. Normal control group was orally administered with PBS. After 8 weeks, the necropsy was compared to compare the lesions. 32 is a schematic diagram of the experimental design.

(4) Administration method of test substance

Omeprazole or omeprazole + amoxicillin + clarithromycin was used as a control drug. Omeprazole is a proton pump inhibitor (PPI), and is known to inhibit the growth of Helicobacter pylori by strongly blocking gastric acid secretion by inhibiting hydrogen ion pump, the last stage of gastric acid secretion. According to the 2009 Korean Society of Gastroenterology Helicobacter Pylori Infection, PPI (Omeprazole, etc., twice daily) + Amoxicillin (1g, twice daily) + clarithromycin (500mg) 2 times a day) is recommended for 1-2 weeks of standard therapy. In this experiment, Sander et. It was administered according to the method of al et al .

Dosages, methods and frequency of test samples and control agents are shown in Table 24 below.

process Dosage Dosing method Number of doses Helicobacter pylori 0.2 (1 × 10 ⁹ cfu /) Gastrointestinal Nutrition Law 2 days a week
3 total intervals Omeprazole 400 mol / kg / day 7 days Amoxicillin 28.6 mg / kg / day 7 days Clarithromycin 14.3 mg / kg / day 7 days Mixed Propolis 350 mg / kg / day 5 Weeks

(5) Statistical processing

For statistical analysis, one-way ANOVA was performed using the GraphPad PRISM program, and post-mortem analysis was performed using the Turkey test to verify the differences between the groups. Statistical significance was determined as p <0.5.

Mortality and clinical symptoms

The clinical symptoms and death / rare animal occurrence were observed once per day during the study period. Records were kept only for individuals with clinical symptoms (not recording if normal), and weekends and public holidays were replaced by one morning observation. However, at least twice a day was observed on the day of administration.

No specific clinical symptoms were observed in all animals during the acclimatization period. The results are shown in Table 25 below.

Test group process The number of animals Our company
The number of animals Mortality rate
(%) Clinical
observe G1 Normal control PBS / PBS 10 - - N G2 HP Control HP / PBS 10 - - N G3 Positive control group HP / Omeprazole 10 - - N G4 HP / Omeprazole + Amoxicillin + Clarithromycin 10 - - N G5 Test group HP / Mixed Propolis 10 - - N * N: Appears normal

2. Weight measurement

Upon receipt, body weights were measured once per week during the acclimation period, during group separation and during the test period. In particular, the weight measurement during the test period was measured using the same scale on the same day and the same time zone (before and after 4 pm) in order to minimize the error in each measurement.

In all G1 to G5, no weight loss was observed for a total of 8 weeks during the trial and there was no difference between the groups. In addition, normal body weight gain was observed during the trial in all groups. The results are shown in Table 26 below.

Test group process Weight (g) Early
(Before HP administration) final
(After HP administration) % Increase G1 Normal control PBS / PBS 15.23 ± 0.18 22.55 ± 0.23 148.9 ± 1.7 G2 HP control HP / PBS 15.62 ± 0.25 21.89 ± 0.22 140.3 ± 2.5 G3 Positive control group HP / Omeprazole 15.45 ± 0.25 22.69 ± 0.16 147.2 ± 2.9 G4 HP / Omeprazole + Amoxicillin + Clarithromycin 15.71 ± 0.18 22.99 ± 0.25 146.5 ± 2.7 G5 Test group HP / Mixed Propolis 15.46 ± 0.17 22.70 ± 0.31 147.1 ± 2.9

3. Autopsy and long-term weight measurement

Helicobacter pylori Eight weeks after the last infection, the abdominal aorta and the abdominal vein were cut open after CO ₂ anesthesia. After visual observation of abnormal internal organs, the absolute organ weight of each organ (liver, spleen, kidney) was measured to calculate the relative organ weight to the fasted body weight before autopsy.

(One) Liver weight

Helicobacter pylori Relative organ weights for fasted body weights were calculated at 8 weeks post-mortem necropsy. No significant difference was observed compared to G2. The result is shown in FIG. 33 (data is shown as mean ± SEM).

(2) Spleen weight

As a result of calculating the relative organ weight of the spleen to the fasted body weight, there was no statistically significant difference. The results are shown in FIG. 34 (data shown as mean ± SEM).

(3) weight

As a result of calculating the relative organ weight of the kidney to the fasted body weight, there was no statistically significant difference. The results are shown in FIG. 35 (data shown as mean ± SEM).

