RESEARCH ARTICLE
Biological activities of extracts from endophytic fungi isolated from Garcinia plants Souwalak Phongpaichit1, Jaru Nikom2, Nattawut Rungjindamai3, Jariya Sakayaroj3, Nongporn Hutadilok-Towatana4, Vatcharin Rukachaisirikul5 & Kanyawim Kirtikara3 1
Natural Products Research Unit, Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand; 2Biotechnology Program, Faculty of Science, Prince of Songkla University, Songkhla, Thailand; 3National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani, Thailand; 4Natural Products Research Unit, Department of Biochemistry, Faculty of Science, Prince of Songkla University, Songkhla, Thailand; and 5Department of Chemistry, Faculty of Science, Prince of Songkla University, Songkhla, Thailand
Correspondence: Souwalak Phongpaichit, Natural Products Research Unit, Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: 166 7444 6661; fax: 166 7444 6661; e-mail:
[email protected] Received 20 May 2007; revised 22 July 2007; accepted 4 August 2007. First published online 20 September 2007. DOI:10.1111/j.1574-695X.2007.00331.x Editor: Alex van Belkum
Abstract Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.
Keywords endophytic fungi; anti-Mycobacterium tuberculosis ; anti-Plasmodium falciparum ; antioxidant; anti-HSV-1; antiproliferation.
Introduction Endophytic fungi have been recognized as possible useful sources of bioactive secondary metabolites (Schulz et al., 2002; Strobel et al., 2004). As part of the authors’ ongoing research program on the detection and identification of bioactive substances from plants and fungi, it was found that some endophytic fungi isolated from various Garcinia plants in southern Thailand could produce antimicrobial substances in their culture broths (Phongpaichit et al., 2006). A further investigation of the extract of a culture filtrate from Eutypella scoparia PSU-D44, one of the endophytic fungi isolated from Garcinia dulcis, led to the isolation and structural elucidation of two new pimarane diterpenes and two new cytochalasins (Pongcharoen et al., 2006). In this report, crude extracts from endophytic fungi were further investigated for other biological activities including antimycobacterial, antimalarial, antiviral, cytotoxic, antiproliferation, and antitoxidant effects. Any strains that showed FEMS Immunol Med Microbiol 51 (2007) 517–525
potentially useful activity were then identified using molecular methods.
Materials and methods Source of endophytic fungi Host plants and isolation methods were as described by Phongpaichit et al. (2006). A total of 51 endophytic fungal isolates were included in this study: five were from Garcinia atroviridis, 23 from G. dulcis, 16 from Garcinia mangostana, six from Garcinia nigrolineata, and one from Garcinia scortechinii. Endophytic fungi that showed bioactivity were identified based on the analysis of the DNA sequences of the ITS15.8S-ITS2, ITS regions of their rRNA gene as described previously (Phongpaichit et al., 2006). A BLAST search was used to search for the closest matched sequences in the GenBank database (Altschul et al., 1990). The authors’ 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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fungal sequences and other related sequences were multiply aligned using BIOEDIT 7.0.5 (Hall, 2005), and the alignments were adjusted manually where necessary to maximize alignments. Phylogenetic relationships were estimated using PAUP v4.0b10 (Swofford, 2002). The ITS sequences of these active endophytic fungi were submitted to GenBank (see accession numbers in Table 4).
Preparation of fungal crude extract Endophytic fungal isolates were grown on potato dextrose agar at 25 1C for 5 days. Three pieces (0.5 0.5 cm2) of mycelial agar plugs were inoculated into 500 mL Erlenmeyer flasks containing 300 mL potato dextrose broth and incubated at room temperature for 3 weeks under a stationary condition. The broth culture was filtered to separate the culture broth and mycelia. All filtrates were extracted three times with ethyl acetate and evaporated to dryness using a rotary evaporator. Some randomly selected mycelia were extracted with methanol, hexane, and ethyl acetate and extracts were again evaporated to dryness. All extracts were kept at room temperature. At least two batches of each isolate were extracted. The resulting extracts from each batch were examined by thin-layer chromatography (TLC), and their 1H nuclear magnetic resonance (NMR) spectra were recorded. Only the extracts with identical TLC chromatograms and 1H NMR signals were selected for bioassays.
