CN100465172C - Macrolides compound of Macrolactin Q with antibacterial activity - Google Patents
Macrolides compound of Macrolactin Q with antibacterial activity Download PDFInfo
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- CN100465172C CN100465172C CNB2007100390182A CN200710039018A CN100465172C CN 100465172 C CN100465172 C CN 100465172C CN B2007100390182 A CNB2007100390182 A CN B2007100390182A CN 200710039018 A CN200710039018 A CN 200710039018A CN 100465172 C CN100465172 C CN 100465172C
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- macrolactin
- water
- methyl alcohol
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Abstract
This invention relates to an anti-bacterium natural product-Macrolactin Q got from the secondary metabolized product of bacillus of east China sea, which has strong suppression action to colibacillus and golden grape cocci, in which, this compound can be produced by applying seawater culture medium to ferment and culture bacillus 201721 strains, and the ferment solution is separated by Sephadex LH-20, middle-pressure liquid phase and RP C8 HPLC.
Description
Technical field
The present invention relates to a kind of new antimicrobial compounds Macrolactin Q that from the secondary metabolite of sea bacillus subtilis, extracts and the application in the preparation antibacterials thereof.
Background technology
About 500,000 kinds of the kind of whole world biology, wherein 80% comes from the ocean, and therefore, the ocean is a huge Biological resources treasure-house.In view of land resources is deficient day by day, the development and use oceanic resources have become the front line science problem that the whole world is paid close attention to.In oceanic resources, Living marine resources are the most important, are the abundant resource treasure-houses of development and use as yet, wherein again with the focus that is developed as of marine pharmaceutical organism resource.Biological resources in the special ecological environment of ocean have become the new space of expanding natural medicine resource.Up to the present, have been found that more than 15,000 kind of marine natural product, wherein more than 200 kind of patent applied for.
Subtilis (Bacillus subtilis) 201721 is a Gram-positive, bacillus.The K.Gustafson of California, USA university teaches and obtained having different antibiotic optionally 6 kinds of macrolides compound Macrolactin A-F in 1989 simultaneously from the secondary metabolite of deep-sea marine bacteria, (K.Gustafson et al.J.Am.Chem.Soc.1989,111,7519), the specific examples of such components of having reported 11 kinds of homogeneous structures later on again in succession is MacrolactinG-N and 7-O-succinoylmacrolactinA and 7-O-succinoylmacrolactin F.
Summary of the invention
The object of the present invention is to provide a kind of a kind of new macrolides compound Macrolactin Q that from the secondary metabolite that grows in the East China Sea genus bacillus, extracts and preparation method thereof and the application in the preparation antibacterials with anti-microbial activity.
The used bacterial classification of the present invention is to separate genus bacillus 201721 bacterial strains that obtain from East China Sea ooze sample, now is stored in Biochemistry and Molecular Biology teaching and research room of The 2nd Army Medical College and Chinese typical culture collection center (CCTCC culture presevation M207008).The present invention separates the new natural product Macrolactin of activity Q of acquisition from genus bacillus 201721, this compound is a yellow oil, molecular formula C
24H
34O
6, high resolution mass spectrum: 419.2454 (M+H)
+,
1H and
13C nuclear magnetic resonance spectrum data see Table 1.
Table 1 Macroactin Q's
1H and
13C nuclear magnetic resonance spectrum data
Adopt the nmr determination of 600MHz, sample dissolution is in deuterated methanol.
