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Antifungal and cytotoxic activities of the secondary metabolites from endophytic fungus Massrison sp.


Keywords: Endophyte Fungus Massrison sp. Antifungal Cytotoxic Metabolite


Three novel compounds with spiro-5, 6-lactone ring skeleton has been isolated from the fermentation broth of Massrison sp. which could be isolated repeatedly from wild Rehmannia glutinosa. Psetariae oryza P-2b was applied to guide fractionation of bioactive compounds produced by Massrison sp. The molecular structures were established by a variety of one- and two-dimensional NMR experiments and the compounds with similar skeleton were reported for the first time from endophytic fungi of terraneous plant. Antifungal and cytotoxic activities of the compounds were tested, compounds 2 and 3 displayed stronger antifungal and cytotoxic activities. The compounds have the potential to be antibiotic against fungal pathogens and tumor cells.

Crown Copyright [C] 2011 Published by Elsevier GmbH. All rights reserved.


Endophytes are microorganisms that live in the intercellular spaces of stems, petioles, roots and leaves of plants causing no discernible manifestation of their presence and have typically gone unnoticed (Strobel and Long 1998). The following hypothesis was developed about endophytes: both the pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least in part based on the secondary metabolites the partners produce. Whereas the pathogen-host interaction is imbalanced and results in disease, that of the endophyte and its host is a balanced antagonism (Schulz et al. 1999). In order to efficiently exploit its ecological niche, endophytes need to compete with other endophytes, pathogens and host; antibiotic secondary metabolites play an important role in the process.

Many fungal endophytes produce secondary metabolites and some of these compounds are antifungal and antibacterial which strongly inhibit the growth of other microorganisms (Gunatilaka 2006). These organisms have proven to be an unusually rich source of novel natural products (Heider et al. 2003), and constitute a valuable source of bioactive secondary metabolites (Li et al. 2000), some of them displayed antibiotics against pathogens and tumor cells to different degrees.

Rehmannia glutinosa is an important traditional herb in China and barefoot doctors of Henan province always apply local fresh herb in the therapy of aphthae and erosion from breast carcinoma. Wang from Zibo Central Hospital has applied fresh herb preparation in aphthae therapy and all 32 patients healed after 6 days caplen-dus. The local folk prescription is widespread in locality though no compounds with conspicuous antibiotic activities were isolated from the herb. The R. glutinosa from Henan Province displayed best therapeutic effect especially the wild herb; there are some microdosis novel compounds which play important roles in the process. In addition, the wild plants were seldom infected by fungal pathogens and displayed stronger resistance against fungal disease than cultivated plants. Bioactive metabolites from endophytes might produce marked effects and the metabolites may have the potential to be applied in medicine field.

In research of endophytes from R. glutinosa, a strain identified as Massrison sp. was isolated repeatedly from wild plant and corresponding extract display evident inhibition against P. oryza P-2b. The antibiotic metabolites were investigated to be massarigenin D, spiromassaritone and paecilospirone. The present study was designed to investigate the main antibiotic metabolites and examine their potential to be antibiotics against fungal pathogens and tumor cells.

Materials and methods

Isolation of endophytes

The roots of R. glutinosa were collected during March in 2007 from Wushe County, Henan Province, where high-quality R. glutinosa has a wide distribution. After spaded out the roots were taken to laboratory in 24h.

The general procedures adopted followed the methodology described by Petrini (Petrini et al. 1992). The roots were washed with water followed by ethanol and then sterilized with mercuric chloride (1 mg/l, 1 min) and washed with sterile water four times. Sterilized tissue segments (1 cm x 1 cm x 1 cm) were pressed on to the surface of PDA medium to ascertain the efficacy of surface sterilization procedure and to confirm endophytic isolations only from internal tissues of the plant segments. The absence of growth of any fungi on the medium confirmed that the surface sterilization procedure was effective in removing the surface fungi (Schulz et al. 1995). The surface-sterilized material was then deposited on a Petri dish containing potato-dextrose-agar medium (PDA) and incubated in the dark at 28[degrees]C in attemperator for one week. Individual hyphae tips of the emerging fungi were removed and placed on PDA (Maier et al. 1997). Isolation frequencies of different endophytes were calculated after the cultures were pure and the colonies symmetrical. 623 strains were obtained from 480 plant tissue segments and only 18 different endophytic fungi were ascertained after checking of the morphology of spores and hyphae. Three fungi could be isolated repeatedly from the plant (isolation frequency>5%) and were identified by Institute of Microbiology Chinese Academy of Sciences as Massrison sp. (isolation frequency = 23.1%), Verticillium sp. (isolation frequency > 27.5%) and Chaetomium sp. (isolation frequency > 14.0%).

Subsample fermentation and screening

Three endophytes obtained were inoculated into the Erlen-meyer flasks (250ml), each containing 100ml PDB (0.4g potato extract, 2.0 g dextrose and 100 ml distilled water, pH 7) which were autoclaved at 125[degrees]C for 20 min. Absorbent cotton was applied to seal the flasks for aerobic respiration and the cultures were incubated for 6 days at 28[degrees]C on rotary shakers at 180 rpm. Biomass was removed by filtration and fermentation broths were extracted by MeOH after vacuum drying.

