KR101645721B1 - Composition comprising mastic for preventing and treating gastric diseases - Google Patents

Abstract

The present invention relates to a perfume-containing composition useful as an effective physiologically active substance for the eradication of Helicobacter pylori, including oriental origin derived from natural products, squalene and olive oil, and useful for the prevention and treatment of gastrointestinal diseases, By weight to 40% by weight, squalene by 10% by weight to 40% by weight, and residual olive oil.

Description

[0001] Composition comprising mastic for preventing and treating gastrointestinal diseases [0002]

The present invention relates to a perfume-containing composition for the prevention and treatment of gastrointestinal diseases, and more particularly to a perfume-containing composition useful as an effective physiologically active substance for the prevention of Helicobacter pylori eradication including scent derived from natural products and squalene and olive oil, ≪ / RTI >

Helicobacter pylori bacteria: and (Helicobacter pylori H. pylori) infection is widespread throughout the world, it is one of the most common chronic infections in humans. Helicobacter pylori, which is infected with 50% of the world's population, was first isolated and isolated from the gastric mucosa by Warren and Marshall in 1983, and has been identified as an important factor in gastritis, gastric ulcer, duodenal ulcer, (See Fig. 1).

The World Health Organization (WHO) defines Helicobacter pylori as the first group of carcinogens in gastric cancer, and Helicobacter pylori is a cause of various gastrointestinal illnesses that persist for a lifetime in most cases unless they are artificially eradicated. Helicobacter pylori infection varies according to age, race, and education level, and is related to socioeconomic conditions in the growing period. Especially in developing countries, H. pylori infection occurs mainly in childhood. In developed countries, however, infections occur in childhood, while infections continue to occur in adulthood, resulting in infection rates of 0.3% to 0.5% per year. Helicobacter pylori infection has not yet been elucidated in terms of chronic infections, infection pathways, and prophylaxis in which lifetime infections persist, in most cases, unless attempts are made to artificially eradicate them. The age-specific characteristics of Helicobacter pylori infection in Korea were found to be low in 17.2% of children under 15 years of age and 66.9% in 16 years of age or older, 2).

The pathogenesis of Helicobacter pylori infection has not been fully elucidated. However, the results of the present study are as follows: first, it is transmitted from person to person; secondly, it is found that infection occurs mainly in the family in childhood; Factors such as sex, body mass index, blood type, education level, economic level, presence of diabetes, presence of fatty liver, peptic ulcer, smoking and drinking ability were analyzed. Helicobacter pylori produces urease, which decomposes urea into carbon dioxide and ammonia, and ammonia neutralizes the acid thereby surviving the stomach where strong acid acts ( 3).

It is known that when infected in the stomach, inflammation occurs directly on the mucous layer or mucous membrane due to adhesion to the mucous layer or mucous membrane, and inflammatory mediators produced by inflammatory cells cause various digestive diseases. Helicobacter pylori is produced by direct contact with epithelial cells of the stomach and various cytokines such as interleukin-8, interleukin-1 and alpha-tumor necrosis factor (TNF-α) Induces the production of chemokines, infiltrating neutrophils, lymphocytes, plasma cells and the like in inflamed areas, resulting in chronic active inflammation of the gastric mucosa. Currently, the US National Institutes of Health has compromised that Helicobacter pylori must be eradicated if a peptic ulcer is present, that Helicobacter pylori found in gastritis patients does not need to be cured, and that the correlation between gastric cancer and Helicobacter pylori is unclear. Among them, Helicobacter pylori antibiotic resistance has increased rapidly in recent years, and antibiotic resistance rates before and after antibiotic treatment can be increased up to 6 times or more. have.

The eradication treatment for eradicating Helicobacter pylori is usually a three to two week treatment with a proton pump inhibitor-amoxicillin-clarithromycin. However, when the triple regimens fail to combine several drugs, the incidence of resistance is high, and the current eradication rate of 70% to 75% is less than the effective eradication rate of 90%. Since the failure of the 3-therapy regimen, clinical trials on 4-drug regimens using the 'proton pump inhibitor-metronidazole-tetracycline-bismuth' are currently underway.

Table 1 below summarizes the combination therapy that is considered to be effective for eradicating Helicobacter pylori.

Medication Duration (days)
Clarithromycin (CLA)
Triple combination therapy PPI + AMOX + CLA
(PPI: omeprazole, lansoprazole, pantoprazole) 10-14 days RBC + AMX + CLA 7 days Metronidazole (MET)
Triple combination therapy BIS + TET + MET 14 days PPI + AMOX + MET With CLA and MET
Triple combination therapy PPI + CLA + MET 7 days RBC + CLA + MET Quadruple therapy PPI + BS + TET + MET 7 days

In Table 1, CLA = clarithoromycin, PPI = proton pump inhinitor, AMOX = amoxicilin, RBC = ranitidine bismuth citrate, BIS = colloidal bismuth citrate Colloidal bismuth citrate, TET = tetracycline, and MET = metronidazole.

As a result of antibiotic resistance analysis of Helicobacter pylori, 'amoxicillin' increased greatly from 0% in 1987 to 18.5% in 2003, and after 2003 decreased to 4.8%, but the resistance rate test showed that the penicillin- Since only the mutation of the protein (penicillin-binding protein) is tested, the actual resistance rate is expected to be higher than this. The resistance rate of 'Clarithromycin' increased from 0% in 1987 to 2.8% in 1994 and increased to 38.5% from 2007 to 2009, and the tolerance of cross-resistance with other antibiotics Is estimated to have occurred. 'Azithromycin', a Macrolide family of antibiotics such as 'Clarithromycin', also showed a remarkable increase in resistance rate to 33.3% in 1994. 'Tetracycline' was also 12.3% in 2003, but it increased sharply to 34.6% in 2007-2009. The resistance rate after treatment of Helicobacter pylori with antibiotics has increased after treatment in all antibiotics except tetracycline. The resistance rate of 'amoxicillin' was 18.3%, the resistance rate after treatment was 31.3% and that of 'cyprofloxacin' increased from 33.8% to 35.8%. Especially, 'clarithromycin' increased from 13.8% to 85.1% The rate of resistance more than doubled increased rapidly.

