Springer 2005
World Journal of Microbiology & Biotechnology (2005) 21:553–558 DOI 10.1007/s11274-004-3273-2
Anti-Helicobacter pylori substances from endophytic fungal cultures Y. Li, Y.C. Song, J.Y. Liu, Y.M. Ma and R.X. Tan* Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China *Author for correspondence: Tel.: +86-25-83592945, Fax: +86-25-83302728, E-mail:
[email protected] Keywords: Anti-Helicobacter pylori, Aspergillus sp. CY725, Cynodon dactylon, endophyte, helvolic acid
Summary The human pathogenic bacterium Helicobacter pylori has been ascertained to be an aetiological agent for chronic active gastritis and a significant determinant in peptic and duodenal ulcer diseases. Endophytic metabolites are being recognized as a versatile arsenal of antimicrobial agents, since some endophytes have been shown to possess superior biosynthetic capabilities owing to their presumable gene recombination with the host, while residing and reproducing inside the healthy plant tissues. A total of 32 endophytic fungi isolated from the medicinal herb Cynodon dactylon (Poaceae) were grown in in vitro culture, and the ethyl acetate extracts of the cultures were examined in vitro for the anti-H. pylori activity. As a result, a total of 16 endophyte culture extracts were identified as having potent anti-H. pylori activities. Subsequently, a detailed bioassay-guided fractionation of the extract of the most active endophyte (strain number: CY725) identified as Aspergillus sp., was performed to afford eventually four anti-H. pylori secondary metabolites. The four isolated compounds were identified through a combination of spectral and chemical methods (IR, MS, 1H- and 13C-NMR) to be helvolic acid, monomethylsulochrin, ergosterol and 3b-hydroxy-5a,8a-epidioxy- ergosta-6,22-diene with corresponding MICs of 8.0, 10.0, 20.0 and 30.0 lg/ml, respectively. The MIC of ampicillin co-assayed as a reference drug against H. pylori was 2.0 lg/ml. Furthermore, preliminary examination of the antimicrobial spectrum of helvolic acid, the most active anti-H. pylori metabolite characterized from the endophyte culture, showed that it was inhibitory to the growth of Sarcina lutea, Staphylococcus aureus and Candida albicans with MICs of 15.0, 20.0 and 30.0 lg/ml, respectively.
Introduction Helicobacter pylori, an aetiologic agent for chronic active gastritis, has been recognized as a significant determinant in peptic and duodenal ulcer disease (Gebert et al. 2003). Sustained infection with this bacterium could lead to development of gastric cancer (Moran & Upton 1986). Unfortunately, H. pylori cannot be easily eradicated after its invasion. In order to control the expansion of its infection, combination therapy utilizing proton pump inhibitors and antibacterial agents has to be applied (Bazzoli et al. 1994; Bayerdo¨rffer et al. 1995; Bell et al. 1995), although an efficient single-agent therapy is highly preferable for better patient compliances with weaker or negligible side effects (Graham et al. 1992; Logan et al. 1994). Furthermore, H. pylori is apt to develop drug-resistance after a period of treatment (Sharara et al. 2002). Accordingly, it is imperative to search for new anti-H. pylori agents. In view of the fact that endophytes, ubiquitous in healthy tissues of almost all plants investigated so far, have been accepted as a rich source of bioactive metabolites (Tan & Zou 2001), we screened for anti-H. pylori
substances the ethyl acetate extracts of cultures of endophytic fungi harboured by Cynodon dactylon (Poaceae), a Chinese medicinal herb used locally for treating hepatitis (Xie et al. 1996). In this paper, we wish to present the screening of the thirty-two endophytic fungal culture extracts, and a detailed fractionation of four anti-H. pylori metabolites from the most active one.
