KR20200058368A - composition for removing helicobacter pyloricomprising extract from pine-tree by-product - Google Patents

Abstract

The present invention relates to a composition having an effect of removing Helicobacter pylori of an extract of Pinus koraiensis by-products. By the composition which extracts Pinus koraiensis by-products with distilled water and formulates the same, H.pyloriIgG antibody is reduced, a treatment rate is high as a result of converting a treatment rate through CLO test in a gastric mucosal tissues, and a result concentration of TNF-α and IL-1β is reduced measured to examine cytokine changes in the gastric mucosa. The present invention has excellent effects in the food industry and the pharmaceutical industry.

Description

Composition for removing helicobacter pylori bacteria containing pine tree byproduct extract {composition for removing helicobacter pyloricomprising extract from pine-tree by-product}

The present invention relates to a composition having a bactericidal effect of Helicobacter pylori containing pine tree by-product extract.

Helicobacter is a gram-negative bacteria and is a pathogen known to cause chronic, acute gastritis, gastric ulcer and gastric cancer in the human body. In the case of industrial society, more than half of the population over the age of 60 are infected, and in developing countries, most of the population are reported to have been infected since childhood. About 10% of infants between the ages of 2 and 8 are infected every year, so most of the teens are infected. As of 2017, in Korea, the infection rate is high among office workers and people with professional occupations, and after 20s, it has a high infection rate of over 70%. Previously, gastrointestinal diseases such as gastritis and gastric ulcer were primarily caused by excess gastric acid, that is, excessive gastric acid secretion, and have been known to cause inflammation due to stress, food, and genetic factors, but have recently been infected by Helicobacter It is the norm that inflammation occurs when. It is known that when the inflammation spreads to the submucosal layer in the stomach, it develops into a gastric ulcer, and does not progress directly to gastric cancer, but when infected with Helicobacter, the probability of progressing to gastric cancer increases by 3-5 times. Until several years ago, antacids and histamine receptor antagonists have been used to treat these diseases. The National Institutes of Health recognized the relationship between Helicobacter and gastrointestinal diseases only in the mid-1990s, allowing the use of antibiotics such as tetracycline or amoxicillin in parallel with other treatments. Recently, triple therapy for the elimination of Helicobacter requires intensive treatment for 2-3 weeks. However, due to the accumulation of bacteria in the human body, side effects, and resistance due to the use of antibiotics, the development of alternative drugs that can ultimately treat gastric diseases is required. Looking at the development status of domestic and foreign gastric disease treatment and prevention agents, the use of immune systems or antibacterial agents, or the production of kits using bacterial cell releasing enzymes is the mainstream, and the use of sugar compounds that are still involved in cell recognition Development is incomplete.

It has been reported that Helicobacter binds mucosal epithelial cells in the gastrointestinal tract, and the binding mechanism is mainly dependent on the interaction between carbohydrates and bacterial surface proteins that recognize them. Particularly recognized carbohydrates include mucin glycoprotein sulfated carbonhydrate, sialyllactose, and fucosylatedlewis b antigen. Recently, efforts have been made to inhibit bacterial cell binding using glycosphingolipids or mucin proteins in needles. Separation and identification of bacterial surface protein receptors has already been made, and the development of sugar complexes using recognized carbohydrates is a very important subject. When using existing antibiotics or chemicals, developmental technologies as well as side effects are cell-destructive, while new substances that inhibit carbohydrate recognition and interaction on the surface of host cells are non-destructive technologies that minimize gastritis by leaking intercellular bonds outside the stomach. It has an important meaning in that.

However, there have been no studies on the extract of pine tree by-products that inhibit the proliferation and activity of Helicobacter pylori in the stomach.

In view of the above points, the present invention is a bioactive substance that inhibits proliferation and activity of Helicobacter pylori in the stomach, and extracts pine tree by-products obtained by extracting pine tree by-products with excipients. It is a solution to provide a composition having an effect.

As a means for solving the above-mentioned problems,

The composition having a bactericidal effect of Helicobacter pylori of the present invention is characterized by having a pine tree by-product extract as an active ingredient.

In addition, it is characterized in that it further comprises a tapioca starch (fine flow; manufactured by Matsuda Nishi, Japan).

The method of disinfecting Helicobacter pylori of the present invention is characterized by selectively inhibiting the proliferation of the Helicobacter pylori by coexisting a pine tree by-product extract in an environment in which Helicobacter pylori is present.

In addition, the pine tree by-product extract is characterized by being mixed with tapioca starch (fine flow; manufactured by Matsuda Nishi, Japan).

When using an existing antibiotic or chemical drug for the treatment of gastric diseases caused by Helicobacter pylori, the development technology is cell destructive, while the composition having the bactericidal effect of Helicobacter pylori of the present invention is a digestive disorder animal As a result of evaluating the effectiveness of administration of the test substance pine tree by-product extract in the model, it is a non-destructive technology that reduces antibodies and reduces inflammatory cytokine of Helicobacter pylori. In the search for the active substance, it is possible to perform quantitative analysis of the substances to be searched as well as the proportional relationship of activity according to the relative concentration of the substance, which has an excellent effect in the food industry and the pharmaceutical industry.