Antimycobacterial assay The antimycobacterial activity was performed using the microplate Alamar blue assay (Collins & Franzblau, 1997). Briefly, 100 mL of the test solution in a Middlebrook 7H9 medium was mixed with 100 mL of the same medium containing 105 CFU mL1 of Mycobacterium tuberculosis H37Ra to give a final concentration of fungal extract of 200 mg mL1 in each microplate well. After incubation at 37 1C for 7 days, 20 mL of Alamar blue was added to the control well. If the dye turned pink, indicating bacterial growth, the dye was then added to all the remaining wells. Growth or no growth was determined on the following day using fluorescence spectroscopy. Active extracts were retested at lower doubling dilution concentrations to determine the minimum inhibitory concentrations (MICs). Standard drugs rifampin, isoniazid and kanamycin gave MICs of 0.0023, 0.1, and 2.5 mg mL1, respectively, which are within the acceptable ranges using this method.
Antimalarial assay Anti-Plasmodium falciparum K1 assays were performed according to the methods of Makler & Hinrichs (1993) and Desjardins et al. (1979). Briefly, 25 mL of the test solution in RPMI-1640 was mixed in microplate wells with 200 mL of a 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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1.5% erythrocyte suspension with 1–2% parasitaemia in the early ring stage to give a final concentration of the fungal extract of 10 mg mL1. Dihydroartemisinin and dimethyl sulfoxide (DMSO) were used as positive and negative controls. The microplates were incubated in a 3% CO2 incubator at 37 1C for 24 h and the malarial lactate dehydrogenase activity was measured (Makler & Hinrichs, 1993). Extracts with antimalarial activity were further tested for a concentration that caused 50% reduction in growth (IC50) using the 3H-hypoxanthine incorporation method (Desjardins et al., 1979). In this method, dihydroartemisinin, the standard antimalarial drug, had an IC50 of 0.0012 mg mL1.
Antiviral assay The antiviral assay was determined colorimetrically as modified from Skehan et al. (1990). Herpes simplex virus type 1 (HSV-1 ATCC VR-260) was maintained in the Vero cell line ATCC CCL-81 (kidney fibroblast of an African green monkey), which was cultured in Eagle’s minimum essential medium with the addition of 10% heat-inactivated fetal bovine serum and 1 mM sodium pyruvate. The test samples were placed in wells of a microtiter plate at final concentrations that ranged from 1 to 50 mg mL1. The viral HSV-1 (30 PFU) was added to wells of a 96-well plate, followed by adding Vero cells (105 cells mL1); the final volume in each well was 200 mL. After incubation at 37 1C for 72 h in a 5% CO2 incubator, the viability of the Vero cells was determined by the sulforhodamine B (SRB) assay. Acyclovir and DMSO were used as positive and negative controls, respectively. Acyclovir inhibited HSV-1 with a typical IC50 of 1.9 0.5 mg mL1.
Cytotoxicity assay The cytotoxicity of the fungal extracts against a normal cell line (Vero cell lines) was determined, using the same procedure as the antiviral assay without addition of virus. The reference inhibitor was ellipticine (IC50 of 0.6 0.3 mg mL1). An extract with an IC50 4 50 mg mL1 was defined as being noncytotoxic. Because the Vero cells were used for both the antiviral assay and the cytotoxicity test, extracts that were cytotoxic to Vero cells were excluded from the antiviral assay.