Materials and methods Isolation and identification of endophytes Following the procedure detailed elsewhere (Lu et al. 2000), a total of 32 cultivable endophytic fungal isolates was obtained from the surface-sterilized fresh leaves of C. dactylon within 3 h after its being collected in early November 2001 from the seashore with certain salinity near Sheyang Port on the Yellow Sea. The endophytic fungus, strain CY725, whose extract afforded the most active anti-H. pylori effects, was then identified as an Aspergillus sp. by Dr. Y.C. Song, through comparison of the morphological characteristics and the ascertained
554 mechanism of sporulation, which are substantially similar to those observed with other Aspergillus species (Frisvad & Samson 1990; Barnett & Hunter 1998). Finally, the endophytic nature of the strain was confirmed by the vital test as described earlier (Lu et al. 2000). Test microorganisms A reference strain of H. pylori (ATCC 43504) and five clinical isolates of the pathogen (obtained from antral biopsies of child and adult patients) were used in this study. Also used herein as test microorganisms for a preliminary investigation of the antimicrobial spectrum were nine human microbial pathogens including five bacteria: Bacillus subtilis, Pseudomonas fluorescens, Escherichia coli, Sarcina lutea and Staphylococcus aureus, as well as three fungi: Aspergillus niger, Trichophyton rubrum and Candida albicans.
Y. Li et al. O2). The MICs were defined as the lowest concentration at which no microbial growth could be observed. The positive reference ampicillin (AMP) was co-assayed at concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 lg/ml. The antibacterial and antifungal activities were evaluated separately on LB (yeast extract 5, peptone 10, NaCl 5 and agar 20 g/l, pH 7.0) and PDA plates seeded with the suspension of each test bacterium and fungus (all at a concentration of 106 cells and/or spores per milliliter) followed by incubation at 37 C for bacteria (48 h) and 28 C for fungi (96 h), respectively. Each of the four metabolites was tested at concentrations 0 (blank control), 10, 20, 30 and 40 lg/ml, respectively, with MICs determined by judging visually the growth of every test microbe. For antifungal evaluations, ketoconazole (KCZ) was co-assessed as positive control at concentrations of 0.1, 0.5, 1, 10, 20 and 50 lg/ml. All experiments were conducted in triplicate.
Antimicrobial screenings Fermentation and preliminary screening The anti-H. pylori screening with the ethyl acetate extracts was performed by the disk diffusion method (DDM) (Zaika 1998). Briefly, a given volume (10 ll) of each of the 32 endophytic extract solutions at 1 mg/ml in dimethyl sulphoxide (DMSO) was dropped on a standard disk (u ¼ 5 mm) which was subsequently placed on a Columbia agar plate pre-inoculated with 0.1 ml H. pylori suspension in Brucella broth (1 · 108 c.f.u./ml). All test plates were then incubated under microaerophilic conditions for 72 h at 37 C, followed by measurement of the diameters of the inhibition zone around each of the extract-carrying disks. The procedure was repeated for the antimicrobial assay against the other test microbes except the incubation for fungi were carried out at 28 C for 96 h. MIC measurements The minimum inhibitory concentration (MIC) was assessed with the agar dilution method (Megraud et al. 1999). Briefly, 1 ml of each stock solutions in sterile water at given concentrations of every isolated fungal metabolite (possibly lower amounts of DMSO were used for the desired intermiscibility) was separately added into petri dishes containing 8 ml of unsolidified Columbia agar base supplemented with 1 ml of horse serum. Final concentrations of each compound in the medium were set to be 40.0, 35.0, 30.0, 25.0, 20.0, 15.0, 10.0, 5.0 and 2.5 lg/ml with a DMSO concentration lower than 1%. Different H. pylori strains taken from the seed cultures were immediately diluted with Brucella broth with the bacterial cells at approximately 1 · 108 c.f.u./ml in the seed liquors. Subsequently, 0.1 ml of each of seed liquors was inoculated onto the surface of the sample-supplemented agar plates, followed by incubation at 37 C for 72 h in an anaerobic jar (containing: 85% N2, 10% CO2 and 5%