Simultaneously, also measured the multiple two dimensional NMR spectrum of this compound (TOCSY, DQCOSY, HMQC, HMBC and NOESY), determined carbon atom and the ownership of hydrogen atom and the chemical structure of this compound that this compound is all, structural formula is as follows:
The preparation method of compound is as follows among the present invention:
(1) 201721 bacterial strains are inoculated into the 250ml triangular flask that fermention medium is housed from the test tube slant carry out seed culture, the bottled 70ml substratum of each triangle, 28 ℃ of shaking table constant temperature culture, rotating speed 90r/min.Cultivate in the triangular flask that after a day seed culture fluid is inoculated into 2000ml and carry out enlarged culturing, every bottled 700ml fermention medium, by the inoculation of 10% inoculum size, the same culture condition is cultivated 4d.Fermentation culture based component: peptone 5.0g, yeast extract paste 1.0g, glucose 6.0g, artificial seawater (or Chen Haishui) 1000ml, pH6.5.Artificial seawater prescription: NaCl 25.0g, Na
2SO
44.0g, KCl 0.7g, NaHCO
30.20g, KBr0.10g, H
3BO
30.03g, NaF 0.003g, 53mL 1.0mol/L MgCl
2Solution, 10mL 1.0mol/lCaCl
2Solution, 0.90ml 0.1mol/L SrCl
2Solution, 1mL trace element (micro-storing solution: EDTA 50g, KOH 31g, FeSO
47H
2O 4.98g, H
2SO
41ml, H
3BO
311.42g, ZnSO
47H
2O 8.82g, MnCl
24H
2O, 1.44g, MoO
30.71g, CuSO
45H
2O 1.57g, Co (NO
3)
26H
2O 0.49g, distilled water 1000mL), distilled water 1000ml, pH is adjusted to 6.5.
(2) the bacterium liquid that will finish the fermentation culture process is collected fermented liquid supernatant after removing thalline after filtration.Fermented liquid supernatant with the extraction of ethyl acetate equal-volume, is repeated 3 times, combining extraction liquid Rotary Evaporators solvent evaporated, with medicinal extract Sephadex LH-20 rough segmentation section, moving phase is chloroform-methanol (4:1).
(3) with behind the gained wash-out phase evaporate to dryness by middle hydraulic fluid phase (the Fraction CollectorC-660 of Switzerland Buchi company, UV-Photomaker C-635, Pump manager C-615, Pump Module C-605) purifying once more, moving phase and elution requirement are: methyl alcohol (0%～40%)-water 60min, methyl alcohol (40%～60%)-water 180min, methyl alcohol (60%～100%)-water 80min, flow velocity 3.0ml/min, 218nm detects, the collection gradient is the elution peak of methyl alcohol (60%～100%)-water, concentrates back HPLC and is further purified, and uses Zorbax partly to prepare C
8Reversed-phase column (9.4mm * 250mm) preparation, moving phase is acetonitrile: water (28:72), flow velocity 2.0mL/min, 218nm detects, and collects t
RFor the elution peak of 21.5min is prepared compound macrolactin Q.
Embodiment
The present invention will be further described below in conjunction with embodiment.
The fermentation culture of embodiment 1 201721 ocean bacterial strains
A.201721 the fermention medium of ocean bacterial strain is as follows: peptone 5.0g, and yeast extract paste 1.0g, glucose 6.0g, artificial seawater (or Chen Haishui) 1000ml, pH 6.5.Artificial seawater prescription: NaCl 25.0g, Na
2SO
44.0g, KCl 0.7g, NaHCO
30.20g, KBr 0.10g, H
3BO
30.03g, NaF 0.003g, 53mL 1.0mol/L MgCl
2Solution, 10mL 1.0mol/l CaCl
2Solution, 0.90ml 0.1mol/LSrCl
2Solution, distilled water 1000ml, 1mL trace element (micro-storing solution: EDTA 50g, KOH 31g, FeSO
47H
2O 4.98g, H
2SO
41ml, H
3BO
311.42g, ZnSO
47H
2O 8.82g, MnCl
24H
2O, 1.44g, MoO
30.71g, CuSO
45H
2O 1.57g, Co (NO
3)
26H
2O 0.49g, distilled water 1000mL), pH is adjusted to 6.5.
