KR20090046425A - Composition for preventing or treating gastritis or gastric ulcer containing cheonma extract - Google Patents

Abstract

The present invention has the effect of protecting the stomach damage (天麻, Gastrodia) It relates to a composition for the prevention or treatment of gastritis or gastric ulcer containing elata Blume) extract as an active ingredient. Chunma extract according to the present invention protects the gastric mucosal cell damage caused by invasive stress and inhibits the synthesis of nitric oxide, which is an inflammation-inducing factor, and thus is effective in preventing or treating gastritis or gastric ulcer. Do.

Cheonma, submersible stress, nitric oxide, nitric oxide synthase, gastritis, gastric ulcer

Description

Composition Comprising the Extract of Gastrodia elata Blume for the Prevention or Treatment of Gastritis or Gastric ulcer}

The present invention has the effect of protecting the stomach damage (天麻, Gastrodia) It relates to a composition for the prevention or treatment of gastritis or gastric ulcer containing elata Blume) extract as an active ingredient.

Inflammatory substances include serotonin (hydroxytryptamine (5-HT), kinins, histamine, prostaglandins, leukotrienes (LTs), platelet-activation factor (PAF) ), Cytokines, oxygen metabolites, and nitric oxides (NO), which are activated upon stimulation and trigger an inflammatory response through interaction.

Nitric oxide is known not only as a pharmacological messenger of the nervous system, but also as an important regulator, such as cellular differentiation or intracellular signal transduction, in addition to inflammatory responses, immune system and cytotoxicity.

Nitric oxide synthase (NOS), which synthesizes nitric oxide, is divided into two types: constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS). Are classified.

Isomers of constitutive nitric oxide synthase include endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), which are endothelial nitric oxide synthase (nNOS). Regulates vascular homeostasis in endothelial cells and platelets (Nishida, K. et. al . , J. Clin . Invest ., 90: 2092, 1992), neuronal nitric oxide synthase produces nitric oxide that acts as a neurotransmitter in the central and peripheral nervous systems (Moncada, S. et. al ., Pharm acol . Rev. , 49: 137, 1997). Isomers of inducible nitric oxide synthase are calcium-dependent enzymes located in macrophages, hepatocytes, mesovascular muscle, neutrophils and endothelial cells (Anggard, E. et al., Lancet , 343: 1199, 1994).

Among them, a large amount of nitric oxide caused by inducible nitric oxide synthase induced by macrophages by stimulator causes an inflammatory response such as vascular permeability and edema or promotes the activity of COX (cyclooxygenase) to induce inflammatory mediators such as prostaglandins. It acts to deepen the inflammatory response due to biosynthesis. This inhibition of nitric oxide production is meaningful in identifying the mechanism of anti-inflammatory agents.

Usually, the gastric wall protects the mucous membranes from hydrogen ions and other toxic substances produced in the stomach, which are hampered by gastric injury (Konturek, PC et. al ., J. Physiol . Pharmacol ., 48: 3, 1997). The gastric microenvironment plays a major role in the protective barrier of the stomach. Obstruction of gastric mucosal perfusion leads to gastric ulcers or gastric damage, and the airfield develops gastric bleeding (Brzozowski, T. et al . , J. Physiol . Pharmacol ., 52: 583, 2001). Gastric bleeding is typically regulated by signaling agents such as prostaglandins and nitric oxide (NO) (Pawlik, M. et. al ., J. Physiol . Pharmacol . , 52: 569, 2001).

Gastritis and gastric ulcers are caused by infections of Helicobacter pylori , which are parasitic in the stomach, and the effects of gastric acid and pepsin, which are factors that cause the gastrointestinal tract to break down, and mucosa, a protective factor that protects the stomach wall from them. Equilibrium is broken between the functions of In addition, many drugs, including non steroidal anti-inflammatory drugs, are considered to be the main causes of gastritis and gastric ulcers because they cause side effects such as gastrointestinal bleeding.

