阅读说明：本技术 用于预防或治疗幽门螺杆菌感染的包含忍冬花水提取物的药物组合物 (Pharmaceutical composition comprising aqueous extract of honeysuckle flower for preventing or treating helicobacter pylori infection ) 是由 金点溶 朴宣奎 张珉廷 李宗勋 林洙焕 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种用于预防或治疗幽门螺杆菌感染的忍冬花水提取物组合物,该组合物包含断氧化马钱子苷作为活性成分。所述提取物包含特定量的断氧化马钱子苷,由此,当用于幽门螺杆菌时表现出优异的抗菌效果,并且当用于感染幽门螺杆菌的小鼠时,在降低血液中幽门螺杆菌IgG抗体的表达、减轻组织病理学病变和减少细胞因子表达方面表现出优异的效果。因此,本发明的忍冬花水提取物可以有效地用作用于预防或治疗幽门螺杆菌感染的组合物。(The invention relates to a honeysuckle flower water extract composition for preventing or treating helicobacter pylori infection, which comprises secologanin oxide as an active ingredient. The extract contains a specific amount of secologlycoside, thereby exhibiting excellent antibacterial effects when used for helicobacter pylori, and excellent effects in reducing the expression of helicobacter pylori IgG antibody in blood, alleviating histopathological changes, and reducing cytokine expression when used for helicobacter pylori-infected mice. Therefore, the aqueous extract of honeysuckle flower of the present invention can be effectively used as a composition for preventing or treating helicobacter pylori infection.)

1. A pharmaceutical composition for preventing or treating helicobacter pylori infection comprises secologanin in the form of an aqueous extract of honeysuckle flower as an active ingredient.

2. The pharmaceutical composition for preventing or treating helicobacter pylori infection according to claim 1, wherein the content of loganin oxide interrupted in the aqueous extract of honeysuckle flower is 0.1 to 10% by weight.

3. The pharmaceutical composition for preventing or treating helicobacter pylori infection according to claim 1, wherein the content of loganin oxide interrupted in the aqueous extract of honeysuckle flower is 0.5 to 5% by weight.

4. The pharmaceutical composition for preventing or treating helicobacter pylori infection according to any one of claims 1 to 3, wherein the pharmaceutical composition is formulated into a pharmaceutical dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent.

5. A health functional food for preventing or improving helicobacter pylori infection comprises secologanin oxide in the form of aqueous extract of Lonicera japonica flower as active ingredient.

6. The health functional food for preventing or ameliorating helicobacter pylori infection according to claim 5, wherein the content of loganin oxide in the aqueous extract of honeysuckle flower is 0.1 to 10% by weight.

7. The health functional food for preventing or ameliorating helicobacter pylori infection according to claim 5, wherein the content of loganin oxide in the aqueous extract of honeysuckle flower is 0.5 to 5% by weight.

8. The health-care functional food for preventing or ameliorating helicobacter pylori infection according to any one of claims 5 to 7, wherein a preparation of the health-care functional food is selected from a tablet, a capsule, a pill or a liquid.

9. A pharmaceutical composition for preventing or treating helicobacter pylori infection comprises secologanin as an active ingredient.

Technical Field

The present invention relates to a pharmaceutical composition for preventing or treating helicobacter pylori infection, comprising an aqueous extract of honeysuckle flower.

Background

Helicobacter pylori (h. pyrori) was first discovered in 1983 by Marshall and Warren in australia. Helicobacter pylori is reported to be a spiral microaerophilic gram-negative bacterium having 4 to 6 flagella and infects more than half of the world population, and in korea, it has been reported that the infection rate of adults over 40 years old is 70% or more. Helicobacter pylori is mainly known to be a pathogenic microorganism closely related to upper gastrointestinal diseases, causing chronic gastritis, gastroduodenal ulcer, gastric cancer and the like. In particular, the international institute for cancer (IARC) and world health organization define helicobacter pylori as a class I carcinogen. In the prevention and diagnosis of gastric cancer, infection with helicobacter pylori is considered to be a very important problem, and identification of the infection factor of helicobacter pylori is being considered to be an important problem concerning national health.

