KR20100055952A - Extract antibiotic to helicobacter pylori extracted from persimmon leaves - Google Patents

Abstract

PURPOSE: An antibacterial extract isolated from persimmon leaf is provided to ensure antibacterial activity to helicobacter pylori and to prevent and treat gastric cancer and gastritis. CONSTITUTION: A persimmon leaf extract is isolated using alcohol, ethyl acetate, chloroform, methyl ethyl ketone, acetone, methylene chloride, or ether. The persimmon leaf extract is additionally purified using hexane:chloroform:ethanol through chromatography. The mixture ratio of hexane, chloroform, and ethanol is 3-7:0.5-1.5:0.5-1.5. A pharmaceutical composition for preventing and treating intestinal disease contains the persimmon leaf extract. The intestinal disease is gastric ulcer, gastric cancer, and gastritis.

Description

Extract Antibiotic to Helicobacter pylori Extracted from Persimmon Leaves}

The present invention relates to a method for extracting an antimicrobial extract from persimmon leaves, an antimicrobial persimmon leaf extract extracted by this method, a pharmaceutical composition and a food composition containing the extract.

Recently percentage of cancers that happen to our country who occupy the gastric cancer is 20.3%, and (Korean Central Center Registry, 2003) , is a country person with high gastric cancer incidence hageteuna due to several factors, such as diet, environment, gastric pathogen is Helicobacter pylori (Helicobacter Pylori infection is one of the important factors. Helicobacter pylori infections, known to be closely related to the development of gastritis, gastric ulcer, peptic ulcer and gastric cancer, accounted for 57.8% of adults aged 19 and older (Cover & Blaser, 2009). ASM News 61: 21-26. 1995).

When infected with Helicobacter pylori, TNF-α, IL-6, IL-1α, and β are secreted from gastric mucosal cells, and IL-8 secretion is the most prominent phenomenon. The secretion of TNF-α, IL-6, IL-1α, β is controversial, but is known to induce the production of IL-8. In the past, there have been reports of high secretion of anti-IL-8 IgA in gastrointestinal patients. Recently, the gastric mucosa of humans infected with Helicobacter pylori has been observed to have higher secretion of IL-8 than those uninfected. It has been shown to be a chemokine involved in neutrophil infiltration and activity, and has been identified as a decisive factor in triggering the inflammatory response caused by Helicobacter pylori infection.

On the other hand, persimmon contains proteins, fats, carbohydrates, sugars, tannins, vitamins A, B1, B2, C, etc., especially vitamin C is known to be much more than tangerine. Persimmon leaf has strong astringent effect, strengthens capillaries, and is effective in people with circulatory diseases such as arteriosclerosis or hypertension, scurvy, hypertension, arteriosclerosis, capillary strengthening, cerebral hemorrhage, diabetes, neurological diseases, ascites, amputees It has been reported to be effective in each edema, hiccups, intoxication, insomnia, prolapse, and diarrhea. Persimmon leaf tea is effective in opening the stomach, cooling alcohol readings, and protecting the liver.

The components of Persimmon Leaves include aspartalin and flavonoid glycosides such as myricitrin, tannins, polyphenols, resins, coumarin compounds, betulic acid and oleanolic acid. ) And organic acids such as ursolic acid and chlorophyll, vitamins A and C are abundant, and vitamin B1, pantothenic acid and folic acid are abundant and are recommended as a good food for preventing adult diseases.

As described above, Helicobacter pylori is a very dangerous microorganism in gastrointestinal diseases, but there is a lack of development of appropriate antibacterial materials using natural materials. Current treatments for Helicobacter pylori include general triple therapy, bismuth preparation, bismuth preparation, omeparazole and tetracycline, which are treated with tetracycline or amoxicillin together with bismuth and metronidazole. It has been reported that quadruple therapy with a combination of metronidazole and helicobacter pylori has an excellent therapeutic effect. However, it has been pointed out that the emergence of resistant strains by antibiotic administration, the penetration of drugs in the stomach, and the regrowth of the bacteria whose growth has been inhibited after treatment. because of this Numerous efforts have been made to search for antimicrobial substances specific to Helicobacter pylori, and recently, interest in bioactive components in plants has been increasing, and the possibility of their bioregulatory function and recovery or prevention of diseases has been suggested. . Recently, the antimicrobial activity of Helicobacter pylori of various natural products, such as thyme, Chinese tea, Cashew apple, joiner and rhubarb, has been reported, but the research is still insufficient. Therefore, the discovery of antimicrobial materials for Helicobacter pylori from edible folk resources, many of which are readily available, is an important alternative to antibiotics such as tetracycline.

