CN101289440B - Polyene macrocyclic compounds, preparation thereof and applications - Google Patents
Polyene macrocyclic compounds, preparation thereof and applications Download PDFInfo
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- CN101289440B CN101289440B CN 200710118178 CN200710118178A CN101289440B CN 101289440 B CN101289440 B CN 101289440B CN 200710118178 CN200710118178 CN 200710118178 CN 200710118178 A CN200710118178 A CN 200710118178A CN 101289440 B CN101289440 B CN 101289440B
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Abstract
The invention discloses a kind of polyene macrocyclic compounds and the preparation method and application thereof, more specifically produce novel polyene macrocyclic compounds and its application using microbial fermentation, belong to field of biotechnology. A kind of polyene macrocyclic compounds, chemical structural formula are as follows: wherein R is methyl or ethyl. The invention has the advantages that novel polyene macrocyclic compounds provided by the invention, the experiment proved that, the growth of the tumour cells such as Pyricularia oryzae, human liver cancer cells Hep G2 is significantly inhibited, and toxicity is lower, it can be used for antimycotic biological pesticide or anti-cancer medicament raw material, with good application prospect; The method of prepare compound provided by the invention is biological fermentation process, and step is simple, cost is relatively low, is conducive to industrialization promotion implementation.
Description
Technical field
The present invention relates to a kind of polyene macrocyclic compounds and preparation method thereof and application, more specifically say so and utilize a kind of novel polyene macrocyclic compounds of Production by Microorganism Fermentation and application thereof, belong to biological technical field.
Background technology
Actinomycetes are the quasi-microorganisms with huge practical value, and about 8000 kinds of biologically active substances of finding from microorganism at present have nearly 70% to be that actinomycetes produce.But because the active substance of finding from the actinomycetes of land is fewer and feweri, people have invested marine actinomycete to sight gradually.It is distributed widely in surface and inner (as sponge, the marine alga etc.) of seawater, marine bottom sediment and marine animal and plant, and, formed the meta-bolites of various active such as having of a large amount of novel structures is antibiotic, antitumor, antiviral, proteases inhibitors because of its special physiological status, genetic background and unique metabolic way.The slogan that proposed " drugs from the sea " from the U.S. in 1967 begins, and " blue medicine " caused that the attention of various countries, short decades just have the novel texture compound of multiple marine actinomycete synthetic biologically active to be found.
(Fenical W such as Fenical, et al., J Am Chem Soc, 2006,8,128 (5), 1622-1632) from a newfound actinomyces Marinispora, extract novel polyenoid poly-hydroxy compounds, its energy effective function is in pathogenic bacteria of drug-resistant such as methicillin-resistant staphylococcus aureus and vancomycin resistance faeciums.(Rajendrap M such as Rajendrap, et al., J Antibiot, 2004,57,17-23) be separated to new compound Trioxacarcins A from marine actinomycete Streptomyces sp., it has all shown stronger anti-tumor activity to tumor cell lines such as SF-268, H-460 and LXFL529L.
Discover that the active substance that many actinomycetes produce not only has single resistance, they often show various active.(Magarvey N A such as Magarvey, et al., Appl Environ Microbiol, 2004,70 (12), 7520-7529) be separated to 2 kinds of actinomycetes novel species with a kind of unique selectivity cultural method from the marine bottom sediment of sea, New Guinea Saloman, its fermented liquid has the activity of inhibition to multiple pathogenic bacteria, tumour cell and vaccinia virus.
According to incompletely statistics, newfound marine microorganism active substance is produced by marine actinomycete more than 50% in recent years, but compares with the land actinomycetes, and people are also backward relatively to the understanding of marine actinomycete and application thereof.Believe that people will find the biologically active substance that more marine actinomycete produces and be applied, so marine actinomycete will be the same with the land actinomycetes, becomes the another important microbe resource of pharmaceutical industries such as microbiotic along with deepening continuously of studying.
Summary of the invention
The technical problem to be solved in the present invention is: the novel polyene macrocyclic compounds that a kind of biologically active is provided.
Another technical problem that the present invention will solve is: provide a strain to be used to prepare the streptomycete of novel polyene macrocyclic compounds.
The 3rd technical problem that the present invention will solve is: the method that a kind of easy novel polyene macrocyclic compounds of preparation is provided.