4. Histopathological Analysis

After euthanasia, the individual stomach was removed and a part of it was fixed in 10% buffered neutral formalin. The fixed tissue was cut to a certain thickness, and then paraffin embedded through a general tissue treatment process to produce 4-5 μm tissue sections, followed by the general staining method, Hematoxylin & Eosin stain (H & E stain). Histopathological findings (histopathological changes such as infiltration of inflammatory cells, structural destruction of epithelial cells, ulcers and bleeding) were observed.

The extracted tissue was checked for damage to the tissue through H & E staining, and the results are shown in FIG. 36. In G1, no abnormalities were observed in all layers including the gastric mucosa, but in G2, inflammatory cells such as macrophages and neutrophils were observed in the mucosa and submucosa. No abnormalities such as inflammation were observed in all other groups.

5. Helicobacter Pylori Stomach adhesion  analysis

The stomach was removed from each mouse and DNA was extracted using a DNA purification kit. Real-time polymerase chain reaction (real-time PCR) was performed using TaqMan probe on 16s DNA of Helicobacter pylori (SS1). Primer sequences used for real time PCR are shown in Table 27 below.

gene primer probe Helicobacter
Pylori
SS1 16S Forward
; 5'-TTT GTT AGA GAA GAT AAT GAC GGT ATC TAA C-3 ' 5'-CAT AGG ATT TCA CAC CTG ACT GAC TAT C-3 ' Reverse
; 5'-CAT AGG ATT TCA CAC CTG ACT GAC TAT C-3 ' GAPDH Forward
; ACA GCA ACA GGG TGG TGG AC 5'-CAG AAG ACT GTG GAT GGC CCT-3 ' Reverse
; TTT GAG GGT GCA GCG AAC TT-3 '

As a result, Helicobacter pylori in G2 compared to G1 The number of infections increased significantly. The results are shown in Figure 37 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 and P when compared to G2. <0.01).

6. In the stomach tissue Oxidative  Damage measurement

When Helicobacter pylori infects epithelial cells, the immune response of the cells first increases reactive oxygen species (ROS), which destroys cellular homeostasis by oxidizing macromolecules such as proteins and lipids in the cells. It also causes fatal damage in cell tissues by killing cells. The protective effect of propolis complex against oxidative damage induced by Helicobacter pylori in epithelial cells was evaluated. The results are shown in Figure 38 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 and P when compared to G2. <0.01).

(One) MDA

Lipid peroxides are the result of tissue damage due to the removal of hydrogen atoms from phospholipid cell membranes in tissues by oxidative stress such as ROS or free radicals. MDA, a lipid peroxidation product of gastric tissue, was measured as an indicator of gastric injury, and the therapeutic effect of propolis complex on gastric tissue damage caused by Helicobacter pylori was measured.

A portion of the tissue was homogenized using a homogenizer by adding four-fold 150 mM KCl. The homogenized tissue was centrifuged at 4,000 rpm for 30 minutes and the supernatant was taken to measure glutathione (GSH), and the remainder was centrifuged again at 20,000 rpm for 1 hour, and then the supernatant was taken to obtain malondialdehyde (MDA). ) Was measured. TBARS assay kit (Cell Biolabs, USA) was used to measure the absorbance at 586nm and MDA was calculated.

As a result, the MDA level was significantly increased in G2 compared to G1, and the MDA level was significantly reduced in comparison to G2 in all groups except G3 (FIG. 38A).

(2) GSH

GSH is one of the antioxidants in vivo that protects against oxidative stress damage caused by free radicals and peroxides.

It was measured using a glutathione assay kit (Cell Biolabs, USA), and the concentration of GSH was calculated after measuring absorbance at 405 nm.

GSH levels were decreased in G2 compared to G1, but there was no significant difference. Only G3 increased GSH levels significantly compared to G2 (FIG. 38B).

7. Measurement of Expression of Inflammatory Cytokines

1 ml of Trizol lysis buffer was dispensed into the tissue and homogenized and dissolved. 200 μl of chloroform was dispensed and inverted for 20 seconds. After centrifugation at 13,200 rpm for 15 minutes, the supernatant was transferred to a tube containing 500 μl of isopropanol. After centrifugation again at 13,000 rpm for 10 minutes, the supernatant was removed, and 1 ml of 70% ethanol was dispensed into each tube, followed by centrifugation at 13,000 rpm for 5 minutes, and then the supernatant was removed and dried at room temperature. 40 μl of D.W. containing DEPC was dissolved to dissolve RNA, and then 1 μl of the RNA solution was taken to measure absorbance at 260/280 nm using a NanoDrop instrument to calculate the total RNA concentration of the solution. CDNA was synthesized using the extracted RNA and RT DriyMIX cDNA synthesis kit.