The problem with the above-mentioned antibiotics is that the antibiotic treatment eliminates Helicobacter pylori, but also removes lactic acid bacteria which are beneficial to the body. When microorganisms such as bacteria and fungi enter the body in this state, they do not have beneficial bacteria against them. Therefore, a large amount of harmful bacteria grows, resulting in toxic diseases caused by harmful bacteria. If Helicobacter pylori strains are resistant to antibiotics (5% to 55%), they can not be treated with antibiotics. Treatment rates of Helicobacter pylori by antibiotic + antacid therapy are at least 65% and up to 95% Is difficult. Even if Helicobacter pylori is completely eradicated with antibiotics, the probability of reinfection within 6 months is over 60%. If the antibiotic is repeated every time it is infected, it will resist antibiotics and eventually give up treatment. Stomach suppressants administered with antibiotics have a significant effect on the pain of ulcer patients infected with Helicobacter pylori, but they do not have the ability to inhibit bacteria, parasites, fungi, etc. in the small intestine and colon by stomach acid.
On the other hand, Korean Patent Laid-Open Publication No. 2014-0097821 discloses an oral cleaning agent containing mastic oil for removing Helicobacter germs and a method for producing the same, wherein the term "oral cleaning agent ", which is extracted from sodium fluoride, phytoncide, mastic oil, Wherein said extract is prepared by dissolving and mixing anthocyanin, myrrh, glycerin, xylitol, carrot extract, green tea extract, propolis extract, sodium citrate, citric acid, methylparabenoxybenzoate, L-menthol, purified water and alcohol. Quot; mastic oil "

Currently, there are side effects such as esophagitis, esophageal cancer, stomach pain, nausea, headache, etc. Therefore, there is a demand for development of a safe and effective therapeutic agent or therapeutic food by natural products.

It is an object of the present invention to provide a perfume-containing composition which is effective for eradicating Helicobacter pylori bacteria including oriental origin derived from natural products and squalene and olive oil as an effective physiologically active substance and which is useful for prevention and treatment of gastrointestinal diseases.

The perfume-containing composition for prevention and treatment of gastrointestinal diseases according to the present invention is characterized by containing oriental, squalane and olive oil.

The perfume-containing composition may contain olive oil as an amount of 10 to 40% by weight of scent, 10 to 40% by weight of squalene, and balance.

The fragrance-containing composition comprising the fragrance, squalene, and olive oil may be a health functional food preparation for prevention and treatment of gastrointestinal diseases, wherein the dosage form is selected from a liquid formulation, a tablet, and a capsule.

Effect of the Invention According to the present invention, there is provided an oriental-based composition useful as an effective physiologically active substance for eradication of Helicobacter pylori bacteria including oriental origin derived from natural materials and squalene and olive oil, and useful for prevention and treatment of gastrointestinal diseases. Thereby effectively eliminating Helicobacter pylori, and contributing to prevention and treatment of gastrointestinal diseases based thereon.

1 is a diagram showing various gastric diseases caused by Helicobacter pylori.
FIG. 2 is a graph showing the serological H. pylori infection rate according to age (described in the Korean Journal of Digestion in 1999).
3 is a diagram schematically showing a principle that Helicobacter pylori is survived from stomach acid.
4 is a diagram showing the molecular structure of squalene.
5 is a diagram schematically showing a Helicobacter Urea Breath test (UBT).
6 is a diagram schematically showing IgG and IgM measurements of serum using the sandwich method for serum antibody testing.
Fig. 7 is photographs showing the results of an anti-Helicobacter pylori comparison experiment of oriental sole.
8 is a photograph showing the results of an antihyperlibacter pylori comparison experiment of the perfume-containing composition according to the present invention.
FIG. 9 is a view showing histopathological observation photographs. FIG.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The perfume-containing composition for prevention and treatment of gastrointestinal diseases according to the present invention is characterized by containing oriental, squalane and olive oil.

The perfume-containing composition may comprise olive oil as the balance 10-40 wt%, squalene 10-40 wt%, and balance 15-35 wt% oriental, 15-35 wt% squalane, and the balance includes olive oil And most preferably 2% by weight fragrance, 20% by weight squalane and the balance olive oil.

The fragrance-containing composition comprising the fragrance, squalene, and olive oil may be a health functional food preparation for prevention and treatment of gastrointestinal diseases, wherein the dosage form is selected from a liquid formulation, a tablet, and a capsule.

Mastic (Scientific name: Pistacia lentiscus LINNE ) is a food additive listed in Natural Additive 272 of Food Additives. It is a viscous natural ginseng with liquid collected from lacquer tree and mastic tree which is native only from Hiaozu Island in south-eastern Greece. The proprietary components are 60 to 70% of resin (resin), 33% of alpha beta -boswellic acid, 33% of olibanoresene, and contain arabic acid and the like . The oriental extract has been known to have anti-inflammatory, antioxidant and anticancer effects. In oriental medicine, it is widely used for the treatment or prevention of inflammatory diseases including arthritis, asthma and inflammatory bowel disease.

