KR20190037926A - 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 at least two kinds of propolis extracts selected from the group consisting of Argentine propolis, Chinese propolis, Brazilian propolis and Korean propolis. It is possible to intake more various active ingredients contained in each propolis when a daily dose of the mixed propolis, manufactured by mixing propolis of various origin, is consumed, compared to when each propolis is ingested alone.

Description

TECHNICAL FIELD The present invention relates to a method for using propolis mixed with propolis extracts of different origin, and a composition for health functional foods containing propolis as an active ingredient.

The present invention relates to a method for using propolis for enhancing efficacy and a composition for health functional food comprising propolis as an active ingredient, and more particularly to a method for mixing two or more kinds of propolis extracts having different origins from each other.

Propolis is a natural waxy mixture made by mixing bees with their saliva to protect the growth spots and wounded areas of plants.

Bees protect themselves from germs and viruses by preventing them from invading enemies such as wasps and rats by spraying propolis in the cracks of the honeycomb to maintain proper habitat for spawning and growth, honey aging and storage. Especially when queen bees spawn, they are used to disinfect and clean the spawning ground.

Propolis, which is used as a folk remedy in various countries including South America, has recently been used as a health functional food for the treatment and prevention of various diseases. Flavonoids and terpenoids ) Have attracted much research and interest in various pharmacological actions due to the antioxidative action of the compound. In particular, the therapeutic effect on skin, mucous membrane and oral infection is caused by various substances which are separated and identified in propolis, and it is believed that at least about 150 terpenes, caffeic acid esters, flavonoids and amino acids Phenylpropane derivatives are known to be involved. These components show antimicrobial and antifungal effects as well as side effects, indicating that they can be used in various areas.

Ethanol and hot water extracts of propolis are used for various purposes as herbal medicines. Propolis and its phenolic components have been shown to inhibit proliferation in human tumor cells (Guarini et al., 1992), anti-inflammatory effects (Dobrowolski et al., 1991), antibacterial effects (Eley 1999; Grange et al., 1990; (Kang et al., 1990) and antioxidant effects (Kang et al., 1990) and antioxidant effects (Steinberg et al., 1996), antiviral effects (Serkedjieva et al. (Starzyk et < RTI ID = 0.0 > al., ≪ / RTI > 1977). In addition, ethanol extracts of propolis have been reported to exhibit 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 been reported to exhibit excellent anti-inflammatory action but do not show adverse effects on the gastrointestinal tract, which is a side effect of the usual non-steroidal anti-inflammatory anti-inflammatory agent (NSAID), and flavonoids Phenolic acid and the like.

These propolis differs in active ingredients, color, and taste depending on the plant species (species) and distribution in each region, because the composition varies depending on the species in which the worker collects tree species and resin.

The color of the propolis varies in green, red, yellow, and black, and there are differences in the active ingredients depending on the color difference. 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 flavon and flavonol, and the active ingredient of black propolis is flavonoid (antozantine). The active ingredient of sweet propolis is carcone, and the active ingredient of bitter propolis is catechin and isoflavone, and the active ingredient of hot flavor propolis is ferulic acid.

At present, propolis is produced worldwide from New Zealand, Brazil, China, and the United Kingdom. Only propolis products produced in each country are produced.

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

The present invention relates to a method of using propolis which improves the efficacy of propolis as a health functional food by allowing a variety of active ingredients to be consumed when a daily intake amount is mixed by using propolis of various origin, And a composition for health functional foods containing such mixed propolis as an active ingredient.

In order to achieve the above object,

Characterized in that, in order to enhance the efficacy of propolis as a health functional food, an extract of propolis having at least two different origins selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis is mixed and used Provides a way to use the Polys.

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

Preferably, the extract of propolis is a water-soluble extract obtained by hydrolyzing an ethanol extract of propolis.

The mixed use of the extract of propolis is preferably a mixture of extracts of propolis from Argentina, propolis from China and propolis from Korea. More preferably, the extract of Argentina propolis, Chinese propolis and Korean propolis is mixed at a weight ratio of 30:40:25 to 35:30:40.

In another embodiment of the present invention, the mixture of the extract of the propolis is preferably a mixture of an extract of an acid propolis of Argentina, a propolis of China, and a propolis of Brazil. More preferably, the extracts of Argentina propolis, Chinese propolis and Brazil propolis are mixed at a weight ratio of 35:45:25 to 35:25 to 35.

Further, in the present invention,

In order to enhance the efficacy of propolis, a composition for a health functional food containing propolis as an active ingredient is mixed with at least one propolis selected from the group consisting of Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis A composition for health functional foods, which comprises a mixed extract.

Further, in the present invention,

A method for producing a health functional food containing propolis as an active ingredient,

In order to enhance the efficacy of propolis, propolis-containing health function characterized by mixing extracts of at least two different propolis selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis A method for producing food is provided.

In the present invention, the term " health functional food " is meant to include both general health functional foods and functional foods including health functional foods in the Health Functional Food Act.

In the present invention, when a daily intake of propolis is mixed with propolis of various origin, dietary propolis can be consumed in a larger quantity than individual propolis alone, Can be increased.

According to the present invention, a composition for health functional foods containing a mixed extract of propolis having different origins can exhibit an improved effect particularly in antioxidative, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy.

1A to 1C are chromatograms of an active ingredient of propolis using LC / MS / MS, wherein FIG. 1A shows a Chrysin, FIG. 1B shows a Galangin, FIG. 1C shows a pinochemin Pinocembrin. ≪ / RTI >
2A to 2H are chromatograms showing an active ingredient of a water-soluble propolis, wherein FIG. 2A shows cinnamic acid, FIG. 2B shows caffeic acid, FIG. 2C shows acacetin, CAPE, 2e is Kaempferol, 2f is gallic acid, 2g is Chrysin, and 2h is ferulic acid.
FIG. 3 is a graph showing the effect of various propolis samples of various origin on the cell viability.
Figure 4 is a graph showing the effect of propolis samples of various origin on NO production.
Figure 5 is a graph showing the effect of mixed propolis on cell viability.
6 is a graph showing the effect of mixed propolis on NO production.
7 is a graph showing the effect of mixed propolis on the cytokine-related gene.
8 is a schematic view of a therapeutic effect test of mixed propolis against alcoholic gastritis.
Fig. 9 shows the result of measuring liver weight in a therapeutic effect test of mixed propolis against alcoholic gastritis.
Fig. 10 shows the result of measuring spleen weight in a therapeutic effect test of mixed propolis against alcoholic gastritis. Fig.
Fig. 11 shows the result of measuring the kidney weight in a therapeutic effect test of mixed propolis against alcoholic gastritis.
Fig. 12 shows histopathological observation results of a therapeutic effect test of mixed propolis against alcoholic gastritis.
Fig. 13 shows the results of examining the gross damage of the gastric mucosa in a therapeutic effect test of mixed propolis against alcoholic gastritis.
FIG. 14 shows the results of a therapeutic effect test of mixed propolis against alcoholic gastritis, wherein A represents the result of measuring the area of gastric injuries, and B represents the gastric lesion inhibition rate.
FIG. 15 shows the result of measuring the oxidative damage of stomach tissue in a therapeutic effect test of mixed propolis against alcoholic gastritis, wherein A is a result of MDA measurement and B is a measurement result of GSH.
FIG. 16 shows the results of confirming the expression of inflammatory cytokines in a therapeutic effect test of mixed propolis against alcoholic gastritis, wherein A is the result of measurement of IL-6, B is the result of measurement of IL-1β, -α.
17 is a schematic view of a therapeutic effect test of mixed propolis against other alcoholic gastritis.
18 shows the result of measuring body weight in a therapeutic effect test of mixed propolis against alcoholic gastritis.
19 shows the result of measuring the organ weight in a therapeutic effect test of mixed propolis against alcoholic gastritis.
20 shows the results of histopathological observation in a therapeutic effect test of mixed propolis against alcoholic gastritis.
FIG. 21 shows the results of examining the gross damage of gastric mucosa in a therapeutic effect test of mixed propolis against alcoholic gastritis.
FIG. 22 shows the results of a therapeutic effect test of mixed propolis against alcoholic gastritis, where A represents the result of measuring the area of gastric injuries, and B represents the gastric lesion inhibition rate.
FIG. 23 shows the result of measuring the oxidative damage of the stomach tissue in a therapeutic effect test of a mixed propolis complex composition against alcoholic gastritis, wherein A is the measurement result of MDA and B is the measurement result of GSH.
FIG. 24 shows the result of confirming the effect of the anti-Helicobacter pylori on the propolis samples by country of origin.
25 shows the activity of mixed propolis and natural product extract against Helicobacter pylori.
Fig. 26 shows the inhibitory activity of urea-decomposing activity of various propolis.
FIG. 27 shows the results of measurement of ALT and AST in serum to confirm the safety of mixed propolis in an anti-Helicobacter pylori animal experiment.
FIG. 28 shows the results of histological examination in an anti-Helicobacter pylori animal experiment.
FIG. 29 shows the result of visual observation of the stomach tissue of an experimental animal in an anti-Helicobacter pylori animal test.
30 shows microscopic observation of the stomach of an experimental animal in an anti-Helicobacter pylori animal test.
FIG. 31 shows the result of confirming the gastric adhesion of Helicobacter pylori in an anti-Helicobacter pylori animal experiment.
32 is a schematic diagram of an anti-Helicobacter pylori animal experiment.
Figure 33 shows the result of liver weight measurement in an anti-Helicobacter pylori animal experiment.
Figure 34 shows the result of measuring the spleen weight in an anti-Helicobacter pylori animal experiment.
35 shows the result of measuring the height of kidneys in an anti-Helicobacter pylori animal test.
FIG. 36 shows histopathological observation of stomach tissues of an experimental animal in an anti-Helicobacter pylori animal experiment.
FIG. 37 shows the results of confirming the gastric adhesion of Helicobacter pylori in an anti-Helicobacter pylori animal experiment.
FIG. 38 shows oxidative damage of the stomach tissues in an anti-Helicobacter pylori animal test, wherein A is MDA, and B is GSH.
FIG. 39 shows the results of confirming the expression of inflammatory cytokines in the anti-Helicobacter pylori animal test. The results of confirming the expression of IL-6, B, TNF-α and C-IL-1β are shown in FIG.

