KR100490224B1 - Compositions for inhibition growth of microorganisms containing CatalpaNP-1 with structure of 4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho[2,3-b]pyran-5,10-dione - Google Patents

Abstract

The present invention relates to a microbial growth inhibitor including a natural antimicrobial active substance isolated from Catalpa ovata GD _ON fruit. To develop natural antimicrobial inhibitors by identifying natural antimicrobial substances from paulownia berry fruit, methanol extract of the paulownia fruit was partitioned into ethyl acetate and phosphate buffer solution to obtain soluble neutral fraction of ethyl acetate, and this active fraction was silica gel adsorption column chromatography. 4,9-dihydroxy-2,2-dimethyl-3 having a molecular formula of C ₁₅ H ₁₄ O ₅ through steps such as Sephadex LH-20 column chromatography, octadecsilane column chromatography, HPLC, and the like. An antimicrobial agent named CatalpaNP-1 having a 4,10-dihydronaphtho [2,3- b ] pyran-5,10-dione structure was isolated and the structure of the active substance was MS. As determined by NMR analysis, the antibacterial activity of catalpa NP-1 showed strong activity against various microorganisms such as Gram-positive bacteria, Gram-negative bacteria, yeast, and fungi. After treatment and scanning electron microscopy, germination was significantly suppressed with spore deformation. Catalpaenpi-1 and extracts containing this material isolated from the paulownia fruit will serve as a natural microbial inhibitor.

Description

Inhibitor of microbial growth including catalfaenpi-1 having the structure of 4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3-b] pyran-5,10-dione {Compositions for inhibition growth of microorganisms containing CatalpaNP-1 with structure of 4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3-b] pyran-5,10-dione}

The present invention relates to 4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2, named catalfaenpi-1, a substance having a naphthoquinone structure isolated from the paulownia fruit. 3- b ] pyran refers to the antimicrobial activity of a substance having a structure of -5,10-dione and a method for separating the same.

Catalpa ovata G. D _ON is a deciduous tree belonging to the Bignoniaceae family and grows in Korea. Fruits have been used to treat diseases such as nephritis. Methanol extract of the paulownia fruit showed antimicrobial activity against various bacteria including fungi, but the antimicrobial activity causing agents were unclear. -Successful separation of a material interpreted as dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3- b ] pyran-5,10-dione, and its antibacterial activity It was confirmed to complete the present invention.

Looking at the conventional research related to the present invention, the study on the chemical composition and physiological activity of the bark of the paulownia bark ( Chem. Pharm. Bull. , 49, 6, 2001, Machida, K. et al., Monoterpene glycosides from the fallen leaves of Catalpa ovata G. D ON, 732-736), flavone glycoside (Phytochemistry, 14, 1975, Okuda , T. et al., Two new flavone glycosides from Catala ovata, 1654-1656), 4- hydroxy Hydroxybenzoic acid, 4-hydroxycinnamic acid ( J. Wood Research Soc ., 8, 1962, Jpn., Imamura, H. et al., 127-130) and naphthoquinones having antitumor activity ( J. Nat Prod ., 61, 1998, Fujiwara, A. et al., Antitumor-promoting naphthoquinones from Catalpa ovata , 629-632). However, there is no report on the antimicrobial activity of catalpaenpi-1 isolated from the present invention.

According to the present invention, when the paulownia berry extract exhibits antimicrobial activity, the antimicrobial activity causative agent is separated from the fruit of the paulownia berry and the structure of the substance is identified, and the antimicrobial activity of the substance is identified, and the separated antibacterial substance and the substance are contained. The purpose of the present invention is to provide the extract of the paulownia fruit.

The present invention is to extract the methanol extract of the paulownia fruit with methanol to obtain a methanol extract, and concentrated by using a solvent fraction, silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, octadecyl column column chromatography, HPLC, etc. The antimicrobial substance was isolated by purification, and the substance was named catalpa NP-1, and the structure of the substance separated by instrumental analysis such as MS and NMR was 4,9-dihydroxy-2. , 2-dimethyl-3,4-dihydronaphtho [2,3- b ] pyran-5,10-dione, which was confirmed that catalpaenpi-1 had antimicrobial activity, catalpaenpi- Extracts containing 1 and catalpaenpi-1 provides microbial growth inhibitors.

Hereinafter, the present invention will be specifically described based on the following examples.

(Example 1) Preparation and antimicrobial activity of paulownia berry extract

5,934 g of dried paulownia fruit was immersed in 97 L of methanol for 24 hours to obtain a methanol extract. The methanol extract was concentrated under reduced pressure at 37 ° C. to obtain an methanol-free extract (670.9 g), which was partitioned into a buffer solution of pH 8.0 (0.2M Na ₂ HPO ₄ -0.2M NaH ₂ PO ₄ ) and ethyl acetate. And ethyl acetate extract was obtained. The antimicrobial activity of the two extracts was determined by the paper disk method (Zaika, LL, J. Food Safety , 9, 1988, Zaika, LL, Species and herbs: Their antimicrobial activity and its determination, 97-118). As a result, the methanol extract and the ethyl acetate extract showed the antimicrobial activity as shown in Table 1.