Helicobacter pylori has 5 to 6 flagella like the tail of an animal as a locomotor, and adheres to the surface of the gastric mucosa with the flagella passing directly through the viscous mucus on the stomach wall. Urease in a subjectIs well conserved genetically, a common feature found in most strains of H.pylori. Urease hydrolyzes urea to ammonia (NH)₃) And carbon dioxide (CO)₂) So that bacteria survive in the gastric mucosa having a low pH at which bacteria cannot survive to increase the surrounding pH and neutralize gastric acid, whereby bacteria survive. The cell damage and mutation in the gastric mucosa are caused by the influence of the produced ammonia, thereby losing the gastric acid defense effect, and the vicious circle of attacking the gastric mucosa by gastric acid is generated, causing gastric tissue lesion and inflammatory reaction. Thus, gastric mucositis caused by various cytotoxic factors secreted by helicobacter pylori induces activation of various inflammatory cells such as lymphocytes and macrophages. It is reported that inflammatory cells have adverse effects such as tissue damage, cytotoxicity, and the like in vivo, and induce inflammatory induction mediators to worsen inflammation.

Helicobacter pylori is a very dangerous microorganism in gastrointestinal diseases, and development of suitable antibacterial substances is lacking. Currently, as a method for treating helicobacter pylori, there is generally a triple therapy of combination treatment of bismuth, metronidazole and tetracycline or amoxicillin; and a tetranect therapy in which bismuth agent, omeprazole, tetracycline and metronidazole are mixed. These treatments are reported to have excellent effects on the treatment of helicobacter pylori. However, it has been reported that repeated use of these drugs causes increased antibiotic resistance and various side effects, and at present, efforts are continuously being made to find extracts and active ingredients capable of inhibiting helicobacter pylori using various natural substances.

Honeysuckle (lonicera floras), a flower of the genus Lonicera of the family Caprifoliaceae, has been used in oriental medicine and non-official medicines for diuresis, stomach strengthening, arthritis, suppurative dermatitis, and bronchitis.

As constituents of honeysuckle flower, tannic acid, inositol, sterol, chlorogenic acid, isochlorogenic acid, etc. are reported, and as flavonoid constituents, luteolin, apigenin, luteolin-7-rhamnoside, luteolin-7-O-rhamnoside, quercetin, etc. are reported. Flavonoid components of honeysuckle flower are known to have anti-inflammatory and anti-mutagenic effects.

As prior art relating to compositions for the prevention or treatment of helicobacter pylori infection comprising aqueous extracts of honeysuckle flowers, the anti-helicobacter pylori effect of various natural aqueous extracts including honeysuckle flower extracts is disclosed in the prior documents [ Ma, F.et al, World J gastroenterol, 16(44), 5629-. Further, in the prior art [ Oku, h.et al, biol.pharm.bull.34 (8),1330-1333,2011], a compound derived from honeysuckle flower comprising secologlycoside (secoxylogenin) and an antiallergic effect thereof are disclosed, and in the prior art [ Xiong, j.et al, Food Chemistry,138,327-333,2013], an antibacterial effect of secologlycoside on escherichia coli and staphylococcus aureus is disclosed.

In studying the water extract of honeysuckle flower, the present inventors found that, among the active ingredients of the water extract of honeysuckle flower, in particular, secologanin oxide exhibits a selective antibacterial action against helicobacter pylori of 20 times or more of the antibacterial action against escherichia coli and staphylococcus aureus under the same conditions. Further, the present inventors found that an aqueous extract of honeysuckle flower containing a predetermined content or more of secologanin oxide has excellent effects of inhibiting and treating helicobacter pylori infection, thereby completing the present invention.