The method of using propolis provided in the present invention is not particularly limited,

In order to enhance the efficacy of propolis as a health functional food, an extract of propolis having at least two different origins selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis is mixed and used.

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

The extract of propolis is not particularly limited, but is preferably an ethanol extract of propolis. And more preferably a water-soluble extract obtained by water-soluble ethanol extract of propolis.

In a preferred embodiment of the present invention, the mixed use of the extract of propolis is a mixture of extracts of propolis, propolis, and propolis. More preferably, the extracts of Argentina propolis, Chinese propolis and Korean propolis are mixed at a weight ratio of 30-40: 25-35: 30-40.

In another preferred embodiment of the present invention, the mixed use of the extract of propolis is a mixture of extracts of propolis, propolis, and propolis. More preferably, the extracts of Argentina propolis, Chinese propolis and Brazil propolis are mixed at a weight ratio of 35: 45: 25 to 35: 25-35.

The composition for a health functional food comprising the propolis as an active ingredient provided in the present invention is characterized in that at least two kinds of origin selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis are added to enhance the potency of the propolis Includes mixed extracts of other propolis.

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

In another preferred embodiment of the present invention, the mixed extract comprises extracts of Argentina propolis, Chinese propolis and Brazil propolis. More preferably, the extract of Argentina propolis, Chinese propolis and Brazil propolis is contained in a weight ratio of 35:45:25 to 35:25 to 35.

The efficacy of the propolis to be improved and the description of the extract are the same as described in the above-mentioned use of propolis.

The method for producing a health functional food comprising the propolis as an active ingredient provided in the present invention is a method for producing a health functional food comprising at least two selected from propolis, Mix propolis extract of different origin.

The mixing of the extract of propolis, the efficacy of the propolis to be improved and the description of the extract are the same as described in the above-mentioned use of propolis.

Propolis varies in composition depending on the type of surrounding plants and the time of collection, and the color, flavor, and functional characteristics of the propolis vary depending on the collection area. In the present invention, the proprietary origin of various origins is used by confirming the characteristics according to each origin, but the effectiveness is kept constant.

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

As used herein, the term " health functional food " means food prepared and processed using raw materials or ingredients having useful functions in the human body. The term " functional " And the like, for the purpose of obtaining a beneficial effect for health use such as exercise. The " health functional food " of the present invention includes both general health food and functional food including health functional food in the Health Function Food Act. The health functional food may be in the form of one of tablet, capsule, powder, granule, liquid, and ring, 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 and are not intended to limit the scope of the present invention.

< Experimental Example  1>

Analysis of Propolis by Country of Origin

Propolis varies in composition depending on the type of surrounding plants and the time of collection, and the color, flavor, and functional characteristics of the propolis vary depending on the collection area. The propolis used in the market has been investigated by the origin (Korean, Chinese, Argentine, Brazilian, etc.), and the propolis ingredient with anti - inflammatory effect was selected.

Production of Propolis Extract by Country of Origin

50 g of each natural propolis of Korea, China, Argentina and Brazil was extracted with two kinds of extraction solvents: 100% solution of 95% fermented alcohol, 150 g of a mixture of 95% fermented alcohol 70% and purified water 25% -SS200) for 6 hours at room temperature with stirring and left for 24 hours. The extracted propolis was filtered using a filter paper (Advantec No. 2).

Analysis of active ingredients

1. Total flavonoid content measurement

Propolis is a high-functioning health functional food, and its total flavonoid (tetal flavonoids) is used as an index component in the health functional food revolution.

(1) Measurement method

The total flavonoid contents were measured according to the functional foods. This is a method of extracting flavonoid substances from samples using ethanol and analyzing them using a spectrophotometer, and measuring the absorbance at 415 nm, which is the maximum absorption wavelength of flavonoids.

Weigh out 0.1-0.5 g (㎖) of propolis extract, add 20 ml of 90% ethanol, dissolve and separate (3,000 rpm, 10 minutes), take the supernatant, extract the residue three times with 8 ml of 80% ethanol, And the total volume was made 50 ml with 80% ethanol. 0.5 ml of this test solution was taken in a test tube, and 1.5 ml of ethanol, 0.1 ml of 10% aluminum nitrate solution, 0.1 ml of 1 M potassium acetate solution and 2.8 ml of water were added and sufficiently stirred. After standing at room temperature for 40 minutes, the absorbance of the liquid layer was measured at 415 nm using a UV / VIS spectrophotometer (Spectrophotometer, Varian, Inc. USA) using a 10 mm cell as a control solution. The total flavonoid content was calculated from the following equation (1) by using the difference in absorbance obtained by subtracting the absorbance of the aluminum nitrate solution added with 0.1 ml of water separately from the absorbance of the sample.

(2) Measurement results

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

origin Solvent (1: 3 weight ratio w: w) Amount of extraction liquid (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 (Green) 95% fermented alcohol 123 0.9 70% fermented alcohol 116 0.93 Brazil (Blend)
95% fermented alcohol 120 0.72 70% fermented alcohol 118 0.83

From the results shown in Table 1, it can be seen that even when extracted under the same conditions, the content of the active ingredient to be extracted differs depending on the origin of propolis, depending on the extraction solvent.

2. LC / MS / MS Of active ingredient ( Chrysin , Galangin , Pinocembrin ) Quantitative analysis

In Table 1, the extracts of Chrysin, Galangin and Pinosembrin (Chrysin), which are anti-Helicobacter pylori effect agents reported in the literature, were used to extract 95% Pinocembrin) were analyzed by LC / MS / MS.

