| Endophytes, the microorganisms colonized in the internal tissues of living plants without causing apparent symptoms of disease, synthesize numerous bioactive compounds. In 1993, Stierle and colleagues firstly isolated paclitaxel-producing endophytic fungi from Pacific yew lead to a concept that there is possibility endophytes may produce secondary metabolites of plant. Resent years, more and more bioactive natural products with the same or similar construct of host metabolites, including alkaloids, steroids, terpenoids, quinones, flavonoids, peptides, etc. were obtained from endophytes which isolated from dozens of medicinal plants. These researches indicate that endophytes have gradually become potential alternative sources of medicinal plants. It also provides a new way against over-harvesting of medicinal plants, environmental pollution of drug industry and adverse effects of chemosynthesis drugs.Dioscorea zingiberensis C. H. Wright that belongs to the family Dioscoreaceae, is the medicinal twining monocotyledonae served as the main source of diosgenin that is an important precursor of steroidal drugs. Previously studies demonstrated that endophytic Fusarium strains from D zingiberensis produce active metabolites and stimulate the diosgenin synthesis of host cells which shows the potential value that the endophytes applied in planting, pre-processing and biological transformation of diosgenin.Endophytes were isolated from inner tissues of D. zingiberensis tuber which were planted in the sandy and clay soil, respectively. Bacillus subtilis isolates were the predominant strains in the inner tissues of the plants from either types of soil. All of the 48 endophytic strains were identified by using morphological, physiological, biochemical and molecular biological methods. We have isolated a Fusarium sp. strain SWM3, and the protein extracts showed broad spectrum antibacterial activity. More interestingly, we have isolated a B. subtilis strain SWB8 that producing diosgenin analogue which is the host secondary metabolite. Both of the (3-1,3-1,4-glucanase and the diosgenin analogue secreted by strain SWB8 showed selective cytotoxicity to cancer cell lines. Besides, the bacterial P-1,3-1,4-glucanase also showed spectrum antimicrobial activities against both bacteria and fungi.1. Isolation and identification of endophytesThe inner tissues of D. zingiberensis tuber which planted in sandy and clay soil were collected. Based on pure culture method,48 strains were isolated, including 46 bacteria and 2 fungi, belonged to genus Bacillus, Staphylococcus, Mesorihizobium, Tsukamurella, Citrobacter and Fusarium, respectively. The results showed that the number of endophytes isolated in sandy soil plants was much more than that in clay soil plants (33:13). B. subtilis isolates were the predominant strains in the inner tissues of the plant (18:48). These isolates could grow on peptone, Gause and Czapek medium, and significantly inhibited the growth of other isolates, for instance, the Staphylococcus. But interestingly, the number of Staphylococcus isolates was more than prediction (9:48), and these strains usually co-distributed with B. subtilis isolates in the same cross-section of the plant tissues. These findings made a breakthrough of endophytic bacteria in Dioscorea and also laid a foundation for the further studying of the interaction between endophytes and their plant host.2. The biologic activity of protein extracts of endophytic Fusarium sp. SWM3The protein extracts of Fusarium sp. SWM3 strain shows broad spectrum antibacterial activity. Especially, Staphylococcus aureus (17.6±0.6 mm) and Enterococcus faecalis (19±1.0 mm) were very sensitive to the extracts. In contrast, the inhibition effect could not be observed in Candida albicans and Cryptococcus neoformans.Methyl thiazolyl tetrazolium (MTT) and flow cytometry (FCM) assay showed that the protein extracts induced apoptosis of A549 cells with IC50 value of 10.9μg/mL. The percentage of apoptotic A549 cells treated with the extracts was significantly increased from 0.76% of the control to 4.29% in a dose dependent manner. Meanwhile, the late apoptotic or necrotic ratio was also significantly increased from 8.42% to 20.9%. Morphological changes, such as intercellular space widening, cell rounding, cytoplasm condensation, crimple and abscission were observed but without cytoplasmic blebbing. Fusarium sp. SWM3 strain could be a potential source of desirable antibiotics or anticancer compounds.3. Biologic activity of the metabolites of endophytic Bacillus subtilis SWB8B. subtilis SWB8 was a gram positive, endospore-forming aerobe or facultative aerobe with growth temperature range from 10℃to 50℃(optimum temperature,32℃), pH range from 6 to 9, salinity range 0.5-9.0%(NaCl) and formed rich foam in liquid medium.1) Identification and bioactivity of (3-1.3-1,4-glucanaseBased on gel permeation chromatography (GPC) and SDS-PAGE methods, protein extracts of B. subtilis strain SWB8 showing five peaks with the retention time of 31.46, 32.36,41.26,43.48 and 52.6 min in GPC were separated to five different gel bands of approximately 70,56,40,30,25 kDa. By mass spectrometry, the partial amino acid sequences of 40,30 and 25 kDa proteins showed the same composition with P-1,3-1,4-glucanase of B. cereus (GB:AAK16547). All the three separated proteins were identified as P-1,3-1,4-glucanase, suggesting that the (3-1,3-1,4-glucanase precursor would be cleaved into three fragments of 40,30 or 25 kDa with the retention time of 41.26,43.48 or 52.6 min in GPC, respectively.