Disclosure of Invention

Technical problem

An object of the present invention is to provide a pharmaceutical composition or health functional food comprising an aqueous extract of lonicera japonica for preventing or treating helicobacter pylori infection.

Technical scheme

The present invention relates to a pharmaceutical composition for preventing or treating helicobacter pylori infection, which comprises secologanin oxide in the form of an aqueous extract of lonicera japonica flower as an active ingredient.

The aqueous extract of honeysuckle flower may be obtained by mixing the honeysuckle flower with 1500 to 2500 parts by weight of water with respect to 100 parts by weight of the honeysuckle flower, and then extracting the mixture at a temperature of 90 ℃ or higher for 1 hour or more, and as the extraction device, a conventional extraction device, an ultrasonic extractor, or a fractionation device may be used. Preferably, the mixture is extracted at a temperature of 90 ℃ or higher for 1 to 5 hours to contain 0.1 to 10% by weight of the active ingredient split loganin oxide.

The secoisolaricoside as an active ingredient contained in the aqueous extract of lonicera japonica flower is contained in an amount of 0.1 to 10% by weight in the aqueous extract of lonicera japonica flower. When the content is less than 0.1% by weight or more than 10% by weight, exceeding the range of the% by weight is not preferable because the effect of inhibiting helicobacter pylori infection is low, or is not preferable because the effect of inhibiting helicobacter pylori infection is not increased much and is uneconomical. Most preferably, in order to effectively treat helicobacter pylori infection, the secologenin as an active ingredient contained in the aqueous extract of honeysuckle flower is contained in an amount of 0.5 to 5% by weight in the aqueous extract of honeysuckle flower.

Helicobacter pylori infection is a disease that infects helicobacter pylori and may be selected from gastritis, gastric ulcer, duodenal ulcer, non-ulcer dyspepsia syndrome, gastric MALT lymphoma, polyps of gastric hyperplasia, gastric cancer, digestive tract cancer, pancreatitis, inflammatory bowel disease, and functional digestive system disorders (abdominal satiety, abdominal pain, hiccups, abdominal distension, initial satiety, nausea, vomiting, reflux, heartburn, anorexia, etc.). The aqueous honeysuckle flower extract of the present invention exhibits a particularly selective therapeutic effect on helicobacter pylori infectious diseases, as compared with helicobacter pylori non-infectious diseases.

The pharmaceutical composition according to the present invention may be formulated in an appropriate form with a commonly used pharmaceutically acceptable carrier. "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions, such as gastrointestinal disorders, dizziness, and the like, or reactions similar thereto, when administered to a human. Further, the composition may be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods; an external preparation; suppositories; and the preparation and use in the form of sterile injection.

Carriers, excipients, and diluents that may be included in the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, but are not limited thereto. The formulations can be prepared by using commonly used diluents or excipients, such as fillers, stabilizers, binders, disintegrants, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and can be prepared by mixing one or more excipients, for example, starch, microcrystalline cellulose, sucrose or lactose, low-substituted hydroxypropyl cellulose, hypromellose and the like, with the extract of the present invention. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral administration may correspond to suspensions, oral liquids, emulsions, syrups and the like, and may contain various excipients, for example, wetting agents, sweeteners, aromatics, preservatives and the like, in addition to water and liquid paraffin, which are generally used as simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilizates and suppositories. As the non-aqueous solution and the suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, ester for injection such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, polyethylene glycol, Tween61, cacao butter, lauryl alcohol, glycerin, gelatin, etc. can be used. For preparation of formulations for parenteral administration, the extract or pharmaceutically acceptable salt is sterilized and/or mixed with preservatives, stabilizers, wettable powders or emulsions, adjuvants such as salts and/or buffers for adjusting osmotic pressure, and other therapeutically useful materials in water to prepare solutions or suspensions, and the prepared solutions or suspensions may be prepared by means of ampules or vial unit dose types.

