| Six strains were isolated from sea water or sludge which was collected from the East China Sea. After the anti-tumor activity of these strains was tested in vitro, a positive strain was selected from them. The selected strain was cultivated in the artificial seawater medium supplied with metal ions at high concentration as a factor of environmental stress, and the chemical diversity of metabolites from the culture was investigated. Some new metabolites which were synthesized under the stress of metal ions were separated, purified and identified. At last, the anti-tumor activity of the separated compounds was tested. The conclusions were summarized as follows:(1) Five fungi strains with differences on the colony morphology were isolated from sea water or sludge which was collected from the East China Sea by using the spread-plate technique. The test of anti-tumor activity was carried on these five strains and Penicillium citrinum MNP12010101, a strain isolated from seas water in previous study. The result revealed that the total extracts of the culture from P. citrinum MNP12010101 showed excellent anti-tumor activity against lung adenocarcinoma cells(A549), the inhibitory rate against A549 was 55.37% when the concentration of total extracts was at 200 μg·mL-1, and IC50 was 141.12 μg·mL-1. The total extracts of culture from strain JHBL-1 also showed anti-tumor activity against A549, the inhibitory rate was 33.46% when the concentration of total extract was at 200 μg·mL-1.The strain JHBL-1 was identified as a strain of Mucor sp. based against morphological characteristics and comparison of 18 S rDNA nucleotide sequences.(2) P. citrinum MNP12010101 was cultivated in artificial seawater medium supplied with metal ion at the high concentration, which was used as environmental stress, and the chemical diversity of metabolites from the culture was analyzed byHPLC. The results showed that when Co2+ at the concentration of 10 mmoL·L-1 was added in artificial seawater medium, more peaks were appeared on the HPLC fingerprint compared with that from culture without Co2+ supplement. This result illustrated that some new metabolites were synthesized. Moreover, the anti-tumor activity of the total extracts was increased obviously, IC50 was changed from 141.12μg·mL-1to 61.23 μg·mL-1. All these results suggested that the concentration of some bioactive metabolites were increased or some new metabolites were synthesized when P. citrinum MNP12010101 was cultivated under the stress of Co2+ at the concentration of 10 mmol·L-1.(3) The cultivation of P. citrinum MNP12010101 was scaled up to 80 L, 16 g of the total extracts from the medium and the cells was harvested. 11 compounds were obtained after the separation was carried out by column chromatography. 7 compounds of them were identified by NMR spectrum, and they are oleic acid(1), cladospolide E(2), ergosterol peroxide,(3) cyclo(gly-pro)(4), cyclo(ala-pro)(5), cyclo(iso- pro)(6)and cyclo(ala-val)(7). It is the first report that the compounds of 3,4,5,6,7 were separated from the metabolites of P. citrinum cells. The anti-tumor test of these compounds showed that 3 has excellent inhibition activity against tumor cells, IC50 was33.12 μg·mL-1 against prostate cancer cells(PC-3). The compound 2 and 4 has little inhibitory activity against tumor cells, IC50 were 199.34 μg·mL-1and 146.22 μg·mL-1against PC-3 cells, 256.22 μg·mL-1 and 178.56 μg·mL-1 against A549 cells respectively.All compounds did not show any inhibitory activity against colorectal carcinoma cells(HCT116). Except of the compound 3, it was the first report that these compounds have inhibition activity against PC-3, A549 and HCT116. |
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