It is known that the ancient Greeks used frankincense as an ingredient of sweeteners or beverages for the purpose of alleviating discomfort in the upper abdomen, stomach cramps, digestive disorders and gastric ulcers. It is used as a food. In Iraq, it is also widely used as a drink or food ingredient. Ahn Deok-gyun's "Clinical application of herbal medicine" is said to be used mainly for analgesic purposes when it causes ulcer due to skin infection caused by trauma, and when it is made into powder and applied to the heart, it promotes the formation of granulation tissue and relieves pain. The fragrance has also been used for skin protection since ancient times, and the ancient text of the 16th-century mummy's coffin describes the effect of wrinkle removal and was also used as a directional ointment for skin diseases in the Franciscan monastery in Jerusalem. Fruit extracts have anticancer effects on leukemia and brain tumors, and are known to affect the proliferation of liver cancer cells and lymphocytes and the migration of white blood cells. Recently, frankincense has been used as a raw material for processed foods. It has been approved by the United States Food and Drug Administration (FDA) as a medicine in 25 countries and became a genuine European natural food. In a study conducted at the University of Nottingham in the United Kingdom, it was found that frankincense spiral germs were eradicated, and especially in 80% of patients, the symptoms were improved by taking 1 g of digestive ulcer from Helicobacter pylori strain for 2 weeks, In 70% of patients, ulcers were healed and no side effects were observed. In the study by Korean researchers, it was noted that it has been known for a long time as a gastrointestinal therapeutic substance excellent in frankincense in the Mediterranean region. Therefore, the inhibitor of Helicobacter pylori strains in the form of a chewing gum has been developed and the density and mucosal neutrophil infiltration of Helicobacter pylori strain . Recent studies have reported the excellent antibiotic effect of Helicobacter pylori on oriental in vitro experiments. The Helicobacter pylori strains and clinical isolates were inoculated in a concentration of 0.0075 to 1.0 mg / dl, Was cultured on an agar-containing medium, it was reported to have a strong anti-bacterial effect against Helicobacter pylori even at the minimum concentration of frankincense. Another report on Helicobacter pylori showed 90% Helicobacter pylori bactericidal activity at an intense concentration of 500 μl / ml. The morphology of Helicobacter pylori was observed with an electron microscope. As a result, the cells were observed to have blebbing, And to induce the same abnormal pattern.

In a study to identify active substances in oriental foods, the active ingredient of oriental fractions was analyzed by gas chromatographic mass spectroscopy. As a result, ten triterpenoid aicd It has been confirmed that these terpenes or terpenoids have antibacterial or antifungal activity. Although the precise mechanism of action against these antibacterial and antifungal effects has not been elucidated, it is presumed that they act as lipophilic components and disrupt membranes of bacteria or fungi. In Korea, it is used for Lotte Confectionery 's "Xylitol Alpha Project" for the prevention of tooth decay, and it is using oriental products for Yakult' s "Tutti" in Korea. As an adjuvant to eradicate Helicobacter pylori, Swanson vitamins in the United States are selling a product that has been refined as a single ingredient.

As described above, frankincense has been known to have an antimicrobial action from ancient times. It has been secured as a natural product widely consumed in Europe due to a private prescription. In the present invention, the frankincense composition containing mixed edible natural products was developed, In order to maximize the efficiency.

Squalene (hexamethyltetracosahexane, C30H50) is an unsaturated oxidized hydrogen system composed of carbon and hydrogen, and is a very important precursor for the biosynthesis of cholesterol, and is synthesized in vertebrates and plants. Squalene was discovered in 1906 by Dr. Yuji Mitsumaru, a humectant chemist in Japan, in the liver of sharks, and then in 1935 Professor Paul Curla of the University of Zurich, Switzerland, uncovered the molecular structure of squalene (see Figure 4). Squalene is contained in large amounts in skin, abdominal fat tissue, subcutaneous fat tissue, lymph node, pancreas and myocardium. It has 6 double bonds and can easily combine with oxygen ion to remove harmful oxygen, Atherosclerosis inhibitory action, myocardial infarction and liver disease.

Sharks are classified into surface-layer sharks and deep-sea sharks according to depths of the inhabited ocean. The best source of squalene is the eye shark, and 71 to 85% of the liver oil is squalane. In general, squalene refers to a product obtained by refining a fat containing squalene components extracted from the liver of a shark. Squalene-containing products are prepared by filling squalene as a main ingredient in a capsule, Refers to a health functional food that meets the standard. In the old man and the sea, a representative work of the American novelist Hemingway (Ernest Hemingway, 1899-1961), who won the Nobel Prize for Literature, the elderly man has been eating healthy shark liver oil every morning, Has widely used liver oil extracted from sharks as folk remedies for various diseases such as weak constitution, wound healing, gastrointestinal diseases. Squalene is also used in the production of cholesterol, reproductive hormone, vitamin D and bile acid, and about 250 mg of squalene is produced every day in the fat mass of the skin. Is secreted as a component of sebum from. Since squalene has excellent fat-solubility and its surface tension is very weak, it penetrates well into cells and tissues, dissolves fat-soluble pesticides, carcinogens, environmental pollutants and heavy metals accumulated in it and releases detoxification out of the tissues. It is known that excretion of waste products and activation of metabolism enable oxygen to be supplied into cells to produce new cells and make them elastic and glossy skin. In addition, it is known that it promotes the function of reticuloendothelial tissue, which removes foreign substances such as bacteria and cancer cells, and strengthens immune function to inhibit cancer cell growth. In particular, it activates anticancer activity by increasing T cell function and phagocytic activity. If you look at the usefulness of squalene products, you can supply deficient oxygen to activate cells, promote the overall function of the body, promote metabolism, revitalize tissues, cleanse body fluids, strong sterilization, And has various advantages when taken along with its function as a Helicobacter pylori eradicator.