Therefore, in order to treat gastritis and gastric ulcers, antibiotics that suppress Helicobacter pylori or administration of drugs that inhibit gastric acid secretion should be administered, depending on the cause. Gastritis and gastric ulcer treatments developed so far to remove or lower the acid secreted in the stomach acts a variety of mechanisms.

Among them, various drugs, especially antibacterial agents, have been tried in order to eradicate Helicobacter pylori bacteria infected with the digestive organs. For example, bismuth preparations, such as colloidal bismuth citric acid and bismuth salicylic acid tea, antibacterial agents such as amoxicillin, ampicillin, clarithromycin, opfloxacin, tetracycline, and antitumor agents such as titazazole and metronitazole Attempts have been made to administer inhibitors of proton pumps alone or in combination of two or three agents.

On the other hand, the drug that has long been used for the treatment of gastrointestinal disorders is antacid. The effect on symptoms is the fastest because it directly neutralizes the stomach acid secreted in the stomach, protecting the stomach and eliminating heartburn. The most representative drug is a suspension prepared from a mixture of aluminum hydroxide and magnesium hydroxide, and a drink system including sodium hydrogen carbonate is on the market. However, antacids do not prevent gastric acid from coming out, and they act to neutralize gastric acid already secreted for a short time. It can cause side effects such as diarrhea and urolithiasis. Therefore, a drug that can prevent the secretion of gastric acid is more fundamentally required, and then acetylcholine receptor inhibitors and prostaglandin receptor active drugs have been developed. However, because they can work throughout the blood and the peripheral nervous system, it acts on nerve cells other than the gastrointestinal tract and shows side effects and is not widely used.

On the other hand, peptic ulcer drugs have been developed as a variety of drugs, and even though various treatment strategies using different drugs may be proposed, they do not fundamentally show a direct healing effect to heal wounds of peptic ulcers.

These drugs only play a role in suppressing the attack from stomach acid to keep the wounds from worsening, and the disappearance of the ulcer is inevitably dependent on the body's natural healing power.

The treatment of peptic ulcer requires a very long time to administer the drug, and therefore, the short term acute toxicity due to drug administration as well as chronic toxicity and its side effects can be considered as a good treatment.

Gastrodia elata Blume (GEB) is a perennial herb belonging to the genus Orchidaceae, and other names such as red roots, guinea pigs, oaks, shoots, and jeongpungcho Also called. Cinnamon is classified as an intermediate product in Shinnongbon Carbide, and it is known as a medicinal herb. The clinical efficacy of Chunma has been widely documented in the herbaceous tree, assimilation and other herbal documents, and is known to be effective for symptoms such as hypertension, headache, paralysis, neurological diseases, diabetes, adult diseases, stress, and fatigue. have. Most of the phenolic compounds contained in Gastrodia are Gastrodin, Phenolic Glycosides, Sulfur-containing Phenolic Compounds, Organic Acids, Sugars and Beta-Sitosterol ( β -sitosterol). (sterols), cholesterol (cholesterol), p -hydroxybenzyl alcohol ( p -hydroxybenzyl alcohol) and vanillin (vanillin) and other ingredients are also known. The water content of raw horses is 81.2%, and the general lyophilized sample contains 61.2% crude protein, 1.50% crude fat, 2.55% crude ash, and 89.74% carbohydrate (Korean Patent No. 10-0625609).