As mobile phase reagents, acetonitrile (HPLC grade, Merck) and formic acid (Sigma Aldrich) were used. Finnigan Surveyor MS pump plus and Autosampler and Finnigan TSQ Quantum Discovery were used as equipment. The analytical conditions of LC / MS / MS are shown in Table 2, 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 (HPLC Pump) Surveyor MS Pump Plus (Finnigan) Autosampler Surveyor Autosampler Plus (Finnigan) software Xcalibur 2.1.0 column Sunniest C18 (2.0 x 100 mm, 3 um) 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) Chrysene (m / z 253.0 to m / z 143.02),
Galangin (m / z 255.0 to m / z 213.05),
(5-NIO, m / z 321.0 to m / z 291.12), &lt; RTI ID = 0.0 & Source / gas / temperature parameters (optimization) Ion Source ESI (HESI, Negative) Ion Spray Voltage (V) 3500 Sheath Gas Pressure 35 Aux Gas Pressure (AuxGas Pressure) 20 CID Gas Pressure 1.5 Evaporator Temperature (Vaporizer Temp.,) 125 Capillary Temp. 315 Tube Lens Offset 105

Compound optimization Chrysene (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 (M / z 269.0 to m / z 169.05) &lt; RTI ID = 0.0 &gt; Collision energy 28 IS (5-NIO) (m / z 321.0 to m / z 291.12) Collision energy 21

The contents of chrysin, galangin and pinosembrine of propolis by the country of origin as the above-mentioned analysis results are shown in Table 4, and the chromatograms of the respective samples are shown in Figs.

Chrysin (ppm) Gallin gene (ppm) (Ppm) Chinese (Hysong) 29.7 38.5 15.7 Made in Korea 47.1 12.3 27.0 Brazil (Green) 0.2 0.2 1.6 Brazilian Mountain (Blend) 0.1 0.1 0.7 Argentina 102.3 26.1 36.4

3. Analysis of Active Ingredient Content of Ethanol Extracted Propolis and Water-Soluble Propolis

Ethanol Extract Propolis is not well soluble in water and sticky, so it sticks to the mouth when it is ingested, sticks to the cup, and is not well removed. To solve these problems, the present invention uses a water-soluble propolis which does not contain ethanol but easily dissolves in water.

The water-soluble propolis used in the present invention is prepared by extracting ethanol, concentrating it, removing ethanol, and then water-soluble with purified water. In order to confirm the difference between the active ingredients of the water-soluble propolis and the ethanol extract propolis of the present invention, quantitative tests for the 15 kinds of active ingredients known as active ingredients were carried out as follows by LC / MS / MS.

Acetonitrile (HPLC grade, Merck) was used as 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 and 0.1 ppm was prepared using a standard product (Sigma) to prepare a calibration curve.

Memantine column Capcell PAK MGII (3 μm, 2.0 × 100 mm) 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, it was confirmed that the content of the active ingredient was not significantly decreased even when the ethanol-extracted propolis was processed into the water-soluble propolis, and that the concentration of the active ingredient was increased through the water-soluble process, .

Soluble propolis such as cinnamic acid, caffeic acid, acacetin, CAPE, kaempferol, gallic acid, chrysin, ferulic acid, Are shown in Figs. 2A to 2H.

< Experimental Example  2>

To determine the cytotoxicity of propolis samples by country of origin

In order to confirm the effect of the propolis samples on the growth of macrophages (toxicity), rosewood cell line RAW 264.7 cells were treated with propolis extracts of Korean, Chinese, Brazilian and Argentinean and 24 hours later MTT assay The toxicity was confirmed. The experiment was repeated 3 times and the results were expressed as the mean ± SD of 3 experiments. The results are shown in Fig.

Domestic and Argentine acid propolis showed toxicity above 50 ㎍ / ㎖. Brazilian propolis showed little toxicity up to a concentration of 100 μg / ml, while Chinese propolis showed little toxicity up to a concentration of 50 μg / ml, but showed weak toxicity at a concentration of 100 μg / ml.

< Experimental Example  3>

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

Raw 264.7 cells were stimulated with LPS and the propolis samples were treated for 24 hours, and the amount of NO produced was measured by greens reaction analysis. The experiment was repeated 3 times and the results were expressed as the mean ± SD of 3 experiments. The results are shown in Fig.

As can be seen from the results of FIG. 4, the propolis produced in Korea and Argentina showed NO production inhibitory activity, but it was thought to be cytotoxic (see FIG. 3) inhibitory activity at a concentration of 50 μg / ml or higher. In the case of Chinese and Brazilian propolis, the inhibitory activity was shown to be dose dependent.

< Example  1>

Mixing ratio of mixed propolis

Since the kind and content of the active ingredient in propolis varies depending on the surrounding environment, the content of active ingredients in the propolis extract is analyzed by LC / MS / MS, The total flavonoid content of the aqueous solution obtained by the hydrolysis process after mixing propolis extract was measured.

The mixture of propolis extract was prepared by adding a mixture of Korean and Brazilian propolis extracts based on a mixture of Argentina and Chinese propolis extracts.

1. Mixture of Argentinean, Chinese and Korean Propolis

Argentina, China, and Korean propolis extracts were mixed in the weight ratios shown in Table 6 below, and the total flavonoid content of the aqueous solution obtained through the water-soluble process was measured.

Ethanol Extraction Propolis Mixing 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 propolis extract was higher than the aqueous solution of the propolis extract mixed with other fractions at a total flavonoid content of 0.8% in the aqueous solution obtained by mixing the propolis extract with the ratio of Argentina: Chinese: domestic = 35:30:35 .

2. Mixture of Argentina, Brazilian and Brazilian Propolis

The propolis spear extracts from Argentina, China and Brazil were mixed at the weight ratios shown in Table 7 below, and the total flavonoid content of the aqueous solution obtained through the aqueous process was measured in the same manner as in Table 6 above.

Ethanol Extraction Propolis Mixing Ratio Total flavonoid content
(%) Argentina Made in China Brazilian mountains 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

The total flavonoid content of the aqueous solution containing the propolis extract mixture mixed with Argentina acid, Chinese acid: Brazil acid = 40: 30: 30 was the highest at 0.99%, and the water soluble with the propolis extract Total flavonoid content was higher than aqueous solution. The propolis aqueous solution prepared from the alcohol extract of Brazilian acid was detected after the spicy taste of Brazil propolis was solubilized, but the spice was weaker than the aqueous solution made from propolis alone.

< Experimental Example  4>

Propolis by Origin and Mixed Propolis  Active ingredient content

The contents of active ingredients of the acid propolis of Argentina, propolis of China, propolis of Korea and mixed propolis mixed therein were confirmed and are shown in Table 8 below.

Mixed propolis samples were prepared by mixing propolis from Argentina, propolis from China and propolis from Korea at a weight ratio of 35:30:35.

ingredient Argentina Made in China Made in Korea mix Flavonon Acacetin 185.5 51.5 154.6 243.6 Krysin 4,505.9 2,284.0 3,062.1 5,036.5 Flavonoid Garangrin 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 Flavonon Hesperrettin 10.8 13.5 35.3 38.0 Pinosembrin 3,491.2 935.2 1,745.1 1,763.6 Phenolic
compound Gallan 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 Shinnam-san 6,988.9 14,911.3 2,508.5 Kumar Mountain 1,417.7 69.7 1,427.0 1180.2

As can be seen from the results of Table 8 above, by mixing propolis of different origin, it is possible to take more effective amounts of each propolis inherent thereto.

< 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 the extracts of Argentina propolis, Chinese propolis and Brazilian propolis at a weight ratio of 40:30:30.

1. Identification of cytotoxicity

To determine the effect of mixed propolis samples on the growth of macrophages (toxicity), Raw 264.7 cells were treated with mixed propolis samples and cell viability was determined by MTT assay after 24 hours.

The experiment was repeated 3 times and the results were expressed as the mean ± SD of 3 experiments. The results are shown in Fig.

As can be seen from the results in Fig. 5, all of the samples showed no toxicity.

2. NO  Confirming production inhibitory activity

To confirm the NO production inhibitory activity of the mixed propolis samples, mixed propolis samples were treated with LPS-treated RAW 264.7 cells by concentration and the amount of NO produced was measured. The experiment was repeated 3 times and the results were expressed as the mean ± SD of 3 experiments. The results are shown in Fig.