Olive oil is a pale yellow nonfat dry oil and fat loss oil extracted from the fruit of olive ( Olea europaea L. , Oleaceae) and has a specific gravity of 0.914 to 0.929 (15 ° C), a refractive index of 1.4654 to 1.4683 (25 ° C.), an acid value of 3.6, a saponification value of 185 to 197 and an iodine value of 70 to 90. It is a vegetable oil obtained by directly compressing or extracting the flesh with the oil contained in the fruit of the olive tree (olive tree). It can be edible without the special purification process after recovering and filtering the oil. Because of its high content of unsaturated fatty acids, it is used for salad oil and canning as well as for soap, fiber lubrication and cosmetics for edible use and as an enema, ointment, and injection solvent for medical use. Olive oil is a relatively edible oil that has been used for a long time in the Old Testament, which is produced mainly in the interventional watershed where there is no cool and cold winter, and it is used extensively throughout the world as the finest product of edible oil. Especially, it is widely used in Greece, . Domestic olive oil is directly imported or refined by importing crude oil, and the market is about KRW20 billion, with the market rapidly expanding since 2000, showing an increase of 30 ~ 40% every year. Studies on olive oil have been conducted mainly on the physicochemical properties and antioxidant properties of the countries with high production and consumption. As for the antioxidant activity, the oxidative stability is good because the duration of autoxidation is longer than other plant seed oils because of the low saturated fatty acid (about 16%) and the high monounsaturated fatty acid (about 70%) content of olive oil. Because it contains a lot, it is different from other seed oil. The fatty acid composition of olive oil is oleic acid, which is the unsaturated fatty acid (65 to 85%), and the saturated fatty acid is palmitic acid. In particular, among the natural antioxidants, phenol compounds present in olive oil act as donors of hydrogen atoms during the chain reaction of lipid oxidation to prevent lipid oxidation. In Korea, recently, the application range such as salad oil and Italy has been expanded, and the effect of physiological activity such as reduction of LDL cholesterol in plasma and prevention of some cancers has been known, Diversification of olive oil varieties is ongoing. Olive oil is called different name depending on the extraction stage, and the purest oil obtained by the first squeezing is called 'extra virgin oil' while 'extra virgin' is greenish yellow and it is mainly used as salad dressing. The purity of the oil after that is called 'pure oil', and it is transparent in light yellow color and has a clear taste. At the final stage of extraction, the solvent is extracted from carbon disulfide (CS ₂ ) oil: sulfur oil ", and is used as a printing agent in the dyeing industry. The ancient Greek Hippocrates called olive leaves 'natural antibiotics', which are known to kill harmful bacteria and viruses and increase immunity. The main effects of these effects are 'olive leaf' and 'olive leaf' (oleuropein). Olla-Europa is an antioxidant, one of the polyphenols, which, like antioxidants like green tea and wine, removes harmful oxygen such as aging, heart disease and cancer.

Currently, premium fermented milk products for Helicobacter pylori eradication and improvement of the intestinal environment in the domestic market are on sale every day, such as Gut of the dairy industry, Lotte liquor, Haitai tantalizing force, and the foundation of the founding milk. Accordingly, the perfume-containing composition of the present invention can be expected to have a sufficient effect as an alternative to the problems encountered in conventional antibiotic disinfection.

Example 1

(MM) according to the present invention was prepared by mixing 20% by weight of oriental, 20% by weight of squalene and 60% by weight of olive oil, which was used in the experiments in the following experimental examples.

Comparative Example

As a comparative example, olive oil alone and squalene alone were used to measure their bactericidal action. However, olive oil exhibited antioxidative action as well as an oriental hydrating action and squalane exhibited bactericidal action. Thus, The bacteriocidal activity of squalene alone is very small and, when mixed with the scent according to the present invention, it is interpreted as a synergistic effect with antimicrobial and antioxidant possessed by scent. That is, the antimicrobial and antioxidant activities of olive oil and squalene alone were very small.

Experimental Example 1: Measurement of Helicobacter pylori antibacterial activity

ATCC 43504 was used as the Helicobacter pylori, a causative agent of gastric and duodenal ulcers. For culture, 0.5 g of peptone, 0.75 g of agar, 0.25 g of sodium chloride (NaCl), 0.25 g of yeast extract, 0.2 g of beef extract and 0.2 g of pyruvic acid ) Was used and the humidity was maintained at 95% or more in a 10% CO2 incubator (Memmert, Schwabach, Germany, Schwabach, Germany) to maintain microaerophilic conditions , And cultured on a flat agar plate at 37 캜 for 48 to 72 hours.

In addition, the antimicrobial activity measurement by the disc method was carried out by dividing 100 μl of Helicobacter pylori culture broth into the optimum medium of Helicobacter pylori plate, plated with a sterilized glass rod, and then sterilized disc paper (φ 8 mm) The extract was sterilized with a 0.45-μm membrane filter (Sigma Chemical Co., St. Louis, MO, St. Louis, Mo.), concentrated using a vacuum evaporator, diluted with sterilized water, ) Was adjusted to 50 to 200 / / 100 ㎕, 100 각 of each extract was absorbed in a disk paper, sterilized water was absorbed in place of the flavor-containing composition according to the present invention as a control, After 24 hours of incubation, a clear zone around the disc was observed.

Experimental Example 2: Minimum inhibitory concentration (MIC) measurement

The minimum inhibitory concentration (MIC) assay for Helicobacter pylori against the perfume containing composition according to the present invention was performed using a broth dilution method (Mann CM and Markham JL 1998, NCLLS 1999, Yu JQ et al. 2004). The test substance was diluted with dimethyl sulfoxide (DMSO) and diluted to a concentration of 0 to 20 / / ml, and each strain was used in this experiment after adjusting the concentration to O.D.600 = 0.2.

100 μl of nutrient broth (NB), 50 μl of the test substance preparation solution and 50 μl of the bacterial dilution were sequentially added to a 96-well plate (Falcon, USA) Were incubated at 37 ° C for 24 hours while measuring absorbance values using an ultraviolet-spectrophotometer.

The minimum concentration at which no bacterial growth (turbidity) is detected on the growth curve of the microorganism is set to MIC. The minimum bactericidal concentration (MBC) was determined by plating 50 μl of each time-zone culture sample for determination of MIC on a NA plate and culturing for 48 hours at 37 ° C, When the number of colonies is observed, MBC is set to the minimum concentration that completely destroys the target bacteria (99%). All experiments performed three independent experiments and synthesized the results.

