KR20050089242A - Anti-helicobactor composition containing extracts or compounds derived from rubus coreanus - Google Patents

Abstract

The present invention relates to an anti-helicobacter composition comprising a bokbunja tree extract or a compound derived therefrom. In particular, the anti-helicobacter composition of the present invention is harmless to the human body and has excellent anti-helicobacter activity, so that it is used as an antibiotic substitute for the conventional Helicobacter pylori, diseases caused by Helicobacter pylori, ie gastritis, gastric ulcer, duodenal ulcer, etc. It can be used to prevent and treat the disease.

Description

ANTI-HELICOBACTOR COMPOSITION CONTAINING EXTRACTS OR COMPOUNDS DERIVED FROM RUBUS COREANUS}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anti-helicobacter composition comprising a bokbunja tree extract or a compound derived therefrom, and more particularly, it is harmless to humans and has excellent anti-helicobacter activity. It relates to an anti-helicobacter composition, which can be used for preventing and treating diseases caused by Helicobacter pylori, that is, gastritis, gastric ulcer, duodenal ulcer and the like.

[Private Technology]

Human parasitic Helicobacter pylori is an aerobic Gram-negative bacterium that infects 30 to 90% of the world's population (Graham, DY, et al. (1991) Dig. Dis. Sci . 36, 1084) . -10; Taylor, DN and Blaser, MJ (1991) Epidemiol. Rev. 13, 42-59). The overall infection of Helicobacter pylori in Korea was 46.6% in 1998, and there was no significant difference between males (47.2%) and females (45.9%) (Kim JH, et al., 2000, The Korean H. pylori Study Group 59 (4): 388-3).

Helicobacter pylori is specific to humans and found primarily in certain places in the stomach, associated with diseases in the upper gastrointestinal tract, including duodenum and gastrointestinal ulcers, and is known to be associated with gastric and non-Hodgkins lymph gland cancer. .

The transmission of Helicobacter pylori is through animals or humans, or through various channels such as carelessness or food during medical practice (Dunn, BE, Cohen, H., and Blaser, MJ (1997).) Clinical Microbiology Reviews, 10 ( 4), 720-7; Kodaira, MS, Escobar, AMU, & Grisi, S. (2002) Revista de Sade Pade Pblica. 36: 356-369), once infected, are not easily eradicated.

Helicobacter pylori treatment includes tetracycline, amoxicillin, metronidazole, clarithromycin, and antibiotics, hydrogen ion pump inhibitors, and three drug treatments. . The triple treatment is a method of treating antibiotics and bismuth or hydrogen ion pump inhibitors, and has been applied to gastroduodenal ulcer disease associated with Helicobacter due to its high eradication rate (Lind, T., et al., (1996). ) Helicobacter 1: 138-144). However, eradication of Helicobacter pylori by tritherapy is not always successful, and repeated use of these agents has been reported to cause side effects (Borody, TJ, Shortis, NP, Chongnan, J., Reyes, E., and O). Shea, JE, (1996), Eradication failure (EF) after H. pylori treatment-further therapies.Gastroenterology 110: A67; Dunn et al., 1997). In other words, Samjae therapy causes mild side effects in about 50% of patients and vaginal infections in about 10% of women (Borody, TJ, Shortis, NP, Chongnan, J., Reyes, E., and O '). Shea, JE, 1996, Eradication failure (EF) after H. pylori treatment-further therapies.Gastroenterology 110: A67). In addition, the continued use of antibiotics leads to the emergence of resistant strains (DKarim, QN, & Maxwell, RH (1989). Journal of Clinical Pathology. 42 (7): 778) and other adverse events such as intestinal bacteria. (Ahn, YJ, et al., (2000) J. Agric. Food Chem. 48, 2744-2748; Zoppi, G., et al., (2001) Curr. Ther. Res. Clin. E. 62, 418-43).

Therefore, there is a need for the development of a plant extract that is harmless to the human body and has antibacterial activity against Helicobacter pylori and at the same time can prevent side effects due to the use of antibiotics made of synthetic compounds.

