JP2010155819A - Antibacterial use of extract from morus australis poir. and kuwanon h compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an extract prepared from root bark of Morus australis Poir. as an antibacterial agent. <P>SOLUTION: Dried root bark of Morus australis Poir. is finely crushed and extracted with an aqueous solution of ethanol under reflux, and the obtained compound of chemical formula (I) is named as kuwanon H and used as a new antibacterial agent. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to novel uses in the treatment of bacterial infections of extracts prepared from the root bark of Shimuwa (Morus australis Poir.) And quanon H compounds isolated from the extract.

  Patent Document 1 includes five compounds isolated from extracts of bark of mulberry trees, such as Yamaguwa (Morus bombycis Koidz), Magwa (Morus alba L.), and Lotus (Morus Lhou Koidz), Marvelofuran C, Antibacterial uses against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and the like have been disclosed for D and G, quanol A and sangenone G.

  Patent Document 2 discloses a quanon G obtained by separating an extract prepared from the root bark of mulberry trees such as Yamagua (Morus bombycis Koidz), Magwa (Morus alba L.), and Lotus (Morus Lhou Koidz). Two compounds, H and H, disclose use for lowering blood pressure in hypertensive patients.

JP-A-10-007555 JP 56-0123979 A

  The object of the present invention is to provide the following chemical formula (I):

And antibacterial use of pharmaceutically acceptable salts thereof.

  For the antibacterial application of the present invention described above, a pharmaceutical composition for the treatment of bacterial infection comprising Quwanon H or a pharmaceutically acceptable salt thereof as an effective ingredient; Use of quanon H, or a pharmaceutically acceptable salt thereof, in the manufacture of the formulation; and administering a therapeutically effective amount of quanon H, or a pharmaceutically acceptable salt thereof, to a subject having a bacterial infection. Also included is a method of treating said subject.

  Another object of the present invention is to provide an antibacterial application of an extract prepared from shimuwa root bark.

  According to the invention, the antibacterial use of the extract comprises a pharmaceutical composition for the treatment of bacterial infections comprising the extract of the invention as an effective ingredient; a formulation for the treatment of bacterial infections in subjects Also included is a method of treating a subject comprising the use of an extract of the invention in the manufacture of; and administering a therapeutically effective amount of the extract of the invention to a subject having a bacterial infection.

  In the present invention, “treating a bacterial infection” broadly means treating any disorder caused by a bacterial infection, or, in a narrow sense, inhibiting bacterial growth.

  “San-Bai-Pi” is a traditional Chinese medicine, dried root bark of Magwa, which has anti-inflammatory uses to relieve cough and diuresis. Magwa plants are rare in Taiwan; however, Simuga can be easily found. As a result, the inventors of the present invention have prepared an extract from Shimagwa and have isolated the compound from an industrially potentially useful extract.

  The present invention relates to chemical formula (I):

And a pharmaceutically acceptable salt thereof is administered to a subject having a bacterial infection. A method of treating the subject is provided.

The present invention provides a method for treating a subject comprising administering to a subject having a bacterial infection a therapeutically effective amount of an extract prepared from shimuwa root bark. At this time, the extract is prepared by a process including the following steps.
a) extracting shimuwa root bark with polar solvent;
b) concentrating the extraction solution obtained in step a);
c) The concentrate obtained in step b) is introduced into the reverse phase chromatography column and the column is eluted in turn with the first and second eluents. At this time, the first eluent is about 50 wt% ethanol aqueous solution (for example, 40 to 70 wt%, preferably 45 to 60 wt% ethanol aqueous solution), and the second eluent is about 95 wt% ethanol aqueous solution (for example, 80 wt% A polarity of ˜98 wt%, preferably 85-97 wt% aqueous ethanol); and d) recovering the second eluate eluting from the column using the second eluent and removing the second eluent from the second eluate. Remove under reduced pressure.

  Preferably, the bacterium is a gram positive bacterium.