Experimental Example 3: Antibacterial activity test using an animal model transfected with Helicobacter pylori

1) Animal experiments

Helicobacter pylori strains were prepared by inserting sufficient activated Helicobacter pylori strains in Brucella agar and then harvesting the strains in a Brucella agar medium with sufficient colonies (approximately 6) using loops The cells were picked, streaked, and cultured in a microaerophilic state at 37 ° C and 10% CO 2. The whole colonies of the cultured 2 plates were harvested and suspended in 1 ml of sterilized deionized water (DW), and then the strains were spread on a Brucella agar plate medium with a spreading of 200 각각 each .

Sprague-Dawley male rats, weighing 150 to 170 g at 5 weeks of age, were purchased from Koatex (Pyeongtaek, Korea), and the temperature during the growing period was 22 ± 2 ° C and the relative humidity was 40 to 70 % And illumination time were adapted to one week of solid form feed (Purina) in an animal chamber maintained for 12 hours and then classified into 10 animals in a randomized complete block design.

As experimental groups, as shown in Table 2 below, the test substance administration group including the normal control, solvent control, and positive drug control groups were divided into four dose groups, namely, 50, 100, 200 and 300 mg / Materials were administered in groups.

Bacteria cultured on a plate medium were collected with a liquid medium, cultured, and then the experimental animals were fasted for 24 hours. One ml of bacteria (over 108 cells) were inoculated three times at 2-day intervals, To confirm the infection.

Infection was checked by urease test (urease test), urea enzyme test (CLO test) (positive from yellow to orange), and then the stomach was cultured (Oxidase activity, catalase activity, nitrate reduction, hydrogen sulfide (H ₂ S), etc.) associated with Helicobactor pylori after plate scraping with a culture swab, And then histopathological findings on the gastric mucosa were examined.

Test Items Test group Number of animals
(Marie) Strain Volume

Helicobacter
Filo
Bactericidal effect Normal control group

10

Non-strain administration menstruum Negative control group
Helicobacter
Filo
inoculation
menstruum
Fermented mixture 50 mg / kg 100 mg / kg 200 mg / kg 300 mg / kg Pantoprazole 30 mg / kg system Group 8 80

2) Identification method of Helicobacter pylori

(1) urease and inhibitory effect experiment

(Asan Pharm Co., Ltd., Korea) based on a gel card and subjected to element resolution at 35 ° C for up to 4 hours.

Urease activity inhibition As an experimental method, 7 ml of urease assay medium is added to a cell culture culture tube, and Helicobacter pylori 5 × 10 4 is added to each cell culture culture tube. The cells were cultured in an Incubator at 37 ° C, 10%, 50%, and 100% humidity for 24 hours, 48 hours, and 72 hours apart. The absorbance at 550 nm was measured using an analyzer. The absorbance of the cultured test tube cultured only with Helicobacter pylori was evaluated as a relative value with respect to the absorbance of the test sample at a ratio of 100%, and the sample tended to absorb light at 550 nm. Thus, the concentration treated with Helicobacter pylori (250 Mu] g / ml) was measured and then corrected.

(2) Helicobacter Urea Breath test (UBT)

As a test principle, as shown in Fig. 5, a helicap including a 14C-element (urea) is taken together with water, and when it is in the presence of Helicobacter pylori, the urease produced by Helicobacter pylori 14C-urea was decomposed into ammonia and carbon dioxide (14CO2), and 14CO2 was transported through the blood to the lungs. This respiration was collected and the amount of carbon dioxide was measured to confirm the presence of Helicobacter pylori infection.

(3) Serum antibody test

This test measures antibodies in the serum produced by Helicobacter pylori infection. The high frequency of Helicobacter pylori immunoglobulin G (IgG) reflects current infection status and Helicobacter pylori immunoglobulin M (IgM) And is available for early diagnosis. Because it is blood-testable, it can be easily tested regardless of the lesion site, and used as epidemiological investigation or screening of infection (see FIG. 6).

(4) Oxidase

After the colonies were plated using a platinum-loop, the cells were immersed in a filter paper and the blue-colored reaction was observed by dropping the p-phenylenediamine dihydrochloride reagent to produce indolphenol blue. Respectively.

(5) Catalase

The colonies cultured on the selective medium were placed on the slide, and 3% hydrogen peroxide (H2O2) was dropped and the cells were read positive when bubbles were generated.

(6) Nitrate reduction

Cultured colonies were inoculated into nitrate broth and incubated at 35 ° C for 48 hours. When the cells were transformed into red purple by adding α-Naphthylamine and sulfanilic acid, the culture was positive.

(7) H ₂ Create S

The central part of the well-separated colonies was taken as a platinum line, punctured at the center of the triple sugar iron (TSI) medium, inoculated to 3 to 5 mm from the bottom of the slope and applied to the slope, Was slightly opened and incubated at 37 ° C for 24 hours. When it turned black, it was judged to produce H ₂ S.

The biochemical characteristics of Helicobacter pylori strains in the above experiments are shown in Table 3 below.