1 is a schematic schematic diagram according to the passage of time in the design of an animal experiment.
2 is a graph showing the results of measuring the mouse weight at each time point.
3 is a graph showing the result of intake of the mouse resignation at each time point.
4 is a graph showing the result of negative mouse intake at each time point.
5 is a graph showing the effect of test substances on serum H. pylori antibody (IgG) test.
Figure 6 is a separate observation image of the tissue on the mouse to determine the effect of the test substance on H.pylori infection.
7 is a histopathological examination image of the mouse to determine the effect of the test substance on H.pylori infection.
8 is a graph showing the inflammation score for each test group in the histopathological examination.
9 is a graph showing the atrophy change score for each test group in the histopathological examination.
10 is a graph showing the total score for each test group in histopathological examination.
11 is a graph showing the CLO score for each test group.
12 is an image showing the CLO test value for each test group.
13 is a graph showing the TNF-α values for each test group.
14 is a graph showing IL-1β values for each test group.
15 is a graph showing the PCR test results for each test group (lane 1 to 10: animal sample, lane P: PCR positive control, lane N: PCR negative control).
16 is a rat fecal antigen test image for each test group.

The pine tree by-product extract of the present invention can be used to sterilize Helicobacter pylori. The present invention relates to a composition having a bactericidal effect of Helicobacter pylori containing the pine tree by-product extract of the present invention as an active ingredient.

The composition of the present invention may further include tapioca starch (fine flow; manufactured by Matsuda Nishi, Japan).

It is preferable that the pine tree by-product extract of the present invention is a pine tree conifer extract.

The composition having a bactericidal effect of Helicobacter pylori containing the pine tree by-product extract of the present invention is a formulation for oral administration such as powder, granule, tablet capsule, suspension, emulsion, syrup, aerosol, sterile injectable solution, suppository and transdermal according to a conventional method It can be used in formulations for administration. As a carrier, excipient and diluent in the composition, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, it is formulated using excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants.

In one aspect, the pine tree by-product extract of the present invention can be formulated into a solid preparation for oral administration. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is negotiated by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

As another aspect, the composition containing the pine tree by-product extract of the present invention may be formulated as a liquid formulation for oral administration. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition, various excipients, such as purified water, ethanol, and liquid paraffin, are commonly used in such liquid preparations. For example, wetting agents, sweeteners, fragrances, preservatives and the like may be included.

As another aspect, the composition containing the pine tree by-product extract of the present invention may be formulated as a parenteral, preferably, a formulation for intraperitoneal administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As a sterilized aqueous solution, an appropriate buffer solution such as Hank's solution, Ringer's solution, or physically buffered saline can be used. As a non-aqueous solvent, propylene glycol, polyethylene glycol, olive oil and The same vegetable oil, injectable esters such as ethyl oleate, etc. can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts and / or buffers for osmotic pressure control can be used. On the other hand, in the case of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc., which are common bases thereof, may be used.

Compositions formulated in the above manner may be administered via various routes including parenteral or oral (dermal, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) in an effective amount. The "effective amount" refers to an amount that exhibits a prophylactic or therapeutic effect when administered to a patient. The dosage of the composition according to the present invention can be appropriately selected according to the route of administration, the target of administration, age, sex, weight, individual differences, and disease state.

Further, as another aspect, the pine tree by-product extract of the present invention may be provided as a food composition by mixing with a food-acceptable carrier. When the pineapple by-product extract of the present invention is used as a food or beverage additive, the pineapple by-product extract is added as it is, or used with other food or food ingredients, and can be suitably used according to a conventional method. The mixed amount of the pine tree by-product extract may be appropriately determined according to its purpose of use (prevention, health or therapeutic treatment).

In the case of long-term intake for health purposes or for health control, the pine tree by-product extract has no problem in terms of safety, so long-term intake is possible.

There are no particular restrictions on the type of food. Examples of foods to which the above substances can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1-Preparation of pine tree by-product extract

The pine tree by-product is put into the extractor tank, and the steam at 85 ° C. is passed through the pine tree by-product (confection) in the extractor tank while maintaining the vapor pressure of 760 mmHg, and the temperature when steam passes through the cooler outside the extractor tank is set to 8 ° C. At this time, the extracted extract is distilled to obtain essential oil components to prepare a pine tree by-product extract.

Example 2- Preparation of Helicobacter pylori growth inhibition composition (test substance) containing pine tree by-product extract

After mixing 40% by weight of pine tree conifer distilled water extract obtained in Example 1 and 60% by weight of tapioca starch (Pineflow; manufactured by Matsuda Nishi, Japan), it was used for the Helicobacter pylori inhibition test.

Experimental Example 1 Experiment on the antibacterial effect of pine tree by-product extract in Helicobacter pylori infected mice

As shown in Figure 1, H. pylori SS1 (Helicobacter Korea) strain was inoculated in Trypticase Soy agar medium to which 5% amount of blood was added and cultured for 2 to 3 days at 10% CO ₂ , 37 ℃ and micro-aerobic conditions. .

C57BL / 6 mice were used for this test, and antacids were administered 2 days before and on the day of infection to increase the H.pylori infection rate. All groups were orally administered with 0.2% per mouse, once per mouse for a total of 3 days with 5% Sodium Bicarbonate (NaHCO ₃ ) using a zoned mouse.

Mice were fasted 12 hours before H. pylori infection. H. pylori cultured in all groups except G1 (negative control group) was matched with the number of 5.0Х10 ⁹ / mL colony-forming unit (CFU), and 0.2 mL of mice was used, 3 times every 2 days using zonde. It was infected by oral administration.

After inducing H. pylori infection, blood was collected from facial vein in all mice 1 week after H. pylori infection to confirm the maintenance of infection. Measurement of H. pylori antibody was performed using the Mouse H. pylori antibody (IgG) ELISA Kit (Cusabio Biotech Co., USA) to select only individuals whose antibody elevation was confirmed by infection.

The composition and administration dose of the test group are as shown in Table 1 below, and the test substance is suspended in DW and administered orally at the same time every 10 ml per kg of the mouse, once a day for 14 days (G3 is 1 week, 1 time, 7 days).