  Preferably, the bacterium is Staphylococcus aureus (Smith) or Streptococcus pneumoniae. More preferably, the bacterium is methicillin-resistant Staphylococcus aureus (Methicillin Resistant). More preferably, the bacterium is Streptococcus pneumoniae. Most preferably, the bacterium is erythromycin and ampicillin resistant Streptococcus pneumoniae (Erythromycin and Ampicillin Resistant).

  Preferably, the extract of the present invention has the following chemical formula (II) in addition to the compound of chemical formula (I):

Sangenon G represented by

  Preferably, the extract contains 1 to 5% of compound (I) and 1 to 5% of compound (II) based on the weight of the extract. More preferably, the extract contains 3.7% of compound (I) and 2.3% of compound (II) based on the weight of the extract.

  Preferably, the first eluent is a 50 wt% ethanol aqueous solution, and the second eluent is a 95 wt% ethanol aqueous solution.

  Preferably, the polar solvent in step a) is water or a 95 wt% aqueous ethanol solution, more preferably a 95 wt% aqueous ethanol solution.

  In the present invention, the following specific examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

  Percentages and other total amounts referred to in this specification are by weight unless otherwise indicated. Percentages can be selected in any range up to a total of 100%.

Example 1 Preparation of Extract EX-1 Sliced dried root bark of Moraceae was finely crushed and screened. 100 g of the obtained powder was mixed with 1000 mL (10: 1 (v / w)) of a 95 wt% aqueous ethanol solution in a round bottom flask, and the resulting mixture was boiled at reflux for 1 hour. Filtration was performed with (# 2) to obtain a filtrate and a residue. The residue was extracted under reflux in the same manner as described above and filtered. The obtained two filtrates were mixed, a part thereof was dried under reduced pressure, and the dry powder weight from the mixed filtrate was calculated to be 10.21 g, and the yield of the crude extract was 10.21 wt%. there were. The mixed filtrate was concentrated to 1/10 of the original weight, and water of the same weight as the obtained concentrate was added. The obtained mixture was introduced into a reverse phase chromatography column packed with polystyrene absorbent resin (Diaion HP20, Mitsubishi Chemical) 30 times the weight of the concentrate of the mixed filtrate, and 50 wt of 2 bed volume (bed volume = 600 mL). Elution was performed using a 1% aqueous ethanol solution (1200 mL) followed by 2 bed volumes of a 95 wt% aqueous ethanol solution (1200 mL). The eluate of 95 wt% aqueous ethanol solution was collected, concentrated in vacuo and dried. 2.73 g of the dried extract EX-1 (code name) was obtained, and the yield was 26.7% with respect to the dry weight of the concentrate of the mixed filtrate. The rate was 2.73%.

Example 2) Separation of Extract EX-1 To a homogeneous solution of 2 g EX-1 dissolved in 10 mL methanol, 10 mL pure water was added, the mixture was shaken vigorously to promote precipitation, followed by 15 Centrifugation was performed at 10,000 rpm for minutes. The solid content obtained by centrifugation was dissolved, precipitated and centrifuged, and this cycle was performed a total of 6 times.

  Six liquids obtained by six centrifugations were mixed, concentrated in vacuo and dried (weight 1.3 g). The obtained dry powder was introduced into a silica gel column (3.2 cm × 48.5 cm) packed with silica gel 60 having a weight 30 times that of the dry powder, and a mixed mobile phase toluene of 15 bed volumes (bed volume = 250): Elute with ethyl acetate = 7: 3 (3750 mL). The eluate was collected in order into bottles with predetermined numbers. The liquid in each container was vacuum-dried, and the obtained dry powder was redissolved by adding a small amount of methanol, and a dextran gel column (1.5 cm × x) packed with Sephadex LH-20 resin 100 times the weight of the dry powder. 40 cm). The column was eluted with 1 bed volume of methanol (30 mL) to obtain fractions of compounds P2-1, P2-2, P3-1, and P3-2. Each fraction was concentrated in vacuo and dried, then purified by preparative HPLC to give compounds P2-1, P2-2, P3-1, and P3-. The yield of P3-1 was 2.3% (46 mg) and the yield of P3-2 was 3.65% (73 mg) based on the weight of EX-1.