Last technical problem that the present invention will solve is: the purposes in the medicine that this novel polyene macrocyclic compounds is antimycotic in preparation or anticancer is grown is provided.
For achieving the above object, the present invention is by the following technical solutions:
A kind of novel polyene macrocyclic compounds, its chemical structural formula is as follows:
Wherein R represent methylidene (compd A) or ethyl (compd B).
The bacterial strain of above-mentioned polyene macrocyclic compounds is produced in one strain, separation is from Sanya, Hainan Island sponge, be the yellowish pink streptomycete of ash Hainan subspecies A429, this bacterial strain is CGMCC NO.2090 at the preserving number of Chinese common micro-organisms DSMZ, on June 14 2007 preservation day, the name of suggestion is called grey yellowish pink streptomycete Hainan subspecies Streptomycesgriseocarneus subsp.hainanensis.
A kind of method for preparing above-mentioned polyene macrocyclic compounds may further comprise the steps:
(1) seed culture of grey yellowish pink streptomycete Hainan subspecies A429 (CGMCC NO.2090):
Seed culture medium: glucose 5-15g, Zulkovsky starch 2-10g, soyflour 5-15g, potassium primary phosphate 0-1g, sodium-chlor 0-20g, distilled water 1000ml;
Culture condition is: pH6.5-7.5, culture temperature 25-32 ℃, incubation time 24-72 hour;
(2) fermentation culture of grey yellowish pink streptomycete Hainan subspecies A429:
Fermention medium: glucose 0-20g, Zulkovsky starch 5-15g, Semen Maydis powder 5-15g, soyflour 2-15g, sodium-chlor 0-20g, distilled water 1000ml;
Fermentation condition is: pH6.5-7.5, leavening temperature 25-32 ℃, fermentation time 96-168 hour, inoculum size 5%-10%;
(3) separate general extractive:
The fermented liquid that step (3) obtains is centrifugal, get supernatant liquor, through chloroform or ethyl acetate extraction, concentrating under reduced pressure gets brown paste substance;
(4) purified extract:
Use the dissolve with methanol extract, utilize half preparation HPLC to carry out separation and purification; Select Waters Nova-PakC18 chromatography column (25mm * 200mm, 6 μ m) for use, half preparation HPLC elution requirement is: room temperature, and 50% methyl alcohol rises to 85% methyl alcohol in the 6min internal linear, and flow velocity 20ml/min detects wavelength 230nm, obtains polyene macrocyclic compounds.
Finally obtain compd A and compd B, outward appearance is pale brown color shape, and through the HPLC analyzing and testing, its purity reaches 97.32% and 97.85% respectively.
Described polyene macrocyclic compounds and the bacterial strain CGMCC NO.2090 purposes in the preparation antifungal drug.
Described polyene macrocyclic compounds and the bacterial strain CGMCC NO.2090 purposes in the medicine of preparation inhibition growth of tumour cell.
Advantage of the present invention is: novel polyene macrocyclic compounds provided by the invention, the experiment proved that, the growth of tumour cells such as, human liver cancer cell SMMC-7721 mould to rice blast has the obvious suppression effect, and toxicity is lower, can be used for antimycotic biological pesticide or anti-cancer medicament raw material, have application promise in clinical practice; The method for preparing compound provided by the invention is a biological fermentation process, and step is simple, cost is lower, help Industry Promotion implements.
The invention will be further described below in conjunction with embodiment, and embodiments of the present invention are not limited to this, every according to content disclosed by the invention or principle, any this area of enforcement be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the HPLC/ESI-MS-MS collection of illustrative plates of compd A.
Fig. 2 is the HPLC/ESI-MS-MS collection of illustrative plates of compd B.
Fig. 3 is a compd A
1The H-NMR collection of illustrative plates.
Fig. 4 is a compd A
13The C-NMR collection of illustrative plates.
Fig. 5 is the H-H COSY collection of illustrative plates of compd A.
Fig. 6 is the HMBC collection of illustrative plates of compd A.
Fig. 7 is the HSQC collection of illustrative plates of compd A.
Fig. 8 partly prepares the HPLC collection of illustrative plates for compd A and B's.
Fig. 9 is that the HPLC of compd A and B detects collection of illustrative plates.
