*2.4. Analyses of the Chemical Structure and Physicochemical Properties*

The purified compounds were prepared at a concentration of 1 mg/mL in MeOH for the measurement of optical rotation, UV spectra, and IR spectra. An optical rotation [α]D of the compound suspension was measured using a P-2200 polarimeter (JASCO, Tokyo, Japan). UV spectra of each compound were recorded with a U-2810 spectrophotometer (Hitachi High-Tech Science Co., Tokyo, Japan), and IR spectra (ATR) were measured using a FT–IR 4600 (JASCO, Tokyo, Japan). The isolated compounds were dissolved in chloroform-*d* (CDCl3) or methanol-*d*<sup>4</sup> (CD3OD) for NMR analyses. NMR spectra of each compound were obtained on a JNM ECP500 NMR spectrometer (JEOL, Tokyo, Japan) with 500 MHz for 1H NMR and 125 MHz for 13C NMR. Chemical shifts (ppm) of CDCl3 (δ<sup>H</sup> 7.26, δ<sup>C</sup> 77.0) and CD3OD (δ<sup>H</sup> 3.30, δ<sup>C</sup> 49.0) were used as references. The accurate mass and molecular formulas of the isolated compounds were established by liquid chromatography–mass spectrometry (LC–MS) analyses. Spectra of electron ionization mass spectrometry (EI–MS) were analyzed using a JMS-AX505 HA spectrometer (JEOL, Tokyo, Japan), while the spectra of electrospray ionization mass spectrometry (ESI–MS) were obtained by a JMS-T100LP spectrometer (JEOL, Tokyo, Japan) equipped with an Agilent1100 HPLC system (Agilent, CA, USA).

### *2.5. Measurement of Antimicrobial Activity*

In the purification process of antimicrobial compounds, every fraction obtained from each fractionation step was tested with representative microbes, *Xanthomonas campestris* pv. *oryzae* KB88, *Kocuria rhizophila* ATCC 9341, *Mucor racemosus* IFO 4581, and *G. boninense* BCC 21330, by paper disk diffusion assay. The antimicrobial activity of the purified compounds was analyzed using the paper disk diffusion method (Ø8 mm disk, Advantec, Co., Ltd., Tokyo, Japan) against fourteen microorganisms. Cell suspensions of *Bacillus subtilis* ATCC 6633 (5 <sup>×</sup> 105 cfu/mL), *Escherichia coli* NIHJ (5 <sup>×</sup> 105 cfu/mL), *K. rhizophila* ATCC 9341 (2 <sup>×</sup> 105 cfu/mL), *Mycobacterium smegmatis* ATCC 607 (5 <sup>×</sup> 105 cfu/mL), *Staphylococcus aureus* ATCC 6538p (5 <sup>×</sup> 10<sup>5</sup> cfu/mL), *Klebsiella pneumonia* ATCC 10031 (5 <sup>×</sup> 105 cfu/mL), *Proteus vulgaris* NBRC 3167 (5 <sup>×</sup> 10<sup>5</sup> cfu/mL), *Pseudomonas aeruginosa* IFO 3080 (1 <sup>×</sup> 10<sup>6</sup> cfu/mL), and *X. campestris* pv. *oryzae* KB88 (1 <sup>×</sup> 10<sup>6</sup> cfu/mL) were individually mixed into the medium containing 0.5% peptone, 0.5% meat extract, and 0.8% agar, while *Aspergillus niger* ATCC 6275 (1 <sup>×</sup> 106 spores/mL), *Candida albicans* ATCC 64548 (2 <sup>×</sup> <sup>10</sup><sup>5</sup> cfu/mL), *G. boninense* BCC 21330 (2 <sup>×</sup> 10<sup>5</sup> cfu/mL), *Mu. racemosus* IFO 4581 (2 <sup>×</sup> 105 spores/mL), and *Saccharomyces cerevisiae* ATCC 9763 (1 <sup>×</sup> 10<sup>6</sup> cfu/mL) were individually mixed into the medium containing 1.0% glucose, 0.5% yeast extract, and 0.8% agar, and poured into Petri dishes. After that, paper disks containing the purified compounds at 50 μg/disk were put onto agar plates of each microorganism with three replicates. All bacterial plates, except *M. smegmatis* ATCC 607 and *X. campestris* pv. *oryzae* KB 88, were incubated at 37 ◦C for 24 h. *M. smegmatis* ATCC 607 was incubated at the same temperature for 48–72 h., whereas *X. campestris*, yeasts, and fungi were incubated at 27 ◦C for 24–48 h. The diameter of the inhibition zone was measured in mm units.