3. Identification of cytokine-related gene inhibitory effect

To confirm the cytokine-related inhibitory effect of mixed propolis samples, mixed propolis samples were treated with RAW 264.7 cells treated with 50% ethanol extract of each concentration for 24 hours without or with 1 μg / MRNA levels of TNF-α, IL-1β, and IL-6, the proinflammatory cytokines produced by LPS stimulation, were measured by quantitative RT-PCR. The experiment was repeated 3 times and the results were expressed as the mean ± SD of 3 experiments. The results are shown in Fig.

< Experimental Example  6>

Therapeutic effect test of mixed propolis for alcoholic gastritis

To evaluate the therapeutic effect of mixed propolis in alcohol - induced gastric injury model, the following tests were conducted.

Test Methods

(1) Test animals

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

The body weight was 150 ~ 180g and male rats. Only one healthy animal was used for the test, with quarantine and refinement periods for one week after receipt. At the start of the administration, the body weight ranged from 180 to 200 g, and a total of 82 animals were used for the test. The mice were kept in a temperature of 20 ± 2 ° C, humidity of 40 to 60%, and 12 hours of night / day period.

(2) induction of alcoholic gastritis

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

(3) Test group  Configuration

Four groups (G1 ~ G4) were used in the test group. Five or seven animals were used in each group. The composition of the test group is shown in Table 9 below.

Test group Gastric damage
(Gastric injury) process Number of animals G1 Normal control group - Saline 5 G2 Alcohol-treated group ethanol Saline solution 10 G3 Positive control group ethanol Omeprazole 10 G4 Experimental group ethanol Mixed Propolis 10

(4) Administration dose and method

The dose and method of administration of the alcohol, the control drug (positive control; omeprazole) and the test drug are shown in Table 10 below.

process Dose Method of administration Number of doses ethanol 5 / kg (anhydrous alcohol) Gut nutrition One Omeprazole 20 mg / kg / day 2 Mixed Propolis 175 mg / kg / day 2

(5) Statistical processing

Statistical analysis was performed by one-way ANOVA using GraphPad PRISM program and posttest was analyzed using the Turkey test to verify the difference between the groups. Statistical significance was determined as p <0.5.

1. Mortality and clinical signs

During the study period, once a day clinical symptoms and mortality / dysphoria were observed for each individual. Records were maintained only for individuals with clinical symptoms (not recorded if normal), and on weekends and holidays, one observation was given in the morning. However, at the day of administration, at least twice a day was observed.

As a result, no clinical symptoms were observed in all animals during the purifying period. The results are shown in Table 11 below.

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

2. Weight measurement

Body weights were measured at the time of acquisition, once a week during the purification period, during the separation of the group and during the test period. In particular, weighing during the test period was measured using the same scales at the same time of day (around 4 pm) on the same day to minimize errors in each measurement.

Weight reduction was observed in the alcohol-treated group (G2 to G4). G2, G3 and G4 groups showed a statistically significant decrease in body weight as compared to G1 group. The results are shown in Table 12 (data are expressed as means ± SEM ^* and ^** are P <0.05 and P <0.01, respectively, when compared to G1).

Test group
process Weight (g) Early (before ethanol administration) Final (Ethanol
After administration) Growth rate (%) G1 Normal control group Saline / saline 319.8 ± 10.5 322.4 + - 9.6 100.9 ± 0.5 G2 Alcohol-treated 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 long-term weight calculation

Twenty-four hours after the alcohol administration, the animals were lavaged under CO ₂ anesthesia, and then the abdominal aorta and the abdominal vein were cut and the blastocysts were sacrificed. The absolute long term weight of each organ (liver, spleen, kidney) And the relative organ weight was calculated based on the body weight fasted before the autopsy.

(One) Liver weight

As a result of calculating the relative weight of liver in relation to the fasted body weight, liver weight increased after administration of alcohol, and liver weight was increased significantly in G2, G3 and G4 groups compared to G1. However, there was no significant difference between G3 and G4 compared to G2. The results are shown in Figure 9 (data are expressed as means ± SEM ^* and ^** are P <0.05 and P <0.01, respectively, when compared to G1).

(2) spleen weight

The relative weight of the spleen for the fasted body weight was calculated. As a result, the weight of the spleen increased after alcohol administration, but there was no statistically significant difference. The results are shown in Fig. 10 (data are shown as means ± SEM).

(3) Height Weight

The weight of the kidney was increased after alcohol administration, and the weight of kidney was significantly increased in G2, G3 and G4 groups compared to G1. However, there was no significant difference between G3 and G4 compared to G2. The results are shown in Figure 11 (data are expressed as means ± SEM ^* and ^** are P <0.05 and P <0.01, respectively, when compared to G1)

4. Histopathological analysis

After euthanasia, the stomachs were removed from each individual and fixed in 10% neutral buffered formalin solution (10% buffered neutral formalin). After fixing the fixed tissue to a certain thickness, the tissue section with 4 ~ 5 ㎛ was prepared by paraffin embedding through general tissue processing, and then subjected to hematoxylin & eosin staining (H & E stain) (Histopathologic changes such as infiltration of inflammatory cells, destruction of epithelial cells, ulceration and hemorrhage) were observed.

H & E staining of the excised stomach was checked for tissue damage. G1 showed no abnormalities in all layers including gastric mucosa, but G2 showed necrosis and hemorrhage as well as gastric mucosa disappeared. HCl-secreting cells (parietal cells and pepsinogen-secreting cells (chief cells). Infiltration of inflammatory cells such as macrophages and neutrophils was observed in the mucosa and submucosa. However, the gastric mucosal damage was reduced in the G3 and G4 groups. The results are shown in Fig.

5. Gastric damage  Measure inhibition rate

The damaged length (mm) and the damaged area (mm ² ) generated after cutting the excised stomach along the Taiwanese part were measured with a microscope and calculated according to the following equation (2). (P <0.05), it was judged to be effective when a significant effect was demonstrated in the control group and the positive control group.

(1) gross damage of gastric mucosa

Alcohol directly stimulates the gastric mucosa and induces edema in the submucosal muscular layer, resulting in transient ischemic conditions, resulting in necrosis of cells due to oxidative damage, and direct stimulation of the gastric mucosa, resulting in hemorrhage and ulceration. In order to evaluate the therapeutic effect of the propolis complex composition on gastric mucosal injury induced by alcohol administration, gross examination of the lesion was carried out by autopsy. As a result, G1 showed no gross abnormality but alcohol- G4) showed a thick band on the gastric mucosa. However, these bleeding sites were reduced in G3 and G4 compared to G2. The results are shown in Fig.

(2) Gastric damage  area

To the gastric mucosal damage length and measures the damage area (mm ²⁾ Comparison of the relative area to the area of the whole gastric mucosa, G1 (control group; 0.0 ± 0.0%) compared to the number in G2 (alcohol group) (20.7 ± 1.7%), and the gastric mucosal injury site was significantly decreased in both G3 and G4 groups compared to G2. The results are shown in A of Figure 14 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

(3) Gastric damage  Inhibition rate

Compared with G2 (group treated with alcohol; 0.0 ± 0.0%), G3 and G4 groups significantly increased gastric mucosal injury inhibition rate. The results are shown in B of Figure 14 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

6. In the above organization Oxidative  Damage measurement

(1) lipid peroxidation

Lipids and oxides are caused by oxidative stresses such as ROS and free radicals, resulting in the loss of hydrogen atoms in the phospholipid membranes in the tissues, resulting in damage to tissues. Lipid peroxidation products increase the permeability of cell membranes, resulting in loss of body fluids and protein changes. MDA, a lipid peroxidation product of liver tissue, was measured as an indicator of gastric injury, and the therapeutic effect of the composite composition on alcoholic stomach injuries was measured.

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

The MDA of stomach tissues was significantly increased in G2 (alcohol treated group; 11.7 ± 1.8 μmol / ㎎ protein) compared to G1 (normal group: 1.2 ± 0.6 μmol / MDA levels were significantly decreased. The results are shown in A of Figure 15 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

(2) GSH

Glutathione assay kit (Cell Biolabs, USA) and the absorbance was measured at 405 nm and the concentration of GSH was calculated.

