1. Introduction
Fungi produce various secondary metabolites, and of the metabolites isolated to date, some have proven useful, and others are harmful mycotoxins [1] [2] [3] [4]. These fungal metabolites are produced as a biological response to medium components, culture temperature, culture medium pH, or the addition of non-nutritional compounds such as epigenetic chemicals (5-azacytidine as DNA methyltransferase inhibitor, suberoylanilide hydroxamic acid, trichostatin A and sodium butyrate as histone deacetylase inhibitors, etc.) [5] [6] [7]. These findings led us to study fungal metabolite production as a biological response to Kampo medicines (traditional Japanese medicines) and their constituent crude drugs.
In our search for factors in Kampo medicines that affect the production of fungal metabolites, we found that Shimbu-to, a Kampo medicine used for gastrointestinal diseases and indigestion, promotes the production of emericellin and related compounds in Aspergillus nidulans [8]. We also reported that the Kampo medicine Shakuyaku-Kanzo-to induced the production of sterigmatocystin, a mycotoxin produced by Emericella nidulans IFM 60678; this inducing activity was enhanced only by peony extract [9].
Aspergillus fumigatus is a common, naturally occurring saprophytic fungus noted for producing a plethora of secondary metabolites (e.g., gliotoxin, helvolic acid, hexadehydroastechrome, trypacidin, endocrocin, neosartoricin, and fumagillin) [10].
In this study, we examined the production of fungal metabolites as biological responses to 120 crude drugs by culturing A. fumigatus CBS101355 and crude drugs, followed by analysis of culture extracts by HPLC. The HPLC chromatograms of culture extracts for nine crude drugs [Kyokatsu (Notopterygium), Kyonin (apricot kernel), Kujin (Sophora root), Goboshi (Burdock fruit), Goma (sesame), Shokyo (ginger), Shin’i (magnolia flower), Togashi (Benincasa seed) and Bukuryo (Poriasclerotium)] exhibited a common new peak (1: tR = 13.8 mim) that was not detected in the control.
Compound 1 was isolated from a culture extract of A. fumigatus grown in medium supplemented with Goboshi (Burdock Fruit) extract, which induced production of the highest amount of 1. Detailed analysis of NMR and MS data for 1 identified the compound as trypacidin (1), an anti-protozoal [11], cytotoxic [12] and antiphagocytic [13] substance produced by A. fumigatus (Figure 1). It is reported that the production of trypacidin and transcription level of trypacidin gene cluster were affected by cultural temperature [14]. These results indicate that nine crude drug extracts can induce the production of trypacidin in A. fumigatus.
2. Materials and Methods
2.1. Experimental Instruments
An LC-20 HPLC system equipped with a DAD detector (Shimadzu, Kyoto, Japan) was used for analytical HPLC. LC-MS was performed using an Agilent 1200 HPLC system and JMS-T100LP ESI-TOF-MS mass spectrometer (JEOL, Tokyo, Japan). Column chromatography was performed using a Sephadex LH-20 (GE Healthcare Japan, Toko, Japan). Preparative HPLC was performed using an LC-20AT pump and SPV-10AV UV detector (Shimadzu, Kyoto, Japan). NMR spectra were recorded on a JEOL ECAII 600 spectrometer (1H: 600 MHz, 13C: 150 MHz) (JEOL) using tetramethylsilane as an internal standard. Optical rotation
was measured using a P-1020 photopolarimeter (JASCO, Tokyo, Japan). All fungi were fermented in a SANYO MIR-554 incubator (SANYO Electric Co., Ltd., Osaka, Japan). Centrifugation was performed using a FORCE 712-100V microcentrifuge (Select BioProducts Inc., New Zealand).
2.2. Fungal Strain
Aspergillus fumigatus CBS101355 was obtained from the Westerdijk Fungal Biodiversity Institute (CBS), Netherlands.
2.3. Screening for Crude Drugs that Affect Fungal Metabolite Expression
For the cultivation of A. fumigatus CBS101355, potato dextrose broth (PDB, Difco, BD, iNJ, Japan) was amended with 120 different crude drug extracts (see Supplementary Table 1) at a final concentration of 2.4 mg/mL. After sterilization, 2 mL of medium containing drug extract was poured into each well of a 24-well plate (TPP, Sweden), and all wells were inoculated with A. fumigatus CBS101355. The plate was then sealed with breathable film (Axygen BF-400-S, Corning, AZ, USA) and cultured at 25˚C for 1 week. After cultivation, each culture broth was lyophilized and extracted with 1.5 mL of methanol at room temperature for 1 day. The extract solution was filtered and dried under air flow.