Helicobacter
Filo Urease + Oxidase + Catalase + Nitrate reduction - Hydrogen sulfide (TSI) -

(8) Gamma-glutamyltranspeptidase (GGT)

GGT is required for the absorption of glutamine, cysteine-glycine amino acids and dipeptides through the cell membrane in animals and is involved in the metabolism of leukotriene D, an inflammatory and allergic reaction , A marker of preneoplastic change and cell aging, is an enzyme involved in glutathione metabolism. To date, GGT has been identified in E. coli, Proteus mirabilis, Bacillus subtilis, Pseudomonas aeruginosa and Helicobacter pylori. In these bacteria, GGT hydrolyzes glutathione to glutamine and cysteine-glycine. GGT of Helicobacter pylori is produced as an enzyme precursor protein of 60 kDa (kilodalton) and decomposed into subunits of 40 kDa and 20 kDa through autolysis. The activity of GGT enzyme per Helicobacter pylori cell lysate protein is more than 100 times stronger than that of E. coli or Proteus mirabilis. Although the genome size of Helicobacter pylori is about 1/3 of that of E. coli, the fact that the activity of GGT is over 100 times higher than that of E. coli suggests that this enzyme plays an important role in the survival of Helicobacter pylori . Since Helicobacter pylori is a micro-oxygenated bacterium, GGT of Helicobacter pylori hydrolyzes glutathione in the gastric mucosa into glutamine and cysteine-glycine and is absorbed into bacterial cells. Then, γ-glutamylcysteine synthetase It is presumed that glutathione is synthesized and used as a defense against intracellular reactive oxygen such as hydrogen peroxide, hydroxyl radical and singlet oxygen generated during metabolic process. Due to the GGT action of this potent Helicobacter pylori, the gastric mucosa is depleted of glutathione, resulting in gastric and duodenal mucosal damage. Therefore, blood samples were collected from the abdominal aorta after the experiment and serum was separated, and the concentration of GGT in the serum was measured by a diagnostic test method.

(9) Alcohol dehydrogenase (ADH)

Among bacteria inhabiting human or animal gut, there are bacteria that produce ethanol. The alcohol dehydrogenase (ADH) of these bacteria converts acetaldehyde to ethanol, but under certain conditions, ethanol is oxidized to acetaldehyde. Acetaldehyde generated here is highly reactive and acts as a toxic substance in the human body. Helicobacter pylori possesses ADH, and acetaldehyde is generated when ethanol is added during bacterial culture. When Helicobacter pylori produces ADH in the gastric mucosa and oxidizes alcohol absorbed in alcohol to acetaldehyde, it will damage the gastric mucosa. Colloidal bismuth subcitrate and omeprazole, which are said to be effective against Helicobacter pylori infection, inhibit the reaction of Helicobacter pylori to form acetaldehyde from ethanol. Patients infected with Helicobacter pylori increase the amount of ADH 4 isoenzymes (ADH class IV isoenzyme) and total ADH enzyme in the serum. Therefore, the infection of Helicobacter pylori causes gastrointestinal lesions. ADH in Helicobacter pylori is classified as cinnamyl alcohol dehydrogenase. This enzyme is the enzyme that acts as a last step in the synthesis of lignin by converting para-hydroxy-innamaldehyde into a plant. Therefore, blood samples were taken from the abdominal artery after the experiment and serum was separated and the concentration of ADH in the serum was measured by a diagnostic test method.

(10) Peroxiredoxine, superoxide dismutase, and catalase.

Helicobacter pylori is a micro-oxygenated bacterium. Oxygen is essential but it means that defensive transfer to active oxygen by oxygen use is incomplete. In addition, the host defense system is activated when the gastric mucosa is inhabited, resulting in a large amount of inflammatory exudates and active oxygen species in the surrounding environment. Helicobacter pylori can grow in the gastric mucosa with a defense mechanism against free radicals, which occur both internally and externally. Unlike other bacteria, Helicobacter pylori has less thiol-dependent redox-active protein. Therefore, redox metabolism differs from general bacteria. In general, peroxiredoxin (Prx) plays a role in protecting oxidative stress in the environment. Catalase (KatA) of Helicobacter pylori is 58.7 kDa and exists as a tetramer and has one heme. There is no peroxidase activity and the pI value is 9.0 to 9.3, which is more alkaline than other bacteria. In fact, Helicobacter pylori catalase binds NADH. These enzymes are enzymes that can fight against the host's defense. This enzyme is explained by neutrophils and monocytes that when Helicobacter pylori is inactivated, they neutralize free radicals such as superoxide anion and hydrogen peroxide generated in these cells, which will interfere with intracellular sterilization, thereby causing neutrophil and macrophage damage . Therefore, in the present study, peroxiredoxin, superoxide dismutase and catalase were measured using an ELISA kit based on the above theoretical background.

Table 4 below summarizes the methods for diagnosing Helicobacter pylori infection.

method of inspection Sensitivity / Specificity (%) Explanation Invasive examination (gastroscopy and biopsy) Rapid urease test
(Rapid Urease Test)

Tissue examination
Culture test 80 ~ 95/95 ~ 100


80 ~ 90/95 or more PPIs, antibiotics, false negatives when using bismuth compounds
Provide histological information by examining tissue samples
It takes time, is expensive, requires experience, but antibiotic susceptibility is known Noninvasive examination Serum antibody test

UBT large-bowel examination
80 or more / 90 or more

90 or more / 90 or more It is cheap and convenient, but it is difficult to make initial diagnosis
Cheap, simple and fast, but false negative by antibiotics

4) Clinical trial

A clinical trial protocol was drawn up and approved by the Clinical Trial Ethics Committee (IRB).

Subjects drop outs for a variety of reasons, so they recruit more subjects than originally needed. Recruitment of subjects was registered as volunteers only from volunteers through the Helicobacter Urea Breath test (UBT).

Since the test group is close to 25 to 40% of the placebo response versus placebo effect in the normal control group, generally in clinical trials, the placebo control and test substance-administered groups were used to test each of the perfume- We recruited 20 people in the group. The composition of the clinical trial group is shown in Table 5 below.

group Test substance Number of patients Duration of administration Normal control group -
20 each
60 days Placebo control Placebo Test substance group The perfume-containing composition of the present invention

All tests were carried out in accordance with the double blinding test and the criteria for all tests in accordance with the Food and Drug Administration clinical trial guidelines.

5) Statistical processing

In order to compare the homogeneity of variance for each experimental result, Levene's test was performed. When the variance was homogeneous, one-way analysis of variance (ANOVA) Dunnett test was performed to identify test groups with significant differences from the control group.

