Figure 1 shows the primary determination of the ustiloxin-like compounds from the water extract of rice false smut balls by LC-ESI-MS. The spectrum with detection wavelength from 190 to 400 nm was employed to obtain more peaks in the HPLC-UV chromatogram. The major detection wavelength of 220 nm was selected. Five peaks (i.e., peaks 1–5) appeared in both HPLC-UV and total ion chromatograms (Figure 1A,B) with retention times of 8.4 min, 13.5 min, 15.4 min, 16.9 min and 24.0 min, respectively. They were selected for further ESI-MS analysis with the results shown in Figure 1. By comparison with the molecular weights of the ustiloxins previously reported [1,10,11], we preliminarily concluded that peaks 1–4 should be ustiloxin-like compounds. At 646.3 m/z (Figure 1C) should be the peak of the [M + H]⁺ ion of ustiloxin B that corresponds to peak 1 in Figure 1A,B. Similarly, at 559.2 m/z (Figure 1D) should be the peak of the [M + H]⁺ ion of ustiloxin C that corresponds to peak at 2, 674.3 m/z (Figure 1E) should be the peak of the [M + H]⁺ ion of ustiloxin A that corresponds to peak 3, and at 495.2 m/z and 517.2 m/z (Figure 1F) should be the peaks of the [M + H]⁺ and the [M + Na]⁺ ions of ustiloxin D that correspond to peak 4 in Figure 1A,B. These ustiloxin-like compounds need to be purified and further confirmed by spectroscopic methods such as NMR and MS. According to the peak areas in the HPLC profile (Figure 1A) and the ion relative abundances in the TIC spectrum (Figure 1B), peaks 1, 3 and 5 should correspond to the three main components in the water extract of rice false smut balls. Peak 5 (in Figure 1A,B) with a major ion peak at m/z 587.2 shown in Figure 1G has not been reported previously. It needs to be purified and further confirmed.

After repeated column chromatographic purification on macroporous adsorption resin HP-20, dialysis, GH gel ODS-AQ, Sephadex LH-20, and Sephadex Q15, the water extract of rice false smut balls afforded two compounds (1 and 2). After comparing their physicochemical and spectrometric data with those reported in the literature, they were identified as ustiloxins A (1) [10], and B (2) [1,11], respectively, whose structures are shown in Figure 2.

With the same conditions as used in Figure 1, both the purified ustiloxins A and B were mixed for LC-ESI-MS analysis. Figure 3 shows the determination of ustiloxins A and B by LC-ESI-MS. At 646.3 m/z (Figure 3C) was the peak of the [M + H]⁺ ion of ustiloxin B that corresponds to peak 1, and at 674.3 m/z (Figure 3D) was the peak of the [M + H]⁺ ion of ustiloxin A that corresponds to peak 2 in Figure 3A,B. The retention times of ustiloxin B (peak 1) and ustiloxin A (peak 2) in Figure 3A,B were 8.3 min and 15.3 min, respectively, which were almost the same as peaks 1 and 3 in Figures 1A,B. The results confirmed that ustiloxins A and B as the two main ustiloxins in rice false smut balls can be determined by an LC-ESI-MS method.

For determination of the main ustiloxins by LC-ESI-MS, liquid chromatography-mass spectrometric analyses were performed using the Agilent 1100 series LC/MSD trap spectrometer (Agilent Technologies, Waldbronn, Germany). MSD, G2446C VL. The HPLC system included a G1311A quaternary pump, a G1313A autosampler, a G1315A detector, a G1379A vacuum degasser with solvent tray and bottles. It was controlled remotely over an Ethernet communication link from a computer using the Hewlett-Packard chemstation system (Agilent Technologies, Waldbronn, Germany). Chromatographic separations were performed at 25 °C and 1 mL/min flow rate using Synergi reversed-phase Hydro-C₁₈ column (250 mm × 4.6 mm, 5 μm, Phenomenex, Torrance, CA, USA). The mobile phase, composed of methanol with water containing 0.02% TFA (15:85, v/v), was eluted at a flow rate of 1.0 mL/min, with UV detection at 220 nm. MS detection was performed on a triple quadrupole analyzer equipped with an ESI source in the positive ion mode. The ESI conditions were as follows: ion source, ESI positive; working mode, SCAN (Scan beginning at 100 m/z and ending at 1000 m/z); nebulizer gas, N₂; nebulizer pressure, 35.00 psi; drying gas temperature, 350 °C; drying gas flow rate, 8.0 L/min; capillary HV, 3500 V. Total time of analysis was 30 min.