The most ideal antimicrobial agent against Helicobacter pylori is not only antibacterial but also irritating to the epithelial cells. The present invention relates to the development of natural antibacterial material with excellent antibacterial effect to Helicobacter without harm to the stomach.

The present inventors have been making efforts to develop natural products that show antimicrobial activity against Helicobacter pylori and do not irritate the stomach, while extracts from persimmon leaves show excellent antimicrobial activity against Helicobacter pylori compared to other plant extracts. It was confirmed that the cells do not induce the production and secretion of IL-8, which does not cause gastrointestinal irritation, and thus the present invention has been completed.

The present invention is to provide a method for extracting the antimicrobial persimmon leaf extract comprising extracting the persimmon leaf with one or more solvents selected from the group consisting of alcohol, ethyl acetate, chloroform, methyl ethyl ketone, acetone, methylene chloride and ether.

In addition, the present invention is to provide an antimicrobial persimmon leaf extract extracted by the extraction method.

In addition, the present invention is to provide a pharmaceutical composition for preventing and treating gastrointestinal diseases containing the antimicrobial persimmon leaf extract.

In addition, the present invention is to provide a food composition containing the antimicrobial persimmon leaf extract.

The present invention relates to a method for extracting antimicrobial persimmon leaf extract comprising extracting persimmon leaf with one or more solvents selected from the group consisting of alcohol, ethyl acetate, chloroform, methyl ethyl ketone, acetone, methylene chloride and ether.

The alcohol may be a lower or higher alcohol, preferably a lower alcohol selected from the group consisting of methanol, ethanol, isopropanol and butanol, and more preferably ethanol. The alcohol may be a mixture with water.

After the extraction step, the obtained extract was purified by chromatography (paper, TLC, column, etc.) using a mixture of hexane: chloroform: ethanol as a carrier and silica or paper as a carrier. It can be carried out. Here, the distribution of the substances in the extract between the water contained in the carrier and the developing solvent occurs, and the substances are separated according to the difference in distribution ratio.

In the developing solvent, normal hexane: chloroform: ethanol is preferably mixed in a ratio of 3-7: 0.5-1.5: 0.5-1.5, most preferably 5: 1: 1.

The chromatography may be thin layer chromatography (TLC) or column chromatography using silica as a carrier. The TLC is a method of applying a thin coating of silica gel to a support such as a glass plate or plastic, dropping the extract thereon and then developing with a developing solvent to purify. The column chromatography is a method in which silica is packed in a column, an extract is loaded thereon, and then developed by using a developing solvent. It is preferable to use column chromatography for mass production of the extract.

In the extraction method of the present invention, when extracts are mass-produced, the persimmon leaf extract is separated into hexane and ethanol between the extraction step and the purification step to obtain an ethanol soluble part, and the ethanol soluble part is fractionated into chloroform and water to obtain a chloroform soluble part. It is preferable to carry out a further step (see FIG. 3). Then, chromatographic purification is performed using the obtained chloroform soluble part.

The present invention also relates to an antimicrobial persimmon leaf extract extracted by the extraction method.

Antimicrobial persimmon leaf extract of the present invention has an antimicrobial activity against Helicobacter pylori bacteria. In addition, IL-8 secretion by the Helicobacter pylori bacterium is suppressed and does not irritate the stomach (see Example 4).

Therefore, the antimicrobial persimmon leaf extract of the present invention can be used to prevent and treat gastrointestinal diseases. The present invention relates to a pharmaceutical composition for preventing and treating gastrointestinal diseases containing the antimicrobial persimmon leaf extract of the present invention.

The pharmaceutical composition containing the antimicrobial persimmon leaf extract of the present invention is a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and other formulations for administration, sterile injectable solutions, suppositories, and transdermal administration according to a conventional method. Can be formulated and used. Carriers, excipients and diluents which may be included in the composition 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. If necessary, it is formulated with diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like.

In one embodiment, the antimicrobial persimmon leaf extract of the present invention may be formulated as a solid preparation for oral administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. Formulated by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used.