It is an object of the present invention to provide a bokbunja tree extract having anti-helicobacter activity.

It is also an object of the present invention to provide a bokbunja tree-derived compound having anti-helicobacter activity.

In order to achieve the above object, the present invention provides an anti-helicobacter composition comprising a bokbunja tree extract as an active ingredient.

The present invention also provides an anti-helicobacter composition comprising dibutyl phthalate or 4- (4-hydroxyphenyl) -2-butanone as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to an anti-helicobacter composition comprising Rubus coreanus extract or a compound derived therefrom as an active ingredient.

The bokbunja tree extract of the present invention is water or bokbunja tree or parts thereof, such as fruit, juice juice, fruit cake, leaves, branches, roots, stems, seeds and flowers, etc. It can be prepared by extraction with organic solvent and / or separation of layers.

The organic solvent may be a low-cost alcohol having 1 to 5 carbon atoms or alcohol dilution water, hexane, chloroform, ethyl acetate and acetone, the low alcohol may be ethanol, methanol, butanol, propanol and isopropanol, but is not limited thereto. . Preferably, the mixture is extracted with methanol or after methanol extraction, hexane, chloroform, ethyl acetate, and butanol are sequentially added, and the layers are separated by each solvent.

The extraction and layer separation method is usually carried out by this method, for example, by adding water or an organic solvent in a weight ratio of 1: 1 to 100 to an extract, a pulverized product or a powder thereof, and then extracting the extract or the separated layers. It can be carried out. Extraction and separation temperature may be 4 to 30 ℃, but is not limited thereto. The extract and the separator may then be filtered under reduced pressure and concentrated in vacuo to remove the solvent.

Bokbunja tree extract of the present invention exhibits a slight difference in anti-helicobacter activity, depending on the extraction site and maturation time of the plant. In particular, depending on the maturity of the fruit, leaves and leaves, not a significant difference between the root and side roots, but there is a difference in antimicrobial activity. For example, in the early stages of maturity, the fruit at the early stage of maturity shows antimicrobial activity at the lowest level, and the leaves have lower antimicrobial activity at the lower level, and even at the lower root level than the root of the roots. Excellent antimicrobial activity. In addition, the extract extracted from juice and gourd of bokbunja fruit also exhibits antimicrobial activity. For example, at 5 mg / disk concentration, chloroform extract and ethyl acetate extract of bokbunja juice had 52 and 26 mm growth inhibition, and hexane, chloroform extract and ethyl acetate extract of bokbunja juice were 15, 24 and 21 mm. Form growth inhibition ring.

In addition, the bokbunja tree extract of the present invention shows a significant difference in antimicrobial activity depending on the extraction solvent. For example, in the crude crude extract of bokbunja and hexane extract, chloroform extract, ethyl acetate extract, butanol extract and water extracts, which were sequentially separated by adding hexane, chloroform, ethyl acetate and butanol to the crude extract, the chloroform extract was the best. It exhibits antimicrobial activity, and hexane and ethyl acetate extracts also exhibit excellent antimicrobial activity. The chloroform extract forms a growth inhibitory ring of 20 mm at 0.5 mg / disk treatment concentration.

The present invention also provides an antimicrobial compound, dibutyl phthalate and 4- (4-hydroxyphenyl) -2-butanone (4- (4-Hydroxyphenyl) -2-butanone) from bokbunja.

Therefore, the bokbunja tree extract of the present invention or the antimicrobial compound derived therefrom can be used for the purpose of sterilizing Helicobacter pylori, the purpose of preventing diseases such as gastritis, gastric ulcer, duodenal ulcer caused by Helicobacter pylori Can be used as In addition, the bokbunja tree extract of the present invention is harmless to the human body and does not cause side effects due to the use of antibiotics.

Accordingly, the present invention provides an anti-helicobacter composition comprising the above-described bokbunja tree extract or a compound derived therefrom as an active ingredient.