Ustiloxin content was analyzed by a Prominence LC-20A high-performance liquid chromatography (HPLC) system (Shimadzu, Japan), which consisted of two LC-20AT solvent delivery units, an SIL-20A autosampler, an SPD-M20A photodiode array detector, and a CBM-20Alite system controller. The chromatography column, mobile phase, flow rate and UV detection were the same as those in LC-ESI-MS analysis except a temperature of 30 °C, and a total analysis time of 20 min. The LC solution multi-PDA workstation was employed to acquire and process chromatographic data.

The anion-exchange resin PA308 and the macroporous adsorption resin HP-20 were purchased from Mitsubishi Chemical Holdings, Japan. Sephadex LH-20 was purchased from Pharmacia Biotech, Sweden. The GH gel ODS-AQ was purchased from Daiso, Japan. The Sephadex G-15 was purchased from GE Healthcare, USA. All other chemicals and reagents were of analytical grade.

HR-ESI-MS spectra were measured on Bruker Apex IV FTMS. NMR spectra (¹H NMR, ¹³C NMR) were recorded on a Bruker Avance DRX-400 NMR spectrometer (¹H at 400 MHz and ¹³C at 100 MHz). The chemical shifts were expressed in ppm as δ values relative to tetramethylsilane (TMS) as an internal standard. Thin layer chromatography (TLC) plates were coated with 0.5-mm layers of silica gel (GF₂₅₄, 300–400 mesh, Qingdao Marine Chemical Company, China). Detection was provided by UV at 254 nm and 365 nm, spraying with 0.2% ninhydrin-acetone (w/v) reagent.

The rice false smut balls, which were mainly composed of the chlamydospores and mycelia of the rice false smut pathogen (Villosiclava virens), were collected from the southwestern part of Shandong Province of China in October 2011. The materials were left to dry in shade at room temperature to a constant weight, and were then stored in sealed plastic bags at −20 °C until required.

The rice false smut balls (800 g) were extracted three times with deionized water (2 L each time) at room temperature. The extract was shaken vigorously. The water solution was concentrated under vacuum at 60 °C by a rotary evaporator to afford a concentrated water extract (245 g) which was divided into two parts. The first part (5 g) of the water extract was subjected to anion-exchange resin PA308 chromatography eluted with water at pH 8.0 to water at pH 2.5. The concentrated part, eluted with water at pH 2.5, was used for LC-ESI-MS analysis. The second part (240 g) of water extract was subjected to chromatography over a macroporous adsorption resin HP-20 column (1 L) eluted with distilled water (6 L), then with 30% aqueous ethanol (6 L) to give 28 g of water fraction (containing ustiloxin B) and 7 g of 30% ethanol fraction (containing ustiloxin A), separately.

After that the 30% ethanol fraction was dialyzed with the dialysis tube (pore size, 3500 Da), the concentrated dialysate was chromatographed on an ODS-AQ column using different percentages of methanol containing 0.02% TFA under isocratic conditions. Crude compound 1 was obtained when 5% methanol containing 0.02% TFA was used as an eluent. It was further chromatographed on a Sephadex LH-20 column with MeOH-H₂O (9:1, v/v) as an eluent, and then on a Sephadex Q15 column with H₂O as an eluent to produce pure compound 1 (90 mg). Compound 2 (12 mg) was obtained from the water fraction by using a similar fractionation procedure as compound 1.