Looking at the prior art related to cheonma, functional beverage composition that can reduce blood cholesterol levels by containing extract extracted by combining the chestnut, black beans, jujube and licorice in a certain ratio (Korean Patent No. 10-0207605) Liver containing rhubarb, astragalus, red peony, schisandra, sesame seed, stone changpo, licorice, sediment box, mountain lion, turmeric, turmeric, white ginseng, gojija, samchil, cheonma, japonica, cheongapsan, sulfur, innerwear, black jima, and liver Herbal extracts for the prevention and treatment of diseases (Korean Patent No. 10-0310979), three hundred seconds, Schisandra chinensis, parsley, Angelica, Cornus, Sesame, Sesame leaf, Brown root, Cheonma and Goji alcohol extracts of two or more herbs selected from the group consisting of Composition for improving brain function and cognitive function (Korean Patent No. 10-0569089), Ginseng 30-80, Male 6-24, Cheonma 6-24, Seokchangpo 5-15, Turmeric 5-15, Kill 5-15, Baekgangjam 5-15, Basement 5-15, Baekbong 5-15, half 5-15, dermis 3-9, licorice 3-9 parts are mixed and extracted with hot water or alcohol and dementia disease prevention and treatment composition using the extract as an active ingredient (Korea Patent No. 10-0382564), 10 to 50 parts by weight of sewage, 1 to 15 parts by weight of cheonma, 1 to 10 parts by weight of Seokchangpo, 1 to 15 parts by weight of turmeric or turmeric, 1 to 8 parts by weight of dead or dead leaves, 1 to 8 parts of silkworm powder 1 to 8 parts by weight of Boo, Ji-sil or tanberry fruit, 5 to 20 parts by weight of Fuling, 2 to 20 parts by weight of dermis, 1 to 5 parts by weight of licorice, and 1 to 10 parts by weight of ginger, characterized in that the extraction method in a conventional manner Alzheimer's disease prevention and treatment composition (Korean Patent No. 10-0360674), 3 to 13% by weight, red rose red 3 ~ 13% by weight, ginseng 3 to 13% by weight, Astragalus 3 to 13% by weight, lignite 1 ~ 10% by weight, Baekjak 1-10% by weight, 1-10% by weight of seaweed, 1-10% by weight, 1-10% by weight of Baeksin, 1-10% by weight of longeye, 1-10% by weight of Baekbokyeong, half 0.5-8 medium %, Dermis 0.5-8% by weight, malt 0.5-8% by weight, creation 0.5-8% by weight, Chunma 0.5-8% by weight, 0.1-6% by weight, new country 0.1-6% by weight, 0.1-6% by weight , 0.1-6% by weight, 0.1-6% by weight of frankincense, 0.1-6% by weight, myrrh 0.1-6% by weight, corydalis 0.1-6% by weight, 0.1-6% by weight, 0.1-6% by weight of Samseong, 0.1-6% by weight, Anti-cancer and cancer metastasis-containing composition (Korean Patent No. 10-0425978) comprising 0.1 to 6% by weight of health, 0.1 to 6% by weight of sulfur and 0.5 to 8% by weight of ginger (Chinese Patent No. 10-0425978) , Pharmaceutical composition for the treatment and prevention of acute rheumatoid, chronic rheumatism, chronic inflammatory arthritis and degenerative osteoarthritis containing gastric gland, Angelica, Angelica, scorpion, safflower, cinnamon, and windproof (Korean Patent No. 10-0540033) , Pharmaceutical composition for the treatment or prevention of hypertension, including extracts of cheonma, dermis, bokyeong, hawthorn, haeju and rhubarb as active ingredients 10-0577674) has been patented.

However, none of the above studies have reported the therapeutic effect of cheonma extract on gastritis and gastric ulcer disease.

Therefore, the present inventors analyzed the components of various traditional herbal medicines that have already been proved to overcome the limitations of conventional gastritis and gastric ulcer treatments, and searched for the effects of treating gastritis and gastric ulcers, the extracts of gastric mucosa caused by inducing stress The present invention was completed by confirming the fact that it protects from damage and inhibits the synthesis of inflammation-causing nitrogen oxide, which has an effect of inhibiting gastritis or gastric ulcer.

It is a main object of the present invention to provide a pharmaceutical composition for the prevention or treatment of gastritis or gastric ulcer, which contains cheonma extract as an active ingredient.