The anti-helicobacter composition of the present invention may include each of the above active ingredients alone, and may further include a pharmacologically acceptable carrier or excipient, depending on the formulation, method of use, and purpose of use. When provided in a mixture, the active ingredient may be included in the anti-helicobacter composition in an amount of 0.1 to 99.9% by weight, but is preferably included in an amount of 0.001 to 10% by weight.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, which may be used one or more, but are not limited to these, and are common carriers And excipients are both available. In addition, when formulating an anti-helicobacter composition, it may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

The anti-helicobacter composition of the present invention can be used as a food, food additive, medicament or health food. When used as a medicament, it can be used orally or parenterally, but preferably oral administration. Since the formulation of the composition may vary depending on the method of use, it is not limited thereto. Examples of formulations include Plasters, Granules, Lotions, Powders, Syrups, Liquids and Solutions, Ointments, Fluidextract, Emulsions, Suspions, Infusions, Tablets, Injections, Capsules and Pills.

The dosage of the anti-helicobacter composition of the present invention is appropriately adjusted according to the use and method of the composition, for example, from 0.01 mg to 5000 mg based on one active ingredient, administered 1 to 5 times a day. can do.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the protection scope of the present invention is not limited to the following examples.

Example 1 Preparation of Methanol Crude Extracts by Regions of Bokbunja Trees

Leaves, berries (early, middle, late), branches, roots, and seeds of the bokbunja trees grown in Gochang-gun, Jeonbuk were collected, and extracts were prepared from the respective samples. Fruits were classified into early, middle and late stages, and the fruits of immature fruit were classified as early and average weight of about 0.7g / piece. Fruits representing the middle, which is dark red of the terminal family of the end weight was used to classify the fruit with the average weight 2.0g / dogs.

Each part of the collected bokbunja trees was dried well, ground and ground to a powder. 50 g of each powder sample was taken into a 500 ml Erlenmeyer flask, 300 ml of methanol was poured, shaken well, and left to stand at room temperature in the dark. After 3 days, the mixture was filtered under reduced pressure and concentrated under reduced pressure at 40 ° C. using a rotary vacuum concentrator (EYELA autojack NAJ-160, Japan) to obtain a crude methanol extract.

Example 2: Preparation of Bokbunja Organic Solvent Extract

A brief manufacturing process is shown in FIG.

2-1. Bokbunja Hexane Extract

20 g of bokbunja methanol crude extract, prepared using the bokbunja fruit (terminal) of Example 1, was dissolved in 800 ml of distilled water, placed in a 2 L separatory filter, and then mixed with the same amount of hexane. Layered into layers. After about 30 minutes, only the hexane layer was obtained using another separatory filter, and 800 mL of fresh hexane was poured into the water layer, and the second hexane layer was extracted in the same manner. The hexane layer was collected and concentrated by a vacuum concentrator to prepare a bokbunja hexane extract.

2-2. Bokbunja Chloroform Extract

After the 2-1 experiment, chloroform was added to the remaining water layer in the same manner to prepare a bokbunja chloroform extract.

2-3. Bokbunja Ethyl Acetate Extract

Ethyl acetate was added to the remaining water layer after the 2-2 experiment in the same manner to prepare a bokbunja ethyl acetate extract.

2-4. Bokbunja Butanol Extract

Butanol was added to the remaining water layer after the 2-3 experiment in the same manner to prepare a bokbunja butanol extract.

2-5. Bokbunja Water Extract

The water layer remaining after the 2-4 experiment was obtained and concentrated by a vacuum concentrator to prepare a bokbunja water extract.

Example 3: Preparation of organic solvent extracts of bokbunja juice and gourd

45 kg of bokbunja sold at the Gochang Bokbunja test site in Jeonbuk was compressed to prepare 21 L of juice and gourd (the rest of the bokbunja except for juice).

Bokbunja gourd was prepared in the same manner as in Example 1 as a crude methanol extract. The methanol crude extract was layered by adding hexane, chloroform and ethyl acetate in the same manner as in Example 2, and concentrated in vacuo to give a hexane extract, a chloroform extract, an ethyl acetate extract and a water extract. A brief manufacturing process is shown in FIG.