Antiproliferation assay A human oral epidermoid carcinoma (KB) cell line ATCC CCL-17 and human small-cell lung cancer cell (NCI-H187) ATCC CRL-5804 were used in antiproliferation assays. Briefly, 10 mL of fungal extract in 10% DMSO was mixed with 190 mL of a cancer cell line suspension (105 cells mL1). After incubation at 37 1C in a 5% CO2 incubator for 72 h, cell growth was determined by the SBR assay for KB cells and FEMS Immunol Med Microbiol 51 (2007) 517–525
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by an MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] assay for NCI-H187 cells (Skehan et al., 1990). Ellipticine and doxorubicin were used as positive controls. The IC50 of ellipticine and doxorubicin for KB and NCI-H187 cells were 0.17 and 0.11 mg mL1, and 0.467 and 0.020 mg mL1, respectively.
Antioxidant assay Three free radical scavenging assays [diphenylpicrylhydrazyl (DPPH) assay, hydroxyl radical scavenging assay, and superoxide anion scavenging assay] were used to determine the antioxidant activity of endophytic fungal extracts.
DPPH assay This was carried out according to Yen & Hsieh (1997). To different concentrations of a sample in methanol (0.5 mL each), 1 mL of a methanolic solution of 0.2 mM DPPH was added. After mixing thoroughly, the mixture was allowed to stand in the dark for 30 min and the absorbance at 523 nm was measured using methanol for the baseline correction. The results were then compared with that of the control prepared as above but without a test sample. Radical scavenging activity was expressed as a percentage and was calculated using the following formula: %Scavenging = [(Acontrol Asample)/Acontrol] 100. For each sample, the result was presented as an IC50 (sample concentration that produced 50% scavenging of the DDPH radical). Butylated hydroxytoluene was used as a positive control.
Hydroxyl radical scavenging assay The method previously described by Murcia et al. (2001) was modified. Various amounts of the test sample were mixed with 134 mL of 30 mM KH2PO4–KOH buffer (pH 7.4); 67 mL of 17 mM deoxyribose, 33 mL of 34 mM H2O2, 33 mL of 1.2 mM EDTA, 67 mL of 300 mM FeCl3, and 67 mL of 600 mM ascorbic acid were then added to start the reaction. After incubation at 37 1C for 1 h, the products of the hydroxyl radical attack on deoxyribose were determined by adding 333 mL of 1% (w/v) thiobarbituric acid (TBA) in 50 mM NaOH, followed by 333 mL of 2.8% (w/v) trichloroacetic acid (TCA). After further incubation at 80 1C for 20 min, the reaction mixtures were centrifuged. The clear supernatants were then measured for their absorption at 532 nm. A parallel assay omitting the test sample was used as a control whereas that without the addition of deoxyribose was used as a sample blank. The results were expressed as percent inhibition of the deoxyribose attack using the following formula: %Inhibition = [Acontrol (Asample Asample blank)/Acontrol] 100. For each sample, the result was presented as an IC50 (sample concentration that produced 50% FEMS Immunol Med Microbiol 51 (2007) 517–525
inhibition of hydroxyl radical). Tannin was used as a positive control.
Superoxide anion scavenging assay For estimating superoxide dismutase activity, the assay was carried out according to the method of Beyer & Fridovich (1987). Test samples were prepared in methanol and 20 mL of each was added to 1 mL of the reagent composed of 9.9 mM L-methionine, 1.72 mM nitroblue tetrazolium (NBT), 1% (w/v) Triton X-100, and 117 mM riboflavin in 50 mM K2HPO4, pH 7.8. After mixing, the reaction mixture was illuminated under a 40 W fluorescent light for 7 min. A control tube in which the test sample was replaced by methanol and a sample blank that contained no riboflavin were also run in parallel. The absorbance at 560 nm was then measured. The results were expressed as percent scavenging of the superoxide radical using the following formula: %Scavenging = [Acontrol (Asample Asample blank)/ Acontrol] 100. For each sample, the result was presented as an IC50 (sample concentration that produced 50% inhibition of superoxide radical). Trolox was used as a positive control.