Real-time PCR was performed to investigate gene expression of IL-1β, IL-6 and TNF-α. Primer sequences used in the experiments are shown in Table 28 below.

gene primer Sequence (5` → 3`) IL-1β Forward AAA AAA GCC TCG TGC TGT CG Reverse GTC GTT GCT TGG TTC TCC IL-6 Forward TCC ATC CAG TTG CCT TCT G Reverse TTC CAC GAT TTC CCA GAG AAC TNF-α Forward AGG TAC AAC CCA TCG CTG G Reverse AAG CCT GTA GCC CAC GTC GTA GAPDH Forward TCG TGG ATC TGA CGT GCC GCC TG Reverse CACCACCCT GTT GCT GTA GCC GTA T

In the early stages of Helicobacter pylori infection, neutrophils in the mucosa proproa are observed, and when chronically progressed, lymphocytes, monocytes, and eosinophils are observed. Increasing neutrophil activity induces the production of several inflammatory cytokines such as chemokines such as IL-8, as well as IL-1β, IL-6, and TNF-α, inducing more inflammatory cells into the gastric mucosa and It causes atrophy and apoptosis of epithelial cells. IL-1β, IL-6, and TNF-α levels were measured in the gastric tissue to quantitatively evaluate the therapeutic effects of gastric inflammatory response and propolis complex due to Helicobacter pylori infection. The results are shown in Figure 39 (data is shown as mean ± SEM. ^* And ^** are P <0.05 and P <0.01 when compared to G1; ^† and ^†† , respectively, P <0.05 and P when compared to G2. <0.01).

(One) IL -6

Expression of IL-6 was significantly increased in G2 compared to G1 (FIG. 39A).

(2) TNF -α

There was no statistical difference in the expression of TNF-α in G2 compared to G1, and there was no significant difference in all test groups compared to G2. However, in comparison to G2 G1 compared using t- tests (t-test), the expression of TNF-α was significantly (B in Fig. 39).

(3) IL -1β

As with the TNF-α results, no significant differences were observed in all groups. However, the expression of IL-1β was significantly increased in G2 compared to G1 when compared using the t- test (FIG. 39C).

8. Comprehensive review of anti-helicobacter potency test of mixed propolis

To investigate the therapeutic effect of mixed propolis on damaged gastric mucosa after Helicobacter pylori infection. It was compared and reviewed, and the results are shown in Table 29 below.

Test group process HP
Upper attachment ^A Anti-inflammatory Antioxidant stress IL-6 ^B TNF-α ^C IL-1β ^D MDA ^E GSH ^F G1 Normal control PBS - - - - - - G2 HP Control PBS - - - - - - G3 Positive control group Omeprazole +++ ++ ++ ++ + +++ G4 Omeprazole + Amoxicillin + Clarithromycin ++ +++ ++ ++ +++ ++ G5 Test group Mixed Propolis + ++ + ++ ++ ++ A: HP stomach attachment> 0.3 times; +, HP stomach attachment = 0.3-0.7 times; ++, HP stomach attachment <0.7 times; +++.
B: IL-6> 5 fold; +, IL-6 = 1-5 fold; ++, IL-6 <1 fold; +++.
C: TNF-α> 2 fold; +, TNF-a = 1-2 times; ++, TNF-a <1 fold; +++.
D: IL-1β> 2 fold; +, IL-1β = 2-1 fold; ++, IL-1β <1 fold; +++.
E: MDA> 15 uM / mg protein; +, MDA = 15-10 uM / mg protein; ++, MDA <10 uM / mg protein; +++.

As shown in Table 29, it was confirmed that the mixed propolis showed anti-Helicobacter pylori attachment, antioxidant and anti-inflammatory effects.

Claims (15)

delete delete delete delete delete delete delete In order to increase the total flavonoid content of propolis,
Including Argentine Propolis, Chinese Propolis, and Brazilian Propolis, including Argentine, Chinese, and Brazilian Propolis in a weight ratio of 40:30:30,
The extract of the propolis is characterized in that the extract of propolis with ethanol and then concentrated to remove the ethanol and then soluble in purified water, water soluble propolis extract, health functional food propolis composition. delete delete delete delete delete delete In order to increase the total flavonoid content of propolis,
Argentine propolis, Chinese propolis, and Brazilian propolis are used in a mixture, which includes mixing extracts of Argentine, Chinese and Brazilian propolis in a weight ratio of 40:30:30,
The extract of the propolis is characterized in that the extract of propolis with ethanol and then concentrated to remove the ethanol and then soluble in purified water, water soluble propolis extract obtained, the health functional food propolis composition.

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