Bokbunja juice was separated by adding hexane, chloroform and ethyl acetate in the same manner as in Example 2, and concentrated in vacuo to give a hexane extract, chloroform extract, ethyl acetate extract and water extract. A brief manufacturing process is shown in FIG.

Example 4 Isolation and Identification of Antimicrobial Compounds from Bokbunja

Chloroform extract of Bokbunja juice prepared in Example 3 was fractionated by chromatography.

10 g of chloroform extract of Bokbunja juice was dissolved in chloroform, and then directly injected into a silica gel column (φ10 × 100 cm), and chloroform and methanol were dissolved as elution solvent 1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 30:70, 50:50, and 7 sequential elution fractions were obtained as BC1 to BC7 after flowing in a concentration gradient of 100% methanol volume ratio. The elution fractions BC1 to BC7 were each concentrated in vacuo and used as the following antimicrobial activity assay samples.

In addition, the BC3 fraction was further subjected to GC-MS to confirm that the dibutyl phthalate of Formula 1 and 4- (4-hydroxyphenyl) -2-butanone of Formula 2 (Fig. 3).

(Formula 1)

(Formula 2)

Experimental Example: Anti-Helicobacter Activity Assay

end. Experiment method

Each extract or compound prepared in Examples 1 to 4 was carried out by the following method to determine anti-helicobacter activity.

Helicobacter pylori (ATCC 43504) stock culture was stored at −80 ° C. with 20% glycerol and 5% calf serum added to Brucella broth and subcultured at Brucella agar when needed. It was. The culture was incubated for 3-5 days at 37 ° C. using an anaerobic incubator at 5% oxygen, 15% carbon dioxide, and 80% nitrogen, and a damp paper towel was placed in the culture vessel to maintain the moisture conditions. The medium used was Brucella broth containing 5% calf serum, 10 μg / ml vancomycin, 5 μg / ml polymyxin B, 5 μg / ml trimethoprim, and 2 μg / ml amphotericin B. All media were checked for contamination after the end of the growth cycle.

The antimicrobial activity assay was performed using a paper disc diffusion method, and microorganisms grown in Brucella agar were taken and mixed in 10 ml of sterile saline solution. The inoculation (0.1 mL) was 1 × 10 ⁷ -8 CFU / mL, and the sample was dissolved in 0.1 mL of acetone or methanol, and then, using a micropipette, a paper disk (ADVANTEC, diameter 8 mm, thickness 1 mm, Tokyo) was used. Roshi, Japan). After evaporation of the solvent, the paper disc was placed on the medium inoculated with the strain. In addition, only methanol and acetone were treated as a control to measure the inhibitory effect of the solvent itself on bacteria. Antimicrobial activity was confirmed by measuring the diameter of the growth-lowering ring (mm), and the results are shown below.

I. Antimicrobial Activity of Extracts by Bokbunja Tree Part of Example 1

Table 1 shows the antimicrobial activity of the extract for each part of the bokbunja tree. In the late stage of fruit, it showed the highest antimicrobial activity of 33 ㎜ at 20 ㎎ / disk treatment, and showed more than 20 ㎜ activity at early stage, deciduous and root at 20 ㎎ / disk treatment. It also showed antimicrobial activity during / disk treatment.

Bokbunja Tree Extract of Example 1 Throughput (mg / disk) 20 10 5 One Bokbunja tree area Fruit early 27 21 14 - Middle fruit 19 15 - - The end of fruit 33 16 - - Leaf 17 18 - - Deciduous 21 17 13 - Root 20 15 - - Side roots 24 19 13 - Seed - - - - Branch 19 18 - -

All. Antimicrobial Activity of Bokbunja Organic Solvent Extract of Example 2

As shown in Table 2, among the bokbunja organic solvent extracts of Example 2, the chloroform extract was very good antibacterial with 40 mm at 10 mg / disk treatment, 25 mm at throughput 25 mg / disk, and 20 mm at 0.5 mg / disk treatment. Activity was shown. In addition, ethyl acetate extract and hexane extract showed high antimicrobial activity.