In another embodiment, the pharmaceutical composition containing the antimicrobial persimmon leaf extract of the present invention may be formulated into a liquid preparation for oral administration. Liquid preparations for oral administration include suspensions, solvents, emulsions, syrups, and the like. In addition to the inert diluents commonly used (e.g., purified water, ethanol, liquid paraffin), various excipients may be used, for example. Wetting agents, sweetening agents, fragrances, preservatives and the like can be included.

In another embodiment, the pharmaceutical composition containing the antimicrobial persimmon leaf extract of the present invention may be formulated as a preparation for parenteral, preferably intraperitoneal administration. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As a sterile aqueous solution, a suitable buffer solution such as Hanks' solution, Ringer's solution, or physically buffered saline can be used. As a non-aqueous solvent, as a suspending agent, propylene glycol, polyethylene glycol, olive oil Vegetable oils such as, injectable esters such as ethyloleate, and the like can be used. If necessary, preservatives, stabilizers, wetting or emulsifying agents, salts for controlling osmotic pressure and / or buffers may also be used. On the other hand, suppositories such as witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Compositions formulated in such a manner can be administered in various amounts, including parenteral or oral (dermal, subcutaneous, intravenous, intramuscular, abdominal, etc.) in an effective amount. As used herein, an "effective amount" refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the composition according to the present invention may be appropriately selected depending on the route of administration, subject of administration, age, sex, weight, individual difference and disease state. Preferably, the composition of the present invention may vary the content of the active ingredient depending on the degree of disease, preferably 1-10000 ㎍ / weight kg / day, more preferably 10 ~ 1000 ㎍ / weight kg / day It may be administered in an effective amount of.

In addition, as another aspect, the antimicrobial persimmon leaf extract of the present invention may be provided as a food composition by mixing with a food acceptable carrier. When the antimicrobial persimmon leaf extract of the present invention is used as a food or beverage additive, the antimicrobial persimmon leaf extract may be added as it is, or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the antimicrobial persimmon leaf extract may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

In the case of prolonged intake for health purposes or health control purposes, the antimicrobial persimmon leaf extract can be taken for a long time because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, drinks, tea, and drinks. Alcoholic beverages and vitamin complexes.

The liquid component added in addition to the antimicrobial persimmon leaf extract when formulated into a beverage is not limited to the above, but may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides (e.g. glucose, fructose and the like), disaccharides (e.g. maltose, sucrose and the like) and polysaccharides (e.g. conventional sugars such as dextrin, cyclodextrin and the like), and xylitol Sugar alcohols such as sorbitol and erythritol. The proportion of said natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention. As flavoring agents other than those described above, natural flavoring agents (taumarin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used. .

In another embodiment, the food composition of the present invention comprises various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The food composition of the present invention may also contain pulp for the production of fruit and vegetable drinks. These ingredients may be used alone or in combination, and the proportion of such additives may be selected in the range of 10 to 20% by weight, based on the total weight of the composition.

The antimicrobial persimmon leaf extract of the present invention shows antimicrobial activity against Helicobacter pylori but does not irritate the stomach, and is useful for preventing and treating various gastrointestinal diseases caused by Helicobacter pylori.

In addition, the antimicrobial persimmon leaf extract of the present invention can be easily obtained by extracting with a solvent having a moderate polarity and further purifying with chromatography.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. It will be obvious to you.

Example 1 Antibacterial Activity of Persimmon Leaves against Helicobacter Bacteria

A. Antimicrobial Activity Test

For the present invention, persimmon leaves native to Korea were collected and dried, and then 50 g of pulverized and extracted into 1000 ml of 50% ethanol and prepared at 100,000 ppm were used for antimicrobial activity experiments.

The strain used in the experiment was Helicobacter pylori (KCTC 12083), which is the causative agent of gastric and duodenal ulcer, and was used by the Gene Bank. For the cultivation of bacteria, brucella broth (Difco, USA) with 10% FBS (fetal bovine serum) was used (Yoon et al., Korean J Biotechnol Bioeng 19: 187-191, 2004). The composition of the bacterium was bacto tryptone 10g, It consists of 10g bacto peptamin, 1g bacto dextrose, 2g bacto yeast extract, 5g sodium chloride and 0.1g sodium bisulfite. Solid medium for measuring the CFU (colony forming unit) of the strain, the disc agar diffusion method (disc agar diffusion method) was also used brucella agar medium, also in this case was prepared by adding 10% FBS, maintaining the aerobic conditions The CO2 incubator was kept at 10% CO ₂ , the humidity was always above 95%, and the temperature was maintained at 37 ° C.

