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Marine actinomycetes for generating antineoplastic compound Norharmane

A technology of marine actinomycetes and compounds, which can be applied in anti-tumor drugs, microorganism-based methods, microorganisms, etc., and can solve problems such as manpower and material resources.

Inactive Publication Date: 2008-02-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the passage of time, the probability of discovering new compounds from terrestrial actinomycetes is getting lower and lower, resulting in a waste of a lot of manpower and material resources, so it is necessary to develop new drug resources

Method used

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  • Marine actinomycetes for generating antineoplastic compound Norharmane
  • Marine actinomycetes for generating antineoplastic compound Norharmane
  • Marine actinomycetes for generating antineoplastic compound Norharmane

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Effect test

Embodiment 1

[0014] 1. Cultivate WBF9 on Gaoshi Synthetic Medium No. 1 by the insert method, observe the mycelium (×400; Figure 1a) under the Olympus BH-2 optical microscope, and observe the sporophytum and Spore morphology (×7000; Fig. 1b). The results are shown in Figure 1. The spore chain presents a regular helical shape, each spore chain has 4-8 helices, and there are more than 10 spores; all spores have short spines on the surface, and the spores are round or oval; the diameter of the spores is about 0.5-1.0 μm, about 0.6-1.4 μm in length, it can be judged that the strain is Streptomyces.

[0015] 2. Cultivate the marine strain WBF9 according to the medium formula recommended by the International Streptomyces Program (ISP) and Waksman, and observe the culture characteristics and physiological and biochemical characteristics.

[0016] Culture characteristics of strain WBF9: strain WBF9 was cultured on a typical actinomycete differential medium (ie: yeast malt extract agar, oat agar, s...

Embodiment 2

[0023] 1. Fermentation experiment of marine actinomycetes WBF9: The medium components used are: glucose 1%, potato 15%, yeast powder 0.6%, calcium chloride 0.12%, natural seawater, pH 7.2. Inoculate the spores of bacterial strain WBF9 into a 250mL Erlenmeyer flask (containing 50mL of medium), and culture it on a shaker at 200r / min at 28°C for 2 days as a seed solution; , cultivated under the same conditions for 10 days, sampled 10 mL every 24 hours, then centrifuged at 4,000 r / min for 10 minutes, and took the supernatant, part of which was used to measure pH value and sugar content, and the other part was sterilized by filtration with a 0.22 μm filter membrane , stored in a refrigerator at 4°C to be tested for anti-tumor activity; and the balls were baked at 90°C until the weight did not change, and the dry weight of the bacteria was measured.

[0024] 2. Antitumor activity detection method of fermented liquid: the tumor cells in logarithmic growth phase were digested with 0.2...

Embodiment 3

[0029] According to the results of the fermentation experiment, the strain WBF9 was fermented, and a total of 40 L of fermentation liquid was collected. The fermentation broth was centrifuged, and the supernatant was collected and concentrated under reduced pressure. The paste was obtained by extraction with ethyl acetate, activity tracking by MTT method, silica gel column chromatography, SephadexLH-20 molecular sieve chromatography and its reversed-phase high-performance liquid phase preparation method to obtain the anti-tumor active compound monomer Norharmane. The physical data and their spectral data are as follows:

[0030] Norharmane: white needle-like solid (ethyl acetate), soluble in organic solvents such as dimethyl sulfoxide, methanol, acetone, ethyl acetate, etc. The reaction of potassium bismuth iodide is orange red, and ESI-MS appears at m / z 169 Molecular ion peak [M+H] + , the molecular formula is C 11 h 8 N 2 , with an unsaturation of 9; MP 198-199° C.; TLC...

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Abstract

The invention relates to assessment and fermentation of marine actinomycete WBF9 that is used for producing anti-tumor compound and the separation and purification of the anti-tumor compound and the cytotoxic activity, and pertains to the microscopic organisms medicine filed. Bacterial strain WBF9, which is streptomyces griseorubens that is separated from the sea for the first time, is identified as one of the new bacterial strains of streptomyces griseorubens by traditionally classification and 16SrDNA genetic analysis. Culture medium (glucose: 1 percent, potato: 15 percent, yeast powder: 0.6 percent, calcium chloride: 0.12 percent and natural seawater, pH value 7.2) is applied by the bacterial strain for fermentation, and the compound extracted from the fermentation liquor, which has strong anti-tumor activity and identified as Norharmane by MS and NMR technique, is the first compound that is acquired by separating marine actinomycete. According to the MTT method, the compound is found to have cytotoxicity against four tumor cells: KB, BGC 803, Hep G2 and SGC 7901, and is a potential anti-tumor medicine.

Description

technical field [0001] The invention relates to the identification and fermentation of a marine actinomycete WBF9 that produces anti-tumor compounds, as well as the separation, purification and cytotoxic activity of the anti-tumor compounds, belonging to the field of microbial medicines. Background technique [0002] As we all know, terrestrial actinomycetes are an important source of antibacterial, antitumor and immunosuppressant drugs. However, with the passage of time, the probability of discovering new compounds from terrestrial actinomycetes is getting lower and lower, resulting in a waste of a lot of manpower and material resources, so it is necessary to develop new drug resources. In recent years, marine actinomycetes have gradually become an important source of drug development. Because they live in a special environment of high salt, high pressure, low temperature and low nutrition, they can produce many unique structures. Compounds with novel effects have advantag...

Claims

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Application Information

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IPC IPC(8): C12P17/18A61P35/00C12R1/465
Inventor 奚涛叶亮那广水邢莹莹
Owner CHINA PHARM UNIV
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