KR20070047743A - Antimicrobial composition which comprising the hibiscus ethanol extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for an antimicrobial agent comprising a hibiscus ethanol extract, more specifically, the antimicrobial activity of the ethanol extract obtained by extracting dry hibiscus with ethanol, and the hibiscus ethanol extract hexane, chloroform, ethyl acetate, n- By sequential fractionation using butanol and water to check the antimicrobial activity of each fraction, it is intended to apply the hibiscus ethanol extract to the pharmaceutical composition for antimicrobial agents.

According to the present invention, by having an antimicrobial activity against various strains, it is possible to provide a pharmaceutical composition for antimicrobial comprising a hibiscus ethanol extract having an excellent effect on acne treatment, food poisoning and food corruption prevention.

Hibiscus, extract, antimicrobial activity, antibacterial agent

Description

Antimicrobial composition which comprises the Hibiscus ethanol extract}

1 is a schematic process diagram of a method for obtaining a solvent-specific fraction of a hibiscus ethanol extract according to the present invention.

Figure 2 is a photograph observing the culture state of the hibiscus extract extracted with 70% ethanol and 50 mg / ㎖ ampicillin solution as a conventional conventional antibiotics in the same content for Escherichia coli ( Escherchia . Coli O-157 ). .

Figure 3 is a hibiscus extract extracted with 70% ethanol and 50mg / ㎖ ampicillin solution as a conventional conventional antibiotic Staphylococcus epidermidis ) is a photograph of the culture state when treated with the same content.

Figure 4 is observed, the culture state at the time when treated with the same content with respect to the hibiscus the fraction of the ethanol extract and prior art as conventional antibiotic E. coli and the solution appeared 50mg / ㎖ Amphitheater (Escherchia. Coli O-157) according to the invention It is a photograph.

Figure 5 is a photograph of observation of the culture state when treated with the same amount of the fractions for the hibiscus ethanol extract according to the present invention and 50 mg / ml ampicillin solution as Staphylococcus epidermidis as a conventional conventional antibiotic to be.

6 is a diagram illustrating a gas chromatography analysis result of a hibiscus ethanol extract according to the present invention.

<Brief description of the major symbols in the drawings>

2A, 3A: 60 µl of 50 mg / ml ampicillin solution

2B, 3B: 60 μl of 50 mg / ml 70% ethanol extract

4A, 5A: 60 µl of 50 mg / ml ampicillin solution

4B, 5B: 60 μl of 100 mg / mL ethyl acetate fraction

4C, 5C: 60 μl of 100 mg / mL hexane fractions

4D, 5D: 60 μl of 100 mg / ml H ₂ O fraction

4E, 5E: 60 μl of 100 mg / mL chloroform fraction

4F, 5F: 60 μl of 100 mg / mL n-butanol fraction

The present invention relates to a pharmaceutical composition for an antimicrobial agent comprising a hibiscus ethanol extract, and more specifically, by having an antimicrobial activity against various gram positive and gram negative strains, a hibiscus having an excellent effect on acne treatment, food poisoning and food corruption prevention, etc. It relates to a pharmaceutical composition for antimicrobial agents comprising an ethanol extract.

Herbs is a term derived from the meaning of 'grass' derived from the Latin herba, which is used as a generic term for plants in which stems, leaves, flowers, and roots are useful to humans. These herbs include perennial and bulbous plants, such as evergreen or deciduous trees, as well as perennial and deciduous trees.In general, these herbs have scents of flowers, leaves, stems, roots, bark and seeds, and regulate the condition of the body. It has been interpreted as belonging to and has been widely used as a spice in western cuisine since ancient times (Lue, KH and Lee, SS The Illustrated Book of New Vegetable, Herbworld Press, Seoul (1998).

Recently, as the health effects of these herbs have been proved, their consumption is increasing as functional vegetables. In addition to preservative spices or health enhancing agents added to foods and drinks, therapeutic drugs, digestive agents, neurostabilizers, odor inhibitors, skin care and fatigue Its use is gradually increasing as a copy.