[Table 1] Antimicrobial Activities of Methanol Extracts and Ethyl Acetate Extracts

Example 2 Isolation of Natural Antibacterial Substance from the Paulownia Fruit

As shown in Example 1, an attempt was made to isolate the antimicrobial active material contained in purified ethyl acetate soluble neutral fraction of methanol extract showing strong antimicrobial activity against various microorganisms. Antimicrobial activity during purification of the active material was performed by assaying the activity against Streptococcus aureus. 68.2 g of ethyl acetate soluble neutral fraction (from 5788 g of Paulownia fruit) was eluted with methanol concentration in chloroform solution by silica gel adsorption column chromatography, resulting in chloroform-methanol 90:10 (v / v) fraction. (48.7 g, at 5748 g of Paulownia fruit) showed activity, and silica gel adsorption column chromatography using hexane-ethyl acetate-methanol solvent system was repeated three times. In other words, hexane-ethyl acetate-methanol 8: 6: 1, 6: 8: 1, 4: 10: 1, 2: 12: 1 (v / v) fractions with solvents (8: 6: 1, 6 : 8: 1 Elution fraction, v / v) 29.4 g (from 5670 g of Paulownia) were obtained, and the fractions were hexane-ethyl acetate-methanol 10: 4: 1, 8: 6: 1, 6: 8: 1, Fractionation with a 4: 10: 1 (v / v) solvent gave 20.8 g (from 5630 g of paulownia) as active fraction (10: 4: 1, 8: 6: 1 eluted fraction, v / v). This was partitioned with hexane-ethyl acetate-methanol 14: 1: 1, 12: 3: 1, 10: 5: 1, 8: 7: 1 (v / v) solvent to obtain an active fraction (14: 1: 1 elution fraction, v / v, 9.7 g, at 5600 g of Paulownia fruit). This was again partitioned with hexane-ethyl acetate 10: 4, 9: 5, 8: 6, 7: 7, 6: 8, 5: 9 (v / v) solvent and the active fraction (10: 4, 9: 5 eluted fraction). , v / v) 3.8 g (from 5570 g of Paulownia berries). Subsequently, the active material was fractionated by Sephadex LH-20 column chromatography (methanol-chloroform, 4: 1, v / v) to assay the antimicrobial activity. Ve / Vt (elution volume / total volume) was 0.74 to 1.00. Activity was seen in the fraction separated at the position of. 554 mg of this active fraction (from 5550 g of Paulownia fruit) was eluted with octadecyl silane column chromatography to gradually increase the methanol concentration in water. / v, at 5530 g of Paulownia fruit). This active fraction was separated by reverse phase column-equipped HPLC (Senshupak, 8 × 250 mm, 60% MeOH, 1.5 mL / min) to pure 76.6 mg of active substance (from 5530 g of paulownia) with a single peak on HPLC. This material was named Catalfaenpi-1.

Example 3 Structure Determination by Instrumental Analysis of Separated Antimicrobial Active Materials

Mass spectrometry (MS) and NMR analysis were performed to determine the structure of catalpaenpi-1 isolated as an antimicrobial substance. The high resolution mass spectrometry of the active substance measured by mass spectrometer (Jeol JMS 700) was 274.0837 with the molecular formula C ₁₅ H ₁₄ O ₅ .

¹ H-NMR analysis measured by instrument (Jeol JNM-AL 400) (400 MHz, DMSO- d ₆ ) δ 1.43 (3H, s, H-12), 1.45 (3H, s, H-11), 1.89 (1H, dd, J = 4.8, 14.5 Hz, H-3a), 2.00 (1H, dd, J = 4.8, 14.5 Hz, H-3b), 4.76 (1H, dt, J = 4.0, 4.8 Hz, H -4), 5.21 (1H, d, J = 4.0 Hz, 4-OH), 7.29 (1H, dd, J = 1.2, 8.2 Hz, H-8), 7.53 (1H, dd, J = 1.2, 7.5 Hz, H-6), 7.74 (1H, dd, J = 7.5, 8.2 Hz, H-8), 11.59 (1H, s, 9-OH), ¹³ C-NMR (100 MHz, DMSO- d ₆ ) measurements are δ25.6 (C-11), 28.4 (C-12), 38.9 (C-3), 56.5 (C-4), 78.5 (C-2), 113.9 (C-9a ), 117.6 (C-6), 120.9 (C-4a), 122.7 (C-8), 131.6 (C-5a), 136.6 (C-7), 152.8 (C-10a), 159.8 (C-9) , 180.0 (C-5) and 183.8 (C-10).

The structure of the antimicrobial active material separated from the above analysis results of MS, ¹ H-NMR, ¹³ C-NMR, etc. is a material having a 1,4-naphthoquinone structure. Hydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3- b ] pyran-5,10-dione).