The inoculum was prepared by introducing the periphery of 7-day-old petri dish cultures of each of the 32 endophytes into 500 ml flasks containing 200 ml of the broth (potato: 200 g, dextrose: 20 g, H2O: 1000 ml), followed by shaking (150 rev/min) continuously for 4 days at 28±1 C. The follow-up culture was accomplished by adding the inoculum (30 ml) into 1000 ml Erlenmeyer flasks containing 500 ml of the same broth, and then shaking for 10 days in the same conditions. The fermentation broth of each endophyte was filtered, and the filtrate concentrated in vacuo into a smaller volume. The concentrated filtrates were extracted exhaustively with EtOAc, and the extracts obtained were tested at a concentration of 1 mg/ml (dry extract/ medium) for the potential anti-H. pylori and other antimicrobial activities as mentioned above. The culture liquor (20 ml) of the endophyte Aspergillus sp. CY725, affording the most promising antibacterial action, was added to 250-ml Erlenmeyer flasks containing 15 g grain, 0.5 g yeast, 0.1 g sodium tartrate, 0.01 g FeSO4Æ7H2O, 0.1 g sodium glutamate, 0.1 ml pure corn oil and 30 ml water. The fermentation, accomplished in a total of 400 Erlenmeyer flasks, was subsequently progressed by standing for another 30 days at 28±1 C. Extraction and fractionation The fermentation broths of Aspergillus sp. CY725 were combined and extracted three times with methanol. In vacuo evaporation of methanol from the extract gave a brown oily residue (314 g), which was suspended in water and partitioned successively with ethyl acetate and n-butanol, respectively. The in vitro test showed that the active compound(s) was (were) in the ethyl acetate fraction (153 g), which was therefore chromatographed
Anti-H. pylori substances from endophytic fungus
555 Results and discussion
on a silica gel column (1000 g, 200–300 mesh) eluting with a CHCl3-MeOH gradient (1:0fi0:1, v/v) to give five fractions (F-1, 103.5 g; F-2, 10.1 g; F-3, 12.8 g; F-4, 10.5 g; F-5, 15.0 g), of which, F-2 and F-3 were further ascertained to be bioactive. Subsequently, F-2 was rechromatographed over silica gel (150 g, 200~300 mesh) using CHCl3-MeOH mixtures of a growing polarity (200:1fi10:1, v/v) to afford 3 (8 mg), 4 (11 mg) and a mixture F-2-1 (5 g), from which 220 mg of 2 were obtained upon further chromatography over a silica gel column (150 g, 200300 mesh) eluting with an isocratic solvent mixture CHCl3-MeOH (10:1, v/v). Repeated separation of F-3 on silica gel column in the same manner followed by gel filtration over Sephadex LH-20 gave helvolic acid (1, 26 mg).
Some endophytes have been confirmed to be excellent producers of bioactive substances such as plant growth regulators, antimicrobial agents and insecticides (Tan & Zou 2001; Carroll 1988). In our preliminary antimicrobial screening of the ethyl acetate extracts originating from thirty-two cultivable endophytes residing in healthy leaves of Cynodon dactylon, sixteen showed pronounced antimicrobial activities against the test H. pylori strains, nineteen against B. subtilis, six against P. fluorescens, six against E. coli, thirteen against T. rubrum, eight against A. niger and twenty against C. albicans (Table 1). This finding reinforced the assumption that endophytes could be a promising source of antimicrobial substances, that may play key and/or helpful roles in protecting the host plants from various phytopathogenetic microbes (Tan & Zou 2001). In order to characterize the anti-H. pylori constituent(s) from the most bioactive culture extract derived from the endophytic fungus under the isolation number CY 725, the identification of the producing organism and a scaled-up fermentation were performed. The endophytic fungal isolate (CY 725) was identified as Aspergillus sp. according to the morphological
Examination of the sterile medium for presence of compounds 1-4 The sterile medium was extracted following exactly the procedure as with that of the solid-substrate culture. The presumable presence of any of the four isolates in the afforded extract was examined by LC-MS comparisons.