P. oryza P-2b was applied to screen extracts with antifungal metabolites, further experiments were considered worthy if minimum morphological deformation concentration (MMDC) value was lower than 500 mg/ml. P. oryzae P-2b inhibitory experiment detects deformations of mycelia germinated from conidia. Morphological deformations included curling,swelling,hyper-divergency and beads shape. Inhibition of the germination was also observed (Kobayashi et al. 1996). Extract from Massrison sp. displayed evident antibiotic activity against Pyricularia oryzae P-2b (MMDC = 7.8 mg/ml) and a serial of follow-up researches were focused on the fungus.

Isolation and identifying of the active metabolites

Massrison sp. were cultivated under the same condition (6 days at 28[degrees]C on rotary shakers at 180 rpm.) in 250 flasks, each containing 100ml PDB (0.4g potato extract, 2.0g dextrose and 100 ml distilled water, pH 7) which were autoclaved at 125[degrees]C for 20 min at first. All fermentation broth (251) was prepared for extraction and antibiotic metabolites were successively extracted with EtOAc. Crude extracts (2.94g, EtOAc) were subjected to size exclusion chromatography (Sephadex LH-20, chloroform:MeOH = l:l, v/v), HPLC semi-preparation (CI8 liquid column chromatography ([H.sub.2]O/C[H.sub.3]OH)) was applied in isolation. P. oryza P-2b was applied to identify active fraction in isolation process. Three compounds with different antibiotic activities have been isolated finally.

All NMR data were acquired on a Bruker DRX500 NMR spectrometer with [ .sup.1]H and [ .sup.13]C nuclear observed at 500 MHz and 125 MHz respectively; the rare spiro-5, 6-lactone ring skeleton have been confirmed based on the data from NMR. TLC were produced by Qingdao Marine Chemical Factory, Qingdao, China, all chemicals used in the study were of analytical grade.

Antifungal activity assay

The compounds were evaluated against a panel of fungal pathogens Candida albicans (ATCC76615), Cryptococcus neoformans (ATCC32609), Trichophyton rubrum and Aspergillus fumigatus. All the fungal pathogens were from Changzheng hospital (Shanghai), Trichophyton rubrum and Aspergillus fumigatus were clinical pathogenic strains from the hospital and were identified using ITS sequence analysis.

Candida albicans and Cryptococcus neoformans were cultured on rotary shakers (250rpm) for 32h at 35[degrees]C in YEPD medium; Sabouraud agar (SDA) plates were inoculated at 28[degrees]C for 14 days for Trichophyton rubrum and at 35 [degrees] C for 7 days for Aspergillus fumigatus. All the cultures were diluted with RPMI1640 to make suspension culture (4 x [10.sup.3]-5 x [10.sup.3] cells/ml). Standardized fungi suspensions (120[micro]l) were inoculated onto microplates and were exposed to tested drugs which were fourfold diluted from 32 to 0.0156[micro]g/ml (final volume= 160[micro]g), a final DMSO concentration [less than or equal to]1%, a well without drugs was set to be negative control. Plates were incubated at 35[degrees]C for Candida albicans (24h), Cryptococcus neoformans (48h) Trichophyton rubrum (7 days) and Aspergillus fumigatus (7 days). Optical density (OD) was tested at 630 nm to measure fungi growth as the negative control showing 100% OD. The MI[C.sub.80] values were calculated as the highest dilution showing 80% growth inhibition of the strains according OD values. Griseofulvin and ketoconazole were used as positive control; all experiments were carried out in triplicate.

In vitro cytotoxic assay

Human normal liver cell (L-02), hepatoma carcinoma cell (HepG-2), breast carcinoma cell (MCF-7), and lung carcinoma (A-2) were obtained from Shanghai Institute of Pharmaceutical Industry. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin and 100 mg/ml of streptomycin.

Cells were maintained at 37[degrees]C in a humidified atmosphere of 5% CO2 in air. Cytotoxicity was measured using the MTT assay which was performed using a modified method described by Plumb et al. (1989). Cells were seeded into 96-well plates (2000 cells/well), after 24h incubation, the cells were treated with the compounds of varying concentrations (2, 4, 8, 16 and 32[micro]g/ml) and mitomycin was used as the positive reference. Cell survival was determined by MTT test according to the method of Mosmann (1983) and modified by Ohno and Abe (1991). 48 h after the drug addition, briefly, 20[micro]1 of MTT solution (5 mg/ml in phosphate buffered saline) was added to each well. Samples were incubated for further 4h at 37[degrees]C in humidified atmosphere with 5% C02-Then, the medium was discarded and the formazan blue, which formed in the cells, was dissolved with 100 ml DMSO. Absorbance was measured at 570 nm 24 h later to achieve cell survival (%), then IC50 values were expressed as the drug concentrations inhibiting cell growth by 50% by setting the viability of untreated cells as 100%.