2.4. DAD-HPLC Analysis Conditions
The culture extracts were dissolved in 1 mL of 50% acetonitrile (CH3CN) and centrifuged at 10,000 rpm for 5 min. The supernatant was analyzed by DAD-HPLC using a Mightysil RP-18 GPII column (3 × 250 mm, 5 μm, Kanto Chemicals, Tokyo, Japan), with the column oven temperature set to 40˚C. The column was eluted using water and CH3CN as mobile phases, and the eluate was analyzed in gradient mode as follows: 0 min: 30% CH3CN, 17 min: 100% CH3CN, 35 min: 100% CH3CN. The flow rate was 0.5 mL/min. The detection range of the DAD detector was 200 - 400 nm, and the chromatograms are shown at 200 nm.
2.5. LC-MS Analysis Conditions
Culture extracts were dissolved in 1 mL of methanol and centrifuged at 10,000 rpm for 5 min. The supernatant was analyzed by LC-MS using an Inertsil ODS-3 column (2.1 × 150 mm, 3 μm, GL Science, Tokyo, Japan), with the column oven temperature set to 40˚C. The mobile phases were 0.5% HCOOH (A) and CH3CN with 0.5% HCOOH (B), and the column was eluted in gradient mode as follows: 0 min, 20% B; 17 min, 95% CH3CN, and 30 min, 95% CH3CN. The flow rate was 0.2 mL/min. The samples were analyzed in positive ESI mode.
2.6. Culture and Extraction of Aspergillus fumigatus on Medium Supplemented with Goboshi (Burdock Fruit) Extract
To 90 g of Goboshi (Burdock fruit) was added 900 mL of water, and the mixture was refluxed for 50 min. The extracted solution was filtered through double gauze and lyophilized to obtain 9.3 g of Goboshi (Burdock fruit) extract.
Aspergillus fumigatus CBS101355 was inoculated into 1 L of PDB with 4.8 mg/mL Goboshi (Burdock fruit) extract, incubated at 25˚C for 1 week, and then lyophilized whole. The lyophilized product was extracted with 150 mL of methanol and filtered. The filtrate was evaporated in vacuo to obtain a methanol extract (2.5 g).
2.7. Isolation of Compound 1
The above methanol extract (2.5 g) was dissolved in 1 L of water and sequentially extracted with hexane, chloroform, ethyl acetate, and 1-butanol (each 1 L × 2) and evaporated in vacuo. The chloroform extract was chromatographed on a Sephadex LH-20 column eluted sequentially with hexane-chloroform (1:4; 200 mL), chloroform-acetone (3:2; 200 mL and 1:4; 200 mL), acetone (200 mL), and methanol (1 L) to obtain 10 fractions. Fraction 2 (4.2 mg) was purified by HPLC on an ODS column eluted with 45% acetonitrile to obtain 1 (2.4 mg) as a white amorphous powder.
3. Results
3.1. Crude Drugs that Affected Fungal Metabolite Expression
To study the effect of crude drug extracts on fungal metabolite expression, A. fumigatus was cultured in PDB supplemented with each of 120 crude drug extracts. As the results of examination for the amounts of crude drugs and cultural time, differences of secondary metabolites were effectively observed at concentration of 2.4 mg/mL crude drugs and 7 days cultural time. The 120 crude drug extracts examined in this study showed no antifungal activity against A. fumigatus at a concentration of 2.4 mg/mL (data not shown).
After 7 days of incubation, the culture extracts were analyzed by DAD-HPLC. A common new peak (1, tR: 13.8 min) not observed in the control appeared in the HPLC chromatograms of PDB culture extracts with 9 of the 120 crude drug extracts (Figure 2). The 9 crude drugs were Kyokatsu (Notopterygium), kyonin (apricot kernel), Kujin (Sophora root), Goboshi (Burdock fruit), Goma (sesame), Shokyo (ginger), Shinni (magnolia flower), Togashi (Benincasa seed), and Bukuryo (Poriasclerotium).
Peak 1 (tR = 13.8 min) showed a maximum UV spectrum at 287 nm by DAD-HPLC analysis. LC-MS analysis of the 9 culture extracts that exhibited common peak 1 confirmed a pseudo–molecular ion peak at m/z 345.11 (Supplementary Figure 1). The highest level of trypacidin was observed in the extract with Goboshi.
3.2. Identification of Compound 1
Aspergillus fumigatus was cultured in PDB supplemented with Goboshi extract, which produced the highest amount of 1 among the 9 herbal extracts.