Results and discussion In vitro bioactivities of endophytic fungi A total of 65 crude extracts from 51 endophytic fungi isolated from Garcinia plants were assessed for various bioactivities including anti-M. tuberculosis, anti-P. falciparum, anti-HSV-1, cytotoxicity to Vero, KB and NCI-H187 cells, and antioxidant activities. The results are shown in Tables 1–3. Fifty-two out of 65 isolates (80.0%) displayed at least one biological activity. Tuberculosis is one of the most serious infections of AIDS patients. Antituberculosis drug resistance is a major public health problem that threatens global tuberculosis control. Therefore, the antimycobacterial activity of the fungal extracts against M. tuberculosis was tested. Table 1 shows the number and percentage of the extracts and endophytic fungi that showed antimycobacterial activity. Fifty out of 65 extracts (76.9%), accounting for 78.4% of the isolates, exhibited antimycobacterial activity, with MIC values that ranged from 6.25 to 200 mg mL1. The percentage of endophytic fungi demonstrating antimycobacterial activity was very high compared with the study of Wiyakrutta et al. (2004), who found that only 92 out of 360 isolates (26%) from 81 plant species were active. Endophytic fungi from Garcinia plants are therefore good sources for exploring the possibility of new antimycobacterial drugs. Among the 50 active extracts, seven displayed strong antimycobacterial activity with MIC values of 6.25–25 mg mL1. These values are comparable with the 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Table 1. Percentages of bioactive fungal extracts and bioactive endophytic fungal isolates from various Garcinia host plants
Host plant Garcinia atroviridis Garcinia dulcis Garcinia mangostana Garcinia nigrolineata Garcinia scortechinii Total
Anti-Mycobacterium tuberculosis
Anti-Plasmodium falciparum K1
7/8 (87.5) 5/5 (100.0)w 31/34 (91.2) 23/23 (100.0)w 6/16 (37.5)z 5/6 (83.3)z 1/1 (100.0)z 50/65 (76.9) 40/51 (78.4)w
1/8 (12.5) 1/5 (20.0) 4/33 (12.1) 4/23 (17.4) 1/16 (6.3) 3/6 (50.0) 0/1 (0.0) 9/64 (14.1) 9/51 (17.6)
Anti-HSV-1
Antioxidant activity
Cytotoxicity (Vero cell)
2/4 (50.0) 2/3 (66.7) 0/14 (0.0) 0/11 (0.0) 2/4 (50.0) 0/2 (0.0) 0/0 (0.0) 4/24 (16.7) 4/20 (20.0)
2/8 (25.0) 2/5 (40.0) 5/32 (15.6) 5/23 (21.7) 3/16 (18.8) 3/6 (50.0) 1/1 (100.0) 14/63 (22.2) 14/51 (27.5)
3/8 (37.5) 3/5 (60.0) 11/24 (45.8) 11/18 (61.1) 3/16 (18.8) 4/6 (66.7) 1/1 (100.0) 22/55 (40.0) 22/46 (47.8)
Antiproliferation KB
NCI-H187
0/8 (0.0) 0/5 (0.0) 4/32 (12.5) 4/23 (17.4) 1/16 (6.3) 2/6 (33.3) 1/1 (100.0) 8/63 (12.7) 8/51 (15.7)
2/8 (25.0) 2/5 (40.0) 2/32 (8.7) 2/23 (8.7) 2/16 (12.5) 1/6 (16.7) 0/1 (0.0) 7/63 (11.1) 7/51 (13.7)
Active extracts/total extracts tested (%). w
Active isolates/total isolates tested (%). One extract per isolate.
