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).

Aspergillus fumigatus CBS101355 was obtained from the Westerdijk Fungal Biodiversity Institute (CBS), Netherlands.

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.

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.

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.

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).

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.

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, t_R: 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 (t_R = 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.

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

obtain 1 (2.4 mg) as a white amorphous powder. The structure of 1 was determined as (-)-trypacidin ([α] _{D ( 20 )} = −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).

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).