B. fermentation culture process: 201721 bacterial strains are inoculated into 12 250ml triangular flasks that fermention medium is housed from the test tube slant carry out seed culture, the bottled 70ml substratum of each triangle, 28 ℃ of shaking table constant temperature culture, rotating speed 90r/min.Cultivate in the triangular flask that after a day seed culture fluid is inoculated into 12 2000ml and carry out enlarged culturing, every bottled 700ml fermention medium, by the inoculation of 10% inoculum size, the same culture condition is cultivated 4d.
The separation and Extraction of embodiment 2 active compounds
With the bacterium liquid of finishing the fermentation culture process after filtration, collect fermented liquid supernatant after removing thalline.Fermented liquid supernatant with the extraction of ethyl acetate equal-volume, is repeated 3 times, combining extraction liquid Rotary Evaporators solvent evaporated, with medicinal extract Sephadex LH-20 rough segmentation section, moving phase is chloroform-methanol (4:1).With behind the gained wash-out phase evaporate to dryness by middle hydraulic fluid phase (the Fraction Collector C-660 of Switzerland Buchi company, UV-Photomaker C-635, Pump manager C-615, Pump Module C-605) purifying once more, moving phase and elution requirement are: methyl alcohol (0%～40%)-water 60min, methyl alcohol (40%～60%)-water 180min, methyl alcohol (60%～100%)-water 80min, flow velocity 3.0ml/min, 218nm detects, the collection gradient is the elution peak of methyl alcohol (60%～100%)-water, concentrates back HPLC and is further purified, and uses Zorbax partly to prepare C
8Reversed-phase column (9.4mm * 250mm) preparation, moving phase is acetonitrile: water (28:72), flow velocity 2.0mL/min, 218nm detects, and collects t
RFor the elution peak of 21.5min is prepared compound macrolactin Q.
The determination of activity result of embodiment 3 active compound Macrolactin Q
Adopt micro-broth dilution method (Dai Ziying, Liu Yukun, the multiple practical antibacterials Shanghai of Wang:: Shanghai science tech publishing house, 1992,7～8) mensuration is to the MIC value of intestinal bacteria and streptococcus aureus.The mould activation measurement of employing rice blast (Hisayoshi K,, Michio N, Takeshi Y, et al.JAntibiotics, 1996,49 (9): 873-878.) measure the compound macrolactin Q inhibition activity mould to rice blast.The result shows that the anticolibacillary MIC value of compound macrolactin Q is 0.2 μ g/ml, and the MIC of anti-streptococcus aureus is 0.7 μ g/ml, and the maximum dilution multiple that suppresses the mould growth of rice blast is 64.
Macrolides compound Macrolactin Q of the present invention all has stronger restraining effect to rice blast is mould with intestinal bacteria and streptococcus aureus, has the potential use as the preparation antibacterials.
Claims (4)
1. macrolides compound Macrolactin Q with anti-microbial activity is characterized in that having following structure:
2. the preparation method of the described Macrolactin Q of claim 1, be made up of following steps successively:
A. 201721 bacterial strains are inoculated into the 250mL triangular flask that fermention medium is housed from the test tube slant and carry out seed culture, the bottled 70mL substratum of each triangle, 28 ℃ of shaking table constant temperature culture, rotating speed 90r/min; Cultivate in the triangular flask that after a day seed culture fluid is inoculated into 2000mL and carry out enlarged culturing, every bottled 700mL fermention medium, by the inoculation of 10% inoculum size, the same culture condition is cultivated 4d;
B. the bacterium liquid that will finish the fermentation culture process is collected fermented liquid supernatant after removing thalline after filtration; Fermented liquid supernatant with the extraction of ethyl acetate equal-volume, is repeated 3 times, combining extraction liquid Rotary Evaporators solvent evaporated, with medicinal extract Sephadex LH-20 rough segmentation section, moving phase is the chloroform-methanol of 4:1;
C. with behind the gained wash-out phase evaporate to dryness by middle hydraulic fluid phase Fraction Collector C-660, UV-Photomaker C-635, Pump manager C-615, Pump Module C-605 is purifying once more, moving phase and elution requirement are 0%～40% methyl alcohol-water 60min, 40%～60% methyl alcohol-water 180min, 60%～100% methyl alcohol-water 80min, flow velocity 3.0ml/min, 218nm detects, the collection gradient is the elution peak of 60%～100% methyl alcohol-water, concentrates back HPLC and is further purified, and uses 9.4mm * 250mm of Zorbax partly to prepare C
8The reversed-phase column preparation, moving phase is acetonitrile and the water of 28:72, flow velocity 2.0mL/min, 218nm detects, and collects t
RFor the elution peak of 21.5min is prepared compound macrolactin Q.