Moreover, it has been found that the use of herbs in the treatment of intractable diseases such as allergies, chronic pain, hyperlipidemia, cancer, arthritis, and heart disease, which are very difficult to treat, has been shown to be effective (Tyler, VE, The Honest Herbal, 3rd edn). . pp: 57-73, Haworth Press Inc Binghampton New York, 1993; Tyler, VE, Herbs of Choice:... The therapeutic use of phytomedicinals pp: 126-145, Haworth Press . New York, 1997; Balchin, ML, Essential oils and "aromatherapy": their modern role in healing. J. Rus . Soc . Health 117: 324-329, 1997), the medical use of these herbs is favored in that they can improve the disease with little side effects, and annual herb sales in the United States exceed $ 2 billion. It's causing it.

Among the chemicals contained in these herbal plants, in addition to carbohydrates, minerals, fats, proteins and vitamins, they are special ingredients such as terpenoids, flavonoids, lignans, sulfides, polyphenols, carotenoids (carotenoids), coumarin (coumarins), saponin (saponin), kureukumin (curcumins), and the like, plant sterols (Phytosterol), butylphthalide (phthalide) (Craig, WJ Health -promoting properties of common herb. Am. J. Clin Nutr . 70: 491-499, 1999; Bouseta, A., Scheirman, V. and Collin, S. Flavor and free amino acid composition of lavender and eucalyptus honeys. J. Food Sci . 61: 683-694, 1996), natural essential oils, which are considered to be the major fraction of herbs, are known to have excellent effects on stress relief, mood stability and antioxidant effects (Kim, SJ, Cho, YO and Park, SW Cytotoxicity). ... of Methanol Extracts of Edible Herbs Against L1210 Cells with the Changes of Antioxidant Enzymes Activities Kor J. Pharmacogn 33 (4): 376-383, 2002).

On the other hand, Hibiscus , which is a type of herb plant, has recently attracted great attention, and is a generic name of a plant belonging to the Dicotyledon Mallow and Mugunghwa, and it is Hawaiian Hibiscus, Chinese Rose, Chinese-Hibiscus, Shoe-flower, Blacking It is a plant called Plant. It is widely used as an edible of hibiscus hibiscus Saab wave bridge Lin (Hibiscus sabdariffa Linn , Roselle), the flower extract of hibiscus sabdariparin not only reduces LDL-C (low-density lipoprotein cholesterol) and lipid formation in blood vessels, but also various varieties such as flavonoids such as red wine, polyphenols, and anthocyanins It is attracting attention for its various uses in that it contains antioxidants (Chen, CC, Chou, FP, Ho, YC, Lin, WL, Wang, CP, Kao, ES, Huang, AC, and Wang, CJ, Inhibitory effects of Hibiscus sabdariffa L extract on low-density lipoprotein oxidation and anti-hyperlipidemia in fructose-fed and cholesterol-fed rats, Journal of the Sci of Food and Agriculture , Volume 84, Issue 15, 2004. Pages 1989-1996).

However, studies to date focus only on the weight loss effect and the antioxidant effect of the hibiscus extract, and there are no studies on the antibacterial effect of the hibiscus extract.

Accordingly, an object of the present invention is to provide an antimicrobial pharmaceutical composition comprising a hibiscus ethanol extract having an excellent effect on the treatment of acne, food poisoning and food corruption by having antibacterial activity against various strains.

The present invention in one embodiment for achieving the above technical problem,

It provides a pharmaceutical composition for an antimicrobial agent, characterized in that the hibiscus ethanol extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a pharmaceutical composition for an antimicrobial agent comprising a hibiscus ethanol extract, more specifically, to confirm the antimicrobial activity of the ethanol extract obtained by extracting dry hibiscus with ethanol, the hibiscus ethanol extract hexane, chloroform, ethyl acetate, By sequentially fractionating using n-butanol and water to check the antimicrobial activity of each fraction, it is intended to apply the hibiscus ethanol extract to the pharmaceutical composition for antimicrobial agents.

Preferably, the hibiscus is Hibiscus sabidaparin ( Hibiscus sabdariffa Linn, Roselle) because the antimicrobial effect of hibiscus sabridarilin is better than that of other hibiscus plants.