Table 1. Antimicrobial effects of ethyl acetate extracts of cultures of 32 endophytic fungi from Cynodon dactylona. Strains
Hp 43504b
Hp 036
CY705b CY705a ZY802 CY 714a ZY 801-2 ZY 802b ZY 801a CY 703b ZY 804-1 CY 703-1 ZY 804a CY 702a’ CY 701c CY 705a CY 704a’’ CY 701-2 ZY 801-3 ZY 803a CY 701a ZY 801a CY 703a CY 701c CY 704a’ CY 703b CY 702b CY 701a CY 703a CY 704a’ CY 7016 CY 701-1 CY 703-1 CY 725
+ ) ) ) + ) ) ) + + + ) + +++ ) + ) ) ) + ) + + ) ++ + ) ) + + ) +++
+ ) ) ) + + + ) + + + ) + ++ ) + ) ) ) + ) + + + ++ ++ ) ) ++ ++ ) +++
b
Bs
b
++ + ++ +++ +++ + ) + + + + ) ) ) + ) ++ +++ + ++ ++ ) ++ + ) ) ) ) ) ) ) +++
Pf
b
+++ ++ +++ + +++ ) ) ) ) ) +++ ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )
Ec
b
) ) ++ ) ) ) ) ) ) +++ + ) + ) ) ) + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ++
Tr
b
) ) ) ) ) + ) ) ) + + ) + + ) + ) ++ ) ++ + + + ) ) ) ) + ) ) ) +
An
b
) ) + ) ) + ) ) ) ++ ++ ) ) ) ) ) ) + ) +++ + + ) ) ) ) ) ) ) ) ) )
Ca
b
) ) + + + + ) + + +++ + ) + ) + + ) + ) ++ + ) + ) ) + ) + ) ) ) +
a Inhibition expressed by the diameter of inhibition zones: ), no inhibition; +, < 10 mm; ++, 10–15 mm; +++, >15 mm. b Hp – Helicobacter pylori; Bs – Bacillus subtilis; Pf – Pseudomonas fluorescens; Ec – Escherichia coli, Sa – Staphylococcus aureus; An – Aspergillus niger; Tr – Trichophyton rubrum; Ca – Candida albicans.
556
Y. Li et al. HOOC
Me
H Me Me
OH
OAc
Me
O
COOMe
H Me
H O
Me
OAc
H Me
OMe
OMe
OH
O 1
2 25 22
21 24 18 20
23
11
26 28
17 19
27
13 15
1
1
9
O
10 3
5
3
7
HO
HO
5
8
6O
3
4
Figure 1. Structures of helvolic acid (1), monomethylsulochrin (2), ergosterol (3) and 3b-hydroxy-5a,8a- epidioxy-ergosta-6,22-diene (4).
Table 2. The MIC values (lg/ml) of compounds 1-4.
H. pylori 43504 H. pylori 001 H. pylori 016 H. pylori 018 H. pylori 019 H. pylori 036 S. lutea S. aureus C. albicans a
1
2
3
4
AMPa
5.0 8.0 5.0 8.0 8.0 8.0 15.0 20.0 30.0
10.0 5.0 10.0 5.0 10.0 10.0 30.0 >100 >100
15.0 20.0 10.0 10.0 10.0 20.0 >100 >100 >100
30.0 5.0 30.0 30.0 30.0 30.0 >100 >100 >100
2.0 2.0 2.0 2.0 2.0 2.0 1.0 2.0
KCZa
1.0
AMP – ampicillin; KCZ – ketoconazole.