The results of the above experiments are summarized below.

1. Helicobacter pylori antibacterial activity measurement

Identification of antibacterial activity of frankincense and natural product mixture

Antimicrobial experiments on malassechia showed that the mixture of frankincense and natural mixture was more effective than that of frankincense alone. That is, the inhibitory effect on malassezia showed a more significant result than the effect of oriental treatment alone, showing a dose-dependent response in the perfume-containing composition according to the present invention (Table 6: Containing antimicrobial composition of the inventive oriental composition according to the present invention).

Concentration (mg)
Low support (mm) frankincense The perfume-containing composition of the present invention 5 22.5 28.5 10 23.4 31.6 25 28.4 35.3

The results of the anti-Helicobacter pylori comparison experiment of oriental soleae are shown in Figs. 7A and 7B. As shown in Figs. 7A and 7B, the average diameter was found to be 24.7 mm < 2 >

The results of the anti-Helicobacter pylori comparison experiment of the perfume-containing composition according to the present invention are shown in FIGS. 8A and 8B. In this figure, an average diameter of 31.8 mm 2 was shown by an experiment on the antibacterial effect of the composition containing an aromatic compound according to the present invention. Which is higher than that of oriental soymilk by about 7.1 mm 2. It is considered that the main component of the perfume-containing composition is more synergistic with the mastic gum, the olive oil containing it and the squalene component than the oriental alone.

Resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), E. coli ( E. coli 0157), and multidrug resistant P. aeruginosa ( P. aeruginosa . The results are shown in Table 7 below. These results suggest that MRSA, VRE, and P. aeruginosa are very important. In the 1970s and 1980s, the spread of hospital infections by MRSA occurred worldwide, resulting in increased use of vancomycin in clinical practice. In 2002, vancomycin resistant staphylococcus aureus (VRSA) was first reported in the United States in 2002 due to increased use of vancomycin for MRSA treatment, which was recognized only as a problem within the hospital. In a recent prospective study of 15 hospitals in Korea, the MRSA isolation rate was 83.7%, and in the multi-center study from 1997 to 2006, 64-72% of MRSA was found to be the most common hospital infection The causative organism was confirmed. The resistance of the antimicrobial agent to the bacterium in the perfume-containing composition has a significant implication. In the future, according to the results of this study, various applications may be possible.

Fructose (1440 占 퐂 / ml, 0.14%) The perfume-containing composition of the present invention
(205 [mu] g / ml, 0.02%), Reaction time (min) 15 30 60 120 15 30 60 120

Strain MRSA + ± ± + + + ± VRE + + + + + + ± ± E. coli + - - - + ± - - P.aeruginosa - - - - - - - -

The results of animal experiments are shown in Table 8 (microbiological biochemistry test results).

Treatment (mg / kg) /
parameter Urease Oxidation
enzyme Catalase nitrate
restoration Hydrogen sulfide
(TSI) Positive rate Normal control group 0/10 0/10 0/10 0/10 0/10 10/10 Solvent control 9/10 10/10 10/10 0/10 0/10 9/10 + MM (50 mg / kg) 7/10 8/10 9/10 0/10 0/10 8/10 + MM (100 mg / kg) 5/10 4/10 6/10 0/10 0/10 6/10 + MM (200 mg / kg) 4/10 3/10 4/10 0/10 0/10 5/10 + MM (300 mg / kg) 2/10 4/10 4/10 0/10 0/10 4/10 Pantoprazole
(30 mg / kg) 2/10 3/10 2/10 0/10 0/10 2/10

A disinfection experiment using an animal model of Helicobacter pylori transplanted

In the CLO test (Campylobacter-Like Organism test), no reaction was observed in the normal control group, whereas in the solvent control group only positive was observed in only one subject, and in the remaining nine subjects, all the positive results were observed. Thus, Helicobacter pylori infection was normal I can observe that it is. On the other hand, a significant dose-dependent finding was observed in the perfume-containing composition according to the present invention, and it was confirmed that the perfume-containing composition had a sterilizing power against Helicobacter pylori (see Table 9: CLO test results).

process
(Mg / kg) One 2 3 4 5 6 7 8 9 10 positivity
ratio Normal control group - - - - - - - - - - 10/10 Solvent control + + + + + + + ± + + 9/10 + MM (50 mg / kg) + + + + + + + + - - 8/10 + MM (100 mg / kg) + + + + - + - - + - 6/10 + MM (200 mg / kg) + ± - - + + - + - + 5/10 + MM (300 mg / kg) ± + - + - + - + - - 4/10 Pantoprazole
(30 mg / kg) + - - - - - - + - - 2/10  Legend: -: negative, ±: weak positive, +: positive

Biochemical Experimental Results

The results of the biochemical experiments are summarized in the following.

Treatment (mg / kg) / Variables GGT
(IU / L) ADH
(pg / ml) Glucose
(Mg / dl) AST
(IU / L) ALT
(IU / L) Normal control group 1.5 ± 0.05 ^c) 15.6 ± 2.8 ^b) 114.2 ± 10.4 ^a) 38.7 ± 5.4 ^a) 48.2 ± 6.2 ^a) Solvent control 22.5 ± 1.2 45.3 ± 7.8 152.4 + - 8.4 62.5 ± 2.3 72.4 ± 4.6 + MM (50 mg / kg) 17.4 ± 1.5 ^a) 35.3 ± 5.1 135.9 ± 13.5 51.5 ± 0.5 65.5 ± 7.7 + MM (100 mg / kg) 15.2 ± 3.7 ^a) 30.2 ± 6.1 142.6 ± 6.8 53.3 ± 6.3 58.1 ± 5.5 + MM (200 mg / kg) 14.6 + - 5.5 ^a) 33.8 ± 3.7 132.5 ± 14.5 48.3 ± 7.6 45.6 ± 3.5 ^a) + MM (300 mg / kg) 10.7 ± 2.6 ^b) 25.6 ± 2.9 ^b) 128.4 ± 12.4 ^a) 45.2 ± 8.2 40.5 ± 6.1 ^a) Pantoprazole
(30 mg / kg) 3.6 ± 2.2 ^c) 19.6 ± 1.3 ^b) 129.6 ± 13.6 ^a) 45.6 ± 3.4 40.5 + 4.6 ^a)  Results were presented as mean ± SD, with a) P <0.05, b) P <0.01, c) P <0.001 for the values of solvent treated Sprague-Dole rats.