Test group Disease induction Name Test substance Dosage
(ml / kg) Number of animals G1 PBS Negative control D.W 10 10 G2 H. pylori Vehicle control D.W 10 10 G3 Positive control AMX + CLR + PPI (omeprazole) 10 10 G4 Positive control 25 mg / kg licorice extract 10 10 G5 Test substance 100 mg / kg pine tree by-product extract 10 10 G6 Test substance 200 mg / kg pine tree by-product extract 10 10 G7 Test substance 400 mg / kg pine tree by-product extract 10 10

G1: Negative control (PBS + D.W), n = 10 G2: Vehicle control (H.pylori + D.W), n = 10

G3: Positive control 1 (H.pylori + 14.25 mg / kg AMX + 7.15 mg / kg CLR + 400 μmol / kg omeprazole), n = 10

G4: Positive control 2 (H.pylori + 25 mg / kg licorice extract), n = 10

G5: Test article (H.pylori + 100 mg / kg pine tree byproduct extract), n = 10

G6: Test article (H.pylori + 200 mg / kg pine tree byproduct extract), n = 10

G7: Test article (H.pylori + 400 mg / kg pine tree byproduct extract), n = 10

AMX: Amoxicillin

CLR: Clarithromycin

PPI: Omeprazole

(1) General symptom observation result

During the test period, the general symptoms and the occurrence of death / diet animals were observed for each individual, and records were kept only for individuals with specific symptoms (if not normal). As a result, no animals with specific symptoms were observed during the test period, and the results are shown in Table 2 below.

Animal clinical observation results Group H. pyloriinfection Positive control
/ Test article Number of mouse Clinical Observations G1 PBS D.W 10 N G2 H. pyloriinfection D.W 10 N G3 AMX + CLR + PPI (omeprazole)
(Positive control 1) 10 N G4 Licorice extract
(Positive control 2) 10 N G5 Pine tree byproduct extract 10 N G6 10 N G7 10 N

N: normal

(2) Confirmation of maintenance of H.pylori infection and group composition results

One week after the induction of infection, blood was collected from facial skin of H.pylori-infected mice, and then separated plasma and blood collected from the abdominal vein at the end of the experiment were separated using plasma mouse H.pylori antibody and IgG ELISA kit, respectively. H.pylork antibody titers in plasma were measured and compared between groups.

As a result of calculating the mean and standard deviation by measuring the H. pylori antibody, as shown in Table 3 below, the non-infected group G1 (negative control group) was 0.21 ± 0.03, and the mean values of the infected groups G2 to G7 were 0.47 ± 0.16. The H. pylori antibody was statistically significantly elevated to 123.8% in the infected group compared to the non-infected group (p <0.01, Table 3).

H.pylori antibody IgG test results Group Number of mouse H. pyloriIgG test Non-Infection 10 0.21 ± 0.03 Infection 60 0.47 ± 0.16 **

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and the p value was set at a level comparable to the non-infected group. ** p <0.01

(3) Measurement results of body weight, feed intake, and negative consumption

Body weight is tested at the time of administration of antacid (-1 week post infection, wpi), after repeated infection with H. pylori 3 times (0 week post infection, wpi), before infection maintenance is confirmed (1 wpi), and after 2 weeks rest period Samples were administered at the time of starting material administration (2 wpi) and for 2 weeks, and body weight was measured at the end of the test (3 w.pi) (see FIG. 1). Feeding and drinking water consumption were measured once a week.

As a result, no significant difference in body weight, feed intake, and negative intake was observed in all groups during the experimental period, which was expressed as the mean ± standard deviation in Tables 4 to 6 and 2 to 4 below.

Weight change during the experiment Group HP Treatment Body weight (g) (-1) w.p.i 0 w.p.i 1 w.p.i 2 w.p.i 3 w.p.i G1 - D.W 13.5 ± 0.53 16.9 ± 0.74 19.9 ± 1.54 21.0 ± 1.41 21.8 ± 1.72 G2 + D.W 14.5 ± 1.09 18.2 ± 0.70 20.8 ± 1.11 21.7 ± 1.57 23.4 ± 1.17 G3 AMX + CLR + PPI 15.1 ± 1.28 19.4 ± 1.06 21.1 ± 1.44 22.8 ± 1.11 24.1 ± 1.83 G4 Licorice extract 14.5 ± 0.25 18.4 ± 1.12 20.7 ± 1.23 21.3 ± 1.30 22.1 ± 1.51 G5 100 mg / kg pine tree by-product extract 14.1 ± 0.92 18.7 ± 0.90 20.8 ± 1.24 21.9 ± 1.51 22.5 ± 1.38 G6 200 mg / kg pine tree by-product extract 15.0 ± 1.20 19.1 ± 1.19 19.9 ± 1.83 21.3 ± 0.97 22.6 ± 1.44 G7 400 mg / kg pine tree by-product extract 15.1 ± 0.90 18.9 ± 1.08 20.7 ± 1.15 21.6 ± 1.62 22.8 ± 1.96

Changes in feed intake during the experiment Group HP Treatment Food consumption (g) (-1) w.p.i 0 w.p.i 1 w.p.i 2 w.p.i 3 w.p.i G1 - D.W 2.1 ± 0.24 3.2 ± 0.05 3.0 ± 0.42 3.1 ± 0.32 3.2 ± 0.17 G2 + D.W 2.6 ± 0.17 3.1 ± 0.47 2.9 ± 0.42 2.9 ± 0.07 3.1 ± 0.69 G3 AMX + CLR + PPI 2.7 ± 0.27 3.1 ± 0.42 2.8 ± 0.24 3.0 ± 0.13 3.3 ± 0.35 G4 Licorice extract 2.4 ± 0.26 3.0 ± 0.09 2.8 ± 0.15 3.0 ± 0.14 3.2 ± 0.10 G5 100 mg / kg pine tree by-product extract 2.2 ± 0.11 2.8 ± 0.29 3.0 ± 0.05 3.2 ± 0.26 3.5 ± 0.17 G6 200 mg / kg pine tree by-product extract 2.2 ± 0.22 2.9 ± 0.16 2.8 ± 0.10 3.4 ± 0.27 3.5 ± 0.10 G7 400 mg / kg pine tree by-product extract 2.4 ± 0.12 3.0 ± 0.25 3.0 ± 0.28 3.5 ± 0.20 3.6 ± 0.10