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

7. Measurement of inflammation-related cytokines in stomach tissue

(1) Total RNA Separation of cDNA  synthesis

1 ml of each of trizolysis buffer was dispensed into the stomach tissue, and homogenized and dissolved. 200 클로 of chloroform was inoculated for 20 seconds, inverted and centrifuged at 13,200 rpm for 15 minutes. The supernatant was transferred to a tube containing 500 이 of isopropanol. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was removed, 70% ethanol was added to each tube in an amount of 1 ml, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was removed and dried at room temperature. The RNA was dissolved in 40 μl of DEW containing D.W. The total RNA concentration of the solution was calculated by measuring the absorbance at 260/280 nm on a NanoDrop instrument by taking 1 μl of the RNA solution. CDNA was synthesized using extracted RNA and RT DriyMIX cDNA synthesis kit.

(2) Real time PCR

IL-1β, IL-6 and TNF-α. The primer sequences used in the experiments are shown in Table 13 below.

gene primer The sequence (5 '- &gt; 3') IL-1? 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-a 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 -6 expression

IL-6 levels were measured in the stomach tissue to evaluate the inflammatory response to alcohol administration and the therapeutic effect of the propolis combination 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 Figure 16 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

② IL -1β

IL-1β expression was significantly increased in G2 compared with G1, and IL-1β expression was decreased in G3 and G4 compared with G2. The results are shown in Figure 16 B (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

③ TNF -α

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

8. Comprehensive Review of the Effect of Mixed Propolis on Alcoholic Gastritis

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

Test group process Microscopic features Anti-oxidant
stress Anti-inflammatory Gastric ulcer
Area ^A Inhibition rate ^B MDA ^C GSH ^D IL-6 ^E IL-1? TNF-α ^G G1 Normal control group Saline / saline - - - - - - - G2 Alcohol-treated 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 &lt;50%; +, Inhibition = 50-70%; ++, inhibition <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 to 5 times; ++, IL-6 &lt;2-fold; +++.
F: IL-1?> 50 fold; +, IL-1? = 20-50 times; ++, IL-1 [beta] < 20 times; +++.
G: TNF-?> 0.5 times; +, TNF-alpha = 0.2-0.5 times; ++, TNF-alpha < 0.2 times; +++.

As shown in Table 14, the mixed propolis was found to exhibit an anti-ulcer, antioxidant and anti-inflammatory effect comprehensively.

< Experimental Example  7>

Therapeutic effect test of mixed propolis for alcoholic gastritis

To evaluate the therapeutic effect of mixed propolis in alcohol - induced gastric injury model, the following tests were conducted.

Test Methods

(1) Test animals

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

The body weight was 150 ~ 180g and male rats. Only one healthy animal was used for the test, with quarantine and refinement periods for one week after receipt. At the start of the study, the body weight ranged from 180 to 200 g, and a total of 75 animals were used for the test. The mice were kept in a temperature of 20 ± 2 ° C, humidity of 40 to 60%, and 12 hours of night / day period.

(2) induction of alcoholic gastritis

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

(3) Test group  Configuration

Four groups (G1 ~ G4) were used in the test group. Five or seven animals were used in each group. The composition of the test group is shown in Table 15 below.

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

(4) Administration dose and method

The dose and method of administration of alcohol, the reference drug (positive control; omeprazole) and the test drug are shown in Table 16 below.

process Dose Method of administration Number of doses ethanol 5 / kg (anhydrous alcohol) Gut nutrition One Omeprazole 20 mg / kg / day 7 Mixed Propolis 175 mg / kg / day 7

(5) Statistical processing

Statistical analysis was performed by one-way ANOVA using GraphPad PRISM program and post-test was performed using a Turkish test (urkey test) to verify the difference between the groups. Statistical significance was determined as p <0.5.

1. Observation of death and clinical symptoms

During the study period, once a day clinical symptoms and mortality / dysphoria were observed for each individual. Records were maintained only for individuals with clinical symptoms (not recorded if normal), and on weekends and holidays, one observation was given in the morning. However, at the day of administration, at least twice a day was observed.

No specific clinical symptoms were observed in all animals during the purifying period, and there were no live animals on the day of testing. There were no dead animals until the test substance (7 days) and alcohol were administered and the autopsy was carried out.

2. Weight measurement

Body weights were measured at the time of acquisition, once a week during the purification period, during the separation of the group and during the test period. In particular, weighing during the test period was measured using the same scales at the same time of day (around 4 pm) on the same day to minimize errors in each measurement.

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

3. Autopsy and long-term weighing

Twenty-four hours after the alcohol administration, the animals were lavaged under CO ₂ anesthesia, and then the abdominal aorta and the abdominal vein were cut and the blastocysts were sacrificed. The absolute long term weight of each organ (liver, spleen, kidney) And the relative organ weight was calculated based on the body weight fasted before the autopsy.

The relative long-term weights of liver for the fasted body weight were not statistically different in all groups (G2 to G4) compared to G1. In addition, the relative long - term weights of spleen and kidney were not significantly different from the fasted body weight. The results are shown in Fig. 19 (data are shown as means ± SEM).

4. Histopathological analysis

After euthanasia, the stomachs were removed from each individual and fixed in 10% neutral buffered formalin solution (10% buffered neutral formalin). After fixing the fixed tissue to a certain thickness, the tissue section with 4 ~ 5 ㎛ was prepared by paraffin embedding through general tissue processing, and then subjected to hematoxylin & eosin staining (H & E stain) (Histopathologic changes such as infiltration of inflammatory cells, destruction of epithelial cells, ulceration and hemorrhage) were observed.

H & E staining of the excised stomach was checked for tissue damage. In G1, no abnormality was observed in all layers including gastric mucosa. G2 showed necrosis and hemorrhage as well as gastric mucosal layer detachment, and damage and atrophy of some wall cells and main cells were confirmed. Infiltration of inflammatory cells such as macrophages and neutrophils in the mucosal and submucosal layers were few. However, these gastric mucosal lesions were reduced in both G3 and G4. The results are shown in Fig.

5. Gastric damage  Measure inhibition rate

The incision length (mm) and the damage area (mm ² ) generated after excising the excised stomach along the Taiwanese part were measured with a microscope and calculated according to the equation (2). (P <0.05), it was judged to be effective when a significant effect was demonstrated in the control group and the positive control group.

(1) gross damage of gastric mucosa

In order to evaluate the therapeutic effect of the propolis complex composition on gastric mucosal injury induced by alcohol administration, gross examination of the lesion was carried out by autopsy. As a result, G1 showed no gross abnormality but alcohol- G4) showed a thick band on the gastric mucosa. However, compared to G2, these bleeding sites were reduced in G3. The results are shown in Fig.

(2) Gastric damage  area

Of gastric mucosal damage length and the damage area (mm ²⁾ was measured by comparing the relative area to the area of the whole gastric mucosa a result, compared to the G1 was the most damage observed in G2, as compared to G2 in the G3 such bleeding The site was significantly decreased. The results are shown in A in Fig. 22 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

(3) Gastric damage  Inhibition rate

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

6. In the above organization Oxidative  Damage measurement

(1) Fractionation of the stomach tissue and lipid peroxidation

MDA, a lipid peroxidation product of liver tissue, was measured as an indicator of gastric injury, and the therapeutic effect of the composite composition on alcoholic stomach injuries was measured.

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

MDA levels in gastric tissues were increased in G2 (alcohol-treated group; 55.3 ± 12.3 μmol / ㎎ protein) compared with G1 (normal group; 20.0 ± 2.9 μmol / ㎎ protein) and MDA levels in both G3 and G4 Respectively. The results are shown in A in Fig. 23 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

(2) GSH

Glutathione assay kit (Cell Biolabs, USA) and the absorbance was measured at 405 nm and the concentration of GSH was calculated.