The pharmaceutical composition comprising the extract as an active ingredient disclosed in the present invention can be administered to mammals such as rats, domestic animals and humans by various routes. All methods of administration are contemplated, for example, the pharmaceutical composition may be administered orally, rectally or intravenously, intramuscularly, subcutaneously, intracerebral dural (intrauterine) injection or cerebrovascular injection. The dosage may vary according to the age, sex, and weight of the subject being treated, the particular disease or condition being treated, the severity of the disease or condition, the time of administration, the route of administration, absorption, distribution, and excretion rates of the drug, the type of other drug used, the judgment of the prescriber, and the like. Dosage determinations based on these factors are within the ability of those skilled in the art, and typically, dosages are in the range of 0.01 mg/kg/day to about 2000 mg/kg/day. More preferred doses are from 1 mg/kg/day to 500 mg/kg/day. Administration may be once a day or several times a day. The dosage does not limit the scope of the invention in any way.

In another aspect, the present invention relates to a health functional food for preventing or ameliorating helicobacter pylori infection, which comprises secologanin oxide in the form of an aqueous extract of honeysuckle flower as an active ingredient.

The health functional food means a food prepared or processed by using raw materials or components having effective functions, and may include, for example, health products, functional foods, nutrients, auxiliary materials, and the like.

The added amount of the extract is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 10% by weight, relative to the total weight of the whole health functional food. The health functional food of the present invention includes forms such as tablets, capsules, pills, or liquid, and the like, and foods to which the extract of the present invention can be added include, for example, various foods, drinks, chewing gums, teas, multivitamins, and the like.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating helicobacter pylori infection, which comprises secologlycoside as an active ingredient.

Advantageous effects

The invention relates to a pharmaceutical composition for preventing or treating helicobacter pylori infection, which comprises water extract of honeysuckle flower with secologanin oxide as an active ingredient. The extract contains a specific content of secologlycoside, thereby exhibiting excellent antibacterial effects when treating helicobacter pylori treatment, and excellent effects in reducing the expression of helicobacter pylori IgG antibody in blood, alleviating histopathological changes, and reducing cytokine expression when treating mice infected with helicobacter pylori. Therefore, the aqueous extract of honeysuckle flower of the present invention can be effectively used as a composition for preventing or treating helicobacter pylori infection.

Drawings

FIG. 1 shows the procedure for preparing mice infected with H.pylori;

FIG. 2 shows the results of confirming the presence of helicobacter pylori in gastric mucosal tissue by PCR based on the administration of a honeysuckle flower extract to mice infected with helicobacter pylori;

FIG. 3 shows the results of confirming the expression of IgG antibody against helicobacter pylori in blood based on the administration of the extract of honeysuckle flower to mice infected with helicobacter pylori;

FIG. 4 shows the results of scoring inflammatory cell infiltration and atrophic changes in stomach tissue based on the administration of honeysuckle flower extract to mice infected with H.pylori;

FIG. 5 shows the results of a rapid urease test according to the administration of a honeysuckle flower extract to mice infected with helicobacter pylori;

FIG. 6 shows the results of confirming the CLO score based on the administration of a honeysuckle flower extract to mice infected with helicobacter pylori;

FIG. 7 shows the results of measuring the expression levels of TNF-. alpha.and IL-1. beta. in gastric mucosal tissues, based on the administration of a honeysuckle flower extract to mice infected with helicobacter pylori.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may also be embodied in other forms. Rather, the description herein should be thorough and complete and is provided to fully convey the concept of the invention to those skilled in the art.

<Example 1 preparation of aqueous extract of honeysuckle flower (I)>

2000ml of water was added to 100g of honeysuckle flower and extracted with hot water at 90 ℃ for 3 hours to obtain 1400ml of honeysuckle flower extract. The extract was filtered through a 5 μm filter paper and then concentrated at 50 ℃ under reduced pressure for 3 hours. Thereafter, the concentrate was dried at 50 ℃ under reduced pressure overnight to obtain 25g of the aqueous extract of honeysuckle flower of example 1 of the present invention.