Example 4 Antimicrobial Activity Assay for Bacteria, Yeast, and Mold of Substances Isolated from Cauliflower Fructus

The antibacterial activity of catalpaenpi-1 isolated from the paulownia fruit was measured by 50% growth inhibition (IC ₅₀ ) targeting various microorganisms by the method described in Example 1. The microorganisms were Gram-positive bacteria, Gram-negative bacteria, two yeasts, and three fungi. In order to measure the IC ₅₀ of the antimicrobial active substance, 180 μl of the medium in which the active substance was dissolved was used, and 20 μl of the culture medium pre-incubated three times for 24 hours at the optimum temperature with the medium, respectively, and the concentration of the active substance was divided into microwell plates. Was finally prepared to be 10, 25, 50, 100, 250, 500 μM. This was incubated at 130 rpm in a shaker for 17 hours (bacteria, yeast) or 24 hours (fungus) at optimum temperature and absorbance at 630 nm using a microspectrophotometer (Microspectrophotometer, EL-800, Bio-Tek Instruments, USA). Was measured. In addition, lapachol (lapachol, Planta Med ., 60, 1994, Guiraud, P. et al., Comparison of antibacterial and antifungal activities of lapachol and β- lapachone, 373-374, 1994) to compare the activity in the same way. This was repeated three times to determine the microbial growth inhibition rate by measuring the absorbance ratio of the active material added to the sample-free addition. As a result, as shown in Table 2, catalpaenpi-1 showed a wide range of antimicrobial activity against microorganisms such as Gram-positive bacteria, Gram-negative bacteria, yeast, mold, etc. It showed microbial growth inhibition effect.

[Table 2] Antimicrobial Activity (50% Growth Inhibition Concentration) of Natural Antibacterial Catalpaenpi-1 and Rapacol Isolated from Paulownia fruit

Example 3 Confirmation of Inhibition of Fungal Growth of Catalpa nP-1 Isolated from the Paulownia Fruit by Scanning Electron Microscope

The catalpaenpi-1 treated group (250 μM) and the fungus spore suspension of the untreated sample were shaken incubated for 24 hours and filtered through filter paper (pore size 0.22 μm, Millipore, USA). Spores of the filter paper were added with 2% glutaraldehyde fixative dissolved in sodium cacodylate buffer, left for 2 hours, washed three times with 0.1 M sodium cacodylate buffer, and sequentially washed with 50, 70, 90, 95, and 100% ethanol. Dehydrated. After lyophilization, gold plating was performed and magnified 3,000 times by scanning electron microscope (S-2400, Hitachi, Japan) to observe the shape of spores. As a result, as shown in Fig. 2, aspergillus plabus, aspergillus paraciticus, and penicillium citrinum were all transformed or spores compared to the control when treated with antimicrobial substances isolated from Paulownia berry. Germination was significantly inhibited, and it was assayed that the isolated substance had the growth inhibitory ability of the fungus.

[Figure 2] Scanning electron micrograph (× 3,000) of fungus treated with catalpaenpi-1 (250μM), an antibacterial substance isolated from paulownia berry.

(A) Spores normally germinated after 24 hours of incubation

(B) Modified spores at 24 hours of incubation after treatment of isolated material

4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3- b ] having a 1,4-naphthoquinone structure, isolated and isolated as an antimicrobial substance from the paulownia fruit Catalpaenpi-1, which has a structure of pyran-5,10-dione, and the paulownia fruit extract containing this substance, showed strong antimicrobial activity against various microorganisms such as bacteria, yeast, and fungi. Extracts and catalpaenpi-1 are expected to be used as inhibitors of microbial growth.

Claims (3)

Paulownia japonica containing catalpaenpi-1 having the structure of 4,9-dihydroxy-2,2-dimethyl-3,4-dihydronaphtho [2,3- b ] pyran-5,10-dione Inhibitor of microbial growth, including fruit extract and catalpaenpi-1. The method according to claim 1, wherein the catalpaenpy-1, the methanol extract obtained by immersing the paulownia fruit in methanol to concentrate under reduced pressure to obtain an extract from which methanol was removed, and the buffer solution of pH 8.0 (0.2M Na ₂ HPO ₄ -0.2M NH ₂ PO ₄ ) and ethyl acetate to partition the aqueous fraction and the ethyl acetate soluble neutral fraction, ethyl acetate soluble neutral fraction using silica gel adsorption column chromatography to increase the methanol concentration in the chloroform solution. Gradually eluting fraction by 10% step by step from 0% to 50% to obtain active fraction, fractionating the active fraction using silica gel adsorption column chromatography using hexane-ethyl acetate-methanol solvent system The active fraction is separated by Sephadex LH-20 column chromatography (methanol-chloroform, 4: 1, v / v). Gel filtration step, and using the octadecyl silane column chromatography to obtain the active fraction by gradually eluting the methanol concentration in water by 10% step by step from 30% to 100% using octadecyl column column chromatography, the above purification The microorganism growth inhibitory prepared by the method comprising the step of separating the active fraction separated by a methanol-water solvent system by HPLC equipped with a reversed phase column. Ethyl obtained according to claim 1, wherein the extract containing catalpaenpi-1 is obtained by extracting methanol of paulownia fruit with methanol, or by concentrating methanol extract to remove methanol, and fractionating with ethyl acetate and phosphate buffer solution of pH 8.0. Inhibitor of microbial growth, characterized in that the acetate extract.