Changes in negative intake during the experiment Group HP Treatment Water consumption (ml) (-1) w.p.i 0 w.p.i 1 w.p.i 2 w.p.i 3 w.p.i G1 - D.W 3.7 ± 0.16 4.2 ± 0.31 4.1 ± 0.12 4.7 ± 0.96 5.3 ± 0.44 G2 + D.W 4.1 ± 1.02 4.3 ± 0.61 4.2 ± 1.05 4.9 ± 0.78 5.3 ± 0.72 G3 AMX + CLR + PPI 3.9 ± 0.71 4.5 ± 0.86 4.6 ± 0.77 5.2 ± 0.82 5.2 ± 1.00 G4 Licorice extract 3.6 ± 0.22 3.9 ± 0.13 3.8 ± 0.19 4.6 ± 0.37 5.2 ± 0.36 G5 100 mg / kg pine tree by-product extract 4.0 ± 0.59 4.1 ± 0.29 4.1 ± 0.30 4.8 ± 0.47 5.5 ± 0.48 G6 200 mg / kg pine tree by-product extract 3.6 ± 0.56 4.1 ± 0.51 4.0 ± 0.21 4.6 ± 0.72 5.1 ± 0.18 G7 400 mg / kg pine tree by-product extract 3.7 ± 0.32 4.7 ± 0.00 4.5 ± 0.60 4.9 ± 0.45 5.5 ± 0.48

(4) Blood Helicobacter antibody IgG titer comparison result

After 1 week of infection, blood was collected from facial skin of H. pylori-infected mice, and then separated plasma and blood collected from the abdominal vein at the end of the experiment were separated and plasma separated using Mouse H. pylori antibody and IgG ELISA kit. My H. pylori antibody titers were measured and compared between groups, and the results are shown in Tables 7 and 5 below. Data were expressed as mean ± standard deviation, statistical analysis was performed using sigma plot statistics, and p values were set at a level comparable to G2. ** p <0.01, ns: not significant.

Helicobacter antibody IgG evaluation results (4 weeks after infection) Group HP Treatment H. pyloriIgG test G1 - D.W 0.25 ± 0.06 ^** G2 + D.W 0.63 ± 0.18 G3 AMX + CLR + PPI 0.41 ± 0.04 ^** G4 Licorice extract 0.33 ± 0.07 ^** G5 100 mg / kg pine tree by-product extract 0.41 ± 0.09 ^** G6 200 mg / kg pine tree by-product extract 0.41 ± 0.11 ^** G7 400 mg / kg pine tree by-product extract 0.44 ± 0.11 ^**

** p <0.01

As a result of calculating the mean and standard deviation by measuring H. pylori antibody in autopsy blood, G1 (negative control): 0.25 ± 0.06, G2 (excipient control): 0.63 ± 0.18, G3 (positive control 1: AMX + CLR + PPI administration group): 0.41 ± 0.04, G4 (positive control group 2: licorice extract administration group): 0.33 ± 0.07, G5 (100 mg / kg pine tree by-product extract group): 0.41 ± 0.09, G6 (200 mg / kg pine tree by-product extract) Administration group): 0.41 ± 0.11, G7 (400 mg / kg pine tree by-product by-product extract administration group): 0.44 ± 0.11.

H. pylori-infected G2 (excipient control) was found to have a statistically significant H. pylori infection by measuring an increase in H. pylori antibody by 152.0% or more compared to H. pylori-uninfected G1 (negative control) ( p <0.01). The positive control substances G3 (positive control 1: AMX + CLR + PPI administration group) and G4 (positive control 2: licorice extract administration group) compared to G2 (excipient control), respectively 34.9%, 47.6% statistically significant H. pylori antibody Decreased (p <0.01). G5 (100 mg / kg administered group), G6 (200 mg / kg administered group), and G7 (400 mg / kg administered group) of the test substance pine tree by-product extract were statistically 34.9%, 34.9%, and 30.2%, respectively, compared to G2 (excipient control group). H. pylori antibody decreased significantly (p <0.01).

G5 (100 mg / kg administration group) and G6 (200 mg / kg administration group) of the test substance by-product by-product byproduct extract exhibited a decrease in equivalent antibody compared to G3 (positive control group 1: AMX + CLR + PPI administration group).

The results of H. pylori antibody test are arranged in the following order.

Licorice extract <100 mg / kg pine tree byproduct extract, 200 mg / kg pine tree byproduct extract and AMX + CLR + PPI <400 mg / kg pine tree byproduct extract.

(5) Observation of histological gross lesions and examination of histopathological analysis

On the day of autopsy, the gastric tissue extracted from the esophagus (esophagus) along the great curvature (great curvature) and incised longitudinally toward the intestine (duodenum, duodenum) was opened to observe specific lesions of the inner mucosa. After observation of visual lesions, the unfolded stomach tissue is fixed in 10% neutral formalin, paraffin embedded using a conventional method for histopathological examination, sectioned into 4 μm thick, stained with hematoxylin and eosin (H & E), and histopathological examination Was performed. The histopathological scores were the degree of infiltration of neutrophils & mononuclear cells (indicated by yellow arrows) and the degree of atrophic gastritis with atrophic deformation (yellow in FIG. 7), which was shown in the entire area of Corpus and Antrum per subject. The dotted line) was observed according to the criteria in Table 8, and the grades of each tissue were converted into scores and recorded (see FIGS. 7 and 8). After the sum of the detailed scores for each organization was obtained, the average score of each group was obtained, and in order to increase objectivity, scores were calculated through observation and consultation of two different researchers.