  Six residues after centrifugation were dissolved in methanol, and the solution was concentrated in vacuo and dried. The dry powder was introduced into a silica gel column (3.2 cm × 48.5 cm) packed with silica gel 60 having a weight 30 times that of the dry powder, and a mixed mobile phase of 2 bed volumes (bed volume = 250) toluene: acetic acid Elute with ethyl = 9: 1 (500 mL). The eluate was collected in order into bottles with predetermined numbers. The liquid in each container was concentrated and dried under vacuum, and the obtained dry powder was redissolved in a small amount of methanol, and a dextran gel column (1.5 cm) packed with Sephadex LH-20 resin 100 times the weight of the dry powder. × 40 cm). The column was eluted with 1 bed volume of methanol (30 mL) to give compounds P3-3 and P3-4.

P2-1: ESI-MS: m / z 603.3 ([M + Na] ⁺ ), m / z 579.2 ([M−H] ⁻ ). NMR analysis was also performed. ESI-MS and NMR data showed that the chemical formula of P2-1 is C ₃₄ H ₂₈ O ₉ . The NMR data was compared with the following paper, and it was confirmed that P2-1 was Marvelofuran C reported in this paper. Marvelofuran C is also disclosed in Japanese Patent Application Laid-Open No. 57-144223.

  Paper: Fukai, T. Hano, Y .; Hirakura, K .; Nomura, T .; Uzawa, J .; Fukushima, K .; , Structure of mulberrofuran J, a 2-arybenzofuran derivative from the mulberry tree (see Morus lhouu Koidz.), 100, Heterocycle.

^{P2-2: ESI-MS: m /} z 715.3 ([M + Na] +), m / z 691.3 ([M-H] -). NMR analysis was also performed. ESI-MS and NMR data showed that the chemical formula of P2-1 is C ₄₀ H ₃₆ O ₁₁ . The NMR data was compared with the following paper, and it was confirmed that P2-2 was Albanin F (Quanon G) reported in this paper. Albanin F (Quanon G) is also disclosed in Korean Patent Application Publication No. 2002-0087225.

  Paper: Nomura, T .; Fukai, T .; Narita, T .; , Hypotensive constituent, Kuwanon H, a new flavone derivative from the root bar of the cultivated mulberry tree, 19 s. 19 c.

P3-1: ESI-MS: m / z 695.4 ([M + H] ⁺ ), m / z 693.7 ([M−H] ⁻ ). NMR analysis was also performed. ESI-MS and NMR data showed that the chemical formula of P3-1 was C ₄₀ H ₃₈ O ₁₁ . The NMR data was compared with the following paper, and it was confirmed that P3-1 was Sangenone G. Sangenon G is also disclosed in JP-A-10-007555.

  Paper: Fukai, T. Hano, Y .; Fujimoto, T .; Nomura, T .; , Structure of sanggenon G, a new Diels-Alder add form from the Chinese crude drug ″ Sang-Bai-Pi ″, Moroc rot barks, 198, 1994.

P3-2: ESI-MS: m / z 761 ([M + H] ⁺ ), 783 ([M + Na] ⁺ ), 759 ([M−H] ⁻ ). The molecular weight of P3-2 was calculated as 760. NMR analysis was also performed. The ESI-MS and NMR data, chemical formula of P3-2 was shown to be _C 45 _H 44 _{O 11.} The NMR data was compared with the following paper, and it was confirmed that P3-2 was quanon H (also referred to as Albanin A or Morasenin A). Japanese Patent Application Laid-Open No. 56-1223979 discloses that Quanon H is effective in lowering blood pressure in hypertensive patients.

  Paper: Nomura, T .; Fukai, T .; Narita, T .; Terada, S .; Uzawa, J .; Iitaka, Y .; Takasugi, M .; Ishikawa, S .; I. Nagao, S .; Masamune, T .; , Confirmation of the Structures of Kuwanons G and H (albanins F and G) by partial synthesis, Tetrahedron Letters, 1981, 22, 2195-2198; Oshima, Y. Konno, C .; Hikino, H .; , Structure of Moracenin A, a hypotensive principal of Morse root bars, Heterocycles 1980, 14, 1287-1290;

  Paper: Oshima, Y .; Konno, C .; Hikino, H .; Matsushita, K .; , Structure of Moracenin B, a hypotensive principal of Morus root bars, Tetrahedron Letters 1980, 21, 3381-3384.