GSH of stomach tissue was significantly decreased in G2 (alcohol treated group; 17.0 ± 2.4 μmol / ㎎ protein) compared with G1 (normal group: 1.2 ± 0.6 μmol / ㎎ protein) group but no significant difference was observed. There was no statistically significant difference between G3 and G4 compared to G2. The results are shown in B of Figure 23 (data represent the average ± SEM ^* and ^** respectively P <0.05 and P <0.01 compared to the G1;. ^† and ^††, respectively, P <0.05 as compared to the G2 and P < 0.01).

7. Comprehensive review of the therapeutic effect of mixed propolis on alcoholic gastritis

To investigate the therapeutic effect of mixed propolis on damaged gastric mucosa after alcohol administration, the results of gross, histologic and oxidative stress analyzes were compared. The results are shown in Table 17 below.

Test group process Microscopic features Antioxidant stress Gastric ulcer
Area ^A Inhibition rate ^B MDA ^C GSH ^D G1 Normal control group Saline / saline - - - - G2 Alcohol-treated 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 <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 antioxidative effects.

< Experimental Example  8>

Anti-Helicobacter Filly  Verify Active

Test Methods

(1) Preparation of propolis sample

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

(2) Helicobacter Filly  Culture of strain

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% newborn calf serum was added to Brucella broth (BD, USA) as a liquid culture medium for pre-culture and main culture of Helicobacter pylori, and solid medium was used for Brucella broth (BD , USA) supplemented with 10% fresh calf serum and bacto-agar (BD, USA). The culture conditions were incubated at 37 ° C in a 10% CO ₂ incubator.

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

1. Of propolis samples by country of origin Anti-Helicobacter Filly  Check the effect

Antibacterial activity against Helicobacter pylori of propolis samples by country of origin was measured by paper disc agar diffusion method. Namely, each of the strains cultured on a plate culture medium was inoculated into 10 ml of the liquid culture medium and 0.1 ml of the culture broth was incubated for 3 to 6 hours. After the cultivation, the cells were inoculated so as to have a cell number of about 1 × 10 ⁷ cells per plate medium and uniformly plated with a sterilized cotton swab. The sterilized filter paper disc (8 mm, Whatman, Japan) was placed on a solid plate medium, and then the sample was absorbed at a concentration of 0.5 mg / disc and incubated at 37 ° C. for 72 hours. Clear zone (mm) diameter was measured. The results are shown in Table 19 and FIG.

On the average, propolis A, C, D, and B showed the highest effect in the order. Especially, the effect of propolis samples of A and C was 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 Filly  Check the effect

Minimum inhibitory concentration method ( MIC assay )

The effect of mixed propolis on Helicobacter pylori was confirmed by MIC (Minimum Inhibitory Concentration) assay. MIC refers to the minimum concentration (mg / ℓ) of a substance that has a developmental inhibitory effect on a specific microorganism, which is a unit used to indicate the antimicrobial activity or developmental inhibition of antibiotics. Above this concentration value, the microorganism does not develop and exhibits a certain number.

The sample mixed propolis was prepared by mixing the extracts of Argentina propolis, Chinese propolis and Brazil propolis at 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 of Helicobacter pylori, each sample was serially diluted with the medium to a concentration of 1 mg / ml, and the cells were reacted while inoculated at OD600 = 0.1 concentration and cultured for 16 hours under micro conditions. After the reaction, the cells were diluted to 10 ^-3 with PBS, loaded onto a plate, cultured for 3 days under micro conditions, and the number of colonies formed was counted.

After the sample was treated, the same amount of urea broth was added, and the reaction was carried out in a 10% CO ₂ incubator at 37 ° C., and the viable cell count was indirectly measured by a 560 nm spectrophotometer.

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

Dish No. The concentration of the sample (/ 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, the mixed propolis showed a high anti-helicobacter activity.

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

Although the pathogenic mechanism of Helicobacter pylori has not yet been fully elucidated, Helicobacter pylori, which is present in the stomach, has the ability to produce strong urease, which causes ammonia (ammonia) It is known that it can survive above neutralization. Currently, urease activity measurement, which is the most commonly used method to measure the antimicrobial activity of Helicobacter pylori, is easy to measure urease activity and Helicobacter pylori The correlation between the number of viable cells and the number of viable cells was also found to be very high.

In order to measure urease activity, Helicobacter pylori was cultured. Cells were separated, disrupted with an ultrasonic sonicator, and subjected to ultrafast centrifugation at 12,000 rpm at 4 ° C for 30 minutes to obtain total protein, &Lt; / RTI &gt; 5 mg / ml of sample and 50 μl of total protein were mixed with 100 μl of urea broth and reacted. The change in the color intensity of phenol-red due to the decomposition of urea was measured at 560 nm using a spectrophotometer (TECAN, USA).

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

B, D, A and C were the inhibition rates of urea degradation activity of propolis samples by country of origin. These results are slightly different from the above-mentioned disk diffusion test. This is because the main substance in the propolis affects the activity of urease and thus shows the activity of anti-Helicobacter pylori but also 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 Low performance (%) 54.99 66.34 34.99 57.93 77.98

< Experimental Example  9>

Anti-Helicobacter pylori animal experiment (preliminary experiment)

In order to test the anti-helicobacter effect of mixed propolis in Helicobacter pylori-induced gastritis model of mice, Helicobacter pylori The stomach adhesion inhibition effect and stomach protection effect were evaluated as follows.

Test Methods

(1) Test substance and test animal

A propolis complex was used as a test substance, and 5-week old female C57BL / 6 mice were used as a test animal.

(2) Helicobacter In filial piety  Gastritis model by

In order to obtain gastritis model by Helicobacter pylori, Helicobacter pylori was prepared at 1 × 10 ⁹ cfu / ml in C57BL / 6 mouse and 0.3 ml per mouse was inoculated by oral gavage three times a week for three days And were observed for 3 weeks. At this time, Helicobacter pylori was adapted from mouse strain (Sydney strain) (SS1, cagA + / vacA +), which was purchased from Ajou University School of Medicine.

(3) Test group  Configuration and dosage setting

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

Test group Helicobacter pylori dose Test substance Number of animals G1 Untreated control group C57BL / 6 mice 3 x 10 ⁸ cfu / mouse - 8 G2 Negative control group 3 x 10 ⁸ cfu / mouse - 8 G3 Positive control group 3 x 10 ⁸ cfu / mouse Omeprazole 8 G4 Experimental group 3 x 10 ⁸ cfu / mouse Mixed Propolis 8

(4) Administration site and administration method

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

(5) Number of administrations

In a model infecting Helicobacter pylori, once / day test substance was administered for 3 weeks.

(6) Inspection items

① Safety evaluation of propolis complex

② Visual and microscopic observation

③ Helicobacter pylori H. pylori colonization

1. Safety evaluation of mixed propolis

(1) Mortality and clinical symptoms

No specific clinical symptoms were observed in all animals during the study period. There were no dead animals and no animals on the day of testing.

(2) Weight measurement

During the 4 weeks of feeding, weight gain in each group showed a normal growth curve in all groups and no statistically significant weight difference was observed.

(3) Serological examination

The ALT and AST in the serum were measured to evaluate the safety of the propolis complex, and the results are shown in Fig. There was no significant difference in ALT and AST levels between G1 group (untreated control group) and G2 group treated with Helicobacter pylori (none treated). There was also no significant difference in G3 group (positive control group) and G4 group (mixed propolis group) compared to G1 group.

(4) Histological examination

Mice were treated with CO ₂ Liver, lung, spleen and kidney were excised and fixed with 10% buffered formalin solution (10% buffered neutral formalin). After the fixed tissue was cut to a certain thickness, the tissue sections were cut into 4-5 μm by paraffin embedding through general tissue processing, and then hematoxylin and eosin stain (H & E stain), which is a general staining method, Respectively. 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 administration of Helicobacter pylori and the test substance, mice were sacrificed and the stomach tissues were excised to observe morphological changes. The results are shown in Fig. As can be seen in FIG. 29, no specific histological lesions were observed in all the groups.