Figure 1 shows a simplified method for obtaining a fraction for each solvent for verifying the antimicrobial activity of the hibiscus ethanol extract according to the present invention.

Hibiscus ethanol extract of the present invention is obtained by immersing the dry hibiscus in ethanol solution and shaking extract, preferably using 70% ethanol of 10 times the weight of the dry hibiscus total weight, at a speed of 150 rpm at a temperature of 50 ℃ It can be obtained by shaking extraction carried out for 48 hours.

The ethanol extract, which has undergone the ethanol extraction step as described above, includes precipitates and extracts, and the precipitates are filtered using a suitable method such as gauze, and then concentrated and dried on the obtained extract. In the concentration and drying process, the process may be concentrated using a concentrator such as, but not limited to, a rotary vacuum evaporator, and then the drying process may be performed by any method known in the art, such as freeze drying. Can be.

After the above process, a solid hibiscus extract is obtained. The solid hibiscus extract is dissolved in water, and then fractionated sequentially using hexane, chloroform, ethyl acetate, n-butanol and water, Finally, the ethyl acetate fraction, n-butanol fraction and water fraction may be concentrated and dried to measure the antimicrobial activity of each fraction of the hibiscus ethanol extract, the active ingredient of the pharmaceutical composition for antimicrobial agents of the present invention.

In the step of dissolving the solid hibiscus extract, the hibiscus is preferably dissolved in a concentration of 50 to 100mg / ml with respect to water, the hibiscus extract dissolved in water is hexane, chloroform, ethyl acetate, n-butanol and water Fractionation sequentially.

As can be seen in Figure 1, two fractionation is achieved by each fractionation step, in the case of fractionation with hexane, for example, hexane and water layers are produced. Subsequent fractionation processes perform fractionation on the water layer rather than the material layer used in the previous fractionation step. For example, in the case of fractionation with chloroform, the water layer except for the hexane layer separated from the previous fractionation step is performed. Fractionation is performed.

Among the fractions prepared through the above process, that is, hexane fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction, the fractions having antimicrobial activity are ethyl acetate fraction, n-butanol fraction and water fraction.

The present invention also provides a hibiscus extract prepared by the above method in another embodiment.

As described above, the hibiscus extract according to the present invention can be usefully used for the treatment of bacterial skin diseases such as acne, food poisoning and food decay due to excellent antibacterial activity.

The present invention also provides a pharmaceutical composition comprising the hibiscus extract in another embodiment.

Pharmaceutical compositions of the invention may include pharmaceutically acceptable additives. The additives may optionally be used one or two or more of conventional excipients, disintegrants, binders, lubricants, suspending agents and the like. For example, when the composition of the present invention is prepared in a solid form such as tablets or hard capsules, microcrystalline cellulose, lactose, low-substituted hydroxycellulose and the like can be used as excipients, sodium starch glycolate, anhydrous as a disintegrant. Calcium monohydrogen phosphate may be used. As the binder, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose and the like can be used. The lubricant can be selected from magnesium stearate, silicon dioxide, talc and the like. In addition, as the suspending agent, surfactants (eg, sorbitan esters, polysorbates, etc.) commonly used in the pharmaceutical field may be used.

The pharmaceutical compositions of the present invention may be formulated in a variety of forms, including solid and liquid forms, which include tablets, capsules (hard or soft), solutions, suspensions, emulsions, syrups, and the like. . Preferably it may have a liquid formulation, such as liquids, suspensions, emulsions, and syrups, and may be prepared as a functional product or a functional food such as a lotion or gum to which the hibiscus ethanol extract is added.

The composition of the present invention may be administered by various methods of administration, including oral or parenteral, and may be preferably administered orally. However, the dosage may be adjusted to an appropriate amount depending on the condition such as age, age and sex and the severity of the disease, such dosage can be easily adjusted by those skilled in the art clinically engaged in the treatment or prevention of patients. have.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention by way of example, the scope of the present invention is not limited only to the following examples.