Results and discussion

Molecular structures of the metabolites.

Chemical studies of extract from the culture broth afforded mas-sarigenin D (15mg), spiromassaritone (24mg) and paecilospirone (38mg). The rare spiro-5,6-lactone ring skeleton found in the three compounds has been reported as fungal secondary metabolites from Massarina tunicata (Oh et al., 2001), Mycosphaerella rosigena (Albinati et al., 1980), Microsphaeropsis sp. (Fukami et al., 1999), Arthropsis truncate (Ayer et al., 1992), freshwater fungus Massarina tunicate (Oh, H et al., 2003) and marine-derived fungus Massarina sp. (Abdel-Wahab et al. 2007). Here, three metabolites with rare spiro-5,6-lactone ring skeleton were isolated from terraneous plant endophytic fungi for the first time.

The molecular structures of all the isolates were determined using spectroscopic evidence and comparison with literature data ([ .sup.13]C, [ .sup.1]H-NMR, NOESY). The compounds 1 and 2 were identified as massarigenin D and spiromassaritone on the basis of MS and [ .sup.13]C, [ .sup.1]H-NMR data. Compound 3 was identified as paecilospirone according to the NMR data (Hirota etal. 1991) (Fig. 1).


Antifungal and cytotoxic assay

The potential of endophytic fungi as sources of novel bioactive substances is now widely recognized.Compounds with rare spiro-5, 6-lactone ring skeleton have been reported to be antibiotic against leukemia of mice and evidently extended live time (Nakayama et al. 1992). Antifungal and cytotoxic activities of three compounds with the rare skeleton were tested and compared sys-tematically.

The in vitro antifungal activity of the three compounds against four fungal pathogens is shown in Table 1. Results showed that three compounds exhibited various levels of antifungal effect against all pathogens tested; MIC values varied from 0.25 to 32[micro]g/ml. Antifungal activities of spiromassaritone and pae-cilospirone were comparable with those of griseofulvin and ketoconazole, spiromassaritone exhibited stronger antagonism against Candida albicans and Cryptococcus neoformans than griseofulvin (Table 1).
Table 1

Antifungal activity of the compounds ([MIC.sub.80], [mu]g/ml).

Drugs             Candida   Cryptococcus  Trichophyton  Aspergillus
                  albicans  neoformans    rubrum        fumigatus

Massarigenin D    >32         16            2             >32
Spiromassaritone    2          4            0.25            1
Paecilospirone      8         16            2               4
Griseofulvin       16        >32            0.25            0.25
Ketoconazole        8          0.25         0.0625          0.0156

The tests of cytotoxicity were performed as described previously; the cytotoxic effect of the three compounds on four tumor cells was investigated by MTT assay. The tested malignant cells showed a good response to the effect of three compounds. Spiromassaritone displayed the strongest cytotoxicity ([IC.sub.50] against HepG-2 = 5.6[microg/ml), but normal human liver cell was also clearly inhibited and no selective toxicity exhibited in the test; cytotoxicity of paecilospirone exhibited selection and inhibited tumor cell stronger (Table 2). The compound was administered i.p. to mice at 2.5 mg/kg/day for 5 days after inoculation of P388 leukemia cells to show %T/C (median survival time for the treated animals/median survival time for the control animals x 100)of 140(Nakayama et al. 1992).
Table 2

Cytotoxic activity of the compounds ([IC.sub.50], [mu]g/ml).

Drugs             L-02  HepG-2  MCF-7  A-549

Massarigenin D    19.6    20.8   11.2   14.4
Spiromassaritone   7.2     5.6    6.8    9.8
Paecilospirone    12.4    10.4    7.6    6.8
Mitomycin         13.6     6.8    3.6    3.2

[IC.sub.50] is defined as the concentration which results in a 50%
decrease in cell number as compared with that of the control cultures
in the absence of an inhibitor. The values represent the mean of five
independent experiments.

Endophytes must synthesize metabolites to compete first with other endophytes and then with pathogens in order to colonize the host (Schulz et al. 2002), most of the metabolites exhibit antimicrobial and cytotoxic activities. As part of our studies on bioactive agents from metabolites produced by endophytic fungi associated with species of traditional herbs, we initially investigated the metabolites and according bioactivities. Microamount bioactive metabolites from endophytes may play important roles in therapy, more researches are required to elucidate if traditional herbs contain more endophytes with novel metabolites.


This work was funded by Microscreen Biotechnology (Shanghai) Limited Company. The authors are grateful to the help from Institute of Microbiology Chinese Academy of Science and Antifungal Research Center of the Second Military Medical University.


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Zhen-Liang Sun, Ming Zhang, Ji-Fa Zhang, Jing Feng *

Branch Hospital in Fengxian of Shanghai No. 6 People's Hospital. No. 9588, Nanfeng Road, Fengxian District, Shanghai, PR China

* Corresponding author.

E-mail address:

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Title Annotation:Short communication
Author:Sun, Zhen-Liang; Zhang, Ming; Zhang, Ji-Fa; Feng, Jing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 15, 2011
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