As a result of observing the gastric tissue extracted after autopsy, specific lesions were not observed in the non-infected group G1 (negative control group) and the infected groups G2 to G7 (FIG. 6).

As a result of histopathology, the findings observed in the above tissues are reference 1 (Lee YJ.No Correlation of Inflammation with Colonization of Helicobacter pylori in the Stomach of Mice Fed High-salt Diet.Journal of Cancer Prevention (2014) 19 (2): 144 As a result of scoring for inflammatory cell infiltration and atrophic change according to the criteria of -151.), Inflammatory cell infiltration in G1 (negative control): 0.75 ± 0.50, and gastritis symptoms due to atrophy deformation were observed. Did not. Total score: 0.30 ± 0.48; In G2 (excipient control), inflammatory cell infiltration: 2.38 ± 0.74, atrophy: 1.00 ± 1.07, Total score: 3.38 ± 1.77; In G3 (positive control group 1: AMX + CLR + PPI administration group), inflammatory cell infiltration: 1.17 ± 0.72, atrophy: 0.08 ± 0.29, Total score: 1.25 ± 0.87; In G4 (positive control group 2: licorice extract administration group), inflammatory cell infiltration: 1.00 ± 0.95, atrophy: 0.17 ± 0.58, Total score: 1.17 ± 1.40; In the case of G5 (100 mg / kg pine tree by-product extract group), inflammatory cell infiltration: 1.17 ± 1.03, atrophic deformation: 0.08 ± 0.29, Total score: 1.25 ± 1.22; In the case of G6 (group administered with 200 mg / kg pine tree by-product extract), inflammatory cell infiltration: 0.92 ± 0.67, atrophic deformation: 0.08 ± 0.29, Total score: 1.00 ± 0.85; In G7 (400 mg / kg pine tree byproduct extract administration group), inflammatory cell infiltration: 0.92 ± 1.16, atrophic deformation: 0.17 ± 0.39, and total score: 1.08 ± 1.51 were observed. As a result of comparing the total score for each group, a statistically significant increase was observed in G2 (excipient control) compared to G1 (negative control) (p <0.01), and the positive control G3 (positive control 1: AMX + CLR + PPI). Administration group) and G4 (positive control group 2: licorice extract administration group) showed a statistically significant decrease of 63.0% and 65.4%, respectively, compared to G2 (excipient control group) (p <0.01). G5 (100 mg / kg administration group), G6 (200 mg / kg administration group), and G7 (400 mg / kg administration group) of the test substance pine tree by-product extract were statistically 63.0%, 70.4%, 68.0%, respectively, compared to G2 (excipient control group). A significant decrease was observed (p <0.01, FIGS. 7 to 10, Table 8).

The results of histopathological examination, H. pylori infection, the findings of inflammation and atrophy deformation by administration of test substance are summarized as follows.

200 mg / kg pine tree byproduct extract <400 mg / kg pine tree byproduct extract <licorice extract <AMX + CLR + PPI and 100 mg / kg pine tree byproduct extract

Score of histopathological examination in gastric tissue Group HP Treatment Histologic Score Inflammation Atrophic change Total score G1 - D.W 0.75 ± 0.50 ^** 0.00 ± 0.00 ^* 0.30 ± 0.48 ^** G2 + D.W 2.38 ± 0.74 1.00 ± 1.07 3.38 ± 1.77 G3 AMX + CLR + PPI 1.17 ± 0.72 ^** 0.08 ± 0.29 ^* 1.25 ± 0.87 ^** G4 Licorice extract 1.00 ± 0.95 ^** 0.17 ± 058 ^* 1.17 ± 1.40 ^** G5 100 mg / kg pine tree by-product extract 1.17 ± 1.03 ^** 0.08 ± 0.29 ^* 1.25 ± 1.22 ^** G6 200 mg / kg pine tree by-product extract 0.92 ± 0.67 ^** 0.08 ± 0.29 ^* 1.00 ± 0.85 ^** G7 400 mg / kg pine tree by-product extract 0.92 ± 1.16 ^** 0.17 ± 0.39 ^* 1.08 ± 1.51 ^**

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were determined at a level comparable to the G2 group. ** p <0.01, * p <0.05

(6) Rapid urease test results

The rapid urease test, when H. pylori is present in the gastric mucosa, secretes urease as bacteria grow in the medium of the test reagent, hydrolyzes the urea present in the test reagent, generates ammonia, and generates ammonia to generate the total pH of the reagent. And the color change (red) of the pH indicator included in the test reagent was examined.

1) Treatment rate calculation

Gastric mucosal tissues extracted on the day of autopsy were aseptically collected and tested using a CLO (campylobacter-like organism) test reagent (Asan Pharm Co., Ltd., Korea). If the color of the result changes from yellow to red after incubation at 37 ° C for 2 hours in the incubator, it is judged as positive and the number of individuals determined to be positive is determined as a percentage to obtain the positive rate, and the treatment rate for H. pylori bacteria by sample treatment is as follows It was obtained by the equation.