<Example>

hibiscus  Preparation of Extract

The dried hibiscus sabdariparin was extracted with 10-fold weight of 70% ethanol (50 ° C., 150 rpm, 48 hours). After separating the pellet and the extract with gauze, concentrated using a rotary vacuum evaporator (Rotary vacuum evaporator N-1000, EYELA, Japan), freeze-dried using a lyophilizer (Ilsin, Korea) and the powder with water It was dissolved and made into 70% ethanol extract.

The extract was partitioned sequentially with hexane, chloroform, ethyl acetate, n-butanol and water, and then concentrated and freeze-dried to obtain a solid. The solid was dissolved in distilled water at a concentration of 100 mg / ml, and then 0.45 μm. Filtration with a PVDF filter (Millipore) was used as a sample for screening antimicrobial activity.

Antimicrobial Activity Measurement

In order to measure the antimicrobial activity, the microorganisms listed in Table 1 were distributed from the Korea Institute of Biotechnology Bioresource Center and the Korea Microbial Conservation Center.

 Classification  Strain  Deposit number  badge   Gram positive Staphylococcus epidermidis 12228 NA Bacillus cereus 11778 NA Clostridium perfringens 13124 RCMA   Gram voice Escherchia coli  O-157: H7 25922 LBA Pseudomonas aeruginosa 29336 TSA Vibrio parahaemolyticus 17802 LBA

Antimicrobial activity was determined for the extract obtained from the 70% ethanol extraction step, followed by hexane, chloroform, ethyl acetate, n-butanol and water fractions respectively.

Screening of dried hibiscus ethanol extracts and their fractions was carried out using paper disc diffusion method (Kim MS, Lee DC, Hong JE, Chang KS, Cho HY, Kwon YK, Kim HY, Antimicrobail effects) .. of ethanol extracts from korean and Indonesian plants Korean J Food Sci Technol 32:. 949-958, 2000.10).

Each test strain was incubated overnight in the corresponding liquid medium, and then 100 μl of the test agar was inoculated in the appropriate agar medium (Difco manual, 1984; Victor, Lorian, 1991) according to each test strain. 60 μl of each sample was loaded into a sterile paper disc (φ10 mm) adhered to the medium, and the culture temperature was adapted to each test strain. The clear zone formed after the culture was confirmed. ATCC 13124 Clostridium Perfringens is an anaerobic strain and anaerobic conditions were prepared using MART II (ANOXOMAT ^™ , Netherlands) (Gas condition: 5% H ₂ , 10% CO ₂ , 85% N ₂ ).

First, using a hibiscus extract, extracted with 70% ethanol to ampicillin and the same amount of 50 mg / ㎖ was measured an antibacterial activity against Escherichia coli (Escherchia. Coli O-157) and Staphylococcus aureus (Staphylococcus epidermidis), after treatment Pictures showing the culture state of each strain are shown in Figures 2 and 3 below.

In Figure 2, 2A is a photograph observing the culture state at the time when processing 60㎕ from a conventional E. coli to ampicillin solution of 50 mg / ㎖ as conventional antibiotics (Escherchia. Coli O-157) , 2B is a 70% ethanol The hibiscus extract 50 mg / ㎖ extracted with E. coli ( Escherchia. Coli O-157 ) 60 μl treatment when observed the culture state.

In addition, in Figure 3, 3A is a conventional conventional antibiotic 50 mg / ml of ampicillin ( Staphylococcus epidermidis ) is a photograph showing the culture state when treated with 60 μl, 3B is 50 mg / ㎖ of hibiscus extract extracted with 70% ethanol Staphylococcus epidermidis ) is a photograph observing the culture state when treated with 60 μl.

2 and 3, it can be seen that the hibiscus extract extracted with 70% ethanol shows an antimicrobial effect against each strain.

On the other hand, Figs. 4 and 5, the E. coli published 50 mg / ㎖ Amphitheater as each fraction and of the prior conventional antibiotics hibiscus ethanol extract according to the invention (Escherchia. Coli O-157) and Staphylococcus aureus (Staphylococcus epidermidis ) shows a photograph observing the culture state when treated with 60ul.