Antioxidant test results

The results of the antioxidation test are shown in Table 11 below.

Treatment (mg / kg) /
variable SOD MDA Catalase GST GSH Normal control group 3.95 ± 0.19 ^a) 2.02 ± 0.19 ^b) 7.48 ± 0.26 ^c) 106.9 ± 5.29 ^b) 17.0 + - 0.27 Solvent control 6.89 + - 0.41 4.05 ± 0.16 3.94 + - 0.15 137.6 ± 17.4 16.6 ± 0.59 + MM (50 mg / kg) 6.46 + - 0.47 3.69 ± 0.33 4.00 ± 0.27 122.4 ± 9.5 15.8 ± 1.78 + MM (100 mg / kg) 6.25 + 0.22 3.89 ± 0.20 4.45 ± 0.46 119.4 ± 10.6 17.16 ± 1.32 + MM (200 mg / kg) 5.26 ± 0.49 4.21 ± 0.43 4.86 ± 0.55 123.7 ± 12.4 16.9 ± 1.26 + MM (300 mg / kg) 4.92 ± 0.63 ^a) 3.71 + - 0.62 ^a) 5.67 ± 0.73 112.6 ± 26.4 16.5 + - 2.42 Pantoprazole
(30 mg / kg) 5.52 + - 0.69 4.06 ± 0.44 4.22 + - 0.34 115.8 ± 16.8 16.23 ± 1.13

PGE2 concentration in stomach tissue

The PGE2 concentrations of the stomach tissues are shown in Table 12 below.

Treatment (mg / kg) PGE2 concentration Normal control group 86.3 + - 2.5 ^c) Solvent control 125.7 ± 16.4 + MM (50 mg / kg) 112.6 ± 13.2 + MM (100 mg / kg) 110.5 15.5 ^a) + MM (200 mg / kg) 105.4 ± 8.6 ^a) + MM (300 mg / kg) 101.8 ± 8.4 ^a) Pantoprazole (30 mg / kg) 91.4 + - 6.4 ^b)  Results were presented as mean ± SD, with a) P <0.05, b) P <0.01, c) P <0.001 for the values of solvent treated Sprague-Dole rats.

Culture of gastric mucosal tissue

As a result of culturing gastric mucosal tissues after the autopsy of the experimental animals, Helicobacter pylori was grown in 9 test animals in the solvent-administered group, while the dose-dependent inhibition was observed in the group containing the oriental composition (MM) according to the present invention It was confirmed that there was a sterilization power. On the other hand, with respect to the perfume-containing composition according to the present invention, significant findings were observed in a dose-dependent manner, and it was confirmed that the perfume-containing composition according to the present invention had a sterilizing power against Helicobacter pylori. On the other hand, in the pantoprazole as a control drug test group, it was observed to be positive in 3 individuals and 2 animals, and thus it was judged that the flavor-containing composition according to the present invention could be used as a functional scavenger in the future (Table 13) &Lt; / RTI &gt; of Helicobacter pylori).

process
(Mg / kg) One 2 3 4 5 6 7 8 9 10 positivity
ratio Normal control group - - - - - - - - - - 0/10 Solvent control + + + + + + - + + + 9/10 + MM (50 mg / kg) + + - + - + + + + - 7/10 + MM (100 mg / kg) + + + - - + - + - + 4/10 + MM (200 mg / kg) + - + - - + + + - - 5/10 + MM (300 mg / kg) - + + + - - - - + + 5/10 Pantoprazole
(30 mg / kg) + - - - - + - - - - 2/10  -: Negative, +: Positive,

Degree of damage of gastric mucosa

The degree of damage of the gastric mucosa is shown in Table 14 below.

Treatment (mg / kg) Damage score above Normal control group 2.5 ± 0.3 ^c) Solvent control 12.5 ± 2.4 + MM (50 mg / kg) 10.3 ± 1.2 + MM (100 mg / kg) 8.2 ± 1.1 ^b) + MM (200 mg / kg) 7.3 ± 0.8 ^b) + MM (300 mg / kg) 5.6 ± 0.7 ^c) Pantoprazole (30 mg / kg) 3.4 ± 0.5 ^c)  Results were presented as mean ± SD, with a) P <0.05, b) P <0.01, c) P <0.001 for the values of solvent treated Sprague-Dole rats.

Histopathological findings

9 (A: normal control group, B: solvent control group, C: MM (100 mg / kg), D: MM (300 mg / kg), E: pantoprazole As compared with the normal histological findings, infiltration phenomena including severe inflammation in the whole tissues including gastric submucosa including cytopathic changes were observed in the case of a solvent in which nothing was administered after infection with Helicobacter pylori On the other hand, the pantoprazole administered with the perfume-containing composition (MM) according to the present invention showed inhibition of the findings observed in the solvent, and as a result, it was interpreted as a meaningful result as a functional material for Helicobacter pylori eradication in the future I will say.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

(Without reference numerals)

Claims (3)

delete A composition for eradicating Helicobacter pylori comprising scent and oil,
Wherein the composition comprising scent and oil comprises 10 to 40% by weight of scent, 10 to 40% by weight of squalane, and the remaining amount of olive oil. The composition for eradication of Helicobacter pylori according to claim 2, wherein the dosage form is selected from liquid formulations, tablets and capsules.

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