Another object of the present invention to provide a health functional food for the prevention or improvement of gastritis or gastric ulcer comprising a cheonma extract and a food supplement acceptable food supplement.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of gastritis or gastric ulcer containing cheonma extract as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating gastritis or gastric ulcer comprising a cheonma extract and a food supplement acceptable food supplement.

Gastrodia elata extract according to the present invention comprises (a) a crushing a thousand miles, and then, a lower ketone of _{3 -6} C lower alcohols, water, C _{3 -6} lower organic acid, a lower alcohol ester of a C _{3 -6, -6} C ₃ of the and a step of extracting with a solvent selected from the group consisting of halogenated hydrocarbons, C _{3 -6;} And (b) may be prepared by the step of recovering the extract, but is not limited thereto.

A lower alcohol of C _{3 -6} will be desirable to use ethanol (ethanol) or methanol (methanol) and using a lower organic acid is a C _{3 -6} acid (butyric acid) or kapro acid (caproic acid) desirable. In addition, the lower alcohol ester of a C _{3 -6} dimethyl ether (dimethyl ether), ethyl ether (methyl ethyl ether), and diethyl ether may be selected from the group consisting of (diethyl ether), lower ketones of C _{3 -6} include dimethyl ketone (dimethyl ketone), ethyl-methyl ketone (methyl ethyl ketone) and diethyl ketone may be selected from the group consisting of (diethyl ketone), a halogenated hydrocarbon group of C _{3 -6} is carbon tetrachloride (carbon tetrachloride) or Trichlorethylene It is preferable to use (trichlorethylene).

In one embodiment of the present invention, after powdering the cheonma, methanol was added to the cheonma, extracted using extraction methods such as hot water extraction, cold sediment extraction, reflux cooling extraction or ultrasonic extraction, and centrifuged to obtain the cheonma extract.

Since the cheonma extract of the present invention is a natural substance, there is no toxicity and can be continuously used in large amounts as a medicine.

  As a result of visually confirming damage to gastric mucosa by inducing invasive stress using a mouse treated with the cheonma extract of the present invention obtained above, mucosal damage and blood clot were not found, thus protecting gastric damage of the cheonma extract. The effect could be confirmed. In addition, in order to confirm the effect of the cheonma extract of the present invention on the production of nitric oxide, an inflammation-inducing factor, the amount of nitric oxide production and the messenger RNA expression of nitric oxide synthase were measured. It was confirmed that messenger RNA expression of the enzyme was suppressed.

The composition comprising the cheonma extract of the present invention can be used in combination or formulated with drugs such as antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics that are already in use.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterile injectable solutions according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided orally. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

The compound containing the extract of the present invention or a pharmaceutically acceptable salt thereof may be used as a main ingredient or additives and auxiliaries of foods in the manufacture of various functional foods and health functional foods.

In the present invention, the "functional food" means a food that has improved the functionality of the general food by adding the extract of the present invention to the general food. Functionality can be roughly divided into physical properties and physiological functions. When the extract of the present invention is added to general foods, the physical properties and physiological properties of general foods will be improved, and the present invention comprehensively describes foods having such improved functions as 'functional foods'. 'Is defined.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract of the present invention may contain a fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical, but is usually selected in the range of 0.01 to 20 parts by weight per 100 parts by weight of the extract of the present invention.

The cheonma extract of the present invention protects the gastric mucosal cell damage caused by invasive stress, and inhibits the synthesis of nitric oxide, an inflammation-inducing factor, and thus suppresses gastritis or gastric ulcer, thus preventing or treating gastritis or gastric ulcer. Or as a health functional food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1:  Preparation of Cheonma Extract

Roots of three-year-old Gastrodia elata Blume (GEB) were purchased from Gyeongsangbuk-do, Korea, washed, sliced, and frozen and dried at -70˚C for freeze-drying.

Methanol was added to the cheonmama, extracted, and then resuspended in 30% methanol, and the supernatant obtained by centrifugation was dried with a rotary evaporator to finally obtain a dark brown powder.