As can be seen from Figures 4 and 5, hexane fractions (4C, 5C) and chloroform fractions (4E, 5E) did not show antimicrobial effects against each strain, but ethyl acetate (4B, 5B), N-butanol ( 4F, 5F) and water fractions (4D, 5D) showed antimicrobial effects on both gram positive and gram negative. 4A and 5A are treated with 60 µl of 50 mg / ml ampicillin solution.

The antimicrobial activity of each of the fractions as described above is shown in Table 2 below.

 Gram positive  Gram voice  ATCC12228  ATCC11778  ATCC13124  ATCC25922  ATCC29336  ATCC17802  Hexane  X  X  X  X  X  X  chloroform  X  X  X  X  X  X Ethyl acetate  O  O  O  O  O  O  n-butanol  O  O  O  O  O  O  water  O  O  O  O  O  O

(X: no antimicrobial activity, O: antibacterial activity)

Thin layer chromatography ( TLC ) And gas chromatography / mass spectrometry

Ethyl acetate, n-butanol and water fractions having antimicrobial effect were developed by spotting by thin layer chromatography (10 × 20, silica gel 60, MERCK, Germany). Butanol: acetate: distilled water (4: 1: 1) was used as a solvent development. After completion of development, after drying, the bands were checked by UV-detector (365 nm) without staining. The band was eluted with 100 μl methanol and filtered through a 0.45 μm PVDF filter (Millipore) to use as GC / MS analysis sample.

GC / MS analysis was performed using a Hewlett-Packard 6890 Series Gas Chromatograph equipped with Agilent Technologies 5973N Mass Selective Detector system (GC-MSD). The front inlet was splitless mode and the initial temperature was 250 ° C. Hewlett Packard 190915-433 cappillary column (30 m × 250 μm id, 0.25 μm thick) was used for the separation of components. Column flow was 1.3 mL, separation temperature was started at 50 ° C. and maximum temperature was fixed at 350 ° C. et al., 2000). The initial temperature of the mass spectrometer was 310 ° C and the scan range was 80-250m / z. Qualitative analysis was made by comparing the mass spectra of the samples with the standard mass spectra provided in the GC-MSD library database.

Hibiscus sabdariffa with antimicrobial effect from GC / MS qualitative analysis Linn , Roselle) extract was separated into a myriad of minor peaks with several main peaks, the shape of the peak is shown in FIG. As can be seen in Figure 6, each peak is 12.32 components of 2,4-di-t-butylphenol (CAS No.96-76-4), 21.92 components of 2,3-dihydroxypropyl hexadecano 8 (CAS No.542-44-9), 1,3-dihydroxypropan-2-yl octadecanoate (CAS No.621-61-4) of 23.48 components, corresponding to each peak Structural formula and chemical formula of the compound are shown in Table 3 below.

 Peak (retention time)  compound   function  constitutional formula     Chemical formula      12.32

2,4-디-t-부틸페놀

2,4-di-t-butylphenol C ₁₄ H ₂₂ O      Anticancer effect      21.92

2,3-dihydroxypropyl hexadecanoate C ₁₉ H ₃₈ O ₄     Antibacterial effect Blood pressure control Inflammation       23.48

1,3-dihydroxypropan-2-yl octadecanoate C ₂₁ H ₄₂ O ₄      Diuretics Hemostatic drugs Pain economy Inflammatory medicine

According to the present invention, by having an antimicrobial activity against various strains, it is possible to provide a novel antimicrobial pharmaceutical composition containing a hibiscus ethanol extract having an excellent effect on the treatment of acne, food poisoning and food corruption.

Claims (4)

Pharmaceutical composition for an antimicrobial agent, characterized in that the hibiscus ethanol extract as an active ingredient. The pharmaceutical composition of claim 1, comprising a pharmaceutically acceptable additive. The pharmaceutical composition of claim 1, wherein the hibiscus is Hibiscus sabdariffa Linn . The method of claim 1, wherein the hibiscus ethanol extract, characterized in that obtained by shaking extraction performed for 48 hours at a rate of 150 rpm at a temperature of 50 ℃ using 70% ethanol of 10 times the total weight of the dry hibiscus Pharmaceutical composition for antimicrobial agents.

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