B. Antimicrobial Activity Assay by Disk Diffusion Method

Disc diffusion was performed by dispensing 100 μl of Helicobacter pylori bacteria (1 × 10 ⁷ CFU / mL) onto a brucella agar medium plate, smearing them with sterile glass rods, and then raising the sterilized disc paper (Φ 10 mm) and membrane filter (0.45 μm). ) And control the concentration of 2.5, 5, 10 mg / mL sterilized persimmon leaf extract, and then absorb 50 μl into disc paper, and control group to absorb sterile water, and then in a CO ₂ incubator at 37 ° C. for 48 hours. After incubation, the presence of a clear zone around the disc was confirmed. At this time, the positive control group was incubated for 48 hours in an incubator at 37 ℃ using tetracycline antibiotics were measured for antimicrobial activity.

As a result, the persimmon leaf extract showed a strong antimicrobial activity against Helicobacter bacteria in a concentration-dependent manner (Fig. 1). Although not specified in the present invention, it showed excellent antimicrobial activity compared to other plants. Although the activity was weak compared to the antibiotic tetracycline used as a control, the plant extract showed a very strong activity, showing high applicability as an antimicrobial agent.

Example 2: Preparation of Extract of Persimmon Leaf

50 g of dried persimmon leaf samples were taken and extracted with 50% ethanol, filtered with Whatman (No. 2) filter paper, and the residue was extracted two times and extracted three times in the same manner. It was dried and used as a sample while storing at -4 degreeC. As a result, persimmon leaves were easily obtained extract with a high yield of 20-30%.

Example 3: Preparation of Active Fraction from Persimmon Leaf Extract

In order to investigate the extraction efficiency according to the extraction solvent was extracted with nucleic acid, chloroform, ethyl acetate, ethanol, water. 5 ml of each solvent was added to 1 g of persimmon leaf dried samples, extracted at 25 degrees for 24 hours, filtered through Whatman (No. 2) filter paper, and the residue was repeated twice in the same manner, and extracted three times in total. The extract was concentrated. Thereafter, the antimicrobial activity was assayed by the disk diffusion method mentioned in Example 1.

As a result, as shown in Table 1, the antimicrobial activity was high in the solvent fraction of medium polarity. Among them, the chloroform fraction showed antimicrobial activity. However, the water and nucleic acid fractions did not show antimicrobial activity, which is considered to be a medium or group of substances of Helicobacter pylori antimicrobial of persimmon leaves.

Extraction Yield and Antimicrobial Activity against Helicobacter Pylori by Solvent Organic solvent yield(%) Antimicrobial activity (mm / disc) Nucleic acid 1.8 ± 0.2 -** chloroform 7.3 ± 0.7 9.3 ± 2.7 ethanol 6.3 ± 1.0 - Ethyl acetate 9.4 ± 0.9 - water 33.1 ± 0.5 -

This active fraction was bound to a silica plate and separated as a developing solvent in which normal hexane: chloroform: ethanol was 5: 1: 1 (v / v / v). As a result, in the long wavelength (365 nm) it can be divided into five fractions according to the degree of coloration. By the long wavelength (365 nm), two fractions (L1, L2) can obtain three fractions (S1, S2, S3) at the short wavelength (254 nm). Among them, antimicrobial activity against Helicobacter pylori was seen in the long-wavelength S2 fraction.

Example 4: Mass preparation of antimicrobial actives from persimmon leaf extract

To prepare the active fraction, 10 kg of persimmon leaves were crushed and ultrasonically extracted from ethanol / water (1: 1 v / v) mixed solution for 1 hour, immersed at room temperature for 2 days, extracted three times, and then the combined extracts were used under reduced pressure. Concentrated. The concentrate was separated into n-nucleic acid and ethanol soluble part in a separatory funnel to take up the nucleic acid soluble part of the upper layer, and discarded. The concentrated ethanol soluble part of the lower layer was concentrated, and the concentrate was partitioned again with chloroform and water, and extracted three times with chloroform. And then concentrated again to obtain a chloroform fraction. In order to purify the active fraction, fractions were purified by silica TLC using a solvent of 5: 1: 1 (v / v / v) in a normal hexane: chloroform: ethanol solution to obtain a pale yellow powder (FIG. 3). This fraction was rediscovered by the above-mentioned thin layer chromatography and found to be a single substance. Therefore, the antimicrobial substance of persimmon leaf can be easily separated by the liquid chromatography method in the present invention without the purification process using a resin such as sephadex and additional purification process such as high performance chromatography (HPLC).