P3-3: ESI-MS: m / z 421 ([M + H] ⁺ ). NMR analysis was also performed. The ESI-MS and NMR data, chemical formula of P3-3 was shown to be _C 25 _H 24 _{O 6.} The NMR data was compared with the following paper, and P3-3 was confirmed to be morsine. Morcine is also disclosed in US Patent Application Publication No. 2008/0286213.

  Paper: Nomura, T .; Fukai, T .; Yamada, S .; Katanagi, M .; , Phenolic constituents of the cultivated mulberry tree (Morus alba L.), Chem. Pharm. Bull. 1976, 24, 2898-2900. See

P3-4: ESI-MS: m / z 447.0 ([M + H] ⁺ ). NMR analysis was also performed. ESI-MS and NMR data showed that the chemical formula for P3-3 is C ₂₉ H ₃₄ O ₄ . NMR data was compared with the following paper, and it was confirmed that P3-4 was Marvelofuran D. Marvelofuran D is also disclosed in JP-A-10-007555.

  Paper: Nomura, T .; Fukai, T .; Shimada, T .; Chen, I .; -S. , Mulberrofuran D, a new 2-arylbenzofuran from the root bars of the mulberry tree (Morus australis poir.), Heterocycles, 1982, 19, 1855.

Antibacterial test In Vitro bacteria: Staphylococcus aureus (Smith strain) and methicillin resistant Staphylococcus aureus (ATCC 33591)
Minimum inhibitory concentrations (MIC) of samples of S. aureus (Smith strain) and methicillin resistant staphylococci (ATCC 33591) were determined using the liquid medium dilution method [Edwards J. R. et al. In vitro antimicrobial activity of SM-7338, a carbapenem antibiotic with stability to dehydreptidase I.M. Antimicrobial Agents Chemotherapy. 33: pp. 215-222, 1989]. Test substances were dissolved (generally in 100% DMSO) and sequentially diluted in a solvent to a predetermined concentration. After separating 0.01 mL (final DMSO concentration of 1%) for each test concentration into 48-well plates, 1-5 × 10 ⁵ CFU / mL of S. aureus (Smith strain) or methicillin-resistant S. aureus ( 0.99 mL medium (Mueller-Hinton Broth (DIFCO, USA)) containing ATCC 33591) was added. As described above, the final maximum DMSO concentration was 1%, and the initial test substance concentration was 100 μg / mL or 30 μM. After incubating the plate at 37 ° C. for 20 hours, the growth, that is, turbidity was suppressed as positive (+), and the growth or turbidity was not evaluated as negative (−). Vehicle-control and active reference agents were used as blank and positive controls. Each concentration was evaluated twice. The results are shown in Table 1.

In Vitro bacteria: Streptococcus pneumoniae (ATCC 6301) and Streptococcus pneumoniae (erythromycin and ampicillin resistant, clinical isolate)
Minimum inhibitory concentrations (MIC) of samples of Streptococcus pneumoniae (ATCC 6301) and Streptococcus pneumoniae (erythromycin and ampicillin resistant, clinical isolates) were determined using the liquid medium dilution method [Edwards J. R. et al. In vitro antimicrobial activity of SM-7338, a carbapenem antibiotic with stability to dehydreptidase I.M. Antimicrobial Agents Chemotherapy. 33: pp. 215-222, 1989]. Test substances were dissolved (generally in 100% DMSO) and sequentially diluted in a solvent to a predetermined concentration. The test substance was dissolved (generally in 100% DMSO) and sequentially diluted in a solvent to a predetermined concentration. Aliquots of 0.01 mL (1% final DMSO concentration) for each test concentration into 48-well plates, followed by 1-5 × 10 ⁵ CFU / mL of Streptococcus pneumoniae (ATCC 6301) and Streptococcus pneumoniae (erythromycin and ampicillin) 7% fetal bovine serum-containing medium (Tryptic Soy Broth (DIFCO, USA)) containing 980 mL of resistant clinical isolates was added. As described above, the final maximum DMSO concentration was 1%, and the initial test substance concentration was 100 μg / mL or 30 μM. After incubating the plate at 37 ° C. for 20 hours, the growth, that is, turbidity was suppressed as positive (+), and the growth or turbidity was not evaluated as negative (−). Vehicle-control and active reference agents were used as blank and positive controls. Each concentration was evaluated twice. The results are shown in Table 1.

