Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof

A compound and cyclic peptide technology, which is applied in the field of preparation of cyclic peptide compound clavatustide A and its producing bacteria, can solve the problem that no one has studied metabolites, and achieve the effects of high product purity, good anti-tumor activity in vitro, and good development prospects

Inactive Publication Date: 2014-04-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This hydrothermal mouth crab was found in Guishan Island, Taiwan (eng, M.-S.; Ng, N.K.; Ng, P.K.L. Nature...

Method used

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  • Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof
  • Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof
  • Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Isolation of fungi from hydrothermal mouth crabs

[0046] The hydrothermal mouth crab Xenograpsus testudinatus was donated by Professor Ye Ying from the Ocean College of Zhejiang University. First, rinse the hydrothermal port crab with sterile seawater 3 times to remove non-attached microorganisms; then separate the shell of the hydrothermal port crab from the tissue, and scrape off the shell powder under aseptic conditions; then add seawater to the shell powder for serial gradient dilution (1:5, 1:25, 1:125, 1:625), oscillate with a vortex shaker for 10 minutes to obtain a suspension; centrifuge the suspension at 5000r / min for 20 minutes, pour off the supernatant; A small amount of sterile seawater was resuspended, and 0.1mL of the resuspension was spread on a GYP medium (1.0g glucose, 0.1g yeast extract, 0.5g peptone, 15g agar, 1L seawater) plate; after culturing at room temperature 20°C for 10 days, A single colony was picked and streaked for purification, the...

Embodiment 2

[0057] (1) Same as part (1) of Example 1.

[0058] (2) Same as part (2) of Example 1.

[0059] (3) Fermentation culture

[0060] The activated Aspergillus clavus C2WU was made into a concentration of 10 9 The cfu / mL spore suspension was inoculated into the fermentation medium with 8% inoculum, and fermented statically at 22°C for 25 days.

[0061] Among them, the formula of the fermentation medium is: starch 3g, bran 14g, yeast extract 6g, KH 2 PO 4 0.5g, MgSO 4 ·7H 2 O0.4g, seawater 1000mL.

[0062] (4) Preparation of cyclic peptide compounds

[0063] After Aspergillus clavus C2WU was fermented and cultivated, take 5L of fermentation culture broth, centrifuge to get the supernatant to obtain fermentation broth; after the fermentation broth was concentrated, mixed with 10g of diatomaceous earth, refluxed with 1L of ethyl acetate, and separated by normal phase silica gel column chromatography (200- 300 mesh, 1kg; silica gel column size L50mm, ), followed by gradient...

Embodiment 3

[0065] (1) Same as part (1) of Example 1.

[0066] (2) Same as part (2) of Example 1.

[0067] (3) Fermentation culture

[0068] The activated Aspergillus clavus C2WU was made into a concentration of 10 7 The cfu / mL spore suspension was inoculated into the fermentation medium with 12% inoculum, and fermented statically at 25°C for 20 days.

[0069] Among them, the formula of the culture solution is: 20.0g of sucrose, 10.0g of yeast extract, 20g of malt extract, MgSO 4 ·7H 2 O0.4g, KH 2 PO 4 0.4g, seawater 1000mL.

[0070] (4) Preparation of cyclic peptide compounds

[0071] After the Aspergillus clavus C2WU is fermented and cultured, the fermentation culture liquid is centrifuged to obtain mycelium and fermentation liquid respectively.

[0072] Take the mycelium and carry out vacuum drying, the dry weight is 253g, extract with methanol (cold soak) 3 times (3×1L), obtain the methanol extract, concentrate to obtain the extract 13g; the extract is suspended in 500mL dis...

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Abstract

The invention discloses a cyclopeptide compound clavatustide A as well as a producing strain, a preparation method and an application thereof. The structural formula of clavatustide A is represented as formula (I): the preparation method comprises steps as follows: activated aspergillus clavatus CCTCC No. M2013421 is inoculated into a fermentation culture solution for fermentation culture; after fermentation is finished, a mycelium and fermentation broth are obtained through separation; a mycelium extract or a fermentation broth extract is subjected to chromatographic separation and purification treatment, and the cyclopeptide compound is prepared; and the application relates to an application of the cyclopeptide compound in preparation of antineoplastic drugs or cancer prevention health food. The cyclopeptide compound has better antitumor activity in vitro, the IC50 value of the cyclopeptide compound to hepG2 cells is 15 mu g/mL, and the cyclopeptide compound has an excellent development prospect; and the preparation method is simple and convenient to operate and suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of active substances of marine fungi, and in particular relates to a cyclic peptide compound clavatustide A and its producing bacteria, preparation method and application. Background technique [0002] Because marine microorganisms live in a stressful environment, their secondary metabolites often have biological activities that terrestrial fungal secondary metabolites do not have, and have become a hot spot in drug development in recent years. Marine microorganisms are not only under great pressure to survive and compete, but the pH range, temperature, pressure, dissolved oxygen, light, nutrition and salinity of the marine environment are different from those of the land environment. This is the reason for the diversity of marine microbial secondary metabolites Fundamentals (Debbab, A.; Aly, A.H.; Lin, W.H.; Proksch, P. Microb. Biotech. 2010, 3, 544–563; Saleema, M.; Ali, M.S.; Hussain, S.; Jabbar, A.; Ashraf, M.; L...

Claims

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Application Information

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IPC IPC(8): C07D273/00A61K31/395A61P35/00C12N1/14C12P17/14C12R1/66
CPCC07D273/00C12N1/14C12P17/14
Inventor 吴斌叶瑛
Owner ZHEJIANG UNIV
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