Bokbunja Organic Solvent Extract of Example 2 Throughput (mg / disk) 10 One 0.5 Organic solvent Hexane 16 13 - chloroform 40 25 20 Ethyl acetate 23 - - Butanol - - - water - - -

la. Antimicrobial Activity of the Organic Solvent Extract of Bokbunja Juice or Gourd of Example 3

The organic solvent extracts extracted from the bokbunja juice or gourd of Example 3 showed antimicrobial activity against Helicobacter pylori. In particular, as shown in Table 3, the chloroform extract of the bokbunja juice was very strong antibacterial activity of 52 ㎜ and 35 ㎜ at 5 mg / disk and 1 mg / disk treatment concentration, ethyl acetate extract was 5 mg / disk treatment It showed an antimicrobial activity of 26 mm. In addition, organic solvent extracts of Bokbunja Park also showed high antibacterial activity as a whole.

Organic Solvent Extract of Example 3 Throughput (mg / disk) 5 One Bokbunja Juice Hexane - - chloroform 52 35 Ethyl acetate 26 - water - - Bokbunja Park Hexane 15 - chloroform 24 - Ethyl acetate 21 12 water - -

hemp. Antimicrobial Activity of the Chromatographic Fractions of Example 4

Chloroform extract of Bokbunja juice was chromatographed, and all fractions except BC2 fraction showed excellent antimicrobial activity (Table 4). In particular, BC3, BC6 and BC7 showed growth inhibition of 18, 15 and 21 mm even at 1 mg / disk treatment concentration.

Chromatographic fractions of Example 4 g / chloroform extract 10g Throughput (mg / disk) 5 One BC1 34.8 14 - BC2 > 100 - - BC3 > 100 32 18 BC4 > 100 12 - BC5 > 100 10 - BC6 19.8 19 15 BC7 11.9 27 21

 bar. Antimicrobial Activity of Bokbunja-derived Compounds

Dibutyl phthalate isolated and identified from BC3 fraction showed antibacterial activity of 19 mm to 30 mm at high concentration of 5 mg / disk or higher, and 4- (4-hydroxyphenyl) -2-butanone was 30 mm The antimicrobial activity was shown above (Table 5).

compound Throughput (mg / disk) 20 10 5 One 0.5 0.1 0.05 Dibutyl phthalate 23 20 20 19 19 16 15 4- (4-hydroxyphenyl) -2-butanone 35 34 33 12 - - - Tetracycline 34 32 Metronidazole 16 -

As mentioned above, the anti-helicobacter composition comprising the bokbunja tree extract of the present invention or a compound derived therefrom as an active ingredient is harmless to the human body and has excellent anti-helicobacter activity. Therefore, the composition of the present invention can be used as a replacement for a conventional Helicobacter pylori therapeutic agent, it is possible to prevent and treat diseases caused by Helicobacter pylori, that is, gastritis, gastric ulcer, duodenal ulcer and the like.

Figure 1 shows the process of extracting the crude extract of Bokbunja tree methanol with an organic solvent.

2 shows a process of extracting bokbunja juice and gourd with an organic solvent.

3 is a GC-MS chromatogram of BC3 fractions isolated from chloroform extract of bokbunja juice.

Claims (7)

An anti-helicobacter composition comprising Bokbunja tree extract as an active ingredient. The composition of claim 1, wherein the bokbunja tree extract is extracted from the bokbunja tree or a portion thereof with methanol. The method of claim 1, wherein the bokbunja tree extract is selected from the group consisting of hexane extract, chloroform extract and ethyl acetate extract; And The hexane extract, chloroform extract and ethyl acetate extract is a composition that is separated by sequentially adding hexane, chloroform and ethyl acetate to the methanol extract extracted from the bokbunja tree or a portion thereof with methanol. The composition of claim 2 or 3, wherein the part of the bokbunja tree is selected from the group consisting of fruit, leaf, root and branch.  The composition of claim 4, wherein the fruit is a juice or cake obtained by juice of the fruit. An anti-helicobacter composition comprising dibutyl phthalate or 4- (4-hydroxyphenyl) -2-butanone as an active ingredient. 7. The composition of claim 1 or 6, wherein the composition is used as a food, food additive, medicament or health supplement.