Example 2: Confirmation of Gastric Protective Effect of Chunma Extract in Inducing Gastric Mucosal Damage

As experimental animals, 8-week-old male BALB / c mice (Samtaco, Korea) weighing 22-25 g were supplied with commercial feed (Samtaco, Korea) and water, and were used in a laboratory environment for 1 week.

The laboratory environment was repeated day and night at 12 hour intervals while maintaining 22 ± 1 ° C and 50 ± 5% humidity.

L-arginine treated group (n = 6) mice were intraperitoneally administered (ip) L-arginine dissolved in saline of isotonic saline (300 mg / kg). Cheonma extract (0.02 ml / g) obtained in oral administration (po). In addition, the negative control group was intraperitoneally administered (n = 6) or orally administered (n = 6) respectively. After administration, mice were stored for 1 hour in a vertical position in each independent cage to ensure sufficient absorption of the drug.

After immersing the mice for 6 hours (Takagi, KY et al., Chem. Pharm. Bull ., 12: 465, 1964) in a water tank maintained at 20 ~ 23 ° C, the mice were ethyl ether (diethyl ether) After anesthesia, the stomach was excised along a large bend, and the gastric mucosa was removed using small scissors. Blood was taken from each mouse and immediately under the vein, and serum was obtained by centrifugation.

As a result of visual observation of spots, linear damage and corrosion on the surface of the gastric mucosa, as shown in Fig. 1, the samples (A and B) of the negative control group (A and B) administered saline intraperitoneally and orally administered were 1 to 2 mm in size. Many mucosal damages and large blood clots were found, whereas only a small amount of mucosal damage and medium blood clots were found in the sample treated with L-arginine, a positive control. On the other hand, in the sample (D) of the mouse treated with cheonma extract, both mucosal damage and blood clot were not found.

Ulcer index measurements, on the other hand, were scored according to the extent of mucosal damage (Nie, SN et al . , World J. Gastroenterol . , 9: 1772, 2003). 1 point for slight circular bleeding corrosion, 2 points for bleeding corrosion smaller than 1mm, 3 points for bleeding corrosion of 1 ~ 2mm long, 4 points for 4 ~ 4mm for bleeding corrosion of 2 ~ 3mm long Longer lengths were scored 5 points. In addition, when the wound had a width of 1 mm or more, the score was doubled. The experimental values were expressed as mean ± standard deviation, and ANOVA (one-way analysis of variance) according to Dunnett's test determined that the p- value was less than 0.05.

The ulcer index of the negative control group was 23.0 ± 4.1 and 23.3 ± 7.8, respectively, whereas the ulcer index of the positive control group was 8.0 ± 5.5 and the ulcer index of the cheonma extract treatment group was 2.5 ± 3.3 (see Table 1).

From the above results, it was found that the cheonma extract has an effect of preventing gastric damage caused by submerged restraint stress.

Pretreatment Ulcer index, points Control (i.p .; intraperitoneal administration) 23.0 ± 4.1 Control (p.o .; oral administration) 23.3 ± 7.8 L-arginine (300 mg / kg, i.p .; intraperitoneal administration) 8.0 ± 5.5 * † Cheonma Extract (0.02 ml / g, p.o.:Oral) 2.5 ± 3.3 * †

The amount of nitric oxide produced in the serum and stomach tissue obtained above was measured as follows. In addition, the amount of nitric oxide produced in the serum was measured by the Griess assay (Dirsch, VM et. al ., AM Planta Med ., 64: 423, 1998).

The serum was added to a 96-well plate containing active agent (LPS 10 ng / well), and the cheonma extract obtained in Example 1 was added to a final concentration of 30 mg / ml. After 24 hours, 100 ml of the supernatant was added with the same amount of grease reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, and 5% phosphoric acid) and left at room temperature for 10 minutes. The optical concentration of each sample was measured at 550 nm using a microplate autometer VICTOR3 (Wallac co., Finland). Measurement curves were prepared using standard sodium nitrite. The experimental values were the results of three repeated experiments, expressed as mean ± standard deviation, and were performed by ANOVA (one-way analysis of variance) according to Dunnett's test, and were determined to be statistically significant when the p value was less than 0.05. .