z
MIC values of normal antituberculosis drugs (pyrazinamide 20 mg mL1, ethambutol 1–5 mg mL1, and streptomycin 8 mg mL1) (Inderlied & Salfinger, 1999). Extracts from isolates M35 and M114 from G. mangostana had the lowest MIC of 6.25 mg mL1. However, the M35 extract was toxic to Vero cells (IC50 8.5 mg mL1), whereas M144 was noncytotoxic (IC50 4 50 mg mL1) (Table 2). Thus, isolate M114 is a good candidate for the isolation of antimycobacterial substances. In addition, an extract from a culture broth of isolate D15 from G. dulcis displayed antimycobacterial activity with an MIC of 12.5 mg mL1 and was noncytotoxic to Vero cells. These findings have led to the isolation and structural identification of one new antimycobacterial substance: phomoenamide, an enamide dimer (MIC 6.25 mg mL1) (Rukachaisirikul et al., 2007). Chomcheon et al. (2005) reported that 3-nitropropionic acid, a potent antimycobacterial agent was found in extracts of several strains of endophytic fungi. Other antimycobacterial-active extracts that were nontoxic to Vero cells should be studied further for the isolation, purification, and determination of their mechanism of action against M. tuberculosis. More than 40% of the world’s population lives in areas where malaria is endemic. There is a demand for new antimalarials in many tropical countries because of the spread of drug-resistant malaria strains (Cowman & Duraisingh, 2001). In this study, the fungal extracts against the multidrug resistant strain of the malarial protozoa P. falciparum K1 were tested. The percentage of active endophytic fungi showing inhibitory activity against P. falciparum was low. Only nine out of 64 extracts (14.1%), accounting for 17.6% of the tested isolates, were active with IC50 values of 1.94–7.87 mg mL1. However, these values are seven times higher than those reported by Wiyakrutta et al. (2004). Extracts from two fungal isolates, A13 and N41 from G. atroviridis and G. nigrolineata, respectively, exhibited potent antimalarial activity, with respective IC50 values of 7.87 and 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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3.97 mg mL1 and were noncytotoxic to Vero cells. These two isolates should be studied further for their mechanism as antimalarial compounds. HSV is endemic in all societies throughout the world and produces year-round infections in all age groups (Costello et al., 2006). Although there are many drugs in clinical use, the search for new antiviral agents for HSV treatment is needed due to the emergence of drug-resistant virus strains (De Clercq, 2001; Morfin & Thouvenot, 2003). It was found that four out of 24 endophytic fungal extracts (16.7%) exhibited weak to moderate antiviral activity against HSV-1. In contrast to the study of Wiyakrutta et al. (2004), 31% of their endophytic fungi were found to have anti-HSV-1 activity. Because the discovery of paclitaxel and some of its derivatives represented the first major group of antiproliferation agents produced by endophytic fungi, many studies have searched for endophytic fungi with anticancer or antitumor activity from various medicinal plants (Li et al., 1996; Strobel et al., 1996, 1997; Wang et al., 2000; Huang et al., 2001; Shrestha et al., 2001; Puri et al., 2005, 2006). In this study, antiproliferation activity against KB and NCIH187 cancer cell lines was rare, being found in only eight (15.7%) and seven (13.7%) out of 51 isolates, respectively. These values are in the same range as those found by Wiyakrutta et al. (2004) but about twice as high as those reported by Huang et al. (2001). Only one extract, that from N24 from G. nigrolineata, displayed antiproliferative activity on both cell lines. The IC50 values of the active extracts against KB and NCI-H187 cells were 0.06–8.44 and 4.16–13.17 mg mL1, respectively. Unfortunately, most of the active extracts against KB cells also showed strong cytotoxicity on Vero cells (IC50 0.03–3.89 mg mL1) except that the one from S9 had weak cytotoxicity (IC50 50 mg mL1). For activity against NCI-H187 cells, two moderately active extracts from A5 and D13 were noncytotoxic FEMS Immunol Med Microbiol 51 (2007) 517–525