Embodiment
Embodiment 1: separation and the evaluation of grey yellowish pink streptomycete Hainan subspecies A429
One. the separation of bacterial classification:
Fresh sponge is collected in marine site, Sanya, Hainan Island.Get in right amount, after aseptic seawater cleaning 3 times, get the 10g sponge sample, shred, add the aseptic seawater of 90ml, smash to the homogenate shape, get in right amount in 55 ℃ of sproutings of handling 6min with the promotion spore with stirrer with sterile scissors.With aseptic seawater step by step 10 times be diluted to 10
-2, 10
-3, adopt improvement Gause I synthetic medium, the plate dilution method coating separates, and is inverted for 28 ℃ and cultivates, and obtains strains A 429.
Two. the evaluation of strains A 429:
(1) form and cultural characteristic: strains A 429 is equal well-grown on multiple solid mediums such as JCM, Sauton ' s, murphy juice, oat juice, Gause I, glycerine asparagine, GYM, form abundant aerial hyphae and substrate mycelium, the gas silk is greyish white to light grey, and the base silk is pale yellow to Vandyke brown.Visible brown pigment diffusion on the murphy juice flat board does not see on other substratum that soluble pigment generates.Aerial hyphae is straight, visible 1～2 grade verticillate, substrate mycelium branch does not rupture, and does not see that spore generates.
(2) physiological and biochemical property: utilize D-glucose, D-fructose, D-wood sugar, sucrose, L-arabinose, D-N.F,USP MANNITOL, inositol etc., do not utilize D-raffinose, L-rhamnosyl, do not grow on the Mierocrystalline cellulose, nitrate reduction, liquefy gelatin does not produce tyrosine oxidase and H
2S, starch hydrolysis, milk no change.
(3) cell wall components analysis:
Amino acid analysis: compare with 2,6 diaminopimelic acids and glycine, A429 strain cell wall hydrolyzate is carried out full cell amino acid analysis, this strain cell wall contains L as a result, and the L-glycine shows that this bacterial strain belongs to cell walls I type.
The sugar analysis of components: A429 strain whole-cell hydrolyzate is analyzed its sugared composition with chromatography and is contained glucose and find no principal character sugar, belongs to sugared C type.
(4) 16S rDNA sequencing and Phylogenetic Analysis
Adopt 16S rDNA special primer,, obtain the nearly full length sequence of its 16S rDNA, altogether 1476bp strains A 429 total DNA pcr amplifications.This sequence is DQ319186 in the accession number of GenBank.Related data among this sequence and the GenBank is carried out the BLAST similarity analysis, find that the bacterial strain that homology is high with it is streptomycete, with S.griseocarneus homology the highest (98.4%).Typical strain with high bacterial strain of these homologys and streptomyces makes up the phylogenetic tree that comprises the relevant kind of 13 strains, and strains A 01059 is in same branch with S.griseocarneus, and the two sibship is nearest.
Strains A 429 belongs to streptomyces through identifying, and is nearest with S.griseocarneus on sibship.But the two exists aspect cultural characteristic and physiological and biochemical property than big-difference, and the active substance of generation is also different.Given this, with this identification of strains be grey yellowish pink streptomycete Hainan subspecies Streptomyces griseocarneus subsp.hainanensis.On June 14th, 2007 was preserved in CGMCC, was numbered CGMCC NO.2090.
Embodiment 2: the preparation of compd A and B
One. the seed culture of grey yellowish pink streptomycete Hainan subspecies A429 (CGMCC NO.2090):
Seed culture medium: glucose 10g, Zulkovsky starch 5g, soyflour 10g, yeast extract paste 10g, potassium primary phosphate 0.5g, sodium-chlor 10g, distilled water 1000ml.
Culture condition is: pH7.2,28 ℃ of culture temperature, incubation time 48 hours.
Two. the fermentation culture of grey yellowish pink streptomycete Hainan subspecies A429 (CGMCC NO.2090):
Fermention medium: glucose 5g, Zulkovsky starch 10g, Semen Maydis powder 10g, soyflour 5g, sodium-chlor 10g, distilled water 1000ml.
Fermentation condition is: inoculum size 5%-10%, pH7.0, leavening temperature 28, fermentation time 144 hours.