<Example 2 preparation of aqueous extract of honeysuckle flower>

2000ml of water was added to 100g of honeysuckle flower and extracted with hot water at 90 ℃ for 1 hour to obtain 1400ml of honeysuckle flower extract. The extract was filtered through a 5 μm filter paper and then concentrated at 50 ℃ under reduced pressure for 3 hours. Thereafter, the concentrate was dried at 50 ℃ under reduced pressure overnight to obtain 25g of the aqueous extract of honeysuckle flower of example 2 of the present invention.

<Example 3 preparation of aqueous extract of honeysuckle flower>

2000ml of water was added to 100g of honeysuckle flower and extracted with hot water at 90 ℃ for 5 hours to obtain 1400ml of honeysuckle flower extract. The extract was filtered through a 5 μm filter paper and then concentrated at 50 ℃ under reduced pressure for 3 hours. Thereafter, the concentrate was dried at 50 ℃ under reduced pressure overnight to obtain 25g of the aqueous extract of honeysuckle flower of example 3 of the present invention.

<Comparative example 1 preparation of ethanol extract of honeysuckle flower for comparison>

The ethanol extract of lonicera japonica thunb of comparative example 1 was prepared in the same manner as in example 1 by using 70% ethanol aqueous solution instead of water as an extraction solvent.

<Comparative example 2 preparation of aqueous extracts of honeysuckle flower for comparison (I)>

The aqueous extract of honeysuckle flower of comparative example 2 was prepared in the same manner as in example 1 by extracting at 60 ℃ for 3 hours instead of hot water at 90 ℃ for 3 hours.

<Comparative example 3 preparation of comparative Water extract of honeysuckle flower->

The aqueous extract of honeysuckle flower of comparative example 3 was prepared in the same manner as in example 1 by extracting at 30 ℃ for 3 hours instead of hot water at 90 ℃ for 3 hours.

<Comparative example 4 preparation of aqueous extract of honeysuckle flower for comparison>

The aqueous extract of honeysuckle flower of comparative example 4 was prepared in the same manner as in example 1 by extracting at 90 ℃ for 30 minutes instead of hot water at 90 ℃ for 3 hours.

<Experimental example 1 confirmation of the content of index component contained in honeysuckle flower extract>

First, a standard solution was prepared to confirm the content of the index component contained in the honeysuckle flower extract. In a 50ml flask, about 5mg of the secologanin standard was added to the standard solution, water was added and completely dissolved by sonication, then the flask was cooled, and the solution adjusted to the marked line was used as a high-concentration (0.1mg/ml) secologanin solution.

Next, about 200mg of each of examples and comparative examples was taken, added to a 50ml flask, added with water and completely dissolved by sonication, the flask was cooled and adjusted to the marked line, and then the filtrate filtered through a filter having a pore size of 0.22 μm was used as the experimental solution (4 mg/ml).

HPLC performance conditions of the standards, examples and comparative examples are shown in the following Table 1, and the content of the loganin oxide as an index component contained in the examples and comparative examples is shown in the following Table 2.

[ Table 1]

[ Table 2]

	Secologanin oxide (mg/g)
		Example 1	9.4
Example 2	8.7
		Example 3	10.5
Comparative example 1	4.4
		Comparative example 2	3.2
Comparative example 3	2.1
		Comparative example 4	4.5

As shown in table 2, it can be seen that the aqueous extract of lonicera japonica thunb in the examples of the present invention has a high content of secologlycoside when the extraction temperature is 90 ℃ or more.

<Experimental example 2 confirmation of antibacterial Activity against helicobacter pylori>

The helicobacter pylori (ATCC43504) strain is applied to a medium containing 10% of the strainBrucella agar medium in horse serum, at 37 deg.C and 10% CO₂The conditions of (3) were cultured in an incubator for 3 days. Thereafter, cultured helicobacter pylori cells were collected and then suspended in a sterilized brucella liquid medium, and a cell suspension having an absorbance at 600nm of 1.0 was prepared.