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Table 2. Biological activities of endophytic fungal extracts (mg mL1) from Garcinia species Fungal code
Extract codew
Anti-Mycobacterium tuberculosis (MIC)
Anti-Plasmodium falciparum K1 (IC50)
Anti-HSV-1 (IC50)z
Antioxidant activity‰
Cytotoxicity (IC50)z
A3
A3CE A3CH A5BE A5CH A13BE A50BE A57BE D1BE D2BE D2CE D2CH D6CE D6CH D9CE D12CH D13CE D15BE D15CH D17CE D17CH D18CE D19CE D19CH D20CE D21CH D25CH D29CE D29CH D31CE D31CH D32CH D36CH D37BE D38BE D38CH D61BE D64BE D69BE D73BE M5BE M7BE M35BE M40BE M100BE M106BE M114BE N2BE N24BE N28BE N40BE N41BE S9BE
200 100 200 100 200 100 25 50 200 25–50 50 200 50 50 50 100 12.5 100 200 200 200 200 200 100 100 200 200 100 200 200 200 200 200 200 100 100 100 200 200 200 6.25 50 200 6.25 200 25 12.5 100 100 100
7.87 6.6 6.93 6.17 NT 3.75 3.74 2.64 1.94 3.97
NA WA MA NA NA NA NA NT NT NA NT NA NT NT NT NA NA NT NA NT NA NA NA NA NT NA NA NA WA MA NA NA NA NA NA
1 1 1 1 NT 1 NT 1 1 1 1 1 1 1 1 1
10.3 4 50 4 50 4 50 4 50 0.42 1.47 4 50 4 50 5.277 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 31.28 NT
A5 A13 A50 A57 D1 D2
D6 D9 D12 D13 D15 D17 D18 D19 D20 D21 D25 D29 D31 D32 D36 D37 D38 D61 D64 D69 D73 M5 M7 M35 M40 M100 M106 M114 N2 N24 N28 N40 N41 S9
30.75 NT 49.69 NT NT 28.89 3.71 NT 44.19 NT 5.78 3.89 4 50 2.05 2.74 4 50 NT 3.18 14.98 8.5 4 50 4 50 4 50 4 50 12.13 6.26 0.025 6.03 4 50 50
Antiproliferationk (IC50) KB
NCI-H187
NT 4.08 8.33 NT 1.53 2.95 4.77 8.44 0.064 0.064
4.16 11.98 4.89 6.52 NT NT 13.17 6.26 4.21 NT
Endophytic fungi isolated from A, Garcinia atroviridis; D, Garcinia dulcis; M, Garcinia mangostana; N, Garcinia nigrolineata; S, Garcinia scortechinii. w
BE, extract from culture broth; CE, extract from fungal cell with ethyl acetate; CH, extract from fungal cell with hexane. WA, weakly active, at highest concentration tested (50 mg mL1) inhibited 25–35% of HSV-1; MA, moderately active, at highest concentration tested (50 mg mL1) inhibited 4 35–50% of HSV-1. ‰ 1, exhibited antioxidant activity for at least two free radical scavenging assays. z IC50 4 50 mg mL1 is defined as noncytotoxic. k IC50 4 20 mg mL1, inactive; 4 10–20 mg mL1, weakly active; 5–10 mg mL1, moderately active; o 5 mg mL1, strongly active. NA, not applicable as extracts were cytotoxic to Vero cells; NT, not tested. z
FEMS Immunol Med Microbiol 51 (2007) 517–525
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Table 3. Antioxidant activity of selected endophytic fungal extracts tested with three free radical scavenging assays Antioxidant activity, IC50 (mg mL1)
Extract
DPPH
OH
A50BE A57BE D1BE D2BE D38BE D64BE D73BE M5BE M7BE M40BE N2BE N24BE N40BE S9BE Butylated hydroxytoluene Tannin Trolox
250 250 180 210 150 30 200 220 40 60 200 140 140 150 30–50
4 2 2 3 3 2 3 4 3 3 4 4 4 3
DPPH assay. IC
w
z
O 2
NA NA 2560 1740 NA NA 2970 7 830 1670 40 2 3 500
2250–2330 850–980 1
, inactive; 4 100–250 mg mL1, weakly active; 4 50–100 mg mL , moderately active; 10–50 mg mL1, strongly active; o 10 mg mL1, very strongly active. w Hydroxyl radical scavenging assay. IC50 4 5000 mg mL1, inactive; 4 4000–5000 mg mL1, weakly active; 4 3000–4000 mg mL1, moderately active; 2500–3000 mg mL1, strongly active; o 2500 mg mL1, very strongly active. z Superoxide anion scavenging assay. IC50 4 3000 mg mL1, inactive; 4 2000–3000 mg mL1, weakly active; 4 1000–2000 mg mL1, moderately active; 800–1000 mg mL1, strongly active; o 800 mg mL1, very strongly active. NA, no activity. 50 4 250 mg mL 1