As a result of calculating the percentage of positive specimens by the CLO Rapid Enzyme Test, G1 (negative control) 0%; G2 (excipient control) 100%; G3 (positive control 1: AMX + CLR + PPI administration group) 40%; G4 (positive control 2: licorice extract administration group) 50%; G5 (100 mg / kg pine tree byproduct extract group) 50%, G6 (200 mg / kg pine tree byproduct extract group) 40%, G7 (400 mg / kg pine tree byproduct extract group) 40%.

As a result of calculating the treatment rate of each test group, G2 (excipient control) 0% was statistically significant. Infection with pylori was confirmed (p <0.01), and the positive control substances G3 (positive control 1: AMX + CLR + PPI administration group) and G4 (positive control 2: licorice extract administration group) were statistically 60% and 50%, respectively. There was a significant treatment rate (p <0.01). Treatment rate of 50%, 60%, and 60% of G5 (100 mg / kg administered group), G6 (200 mg / kg administered group), and G7 (400 mg / kg administered group) of test substance by-product by-product extract, respectively, compared to G2 (excipient control group) And statistical significance was confirmed (p <0.01, Table 9-10).

As a result of the CLO test, the treatment rates by administration of test substances in H. pylori infection are summarized as follows.

400 mg / kg pine tree byproduct extract, 200 mg / kg pine tree byproduct extract and AMX + CLR + PPI> 100 mg / kg pine tree byproduct extract and licorice extract.

Sample of rapid urease (CLO) test Group HP Treatment Animal No. Ratio One 2 3 4 5 6 7 8 9 10 Positive Partially
positive Negative G1 - D.W ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ 0/10 0/10 10/10 G2 + D.W ● ● ● ● ● ● ● ● ● ● 10/10 0/10 0/10 G3 + AMX + CLR + PPI ○ ● ○ ◐ ○ ○ ○ ○ ◐ ● 2/10 2/10 6/10 G4 + Licorice extract ◐ ● ◐ ○ ○ ○ ○ ○ ◐ ● 2/10 3/10 5/10 G5 + 100 mg / kg pine tree by-product extract ○ ○ ○ ◐ ◐ ◐ ○ ● ● ○ 2/10 3/10 5/10 G6 + 200 mg / kg pine tree by-product extract ○ ○ ○ ◐ ◐ ○ ○ ○ ● ◐ 1/10 3/10 6/10 G7 + 400 mg / kg pine tree by-product extract ○ ○ ◐ ● ◐ ○ ○ ◐ ○ ○ 1/10 3/10 6/10

○, negative; Partially, partially positive; ●, positive

Treatment rate of rapid urease (CLO) in gastric tissue Group HP Treatment Number of mouse Positive Number Therapeutic G1 - D.W 10 0 100% ^** G2 + D.W 10 10 0% G3 AMX + CLR + PPI 10 4 60% ^** G4 Licorice extract 10 5 50% ^** G5 100 mg / kg pine tree by-product extract 10 5 50% ^** G6 200 mg / kg pine tree by-product extract 10 4 60% ^** G7 400 mg / kg pine tree by-product extract 10 4 60% ^**

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were determined at a level comparable to the G2 group. ** p <0.01

2) CLO score

After the CLO test, the average and standard deviation of each group were determined by measuring 0 points for no color change of the medium, 1 point for slightly red, 2 points for light purple, and 3 points for dark purple. The difference in values between the groups is compared and shown in Table 11 and FIGS. 11-12 below. Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were determined at a level comparable to the G2 group. (** p <0.01, ns: not significant)

As a result of calculating the average and standard error by measuring rapid urease, G1 (negative control): 0.00 ± 0.00; G2 (excipient control): 3.00 ± 0.00; G3 (positive control 1: AMX + CLR + PPI administered group): 0.90 ± 1.29; G4 (positive control group 2: licorice extract administration group): 1.10 ± 1.29; G5 (100 mg / kg pine tree by-product extract group): 1.10 ± 1.29; G6 (200 mg / kg pine tree by-product extract group): 0.60 ± 0.97; G7 (400 mg / kg pine tree by-product extract group): 0.70 ± 1.06, respectively (Figure 11, Table 10, Appendix 1). G1 (negative control) compared to G2 (excipient control) showed a statistically significant increase. (P <0.01), positive control substances G3 (positive control 1: AMX + CLR + PPI administration group), G4 (positive control 2: licorice extract administration group) compared to G2 (excipient control), respectively 70.0%, 63.3% statistically There was a significant decrease (p <0.01). G3.3 (100 mg / kg administration group), G6 (200 mg / kg administration group), and G7 (400 mg / kg administration group) of the test substance by-product by-product extract were statistically 63.3%, 80.0%, 76.7%, respectively, compared to G2 (excipient control group). Showed a significant decrease (p <0.01).

The CLO scores are as follows.

200 mg / kg pine tree byproduct extract <400 mg / kg pine tree byproduct extract <AMX + CLR + PPI <100 mg / kg pine tree byproduct extract and licorice extract

Rapid urease (CLO) score Group HP Treatment CLO score G1 - D.W 0.00 ± 0.00 ^** G2 + D.W 3.00 ± 0.00 G3 AMX + CLR + PPI 0.90 ± 1.29 ^** G4 Licorice extract 1.10 ± 1.29 ^** G5 100 mg / kg pine tree by-product extract 1.10 ± 1.29 ^** G6 200 mg / kg pine tree by-product extract 0.60 ± 0.97 ^** G7 400 mg / kg pine tree by-product extract 0.70 ± 1.06 ^**

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were determined at a level comparable to the G2 group. ** p <0.01

(7) Analysis of cytokine in gastric mucosal tissue (TNF-α, IL-1 β ) result

To measure the pro-inflammatory cytokine in gastric mucosal tissues, the gastric tissues collected aseptically were finely crushed with liquid nitrogen, and proteins were extracted using cell lysis buffer for analysis. The isolated protein was analyzed for TNF-α (tumor necrosis factor-α) and Interleukin-1β using an ELISA kit purchased from the R & D system (Minneapolis, MN, USA).