14g of Brucella medium was dissolved in 450ml of purified water, 6g of agar was added, suspended and sterilized at 121 ℃ for 15 minutes. 50ml of horse serum was mixed in a sterilized medium at about 40 ℃ and 25ml was dispersed on a plate having a diameter of 90mm to harden the agar medium, and then 0.2ml of helicobacter pylori cell suspension was smeared on the agar medium.

The examples and comparative examples were dissolved in water at a concentration, sterilized and filtered at 0.2 μm, and then sterilized paper discs (6 mm in diameter) were treated with 20 μ l, respectively, and placed on plates smeared with the strains. The examples and comparative examples were conducted at 37 ℃ and 10% CO₂The culture was performed for 72 hours in an incubator under the conditions of (1), and then the diameter of the resulting transparent region was measured, and the diameter of the pure zone of inhibition from which the diameter of the perforation was removed is shown in table 3.

[ Table 3]

As can be seen from the above Table 3, when helicobacter pylori was treated with the aqueous extract of Japanese honeysuckle flower of examples 1 to 3, which contained 0.5% by weight or more of secologanin oxide as an index ingredient, a transparent region of 10mm or more was exhibited, thereby having excellent antibacterial activity.

Specifically, the present inventors confirmed the inhibitory effect of oral administration of 100mg/kg on helicobacter pylori infection in mice by using the aqueous extract of lonicera japonica flowers according to the content of secologanin, respectively. As a result, unlike comparative examples 1 to 4, it was confirmed that the effect of inhibiting helicobacter pylori infection was exhibited only in the aqueous extract of lonicera japonica flower containing 0.5% by weight or more of secologanin in examples 1 to 3. Therefore, thereafter, in an animal experiment, it was confirmed that the effect of inhibiting helicobacter pylori infection was exhibited by orally administering the composition of example 1 to mice infected with helicobacter pylori at doses of 100mg/kg, 200mg/kg and 400 mg/kg.

<Experimental example 3 confirmation of Effect of inhibiting helicobacter pylori infection>

Experimental example 3-1 preparation of mice infected with helicobacter pylori

First, Helicobacter pylori strain (H.pylori SS1, Korea Helicobacter Bank) was inoculated in trypticase casein soy agar medium added with 5% sheep blood, and then in 10% CO₂Incubated at 37 ℃ for 2 to 3 days under microaerophilic conditions.

To increase the infection rate of H.pylori, mice were administered antacid 2 days before and on the day of H.pylori infection, and in all groups, 0.2ml of 5% sodium bicarbonate (NaHCO) was orally administered to each mouse using a mouse probe (zonde)₃) Once for 3 days.

Mice were fasted for 12 hours before H.pylori infection, and in all groups except the negative control group G1, according to 5.0X 10⁹Bacterial count per ml Colony Forming Unit (CFU), 0.2ml of H.pylori culture was orally administered at 2 day intervals using a mouse probe and infected.

To confirm the maintenance of infection after induction of H.pylori infection, blood was collected from the facial veins of all mice 1 week after H.pylori infection and plasma was isolated. In helicobacter pylori antibody measurements, individuals with increased antibody due to infection were identified and used in the experiment by only the mouse h.

All experimental groups were suspended in distilled water and administered orally at a dose of 5ml per mouse at the same time every day for 28 days 1 time per day (in the positive control group 1, 1 week, 3 weeks, 1 time per day, 14 days).

[ Table 4]

Experimental example 3-2 PCR experiment of helicobacter pylori in gastric mucosa

Genomic DNA was collected from the sterilized and extracted gastric mucosal tissue, and PCR experiments of helicobacter pylori were performed under the conditions of Table 5 below. The target gene used in the experiment was CagA, a toxic gene which is present only specifically in H.pylori, and a gene which is not present in humans or mice. Thus, FIG. 2 shows that in mice infected with H.pylori, specific bands generated by treatment of each experimental group were identified and positive individuals were determined.