As a result, as shown in Figure 2, the amount of nitric oxide produced in the serum and gastric tissue was confirmed that the L- arginine treated group and the cheonma extract treated group 20 to 50% of the production amount of the negative control group.

From the above results, it was found that the cheonma extract of the present invention reduces the production of nitric oxide in serum and stomach tissue.

Example 3:  Screening of Nitric Oxide Inhibitory Activity of Chunma Extract

Changes in nitric oxide production in serum and gastrointestinal tissues include endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase To investigate the effect on the change in the expression level of oxide synthase (iNOS), messenger RNA transcription levels encoding the enzymes were measured via RT-PCR using primers described in Table 2 below.

Messenger RNA expression was based on glyceraldehyde phosphatase (GAPDH) and was tested using Quantity One Version 4.6.1 (Bio-Rad, Philadelphia, PA).

Frozen gastric tissue (about 100 mg) was homogenized using 1 ml Trizol ^® reagent and total RNA (Roos-van Groningen et al ., JA Kidney Int ., 65:97, 2004) were measured at 260 nm with a spectrophotometer (Perkin Elmer, US).

The RNA was examined by staining with ethidium bromide (EtBr) after 1% formaldehyde-agarose electrophoresis. RNA was stored at -80 ° C until use, RT-PCR was performed using Maxime RT-PCR PreMix and the primers (Bioneer Co., Korea) of Table 2 below. The primer was used Primer3 software (Whitehead Institute, MIT Center for Genome Research, US) to analyze the nucleotide sequence stored in the NCBI GenBank database.

 Target genes Primer sequence (sense / antisense) SEQ ID NO: Product size (bp)  eNOS 5’-TACGCACCCAGAGCTTTTCT-3 ’ One 307 5’-GCAGGATGCCCTAACTACCA-3 ’ 2  nNOS 5’-CCATCAGCTCCTCTCCAGAC-3 ’ 3  511  5’-ATATGGCCTCCTTGCTCCTT-3 ’ 4  iNOS  5’-AGACCTCAACAGAGCCCTCA-3 ’ 5  305 5’-GCAGCCTCTTGTCTTTGACC-3 ’ 6 GAPDH 5’-ATGTTCCAGTATGACTCCAC-3 ’ 7  371 5’-GCCAAAGTTGTCATCTGATGA-3 ’ 8

Single stranded cDNA was synthesized by reacting 500 ng of total RNA at 45 ° C. for 30 minutes and 95 ° C. for 5 minutes.

PCR conditions were allowed to react at 95 ° C for 45 seconds, then endothelial nitric oxide synthase, neuronal nitric oxide synthase and inducible nitric oxide synthase were 32 cycles at 54 ° C / 55 ° C / 53 ° C, respectively. , Glyceraldehyde phosphatase was performed 27 cycles at 54 ° C, and then recovered for 60 seconds at 72 ° C. After the PCR procedure, 20 ml of each sample of the product was analyzed by 1.5% agarose gel electrophoresis.

The predicted position of the product was confirmed by comparison with 1kb DNA ladder (Solgent, Korea). Based on glyceraldehyde phosphatase gene expression for approximate quantitation, each sample is then compared to the corresponding amount of cDNA relative to the molecular image Gel Doc XR system (Bio-Rad Co., Italy). And Quantity One 1-D analysis software (Bio-Rad Co., US).

The experimental values were expressed as mean ± standard deviation, and ANOVA (one-way analysis of variance) according to Dunnett's test was considered to be statistically significant when the p value was less than 0.05.