to Vero cells. These extracts may show selective cytotoxicity against this cancer cell line but not against normal Vero cells. Further investigations will be conducted on these three isolates. The discoveries of two antioxidants, pestacin and isopestacin from an endophytic fungus Pestalotiopsis microspora residing in Terminalia morobensis in Papua New Guinea (Strobel et al., 2002; Harper et al., 2003), also led to the search for endophytic fungi with an antioxidant potential. When all extracts were screened for their antioxidant activity in the DPPH assay, 14 out of 64 extracts (22.2%) were active. As shown in Table 3, a high antioxidant capacity (IC50 4 10–50 mg mL1) was found in the extracts of D64 from G. dulcis and M7 from G. mangostana with IC50 of 30 and 40 mg mL1, respectively, whereas moderate activity against DPPH (IC50 4 50–100 mg mL1) was obtained from M40 (IC50 = 60 mg mL1). The other tested extracts, however, displayed a rather low activity with IC50 ranging from 140 to 250 mg mL1. Also in Table 3, all 14 extracts, in our assay system, showed a remarkable ability to inhibit hydroxyl radicals generated via the Fenton reaction. Their IC50 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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values ranged from 2 to 4 mg mL1, about 1000-fold lower concentrations than that of the standard substance the authors used, tannin (IC50 c. 2000 mg mL1). As most extracts were not so effective in the DPPH system, their strong scavenging action toward hydroxyl radicals observed in this study may depend largely on iron chelation rather than direct quenching of the radicals. It is also plausible that D64, M7, and M40 may contain active materials that can donate electrons and react with free radicals in high amounts, resulting in their prominent DPPH scavenging effect compared with the others. In addition to DPPH and hydroxyl radical inhibition, M5, N2, N24, N40, and S9 showed a remarkable ability to scavenge for superoxide anions, whereas M7 was comparable with the standard substance, trolox (Table 3). The fact that these extracts, except M7, are not potent DPPH scavengers indicates that their superoxide inhibition may be attributed to compounds of extracts possessing superoxide dismutase-like properties. Among all tested extracts, M7 showed the strongest antioxidant activity in all three assays but unfortunately exhibited cytotoxicity on Vero cells. Only D2, M40, N40, and S9 displayed antioxidant activity and were noncytotoxic on Vero cells, and so these could be good candidates for further study. Although numerous bioactive compounds have been discovered, new products applicable to human therapy are rare, owing to their cytotoxic effects. In this study, extracts on growth inhibition of Vero cells were tested to indicate cytotoxicity and it was found that half of the extracts tested (22/44, 50%), which showed activity of one sort or another and accounted for 47.8% of the fungal isolates, exhibited cytotoxicity. Only noncytotoxic extracts should be selected for further studies.
Identification of bioactive endophytic fungi Fifteen endophytic fungal isolates were identified based on an analysis of their ITS sequences. The identities and GenBank accession numbers of the active endophytes are provided in Table 4. They belong to nine genera: Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis. One isolate (N24) was matched with an unidentified fungal endophyte. Botryosphaeria spp. are common endophytes of various plants including Garcinia spp. (Camatti-Sartori et al., 2005; Mohali et al., 2006; Phongpaichit et al., 2006; Wang et al., 2007). In a previous report, it was found that Botryosphaeria sp. M76 from G. mangostana displayed antistaphylococcal and anti-M. gypseum activities (Phongpaichit et al., 2006). One bioactive isolate D13 was identified as Fusicoccum sp. Mohali et al. (2006) reported that Botryosphaeria spp. had numerous anamorphs and Fusicoccum species are among the most common. Fusicoccum sp. D13 may be one of the anamorphs of Botryosphaeria in Garcinia spp. FEMS Immunol Med Microbiol 51 (2007) 517–525
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Table 4. Identity of endophytic fungal species having bioactivities
Code
Species
GenBank accession no.