Three. separate general extractive:
With the centrifugal 10min of above-mentioned fermented liquid 8000r/min, get supernatant liquor, through ethyl acetate extraction three times, concentrating under reduced pressure gets brown paste substance.
Four. purified extract:
With an amount of 50% dissolve with methanol extract, utilize half preparation HPLC to carry out separation and purification.Select WatersNova-Pak C18 post (25mm * 200mm, 6 μ m) for use, half preparation HPLC elution requirement is: 50% methyl alcohol rises to 85% methyl alcohol in the 6min internal linear, and flow velocity 20ml/min detects wavelength 230nm.From collection of illustrative plates, isolate 9 components (Fig. 8) altogether, collect each component, adopt disk diffusion method, measure the dissolve with methanol thing inhibition situation mould of 9 components to rice blast, compare with methyl alcohol.Cultivate to observe behind the 24h for 28 ℃ and find, the equal non-activity in 1～7 peak, 8,9 peak elutriants show strong inhibition activity to rice blast is mould, and contrasting pure methyl alcohol does not have influence to the sprouting of the mould spore of rice blast.Therefore, 8,9 peaks are our interested active peaks.Equally, adopt mtt assay to detect the inhibition effect of 9 components to tumour cell, the result shows that 8,9 peaks have anti-tumor activity.
Collect 8,9 peaks, concentrating under reduced pressure obtains compd A and compd B, and outward appearance is pale brown color shape, and through the HPLC analyzing and testing, its purity reaches 97.32% and 97.85% (Fig. 9) respectively.
Five. identify
Adopt spectral method to carry out the evaluation of chemical structure.Result such as Fig. 1 are to shown in Figure 7, the testing data of compd A and B: the mass signal m/z509/[M+H of HPLC/ESI-MS gained A]+and 507/[M-H]-, the m/z523/[M+H of B]+and 521/[M-H]-, calculate that through peacekeeping two dimensional NMR data the A molecular formula is C again
32H
44O
5, coincide with mass-spectrometric data.Comprehensive above spectroscopic analysis infers that the structure of this compd A belongs to the polyene macrocyclic class, and systematic naming method is: 5-methyl-6-[3-methyl-5-(8-ethanoyl-12-hydroxyl-2,4; 11,13-tetramethyl--ring 13 carbon-3,5; 8-three ketenes)-six carbon-1, the 3-diene]-5,6-dihydro-pyran-2-one.And C, H signal and A in the 1H NMR of B and the 13C NMR collection of illustrative plates are extremely similar, the two is the compound of same type, the ESIMS mass spectrum shows that the two molecular weight differs 14 in the one-level mass spectrum, fragment main in the second order ms also all differs 14, infers that compd B is than Duoing a methylene radical in the A molecular structure.Analogy the two C, H the spectrum data, B is that the R bit substituent is an ethyl in the structural formula.The 1H of compd A and 13C nuclear magnetic resonance data see Table 1.
The 1H NMR (400MHz) of table 1 compd A and 13C NMR (100MHz) data (Me2CO-d6)
* overlapped signal can't be differentiated integrated value and coupling constant.
Embodiment 3: anti-rice blast mould (Pyricularia oryzae) activity test
The centrifugal 10min of 8000r/min collects fermented supernatant fluid.Adopt disk diffusion method, application of sample amount 10 μ L measure antibacterial circle diameter behind the cultivation 24h.Measure anti-microbial activity again after the fermented liquid of this bacterial strain diluted, still have obvious inhibition effect when finding 30 times of dilutions, antibacterial circle diameter is 10mm.Simultaneously, cultivate to observe find that all dilutions and undiluted fermented liquid cause that all the mould spore of rice blast do not sprout or gross distortion (table 2) by 96 orifice plates.
The fermented liquid of the different extension rates of table 2 is to the bacteriostatic activity of rice blast mould (rice CGMCC3.3283)
Embodiment 4: anti-tumor activity test
With the centrifugal 10min of fermented liquid 8000r/min of this bacterial strain, collect fermented supernatant fluid, and, detect the fermented liquid of different concns to human liver cancer cell SMMC-7721, murine sarcoma cell S by mtt assay with 10～1000 times of its dilutions
180, human liver cell HL-02 the inhibition activity.Cell strain is the biochemical and cell research institute available from Chinese Academy of Sciences Shanghai all.