3. Macrolactin Q preparation method according to claim 2 is characterized in that described fermentation culture based component is: peptone 5.0g, yeast extract paste 1.0g, glucose 6.0g, artificial seawater or Chen Haishui 1000mL, pH6.5;
Artificial seawater prescription wherein: NaCl 25.0g, Na
2SO
44.0g, KCl 0.7g, NaHCO
30.20g, KBr 0.10g, H
3BO
30.03g, NaF 0.003g, 53mL 1.0mol/L MgCl
2Solution, 10mL 1.0mol/L CaCl
2Solution, 0.90mL 0.1mol/L SrCl
2Solution, distilled water 1000mL, and 1mL is by EDTA 50g, KOH 31g, FeSO
47H
2O 4.98g, H
2SO
41ml, H
3BO
311.42g, ZnSO
47H
2O8.82g, MnCl
24H
2O, 1.44g, MoO
30.71g, CuSO
45H
2O 1.57g, Co (NO
3)
26H
2The micro-storing solution that O 0.49g, distilled water 1000mL are mixed with, pH is adjusted to 6.5.
4. the application of the described macrolactin Q of claim 1 in the preparation antibacterials.
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| CN100465172C true CN100465172C (en) | 2009-03-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008465A (en) * | 2010-11-19 | 2011-04-13 | 中国人民解放军第二军医大学 | Application of macrolide compound from marine microorganisms in preparing anti-tumor medicines |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333206B (en) * | 2007-06-27 | 2012-12-12 | 国家海洋局第一海洋研究所 | Macrolide compounds, preparation and application thereof |
| CN101830884B (en) * | 2009-11-25 | 2012-12-26 | 中国科学院南海海洋研究所 | Macrolide as well as preparation method and antibacterial application thereof |
| CN103320409B (en) * | 2013-07-08 | 2014-11-05 | 中国海洋大学 | Glucosyltransferase and application thereof |
| CN106831696B (en) * | 2017-02-20 | 2020-05-19 | 广西大学 | Macrolide derivative and preparation method and application thereof |
| CN112961817B (en) * | 2021-04-06 | 2021-11-05 | 广西中医药大学 | Method for screening high-yield Macrolactins marine bacillus by using osmotic pressure stress of sea salt |
-
2007
- 2007-03-30 CN CNB2007100390182A patent/CN100465172C/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| A stereoselective synthesis of the C(3)-C(13) and C(14)-C(24)fragments of macrolactin A.. 李树坤等.Chines Journal of Chemistry,Vol.18 No.6. 2000 * |
| Recent process in bioactive metabolites ofmarinemicroorganisms.. Liu，Xiao-hong，et，al.中国抗生素杂志，第19卷第8期. 2004 * |
| The Macrolactins, a novel class of antiviral and cytotoxicMacrolides from a Deep-Sea marine bacterium.. K. Gustafson et al.J. Am. Chem. Soc.,Vol.111 No.19. 1989 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008465A (en) * | 2010-11-19 | 2011-04-13 | 中国人民解放军第二军医大学 | Application of macrolide compound from marine microorganisms in preparing anti-tumor medicines |
| CN102008465B (en) * | 2010-11-19 | 2011-12-21 | 中国人民解放军第二军医大学 | Application of macrolide compound from marine microorganisms in preparing anti-tumor medicines |
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