Example 5 Assay for IL-8 Production in Epithelial Cells

A. Measurement of IL-8 (Interleukin-8) Production

The epithelial cell used in this invention was AGS, a human epidermal cell line. This cell line was incubated in 37 ° C., 10% CO ₂ incubator in RPMI-1640 (Sigma) medium supplemented with 10% FBS, 100 U / ml penicillin and 100 g / ml streptomycin.

AGS was incubated with Helicobacter pylori and infected, and IL-8 levels in the culture supernatants were measured by ELISA. The bacterial cultures incubated for 48 hours in liquid medium were centrifuged twice with RPMI-1640 without the addition of antibiotics to make a 2 × 10 ⁸ / mL bacterial suspension. AGS was cultured by infecting the bacteria with no antibiotics so that the ratio of gastric epithelial cells to bacteria was 1: 100 after 95% survival by trypan blue dye exclusion test. IL-8 levels were measured in culture supernatants at 1, 3, 6, 9 and 12 hours after incubation.

IL-8 was measured using a DuoSet kit (Genzyme diagnostics, Cambridge, MA, USA). Mouse anti-human IL-8 was used as a capture antibody, and rabbit anti-human IL-8 biotinylated antibody was used as a secondary antibody. IL-8 was measured using an ELISA reader at a wavelength of 450 nm to obtain IL-8 levels using a standard curve previously obtained. The minimum measurement of IL-8 levels in this example was 6 pg / ml.

As a result, the AGS cells by the Helicobacter pylori, the time-dependent IL-8 secretion of IL-8 secretion was started at 3 hours of bacterial treatment and reached almost equilibrium at 12 hours and 24 hours (Fig. 2). Therefore, in the present Example, the experiment was conducted with the Helicobacter pylori treatment time as 24 hours.

On the other hand, as a result of examining the effect of persimmon leaf extract on the secretion of 1L-8 increased by Helicobacter pylori treatment, it was possible to measure the inhibitory effect of IL-8 in a concentration-dependent manner, especially at 5 and 10 ㎎ / ㎖ An effect was observed (below Figure 2).

Figure 1 shows the antimicrobial activity of Helicobacter pylori bacteria of the antimicrobial persimmon leaf extract according to the present invention (A: 50% ethanol extract × 50,000 mother liquor 50μl, B: control (ethanol) 50μl drop, C: 50% ethanol 50 μl drop of extract × 100,000 mother liquor, D: antimicrobial activity upon 50 μl drop of tetracycline × 1,000 mother liquor).

Figure 2 shows the levels of IL-8 secreted from epithelial cells infected with Helicobacter pylori (top) and when the antimicrobial persimmon leaf extract is treated (bottom).

Figure 3 shows the mass extraction method of antimicrobial persimmon leaf extract according to the present invention.

Claims (9)

Extracting persimmon leaves with at least one solvent selected from the group consisting of alcohol, ethyl acetate, chloroform, methyl ethyl ketone, acetone, methylene chloride and ether. The method of claim 1, After the extraction step, the extract is characterized in that the extraction step further comprises the step of performing purification by chromatography using a mixed solution of silica or paper as a carrier, hexane: chloroform: ethanol as a developing solvent . 3. The method of claim 2, The hexane: chloroform: ethanol is an extraction method, characterized in that the ratio of 3-7: 0.5-1.5: 0.5-1.5. 3. The method of claim 2, Extracting persimmon leaf extract between hexane and ethanol between the extraction step and the purification step to obtain an ethanol soluble part, and further comprising the step of fractionating the ethanol soluble part with chloroform and water to obtain a chloroform soluble part. Way. Antimicrobial persimmon leaf extract extracted by the extraction method according to any one of claims 1 to 4. The method of claim 5, Extract characterized by having antimicrobial activity against Helicobacter pylori. A pharmaceutical composition for preventing and treating gastrointestinal diseases, comprising the extract of claim 5. The method of claim 7, wherein The gastrointestinal diseases are gastritis, gastric ulcer, gastric cancer caused by Helicobacter pylori bacteria. Food composition containing an extract according to claim 5.

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