(2) Microscopic observation

The stomach was removed, and a part of the stomach was fixed with 10% neutral buffer formalin solution. Histochemical staining was performed to observe histopathologic findings (histopathological changes such as infiltration of inflammatory cells and destruction of epithelial cells) Is shown in Fig. 30, As can be seen, after 3 weeks of infection with Helicobacter pylori, the stomach of the mouse showed some infiltration of inflammatory cells in the G2, G3 and G4 groups, but no other tissue lesions were observed.

3. Helicobacter Filial Stomach Attachment ( H. pylori colonization )

Helicobacter pylori The mechanism by which the infection causes gastrointestinal disease is that Helicobacter pylori permeates through the mucous layer of the stomach and attaches to the gastric mucosal layer (colonization), thereby causing inflammation, and inflammatory mediators produced by inflammatory cells cause diseases. The amount of Helicobacter pylori attached to the gastric mucosa was measured to see how the propolis and propolis complexes play a role in the gastric attachment of such Helicobacter pylori. After removing the stomach from each mouse, DNA was extracted and the 16s DNA of Helicobacter pylori was subjected to real-time PCR to quantitate Helicobacter pylori. The results are shown in Fig.

As a result, compared to the G2 group treated with Helicobacter pylori, The number of infections was slightly decreased, but no significant difference was observed.

4. Comprehensive examination of anti-helicobacter effect test of mixed propolis

Helicobacter pylori-induced gastritis in mice Model of mixed propolis Helicobacter pylori As a result of evaluating the stomach adhesion inhibition effect and stomach protection effect, there was no significant difference in the group (G4) administered with mixed propolis to the group (G2) infected with Helicobacter pylori without the drug treatment Similar effects were observed with omeprazole (G3), which is used as a therapeutic agent.

< Experimental Example  10>

Anti-Helicobacter pylori Animal Experiment

Helicobacter pylori-induced gastritis in mice Model of mixed propolis Helicobacter pylori The stomach adhesion inhibition effect and stomach protection effect were evaluated as follows.

Test Methods

(1) Test animals

As a test animal, C57BL mouse was used and clean mice not infected with Helicobacter were supplied from Korea Biotechnology Research Institute.

The mice weighing 9-14 g were 4-week-old male mice. Only one healthy animal was used for the test, with quarantine and refinement periods for one week after receipt. At the start of the administration, the animals were 5 weeks old and the body weight ranged from 10 to 15 g, and a total of 120 animals were used for the test. The mice were kept in a temperature of 20 ± 2 ° C, humidity of 40 to 60%, and 12 hours of night / day period.

(2) Test group  Configuration

Ten groups of animals were used in each test group, and the composition of the test group is shown in Table 23 below.

Test group Helicobacter
Filly Test substance Number of animals G1 Normal control group - PBS (phosphate buffered saline) 10 G2 HP control + PBS 10 G3 Positive control group + Omeprazole 10 G4 + Omeprazole + amoxicillin + clarithromycin &lt; RTI ID = 0.0 &gt; 10 G5 Test group + Mixed Propolis 10

(3) Helicobacter In filial piety  Induced gastritis by

The mice were fasted for 24 hours and Helicobacter pylori was administered by gavage.

Helicobacter pylori has been used by the University of Ajou University for the Sydney Strain (SS1, cagA + / vacA +), which has a high infection rate in mice.

Helicobacter pylori was prepared at 1 × 10 ⁹ cfu / ml in C57BL / 6 mice, and the mice were inoculated three times a day for 1 week with 0.2 ml of mouse per day by gavage. The normal control group was orally administered with PBS. 8 weeks later, autopsy was performed to compare the above lesions. A schematic diagram of the experimental design is shown in Fig.

(4) Method of administration of the test substance

Omeprazole or omeprazole + amoxicillin + clarithromycin was used as the reference drug. Omeprazole is a proton pump inhibitor (PPI) that is known to inhibit the growth of Helicobacter pylori by strongly inhibiting gastric acid secretion by inhibiting the hydrogen ion pump, the last stage of gastric acid secretion. According to the Guidelines for the Diagnosis and Treatment of Helicobacter pylori Infection in the Korean Society of Gastroenterological Surgery 2009, PPI (omeprazole etc. standard dose twice a day) + amoxicillin (1 g twice a day) + claritromycin (500 mg , Twice a day) standard triple therapy is recommended for 1-2 weeks. In this experiment, considering the weight of the mouse, Sander et al ., &lt; / RTI &gt;

The dose, method and frequency of administration of the test sample and the reference drug are as shown in Table 24 below.

process Dose Method of administration Number of doses Helicobacter pylori 0.2 (1 x 10 &lt; ⁹ &gt; cfu / Gut nutrition 2 days a week
3 times in total Omeprazole 400 mol / kg / day 7 days Amoxicillin 28.6 mg / kg / day 7 days Claritromycin 14.3 mg / kg / day 7 days Mixed Propolis 350 mg / kg / day 5 weeks

(5) Statistical processing

Statistical analysis was performed by one-way ANOVA using GraphPad PRISM program and posttest was analyzed using the Turkey test to verify the difference between the groups. Statistical significance was determined as p <0.5.

1. Mortality and clinical signs

During the study period, once a day clinical symptoms and mortality / dysphoria were observed for each individual. Records were maintained only for individuals with clinical symptoms (not recorded if normal), and on weekends and holidays, one observation was given in the morning. However, at the day of administration, at least twice a day was observed.

No specific clinical symptoms were observed in all animals during the purifying period, and there were no live animals on the day of testing. The results are shown in Table 25 below.

Test group process Number of animals Our company
Number of animals Mortality rate
(%) Clinical
observe G1 Normal control group 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

Body weights were measured at the time of acquisition, once a week during the purification period, during the separation of the group and during the test period. In particular, weighing during the test period was measured using the same scales at the same time of day (around 4 pm) on the same day to minimize errors in each measurement.

In G1 to G5, no reduction in body weight was observed for a total of 8 weeks and there was no difference between the groups. Normal weight gain was also observed in all groups during the study period. The results are shown in Table 26 below.

Test group process Weight (g) Early
(Before HP administration) final
(After HP administration) Growth rate (%) G1 Normal control group 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 weighing

Helicobacter pylori After 8 weeks of the last infection, the catheter was lavaged under CO ₂ anesthesia, and then the abdominal aorta and the abdominal vein were cut, and the catheter was lysed. After observing the presence or absence of internal organs visually, the absolute organ weights of the organs (liver, spleen, kidney) were measured and the relative organ weights were calculated based on the fasted body weight before the autopsy.

(One) Liver weight

Helicobacter pylori The relative long term weight of liver for the fasted body weight at 8 weeks after administration was calculated. There was no significant difference compared to G2. The results are shown in Fig. 33 (data are expressed as mean ± SEM).

(2) spleen weight

There was no statistically significant difference in the relative long-term weight of the spleen relative to the fasted body weight. The results are shown in Figure 34 (data are expressed as mean ± SEM).

(3) Height Weight

There was no statistically significant difference in the relative long-term weight of fasting relative to fasted body weight. The results are shown in Figure 35 (data are expressed as mean + -SEM).

4. Histopathological analysis

After euthanasia, the stomachs were removed from each individual and fixed in 10% neutral buffered formalin solution (10% buffered neutral formalin). After the fixed tissue was cut to a certain thickness, the tissue sections were cut into 4-5 μm by paraffin embedding through general tissue processing, and then hematoxylin and eosin stain (H & E stain), which is a general staining method, (Histopathologic changes such as infiltration of inflammatory cells, destruction of epithelial cells, ulceration and hemorrhage) were observed.

The extracted stomach tissue was stained with H & E staining to confirm the degree of tissue damage, and the results are shown in Fig. In G1, no abnormal findings were observed in all layers including gastric mucosa. In G2, infiltration of inflammatory cells such as macrophages and neutrophils was observed in mucosa and submucosa. No abnormal findings such as inflammation were observed in all other groups.