As a result of calculating the mean and standard error by measuring TNF-α in gastric mucosal tissue, G1 (negative control): 5.02 ± 0.44; G2 (excipient control): 7.41 ± 1.66; G3 (positive control 1: AMX + CLR + PPI administered group): 5.00 ± 1.19; G4 (positive control group 2: licorice extract administration group): 4.89 ± 1.10; G5 (100 mg / kg pine nut by-product extract group): 4.32 ± 0.86; G6 (200 mg / kg pine tree byproduct extract group): 3.83 ± 0.69; G7 (400 mg / kg pine tree by-product extract administration group): 4.08 ± 0.84, respectively (FIG. 13, Table 12). G1 (negative control) compared to G2 (excipient control) showed a statistically significant increase of 47.6%, (p <0.01), positive control substances G3 (positive control 1: AMX + CLR + PPI administration group), G4 (positive control 2: licorice extract administration group) compared to G2 (excipient control) 32.5%, 34.0% statistically significant There was one decrease (p <0.01). Statistically, 41.7%, 48.3%, and 44.9% of G2 (100 mg / kg administered group), G6 (200 mg / kg administered group), and G7 (400 mg / kg administered group) of test substance pine tree by-product extract were compared with G2 (excipient control), respectively. Showed a significant decrease (p <0.01). The test substances G6 (200 mg / kg administration group) and G7 (400 mg / kg administration group) were statistically significantly reduced by 23.4% and 18.4%, respectively, compared to G3 (positive control group 1: AMX + CLR + PPI administration group) (p < 0.01, p <0.05), and G4 (positive control group 2: licorice extract-administered group) were statistically significantly reduced by 21.7% and 16.6%, respectively (p <0.05).

The TNF-α values are as follows.

200 mg / kg pine tree byproduct extract <400 mg / kg pine tree byproduct extract <100 mg / kg pine tree byproduct extract <licorice extract <AMX + CLR + PPI

As a result of calculating the mean and standard error by measuring IL-1β in gastric mucosal tissue, G1 (negative control): 17.91 ± 3.43; G2 (excipient control): 25.26 ± 4.77; G3 (positive control 1: AMX + CLR + PPI administered group): 16.2 ± 2.76; G4 (positive control group 2: licorice extract administration group): 18.47 ± 2.86; G5 (100 mg / kg pine tree byproduct extract group): 13.42 ± 3.55; G6 (200 mg / kg pine tree by-product extract group): 14.06 ± 3.57; G7 (400 mg / kg pine tree by-product extract administration group): 11.98 ± 2.71, respectively (FIG. 14, Table 13).

Compared to G1 (negative control), G2 (excipient control) showed a statistically significant increase of 41.0% (p <0.01), positive control substance G3 (positive control 1: AMX + CLR + PPI administration group), G4 (positive) Control group 2: Licorice extract administration group) showed statistically significant reduction of 35.9% and 26.9%, respectively, compared to G2 (excipient control group) (p <0.01). G5 (100 mg / kg administration group), G6 (200 mg / kg administration group), and G7 (400 mg / kg administration group) of the test substance pine tree by-product extract were 46.9%, 44.3%, and 52.6% statistically, respectively, compared to G2 (excipient control group). Showed a significant decrease (p <0.01). G5 (100 mg / kg administration group) and G7 (400 mg / kg administration group) showed statistically significant decreases of 17.2% and 26.0%, respectively, compared to G3 (positive control group 1: AMX + CLR + PPI administration group) (p < 0.05, p <0.01), G5 (100 mg / kg administration group), G6 (200 mg / kg administration group) and G7 (400 mg / kg administration group) compared to G4 (positive control group 2: licorice extract administration group), respectively, 27.3%, The statistically significant decrease was 23.9% and 35.1% (p <0.01).

The IL-1β values are as follows.

400 mg / kg pine tree byproduct extract <100 mg / kg pine tree byproduct extract <200 mg / kg pine tree byproduct extract <AMX + CLR + PPI <licorice extract

TNF-α cytokine values in gastric tissue Group HP Treatment TNF-α
(pg / μg tissues) G1 - D.W 5.02 ± 0.44 ^** G2 + D.W 7.41 ± 1.66 G3 AMX + CLR + PPI 5.00 ± 1.19 ^** G4 Licorice extract 4.89 ± 1.10 ^** G5 100 mg / kg pine tree by-product extract 4.32 ± 0.86 ^** G6 200 mg / kg pine tree by-product extract 3.83 ± 0.69 ^{**, §§, ¶} G7 400 mg / kg pine tree by-product extract 4.08 ± 0.84 ^{**, §, ¶}

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were compared with the G2 group ** p <0.01, compared with the G3 group ^§§ p <0.01, ^§ p <0.05, compared with the G4 group ^¶ p <0.05.

IL-1β cytokine values in gastric tissue Group HP Treatment IL-1β
(pg / μg tissues) G1 - D.W 17.91 ± 3.43 ^** G2 + D.W 25.26 ± 4.77 G3 AMX + CLR + PPI 16.20 ± 2.76 ^** G4 Licorice extract 18.47 ± 2.86 ^** G5 100 mg / kg pine tree by-product extract 13.42 ± 3.55 ^{**, §, ¶¶} G6 200 mg / kg pine tree by-product extract 14.06 ± 3.57 ^{**, ¶¶} G7 400 mg / kg pine tree by-product extract 11.98 ± 2.71 ^{**, §§, ¶¶}

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were compared with the G2 group ** p <0.01, compared with the G3 group ^§§ p <0.01, ^§ p <0.05, compared with the G4 group ^¶¶ p <0.01.