The culture extract was purified by various chromatographic methods to
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Figure 2. HPLC chromatograms of extracts of A. fumigatus cultured on PDB supplemented with crude drug extracts. Chromatograms were recorded at 200 nm. a: culture extract without crude drug; b: culture extract with Kyokatsu (Notopterygium); c: Kyonin (apricot kernel); d: Kujin (Sophora root); e: Goboshi (Burdock fruit); f: Goma (sesame); g: Shokyo (ginger); h: Shin’i (magnolia flower); i: Togashi (Benincasa seed); j: Bukuryo (Poria sclerotium).
obtain 1 (2.4 mg) as a white amorphous powder. The structure of 1 was determined as (-)-trypacidin ([α] D = −68.1, c = 0.05, MeOH) by comparison of MS and NMR spectral data and optical rotation in reference to previous data [11], [15] and detailed analyses of 2D-NMR data in DMSO-d6 (Table 1 and Supple. Figures S1-S7).
3.3. Factors in Crude Drug Extracts that Induce Trypacidin Production
Aspergillus fumigatus produced trypacidin (1) in the presence of the 9 crude drug extracts. These results suggested that these crude drug extracts contain inducers of trypacidin (1) production in A. fumigatus. To identify the trypacidin inducers in the 9 crude drug extracts, each of the extracts was sequentially partitioned into liquid solutions using chloroform, ethyl acetate, and 1-butanol. Then, A. fumigatus was cultured in PDB supplemented with these partitioned extracts.
DAD-HPLC analysis confirmed the production of trypacidin only when the aqueous fraction of all 9 herbal extracts was added, and no trypacidin production was observed when other extract fractions were added to the culture (Figure 3).
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Table 1. NMR data of trypacidin (1).
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(b)
Figure 3. Separation procedure for crude drug extracts and HPLC chromatograms of Aspergillusfumigatus culture extracts supplemented with water fractions of crude drug extracts. (a) Separation procedure; (b) HPLC chromatograms. Chromatograms were recorded at 200 nm. a: culture extract without crude drugs, b: culture extract with Kyokatsu (Notopterygium), c: Kyonin (apricot kernel), d: Kujin (Sophora root), e: Goboshi (Burdock fruit), f: Goma (sesame), g: Shokyo (ginger), h: Shin’i (magnolia flower), i: Togashi (Benincasa seed), j: Bukuryo (Poriasclerotium).
4. Discussion
In the present study, A. fumigatus was cultured in medium supplemented with 120 crude drug extracts derived from components of Kampo medicine in order to explore the factors that influence the production of secondary metabolites in A. fumigatus.
Our analyses confirmed that 9 crude drug extracts [Kyokatsu (Notopterygium), Kyonin (apricot kernel), Kujin (Sophora root), Goboshi (Burdock fruit), Goma (sesame), Shokyo (ginger), Shin’i (magnolia flower), Togashi (Benincasa seed), and Bukuryo (Poriasclerotium)] commonly induced A. fumigatus to produce trypacidin (1). Furthermore, only the aqueous fraction of the 9 crude drug extracts induced the production of trypacidin. These results indicate that the factor that induces the production of 1 is a water-soluble substance.
Since the aqueous fraction contained glucose, fructose, and sucrose as major components, the ability of these sugars to induce the production of trypacidin by A. fumigatus was examined. None of these sugars induced the production of trypacidin (data not shown).
Trypacidin was first isolated from A. fumigatus and reported as an anti-protozoal agent [11] that is also cytotoxic to human A549 lung cells [12]. These biological activities are thought to play a role in predator avoidance, as trypacidin is present in the conidia of A. fumigatus. However, trypacidin is also a clinically important chemical because its bioactivity is thought to play a role in the pathogenesis of pulmonary aspergillosis caused by A. fumigatus [13] .
To date, 13 gene clusters involved in the biosynthesis of trypacidin have been reported [13] [16]. Lind also reported that these biosynthetic gene clusters are regulated by the global regulators LaeA and BrlA [17].
Based on the results of the present study, we hypothesized that the increase in trypacidin production induced by the 9 crude drug extracts is due to chemical components in the extracts that affect either the trypacidin biosynthesis genes or the regulator genes.
To our knowledge, there have been no reports of chemicals that control the production of trypacidin, although some studies have described the effects of physical factors such as temperature and light or modification of gene clusters using molecular biological techniques. Identifying the substances in the aqueous fraction that affect the production of trypacidin may aid in the development of methods to control the production of trypacidin by A. fumigatus.
5. Conclusion
In this study, we found that the aqueous fractions of 9 crude drug extracts induce the production of trypacidin in A. fumigatus. The identification of trypacidin producing substances in these aqueous fractions is expected to facilitate the development of methods to inhibit trypacidin production in A. fumigatus in the future.
Acknowledgements
This research was supported by a Grant-in-Aid for the Cooperative Research Project from Institute of Natural Medicine, University of Toyama, in 2014.
We would like to thank Mr. Norifumi Kishida for chromatographic analyses.
Supplementary Tables and Figures
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Table S1. Crude drugs used in this study.
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Figure S1. UV and MS spectra of compound 1. (a) UV spectrum of 1 from DAD-HPLC analysis, (b) MS spectrum.