D2 M35 M114 D12 D13 A5 D9 D31 M7 N28 A13 M5 A57 D15 N24
Aspergillus aculeatus Botryosphaeria rhodina Botryosphaeria rhodina Curvularia sp. Fusicoccum sp. Guignardia mangiferae Guignardia mangiferae Muscodor sp. Muscodor sp. Muscodor sp. Penicillium sclerotiorum Pestalotiopsis sp. Phomopsis sp. Phomopsis sp. Fungal endophyte
DQ480347 EF564146 EF564147 DQ480350 DQ480351 DQ480343 DQ480349 EF564148 EF564149 EF564150 EF564151 EF564152 EF564153 DQ480353 DQ480361
Anti-TB
Anti-Pf
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1
w
Anti-HSV-1
Antioxidant
Cytotoxicity (Vero cells)
1 1 1 1 1
1/ 1 1 1 1 1 1 1
Antiproliferation KB
NCI-H187
1 1 1 1
1 1 1 1 1 NT 1
Anti-Mycobacterium tuberculosis. w
Anti-Plasmodium falciparum K1. NT, not tested.
Teleomorph–anamorph connections between the two species should be further investigated. Isolate D12 was identified as Curvularia sp. Curvularia species is an endophyte that has been isolated from Ocotea corymbosa (Lauraceae). It produced benzopyrans with a weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides (Teles et al., 2005). Another group of active isolates identified as Muscodor sp. Muscodor albus is reported to be the most useful fungus of this genus. It is a newly described endophytic fungus obtained from Cinnamomum zeylanicum (Worapong et al., 2001). It has been shown to produce a mixture of volatile antibiotics with antibacterial and antifungal activities (Ezra et al., 2004). This microorganism is already on the market for the decontamination of human wastes. It can be used as a mycofumigant to treat soil, seeds, and plants (Strobel et al., 2004). Therefore, this group of fungi seems to be a good source of bioactive compounds and should be studied further. Isolate M5 was identified as Pestalotiopsis sp. Pestalotiopsis species are common endophytes found on the world’s yew (Taxus) species (Strobel et al., 1996). Pestalotiopsis microspora from Taxus wallichiana and from bald cypress as well as Pestalotiopsis guepini from Wollemia nobilis can produce paclitaxel (Li et al., 1996; Strobel et al., 1996). Moreover, P. microspora from T. morobensis produced pestacin and isopestacin, which both possess antioxidant and antifungal activities (Strobel et al., 2002; Harper et al., 2003). The last group of active fungi in this study was identified as a Phomopsis sp. Phomopsis species are another common group of endophytes found in various plants (Jordaan et al., 2006; Promputtha et al., 2007; Wang et al., 2007). Many active FEMS Immunol Med Microbiol 51 (2007) 517–525
metabolites from these species have been reported including phomosichalasin (antibacterial and anti-Candida), phomoxanthones A and B, two new xanthones with significant antimalarial, antimycobacterial activities and cytotoxicity, and phomoenamide, a new enamide dimer from D15 possessing antimycobacterial activity (Horn et al., 1995; Isaka et al., 2001; Rukachaisirikul et al., 2007). The results from this study have clearly confirmed the high diversity of endophytic fungi producing bioactive compounds residing in Garcinia spp.
Acknowledgements This work was supported by the Thailand Research Fund and National Center for Genetic Engineering and Biotechnology, under the BRT’s Bioresources Utilization Program (Grant number BUP 005 G-47) and Bioresources Research Network (Grant number BRN-002 G-49). J.N. thanks the Thailand Advanced Institute of Science and Technology’s Pilot Project (THAIST) for financial support. The authors are grateful to Miss Pratum Ritthisunthorn for technical assistance in the antioxidant assays and Dr Brian Hodgson for critically reviewing this manuscript.
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