(8) Gastric mucosal H.pylori PCR (polymerase chain reaction) test results

After the test, genomic DNA was collected from the gastric mucosal tissue collected aseptically and Helicobacter PCR test was performed under the following conditions.The target gene used in this experiment was human, CagA, a toxin type gene specific to H. pylori. Or a gene that does not exist in mice. Therefore, the target size of H. pylori PCR was 298 bp specific band, and specific bands were identified for each group and positive individuals were determined.

1) PCR information-primer

H-cagA-F (5'-ATA ATG CTA AAT TAG ACA ACT TGAA GCG A)

H-cagA-R (5'-TTA GAA TAA TCA ACA AAC ATC ACG CCA T)

2) PCR information-reaction conditions

Denaturation at 94 ° Cfor 5 min 298bp 95 ° C for 1 min 35 cycles 57 ° Cfor 30 s 72 ° C for 30 s Final extension step72 ° C for 10 min,

In gastric mucosal tissue H. As a result of measuring the presence or absence of pylori by PCR, the percentage of positive specimens is as follows. G1 (negative control) 0%; G2 (excipient control) 100%; G3 (positive control 1: AMX + CLR + PPI administered group) 40%; G4 (positive control group 2: licorice extract administration group) 30%; G5 (100 mg / kg pine tree by-product extract group) 60%, G6 (200 mg / kg pine tree by-product extract group) 30%, G7 (400 mg / kg pine tree by-product extract group) 30%. As a result of calculating the treatment rate of each test group, G2 (excipient control) 0% was statistically significant. Infection with pylori was confirmed (p <0.01), and the positive control substances G3 (positive control 1: AMX + CLR + PPI administration group) and G4 (positive control 2: licorice extract administration group) were statistically 60% and 70%, respectively. There was a significant treatment rate (p <0.01). Test substance pine nuts or by-product extracts G40 (100 mg / kg administered group), G6 (200 mg / kg administered group), and G7 (400 mg / kg administered group) compared to G2 (excipient control group) 40%, 70%, 70% respectively Treatment rate was shown and statistical significance was confirmed (p <0.01, FIG. 15, Table 15-16).

As a result of PCR test, the treatment rate by administration of test substance in H. pylori infection is summarized as follows.

200 mg / kg pine tree byproduct extract, 400 mg / kg pine tree byproduct extract and licorice extract> AMX + CLR + PPI> 100 mg / kg pine tree byproduct extract.

PCR test samples Group HP Treatment Animal No. Ratio One 2 3 4 5 6 7 8 9 10 Positive Partially
positive Negative G1 - D.W ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ 0/10 0/10 10/10 G2 + D.W ● ● ● ● ● ● ● ● ● ● 10/10 0/10 0/10 G3 + AMX + CLR + PPI ○ ● ○ ◐ ○ ◐ ◐ ○ ○ ○ 1/10 3/10 6/10 G4 + Licorice extract ○ ○ ● ○ ● ○ ○ ● ○ ○ 3/10 0/10 7/10 G5 + 100 mg / kg pine tree by-product extract ● ● ○ ● ● ◐ ● ○ ○ ○ 5/10 1/10 4/10 G6 + 200 mg / kg pine tree by-product extract ○ ● ○ ○ ● ○ ○ ○ ○ ● 3/10 0/10 7/10 G7 + 400 mg / kg pine tree by-product extract ○ ○ ○ ● ○ ● ○ ○ ● ○ 3/10 0/10 7/10

○, negative; Partially, partially positive; ●, positive

PCR test treatment rate Group HP Treatment Number of mouse Positive Number Therapeutic G1 - D.W 10 0 100% ^** G2 + D.W 10 10 0% G3 AMX + CLR + PPI 10 4 60% ^** G4 Licorice extract 10 3 70% ^** G5 100 mg / kg pine tree by-product extract 10 6 40% ^** G6 200 mg / kg pine tree by-product extract 10 3 70% ^** G7 400 mg / kg pine tree by-product extract 10 3 70% ^**

Data are expressed as mean ± standard deviation. Statistical analysis was performed using sigma plot statistics, and p-values were determined at a level comparable to the G2 group. ** p <0.01

(9) Helicobacter bacterial antigen test results in feces

To confirm whether H. pylori bacteria are excreted in the feces, the mouse feces collected at the end of the test are collected to obtain SD BiolineH. Fecal antigen testing was performed using a pylori Ag kit (Standard Diagnostics, Inc).

To determine whether H. pylori bacteria are excreted in the feces, the results obtained by taking and measuring mouse feces collected at the end of the test are G1 (negative control), G2 (excipient control), and G3 (positive control 1: AMX + CLR + PPI administration group). ), G4 (positive control group 2: licorice extract administration group), G5 (100 mg / kg pine tree byproduct extract administration group), G6 (200 mg / kg pine tree byproduct extract administration group), G7 (400 mg / kg pine tree byproduct extract administration group) all individuals In the H. pylori Stool Ag test test, a positive individual could not be found (FIG. 16).

As a result of the disinfection test, when the pine tree by-product extract was administered orally, H. pylori antibody was reduced and the treatment rate was increased in H. pylori-infected mouse models, histopathological lesions were improved, and inflammatory cytokine was reduced, which was effective for H. pylori disinfection. Appeared.

That is, it was found that in the environment in which Helicobacter pylori is present, coexistence of a pine tree by-product extract has an effect of selectively inhibiting the proliferation of Helicobacter pylori.

Claims (1)

Functional food composition for improving bactericidal effect of Helicobacter pylori which contains 40% by weight of pine tree conifer distilled water extract and 60% by weight of tapioca starch.

Images