characters below. The newly isolated mycelium grew well on PDA to produce fruiting bodies easily. Colonies with a regular margin attained 35–45 mm in diameter after incubation on PDA at 28 C for 4 days and took on a weak green colour in PDA plates. Conidiophores were upright, simple, terminating in a globose swelling, bearing phialides at the apex; condia (phialospores)1celled and globose. These morphological observations of the endophytic fungus were nearly identical with those described for other Aspergillus species (Frisvad & Samson 1990; Barnett & Hunter 1998). A living culture is being maintained in our institute. Through a bioassay-guided fractionation of the ethyl acetate extract of the endophytic culture, four main antiH. pylori secondary metabolites (1–4) were obtained. On the basis of spectral and physical data, compounds 1–4 were identified as helvolic acid (Oxley 1966; Okuda et al. 1967), monomethylsulochrin (Turner 1965), ergosterol (Cushley & Filipenko 1976), 3b-hydroxy-5a,8a-epidioxy-ergosta- 6,22-diene (Ma et al. 1994), respectively (Figure 1). To exclude the possibility that any of the four isolated metabolites might have originated from the
medium materials used in the study, an LC-MS examination was therefore conducted with the ethyl acetate extract of the blank sterile medium treated identically to with fungal cultures. Howevert, none of the four fungal metabolites (1–4) could be detected, indicating that all were actually produced by the endophytic fungus. Concerning the results of antimicrobial assays, the four identified metabolites 1–4 displayed significant growth inhibition against all the six strains of H. pylori with the MICs of 8.0, 10.0, 20.0 and 30.0 lg/ml, respectively (Table 2). For a preliminary understanding of the antimicrobial spectrum, the four compounds were tested additionally for the inhibitory effects on other human pathogens including five bacteria: B. subtilis, P. fluorescens, E. coli, S. lutea and S. aureus, as well as three fungi: T. rubrum, A. niger and C. albicans. As summarized in Table 2, helvolic acid (1) was bacteriostatic to S. lutea and S. aureus, and fungistatic to C. albicans whereas monomethylsulochrin (2) was bacteriostatic to S. lutea only. However, ergosterol (3) and 3b-hydroxy-5a, 8a-epidioxy-ergosta-6,22-diene (4) did not show any discernible inhibitory effects on the six test
Anti-H. pylori substances from endophytic fungus microbes. The results observed with insusceptible microbes are not tabulated. As to the magnitude of the antimicrobial action, the presently ascertained MIC values of helvolic acid (1) (8.0 lg/ml) and monomethylsulochrin (2) (10.0 lg/ml) demonstrated that both compounds are fairly comparable to the promisingly potent anti-H. pylori natural products reported previously, such as alkaloids (Hamasaki et al. 2000), flavonoids (Bae et al. 1999; Ohsaki et al. 1999), quinines (Dekker et al. 1998; Taniguchi et al. 2002), peptides (Iwahori et al. 1997) and rotenoid (Takashima et al. 2002). Helvolic acid (1), characterized previously from the culture broth of Cephalosphorium caerulens, shows antibiotic activity against a wide range of microorganisms including Streptococcus (MIC 6.25 lg/ml), Salmonella typhi (MIC 100.0 lg/ml), Shigella lutea (MIC 0.8 lg/ml) (Okuda 1967; Cole 1981). Furthermore, monomethylsulochrin (2), produced by the fungi belonging to the genera Aspergillus (Turner 1965; Inamori 1983), Penicillium (Mahmoodian 1964), Oospora (Curtis 1966) and Rhizoctonia (Ma et al. 2004), has been shown to inhibit eosinophils (IC50 0.3 lM), which may play important roles in allergic diseases such as asthma and atopic dermatitis (Ohashi 1999). 3b-Hydroxy-5a, 8a- epidioxy-ergosta-6,22-diene (4), widely distributed in fungi and lichens, shows potent cytotoxity (LD50 11.7 lg/ml) against mouse lymphaemia L-1210/v/ c strain, and to KB cell (LD50: 12.3 lg/ml) derived from a human epidermoid carcinoma of the mouth (Gunatilaka 1981; Matsueda 1985). In conclusion, the firsttime characterization of four anti-H. pylori metabolites, helvolic acid (1), monomethylsulochrin (2), ergosterol (3) and 3b-hydroxy-5a,8a-epidioxy- ergosta-6,22-diene (4) from a Cynodon dactylon endophyte culture highlights the possibility that some endophytes in nature could be efficient producers of anti-H. pylori and/or bacterial ulcer-treating compounds; the compounds may have clinical potential in the future.
Acknowledgements The work was co-financed by grants for RXT from the National Natural Science Foundation of China (No. 30171104) and from the Ministry of Science & Technology-National Marine 863 projects (Nos. 2003AA624010 and 2003AA 624110).
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