β-1,3-1,4-glucanase showed broad antimicrobial spectrum activity. E. faecalis (21.8±0.3 mm), E. coli (19.0±1.0 mm), C. albicans (22.5±0.6 mm) and C. neoformans (24±1 mm) were more sensitive toβ-1,3-1,4-glucanase.MTT and FCM assay showed significantly anticancer activity against A549 cells ofβ-1,3-1,4-glucanase with IC50 value of 11.5μg/mL. The percentage of apoptotic A549 cells treated with different concentrations ofβ-1,3-1,4-glucanase was significantly increased from 4.43% of the control to 43.1% of 19.2μg/mL in a dose dependent manner. Meanwhile, the late apoptotic or necrotic ratio was also increased from 2.57% to 14.7%. Morphological changes, such as intercellular space widening, shrinking, cytoplasmic blebbing, cytoplasm condensation and abscission were observed. In contrast, these changes could not be observed in the treated human bone marrow mesenchymal stem cells (MSCs).Perhaps, the B. subtilis strain SWB8 or itsβ-1,3-1,4-glucanase product could be a potential source of desirable antimicrobial and anticancer compounds.2) Identification and bioactivity of diosgenin analogueThe organic extract of B. subtilis SWB8 showed the same UV absorption peak in the range of 200-280 nm and 304 nm and similar waveform as the standard diosgenin. Thin-laver chromatography showed the similar Rf values of the extract (0.49) and standard diosgenin (0.46). Infrared (IR) spectra showed that the extract and standard diosgenin exhibited almost the same stretching and bending vibration and thus revealed the same functional groups in the region from 4500 to 500 cm-1. Electrospray ionization mass spectrum (ESI-MS) of the extract produced an major ion peak of m/z 437.1 [M+Na]+, which is consistent with 437.3 [M+Na]+of diosgenin, both of the samples formed precursor ion groups of m/z 453[M+H2O+Na]+,475,495,569 and fragment ions of m/z 158.6 and 302.6, suggesting the same molecular structural moiety. However, extract formed other different fragment ions of m/z 305.3,321.2,337.1, 358.2,371.9,377.8 and 379.8 in comparing with diosgenin fragment ions of m/z 182.5, 214.4,225.5,252.7,270.8,347.7,396.5 and 425.2. These differences are probably due to the same molecular skeleton but different latent chains. Nuclear magnetic resonance (’H-NMR) spectroscopy analysis showed that the extract and standard diosgenin produced the same hydrogen signals in the range of 1-5.5 ppm, suggesting the similar molecular structure between the analogue and the standard diosgenin.These results validated that B. subtilis SWB8 extract possesses the similar molecular structure of diosgenin and could be designated as diosgenin analogue (C27H42O3). Diosgenin analogue showed certainly antibacterial activity.The results of MTT assay showed selective cytotoxicity of diosgenin analogue that significantly acted on A549 cells. The inhibition ratio of A549 cells indicated positive correlation with the increasing concentrations of diosgenin analogue. A549 cells were intensely inhibited by the extract with an IC50 value of 13.09μg/mL, compared with the higher tolerance of MSCs cells that the IC50 value of 28.81μg/mLFCM analysis showed that the percentage of apoptotic A549 cells which treated with different concentrations of the extract (0,2.54,7.62 and 12.7μg/mL) was significantly increased from 3.59% to 24.4% in a dose dependent manner, but the late apoptotic or necrotic ratio was only increased from 5.44% to 10.2%. In contrast, these changes could not be observed in MSCs cells.Laser scanning confocal microscopy (LSCM) analysis indicated that the apoptosis-like morphologic changes, such as cell shrink, cytoplasmic blebbing and condensation were occurred on induced A549 cells. These results reconfirmed that diosgenin analogue inhibited A549 cells mainly by inducing apoptosis. Perhaps, diosgenin analogue from B. subtilis SWB8 could be a potential source of desirable anticancer compounds and alternative plant steroids.B. subtilis SWB8 has an application value in pre-processing of diosgenin production. On the other hand, it also could be a valuable model to study the mechanism that endophytic bacteria synthesizing the host secondary metabolite. |
PDF Full Text Request