[ Table 5]

Fig. 2 shows that, as a result of measuring the presence of helicobacter pylori in gastric mucosal tissue by PCR and calculating the treatment rate of each experimental group, the lonicera japonica extracts G5 to G7 in example 1 of the present invention showed a treatment rate of 40% to 60% compared to the infection group G2 for each concentration. Thus, it can be seen that the honeysuckle flower extract of the present invention is a composition for reducing the expression of a specific gene in the gastric mucosa increased by infection with helicobacter pylori.

Experimental examples 3-3 comparison of IgG titer of helicobacter pylori antibody in blood

In experimental example 3-1, in plasma isolated from the facial vein of mice 1 week after induction of H.pylori infection and plasma isolated after blood collection from the abdominal vein at the end of the experiment, the H.pylori antibody titer in each plasma was measured by the Mouse H.pylori antibody (IgG) ELISA Kit and is shown in FIG. 3.

In FIG. 3, as a result of measuring the H.pylori antibody in blood during autopsy, it can be seen that the H.pylori antibody was reduced in a concentration-dependent manner in examples G5 to G7 of the present invention as compared with the infection group G2.

From this, it can be seen that the aqueous extract of lonicera japonica of the present invention containing secologlycoside as an effective ingredient is a composition having an excellent effect of inhibiting helicobacter pylori infection.

Experimental examples 3-4 comparison of Observation of visible lesions in stomach tissue and histopathological analysis

Stomach tissue extracted on the day of autopsy was incised vertically from the esophagus along the greater curvature toward the duodenum, and specific lesions of the inner mucosa were observed. After observing the visible lesion, the incised stomach tissue was fixed in 10% neutral formalin, paraffin-embedded using a conventional method for histopathological examination, then sectioned to 4 μm thickness, and stained with hematoxylin and eosin (H & E), after which histopathological examination was performed. After observing the degree of infiltration (marked with yellow arrows) of inflammatory cells (neutrophils & monocytes) and the degree of atrophic gastritis with atrophic changes shown for each individual in combination in the overall region of Corpus and Antrum, histopathological scores are shown in fig. 4 by converting the grade of each tissue into a score according to the criteria of table 6.

[ Table 6]

Referring to fig. 4, the results observed in stomach tissue as a result of histopathology were scored for inflammatory cell infiltration and atrophic changes according to the criteria of the prior art [ Lee, j.y.et al, J Cancer prev.,19(2), 144-. In the infection group G2, the inflammation and atrophic changes in the stomach tissue increased due to helicobacter pylori infection compared to the normal group, but in examples G5 to G7, the inflammation and atrophic changes in the stomach tissue decreased in a concentration-dependent manner.

It can be seen that the honeysuckle flower extract of the present invention alleviates the symptoms of gastritis caused by infiltration and atrophic changes of inflammatory cells upon helicobacter pylori infection.

EXAMPLES 3-5 Rapid urease test (CLO test)

When helicobacter pylori is present in the gastric mucosa, urease is secreted when helicobacter pylori proliferates in the test agent medium, and urea in the test agent is hydrolyzed to produce ammonia. As a result, the total pH of the test agent is raised and a rapid urease test is performed by the color change (red) of the pH indicator, the test results being represented by the treatment rate and CLO score.

In the rapid urease test, gastric mucosa extracted on the day of autopsy was collected and tested by sterilization using campylobacter-like microorganisms (CLO) (korean Asan Pharm co., Ltd.) as a test reagent. Collected gastric mucosa was incubated in an incubator at 37 ℃ for 2 hours and then determined to be positive when the color of the reagent changed from yellow to red. The number of individuals determined to be positive was obtained from the percentage to calculate the positive rate, and the treatment rate for helicobacter pylori sterilization by sample treatment was calculated by the following equation and shown in fig. 5.