5. Helicobacter Filly Stomach Attachment  analysis

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

gene primer probe Helicobacter
Filly
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, compared to G1, The number of infections was significantly increased. The results are shown in Figure 37 (data represent the average ± SEM ^* and ^** is compared to the G1, respectively P <0.05 and P <0.01; . † and ††, as compared to the G2, respectively P <0.05 and P &Lt; 0.01).

6. In the above organization Oxidative  Damage measurement

When Helicobacter pylori is infected with phagocytic cells, reactive oxygen species (ROS) first increase due to the immune response of the cells. This ROS destroys the homeostasis of the cells by oxidizing macromolecules such as proteins and lipids in the cells , And self-destructive action of the cells. The protective effect of propolis complex on oxidative damage induced by Helicobacter pylori on phagocytic cells was evaluated. The results are shown in Figure 38 (data represent the average ± SEM ^* and ^** is compared to the G1, respectively P <0.05 and P <0.01; . † and ††, as compared to the G2, respectively P <0.05 and P &Lt; 0.01).

(One) MDA

Lipid peroxides are caused by oxidative stress such as ROS and free radicals, resulting in the loss of hydrogen atoms in the phospholipid membranes in the tissues, resulting in damage to tissues. Lipid peroxidation products increase the permeability of cell membranes and cause loss of body fluids and protein changes. MDA, a lipid peroxidation product of stomach tissue, was measured as an index of gastric injury, and the therapeutic effect of propolis complex was measured for gastric tissue damage caused by Helicobacter pylori.

A part of the stomach tissue was homogenized with a homogenizer by adding 4 times 150 mM KCl. The homogenized tissue was centrifuged at 4,000 rpm for 30 minutes, and the supernatant was measured for glutathione (GSH). The rest of the supernatant was centrifuged at 20,000 rpm for 1 hour, and the supernatant was recovered. The malondialdehyde, MDA ) Were measured. (Cell Biolabs, USA). The absorbance at 586 nm was measured and the MDA was calculated.

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

(2) GSH

GSH is one of the in vivo antioxidants that prevent oxidative stress-induced damage caused by free radicals and peroxides.

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

GSH level was decreased in G2 compared to G1, but no significant difference was observed. GSH level of G3 was significantly increased compared with G2 (FIG. 38B).

7. Expression of inflammatory cytokines

Trisolysis buffer (Trizol lysis buffer) was dispensed into each of the above tissues, followed by homogenization and dissolution. 200 클로 of chloroform was inoculated for 20 seconds, inverted and centrifuged at 13,200 rpm for 15 minutes. The supernatant was transferred to a tube containing 500 이 of isopropanol. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was removed, 70% ethanol was added to each tube in an amount of 1 ml, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was removed and dried at room temperature. The RNA was dissolved in 40 μl of DEW containing D.W. The total RNA concentration of the solution was calculated by measuring the absorbance at 260/280 nm on a NanoDrop instrument by taking 1 μl of the RNA solution. CDNA was synthesized using extracted RNA and RT DriyMIX cDNA synthesis kit.

IL-1β, IL-6 and TNF-α. The primer sequences used in the experiments are shown in Table 28 below.

gene primer Sequence (5` → 3`) IL-1? 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-a 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, there is an increase in neutrophils in the lamina proproa, and an increase in lymphocytes, plasma cells, eosinophils, and the like is observed in chronic progression. The increase in neutrophil activity induces the production of inflammatory cytokines such as IL-1β, IL-6, and TNF-α as well as chemokines such as IL-8, thereby allowing more inflammatory cells to enter the gastric mucosa, And atrophy and apoptosis of phase epithelial cells. IL-1β, IL-6 and TNF-α levels were measured in stomach tissues to quantitatively assess the inflammatory response caused by Helicobacter pylori infection and the therapeutic effect of propolis complexes. The results are shown in Figure 39 (data represent the average ± SEM ^* and ^** is compared to the G1, respectively P <0.05 and P <0.01; . † and ††, as compared to the G2, respectively P <0.05 and P &Lt; 0.01).

(One) IL -6

The expression of IL-6 was significantly increased in G2 compared to G1 (Fig. 39A).

(2) TNF -α

There was no statistically significant difference in expression of TNF-α in G2 compared to G1, and there was no significant difference in all test groups compared with 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

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

8. Comprehensive examination of anti-helicobacter efficacy test of mixed propolis

To investigate the therapeutic effect of mixed propolis on damaged gastric mucosa after H. pylori infection, we analyzed the histopathological findings, the expression of Helicobacter pylori, the expression of pro-inflammatory cytokines, and the results of oxidative stress analysis. 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-1? ^D MDA ^E GSH ^F G1 Normal control group PBS - - - - - - G2 HP control PBS - - - - - - G3 Positive control group Omeprazole +++ ++ ++ ++ + +++ G4 Omeprazole + amoxicillin + claritromycin ++ +++ ++ ++ +++ ++ G5 Test group Mixed Propolis + ++ + ++ ++ ++ A: HP stomach attachment> 0.3 fold; +, HP stomach = 0.3-0.7 fold; ++, HP stomach attachment <0.7x; +++.
B: IL-6> 5 times; +, IL-6 = 1 to 5 times; ++, IL-6 &lt;1x; +++.
C: TNF-α> 2 times; +, TNF-a = 1 to 2 times; ++, TNF-a &lt;1x; +++.
D: IL-1?> 2 fold; +, IL-1 [beta] = 2 to 1 fold; ++, IL-1 [beta] &lt;1x; +++.
E: MDA > 15 uM / mg protein; +, MDA = 15 to 10 uM / mg protein; ++, MDA < 10 uM / mg protein; +++.

As shown in Table 29, it was confirmed that mixed propolis has anti-Helicobacter pylori attachment, antioxidant and anti-inflammatory effects in a comprehensive manner.

Claims (15)

Characterized in that, in order to enhance the efficacy of propolis as a health functional food, an extract of propolis having at least two different origins selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis is mixed and used How to use the Police. The method according to claim 1,
Wherein said efficacy is one of antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy. The method according to claim 1,
Wherein the propolis extract is a water-soluble extract obtained by hydrolyzing an ethanol extract of propolis. 4. The method according to any one of claims 1 to 3,
The method of using propolis according to claim 1, wherein the mixture is used in combination with an extract of propolis, propolis, and propolis. 5. The method of claim 4,
Wherein the mixed use comprises mixing the extracts of propolis, propolis, and propolis of Korea at a weight ratio of 30-40: 25-35: 30-40. 4. The method according to any one of claims 1 to 3,
The method of using propolis according to claim 1, wherein the mixed use comprises mixing Argentina propolis, Chinese propolis and Brazil propolis extract. The method according to claim 6,
The method of using propolis according to claim 1, wherein the mixture is used in an amount of 35 to 45:25 to 35:25 to 35 by weight of extracts of propolis, propolis, and propolis. In order to enhance the efficacy of propolis, a composition for a health functional food containing propolis as an active ingredient is mixed with at least one propolis selected from the group consisting of Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis Wherein the composition contains a mixed extract. 9. The method of claim 8,
Wherein said efficacy is any one of antioxidant, anti-inflammatory, anti-ulcer and anti-Helicobacter pylori efficacy. 9. The method of claim 8,
Wherein the mixed extract is a water-soluble extract obtained by hydrolyzing an ethanol extract of propolis. 11. The method according to any one of claims 8 to 10,
Wherein the mixed extract comprises extracts of propolis, propolis, and propolis from Argentina. 12. The method of claim 11,
Wherein the mixed extract comprises extracts of propolis, propolis, and propolis, in a weight ratio of 30-40: 25-35: 30-40. 11. The method according to any one of claims 8 to 10,
Wherein the mixed extract comprises extracts of propolis, propolis and propolis from Argentina. 14. The method of claim 13,
Wherein the mixed extract comprises extracts of propolis, propolis, and propolis of Brazil in a weight ratio of 35:45:25 to 35:25 to 35. A method for producing a health functional food containing propolis as an active ingredient,
In order to enhance the efficacy of propolis, propolis-containing health function characterized by mixing extracts of at least two different propolis selected from Argentina propolis, Chinese propolis, Brazilian propolis and Korean propolis Method of manufacturing food.

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