Compound 1 was isolated as a white amorphous powder (MeOH). The molecular formula C₂₈H₄₃N₅O₁₂S was assigned by HR-ESI-MS, m/z 674.26859 [M + H]⁺ (calcd. 674.27017). ¹H NMR (D₂O, 400 MHz) δ (ppm): 7.60 (1H, s, H-13), 7.08 (1H, s, H-16), 4.93 (1H, d, J_{10, 9} = 10.0 Hz, H-10), 4.84 (1H, s, H-3), 4.35–4.40 (1H, m, H-3′), 4.28 (1H, d, J_{9, 10} = 10.0 Hz, H-9), 4.14 (1H, d, J_{6, 24} = 10.2 Hz, H-6), 3.99 (1H, dd, J _{5′, 4′} = 4.0 Hz, 7.6 Hz, H-5′), 3.77 (2H, s, H-19), 3.33 (1H, dd, J _{2′, 2′} = 13.2 Hz, J _{2′, 3′} = 9.6 Hz, H-2′), 3.04 (1H, dd, J _{2′, 2′} = 13.2 Hz, J _{2′, 3′} = 2.8 Hz, H-2′), 2.77 (3H, s, NCH₃-9), 2.17–2.25 (2H, m, H-22, H-4′), 2.12 (1H, ddd, J _{4′, 3′} = 2.8 Hz, J _{4′, 4′} = 14.2 Hz, J _{4′, 5′} = 7.6 Hz, H-4′), 1.86–1.92 (1H, m, H-24), 1.76 (1H, s, H-21), 1.68–1.73 (1H, m, H-22), 1.09 (3H, t, J = 7.2 Hz, H-23), 0.88 (3H, d, J _{26, 24} = 7.0 Hz, H-26), 0.78 (3H, d, J _{25, 24} = 7.0 Hz, H-25). ¹³C NMR (D₂O) δ (ppm): 176.0 (C-20), 174.1 (6′-C), 170.7 (C-5), 170.0 (C-17), 166.0 (C-8), 151.9 (C-14), 145.7 (C-15), 136.1 (C-12), 127.7 (C-11), 123.9 (C-16), 113.7 (C-13), 86.9 (C-2), 73.7 (C-10), 66.4 (C-9), 64.5 (2′-C), 63.5 (3′-C), 59.8 (6-C), 59.3 (3-C), 52.5 (5′-C), 43.5 (19-C), 36.4 (4′-C), 32.0 (NCH₃-C), 31.8 (22-C), 28.4 (24-C), 20.9 (21-C), 18.0 (26-C), 17.7 (25-C), 7.5 (23-C). The data were consistent with the literature [1]. Thus, compound 1 was identified as ustiloxin A.

Compound 2 was isolated as a white amorphous powder (MeOH). The molecular formula C₂₆H₃₉N₅O₁₂S was assigned by HR-ESI-MS, m/z 646.23751 [M + H]⁺ (calcd. 646.23887). ¹H NMR (D₂O, 400 MHz) δ (ppm): 7.57 (1H, s, H-13), 7.39 (1H, s, H-16), 4.98 (1H, d, J_{10, 9} = 10.0 Hz, H-10), 4.71 (1H, s, H-3), 4.46 (1H, q, J_{6, 24} = 7.0 Hz, H-6), 4.33–4.38 (1H, m, H-3′), 4.21 (1H, d, J_{9, 10} = 10.0 Hz, H-9), 3.97 (1H, dd, J _{5′, 4′} = 4.0 Hz, 8.0 Hz, H-5′), 3.81 (1H, d, J_{19, 19} = 17.0 Hz, H-19), 3.75 (1H, d, J_{19, 19} = 17.0), 3.37 (1H, dd, J _{2′, 2′} = 13.4 Hz, J _{2′, 3′} = 10.0 Hz, H-2′), 3.03 (1H, dd, J _{2′, 2′} = 13.4 Hz, J _{2′, 3′} = 2.4 Hz, H-2′), 2.76 (3H, s, NCH₃-9), 2.04–2.19 (3H, m, H₂-4′, H-22), 1.74 (3H, s, H-21), 1.65–1.70 (1H, m, H-22), 1.19 (3H, d, J _{24, 6} = 7.0 Hz, H-24), 0.97 (3H, t, J _{23, 22} = 7.2 Hz, 7.2 Hz, H-23). ¹³C NMR (D₂O) δ (ppm): 176.2 (C-20), 174.1(6′-C), 171.8 (C-5), 169.8 (C-17), 165.6 (C-8), 152.0 (C-14), 145.6 (C-15), 136.5 (C-12), 127.8 (C-11), 123.9 (C-16), 113.7 (C-13), 87.0 (C-2), 73.3 (C-10), 66.1 (C-9), 64.5 (2′-C), 63.5 (3′-C), 59.6 (3-C), 52.4 (5′-C), 49.4 (6-C), 43.5 (19-C), 36.4 (4′-C), 31.7 (NCH₃-C), 30.9 (22-C), 21.6 (21-C), 15.2 (24-C), 7.7 (23-C). The data were consistent with the literature [1]. Therefore, compound 2 was identified as ustiloxin B.