[ equation ]

{ (number of samples-number of positive samples)/number of samples }. times.100

In addition, after the CLO experiment, CLO scores were shown in fig. 6, measured as 0 point in the case where the color of the medium was not changed, 1 point in the case of reddish color, 2 points in the case of light purple color, and 3 points in the case of dark purple color, and the mean value and standard deviation of each group were calculated and the differences between the groups were compared.

When describing the treatment rate and the CLO score results obtained by the rapid urease test of fig. 5 and 6, the lonicera japonica extract G5 to G7 in example 1 of the present invention showed a treatment rate of 40% to 60% and showed an effect of reducing the CLO score by 60% to 90% compared to the infection group G2.

Thus, it can be seen that the honeysuckle flower extract of the present invention is a composition for reducing the expression of fast urease in the gastric mucosa increased by infection with helicobacter pylori.

Experimental examples 3-6 analysis of cytokines in gastric mucosal tissues

To measure proinflammatory cytokines in gastric mucosal tissue, the sterilized collected gastric tissue was pulverized with liquid nitrogen and proteins were extracted using cell lysis buffer for analysis. Among the isolated proteins, tumor necrosis factor-alpha (TNF-. alpha.) and interleukin-1 beta (IL-1. beta.) were analyzed using ELISA kit (R & D system, Minneapolis, MN, USA), and the results are shown in FIG. 7.

Referring to FIG. 7, the honeysuckle flower extract of example 1 of the present invention decreased the expression of TNF-. alpha.and IL-1. beta. in gastric mucosal tissues increased by infection with helicobacter pylori in a concentration-dependent manner.

<Preparation example 1 preparation of tablets>

20g of the honeysuckle flower extract of example 1 of the present invention was mixed with 175.9g of lactose, 180g of potato starch and 32g of colloidal silicate. To the mixture was added a 10% gelatin solution, which was then pulverized and passed through a 14 mesh screen. The mixture was dried and 160g of potato starch, 50g of talc and 5g of magnesium stearate were added to prepare tablets.

<Preparation example 2 preparation of capsules>

100mg of the honeysuckle flower extract of example 1 of the present invention, 100mg of corn starch, 100mg of lactose and 2mg of magnesium stearate were mixed, and then the ingredients were mixed and filled into capsules according to a conventional capsule preparation method to prepare capsules.

<Preparation example 3 preparation of injection>

1g of the honeysuckle flower extract of example 1 of the present invention, 0.6g of sodium chloride and 0.1g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was placed in a bottle, heated and sterilized at 20 ℃ for 30 minutes.

<Preparation example 4 preparation of health functional food>

20g of the honeysuckle flower extract of example 1 of the present invention, an appropriate amount of vitamin mixture, 70 μ g of vitamin A acetate, 1.0mg of vitamin E, 0.13mg of vitamin B1, 0.15mg of vitamin B2, 0.5mg of vitamin B6, 0.2 μ g of vitamin B12, 10mg of vitamin C, 10 μ g of biotin, 1.7mg of nicotinamide, 50 μ g of folic acid, 0.5mg of calcium pantothenate, an appropriate amount of mineral mixture, 1.75mg of ferrous sulfate, 0.82mg of zinc oxide, 25.3mg of magnesium carbonate, 15mg of monopotassium phosphate, 55mg of dicalcium phosphate, 90mg of potassium citrate, 100mg of calcium carbonate and 24.8mg of magnesium chloride are mixed to prepare granules, but may be variously changed according to the purpose and prepared into various formulations. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily adjusted, and the food may be prepared by mixing the above components according to a conventional preparation method of a health functional food.

<Preparation example 5 preparation of functional drink for health>

1g of the honeysuckle flower extract of example 1 of the present invention, 0.1g of citric acid, 100g of fructo-oligosaccharide and 900g of purified water were mixed and stirred, heated, filtered, sterilized and refrigerated according to a conventional preparation method of a drink to prepare a drink.