Can find out that from table 4 active substance that this bacterium produces all has two kinds of tumours and suppresses active, when 100 times of dilutions to human liver cancer cell SMMC-7721, murine sarcoma cell S
180, human liver cell HL-02 inhibiting rate be respectively 80%, 81%, 5%.When diluting 1500 times, still can reach 50% to the inhibiting rate of tumour cell.In addition, the fermented liquid of this each concentration of bacterium all is not higher than 14% to the inhibiting rate of normal liver cell HL-02, illustrates that active substance has the selection restraining effect to tumour cell.
The inhibiting rate of the fermented liquid pair cell of the different extension rates of table 4
Claims (4)
1. polyene macrocyclic compounds, it is characterized in that: its chemical structural formula is as follows:
Wherein R is methyl or ethyl.
2. method for preparing the described polyene macrocyclic compounds of claim 1 is characterized in that may further comprise the steps:
(1) grey yellowish pink streptomycete Hainan subspecies A429 preserving number is the seed culture of CGMCC NO.2090:
Seed culture medium: glucose 5-15g, Zulkovsky starch 2-10g, soyflour 5-15g, potassium primary phosphate 0-1g, sodium-chlor 0-20g, distilled water 1000ml;
Culture condition is: pH6.5-7.5, culture temperature 25-32 ℃, incubation time 24-72 hour;
(2) fermentation culture of grey yellowish pink streptomycete Hainan subspecies A429:
Fermention medium: glucose 0-20g, Zulkovsky starch 5-15g, Semen Maydis powder 5-15g, soyflour 2-15g, sodium-chlor 0-20g, distilled water 1000ml;
Fermentation condition is: pH 6.5-7.5, leavening temperature 25-32 ℃, fermentation time 96-168 hour, inoculum size 5%-10%;
(3) separate general extractive:
The fermented liquid that step (2) obtains is centrifugal, get supernatant liquor, through chloroform or ethyl acetate extraction, concentrating under reduced pressure gets brown medicinal extract;
(4) purified extract:
Use the dissolve with methanol extract, utilize half preparation HPLC to carry out separation and purification; Half preparation HPLC elution requirement is: room temperature, and 50% methyl alcohol rises to 85% methyl alcohol in the 6min internal linear, and flow velocity 20ml/min detects wavelength 230nm, obtains polyene macrocyclic compounds.
3. the purposes of the described polyene macrocyclic compounds of claim 1 in preparation anti-rice blast mould medicine.
4. the described polyene macrocyclic compounds of claim 1 suppresses purposes in the medicine of growth of tumour cell in preparation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3814795A (en) * | 1970-09-07 | 1974-06-04 | Asahi Chemical Ind | Antibiotic s 15-1 and process for producing the same |
| CN1193979A (en) * | 1995-07-04 | 1998-09-23 | 诺瓦蒂斯有限公司 | Macrolides |
| CN1232030A (en) * | 1998-03-05 | 1999-10-20 | 斯索恩有限公司 | Process for producing simvastatin and/or its derivatives |
| US6124453A (en) * | 1995-07-04 | 2000-09-26 | Novartis Ag | Macrolides |
-
2007
- 2007-06-29 CN CN 200710118178 patent/CN101289440B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3814795A (en) * | 1970-09-07 | 1974-06-04 | Asahi Chemical Ind | Antibiotic s 15-1 and process for producing the same |
| CN1193979A (en) * | 1995-07-04 | 1998-09-23 | 诺瓦蒂斯有限公司 | Macrolides |
| US6124453A (en) * | 1995-07-04 | 2000-09-26 | Novartis Ag | Macrolides |
| CN1232030A (en) * | 1998-03-05 | 1999-10-20 | 斯索恩有限公司 | Process for producing simvastatin and/or its derivatives |
Non-Patent Citations (2)
| Title |
|---|
| R. Cruz et al..Nutritional requirements for the production ofpyrazoloisoquinolinone antibiotics by streptomycesgriseocarneus NCIMB 40447.Appl. Microbiol. Biotechnol.53 1.1999,53(1),115-119. |
| R.Cruz et al..Nutritional requirements for the production ofpyrazoloisoquinolinone antibiotics by streptomycesgriseocarneus NCIMB 40447.Appl. Microbiol. Biotechnol.53 1.1999,53(1),115-119. * |
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