As a result, as shown in Figures 3 and 4, messenger RNA of inducible nitric oxide synthase was significantly decreased in the gastric tissue of the mouse induced the submerged restraint stress, endothelial nitric oxide synthase and neuronal Nitric oxide synthase was similar in all experimental groups irrespective of treatment, and glyceraldehyde phosphatase, a housekeeping gene, showed no significant difference in expression level. This means that the damage of gastric mucosa is reduced due to the decrease in the expression of inducible nitric oxide synthase during the expression of the three kinds of nitric oxide synthase caused by the submerged restraint stress, and compared with Example 2 When inducible nitric oxide is an important molecular marker for gastric mucosal damage. In conclusion, it was found that the synthesis of inducible nitric oxide was inhibited by the treatment of cheonma extract to reduce gastric mucosal damage.

Examples of the formulation of the pharmaceutical composition comprising the cheonma extract of the present invention will be described, but the present invention is not intended to limit this, but is intended to explain in detail only.

Formulation example  One: Powder  Produce

Cheonma Extract 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2: preparation of tablets

Cheonma Extract 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional manufacturing method of the tablet.

Formulation example  3: Manufacture of capsule

Cheonma Extract 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4: Preparation of Injection

Cheonma Extract 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

According to the conventional method for preparing an injection, the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).

Formulation example  5: Liquid  Produce

Cheonma Extract 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The solution was prepared by sterilization.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the state of mucosal damage in the gastric tissue of the mouse induced submerged stress.

Figure 2 shows the amount of nitric oxide in the serum and stomach tissue of mice exposed to immersion stress.

Figure 3 shows messenger RNA expression of endothelial nitric oxide synthase, neuronal nitric oxide synthase and inducible nitric oxide synthase in gastric tissues of mice exposed to submerged stress (marker: size of synthetase chain reaction). Marker; column 1: control group administered intraperitoneally; row 2: negative control group administered orally; row 3: positive control group treated with L-arginine;

Figure 4 shows the messenger RNA expression ratio of endothelial nitric oxide synthase, neuronal nitric oxide synthase and inducible nitric oxide synthase in gastric tissues of mice exposed to submersible stress.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition Comprising the Extract of Gastrodia elata Blume for          the Prevention or Treatment of Gastritis or Gastric ulcer <130> P07-B213 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense for eNOS primer <400> 1 tacgcaccca gagcttttct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense for eNOS primer <400> 2 gcaggatgcc ctaactacca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense for nNOS primer <400> 3 ccatcagctc ctctccagac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense for nNOS primer <400> 4 atatggcctc cttgctcctt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense for iNOS primer <400> 5 agacctcaac agagccctca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense for iNOS primer <400> 6 gcagcctctt gtctttgacc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense for GAPDH primer <400> 7 atgttccagt atgactccac 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense for GAPDH primer <400> 8 gccaaagttg tcatctgatg a 21  

Claims (5)

 A pharmaceutical composition for the prevention or treatment of gastritis or gastric ulcer, which contains cheonma extract as an active ingredient. The pharmaceutical composition for preventing or treating gastritis or gastric ulcer according to claim 1, wherein the cheonma extract selectively inhibits the expression of inducible nitric oxide synthase. The pharmaceutical composition according to claim 1, wherein the cheonma extract is prepared by the following steps: (a) a crushing a thousand miles, and then extracting with a lower alcohol, water, a lower organic acid, a lower alcohol ester, a lower ketone and the solvent, selected from the group consisting of halogenated hydrocarbons, C _{3 -6;} And (b) recovering the extract. A health functional food for the prevention or amelioration of gastritis or gastric ulcer comprising a cheonma extract and a food supplement acceptable food supplement. The health functional food according to claim 3, wherein the cheonma extract is prepared by the following steps: (a) a crushing a thousand miles, and then extracting with a lower alcohol, water, a lower organic acid, a lower alcohol ester, a lower ketone and the solvent, selected from the group consisting of halogenated hydrocarbons, C _{3 -6;} And (b) recovering the extract.

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