  Referring to Table 1, it can be seen that Compound P3-2 is effective in inhibiting the growth of SA (Smith strain) and MRSA at a concentration of 0.3 μg / mL. In contrast, the MIC of gentamicin to MRSA is 10 times that of SA (Smith strain).

  Compound P3-1 had an MIC against SA (Smith strain) and MRSA of 1 μg / mL and 3 μg / mL, respectively, and was shown to be effective in inhibiting growth. Compound P3-1 has MICs for SP and SP (erythromycin and ampicillin resistance) of 30 μg / mL and 10 μg / mL, respectively, and has a relatively low growth inhibitory effect.

  Surprisingly, considering that it contains 3.65 wt% and 2.3 wt% of P3-2 and P3-1 respectively, the extract EX-1 of the present invention has a MIC for SA and MRSA of 0 .3 μg / mL and 1 μg / mL, which were shown to be very effective in inhibiting growth. It is clear that the components contained in the extract EX-1 act synergistically in suppressing the growth of SA and MRSA, and therefore the MIC value of the extract EX-1 for SA and MRSA is very high. It is low.

  Although the present invention has been described in detail in the present specification using some examples, the scope of the present invention is not limited to the claims described below. Various changes and modifications are possible in light of the above disclosure.

Claims (14)

The following chemical formula (I):

Or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for the treatment of bacterial infection. The following steps:
a) extracting shimuwa root bark with polar solvent;
b) concentrating the extraction solution obtained in step a);
c) The concentrate obtained in step b) is introduced into a reverse phase chromatography column, and the first eluent and the second eluent are filled in the column in this order. At this time, the first eluent is about 50 wt% aqueous ethanol solution. And the second eluent is about 95 wt% ethanol aqueous solution; and d) recovering the second eluate eluting from the column using the second eluent and second elution from the second eluate. Remove the liquid under reduced pressure,
A pharmaceutical composition for the treatment of bacterial infection, comprising an extract prepared from shimuwa root bark by a process comprising: The extract is a compound represented by the chemical formula (I) according to claim 1 and the following chemical formula (II):

The pharmaceutical composition of Claim 2 containing the compound represented by these.   The pharmaceutical composition according to claim 3, wherein the extract contains 1 to 5 wt% of the compound (I) and 1 to 5 wt% of the compound (II) with respect to the weight of the extract.   The pharmaceutical composition according to claim 4, wherein the extract contains 3.7 wt% of the compound (I) and 2.3 wt% of the compound (II) based on the weight of the extract.   The pharmaceutical composition according to any one of claims 2 to 5, wherein the first eluent is a 50 wt% aqueous ethanol solution and the second eluent is a 95 wt% ethanol aqueous solution.   The pharmaceutical composition according to any one of claims 2 to 6, wherein the polar solvent in step a) is water or a 95 wt% aqueous ethanol solution.   The pharmaceutical composition according to claim 7, wherein the polar solvent is a 95 wt% aqueous ethanol solution.   The pharmaceutical composition according to any one of claims 1 to 8, wherein the bacterium is a Gram-positive bacterium.   The pharmaceutical composition according to claim 9, wherein the bacterium is S. aureus (Smith strain) or S. pneumoniae.   The pharmaceutical composition according to claim 10, wherein the bacterium is Staphylococcus aureus (Smith strain).   The pharmaceutical composition according to claim 11, wherein the bacterium is methicillin-resistant Staphylococcus aureus.   The pharmaceutical composition according to claim 10, wherein the bacterium is Streptococcus pneumoniae.   14. The pharmaceutical composition according to claim 13, wherein the bacterium is erythromycin and ampicillin resistant Streptococcus pneumoniae.