Mass Spectrometric Analysis of Cucurbitacins and Dihydrocucurbitacins from the Tuber of Citrullus naudinianus

Abstract

The vast pool of structurally and functionally distinct secondary metabolites (i.e., natural products (NPs)) is constantly being expanded, a process also driven by the rapid progress in the development of analytical techniques. Such NPs often show potent biological activities and are therefore prime candidates for drug development and medical applications. The ethyl acetate extract of the tuber of Citrullus naudinianus (C. naudinianus), an African melon with edible fruits and seeds, shows in vitro immunomodulatory activity presumably elicited by cucurbitacins that are known major constituents of this plant. Further potentially immunomodulatory cucurbitacins or cucurbitacin derivatives were assumed to be in the tuber. Given the typically high content of cucurbitacins with similar physicochemical features but often distinct bioactivities, an efficient and reliable separation process is a prerequisite for their detailed characterization and assessment in terms of bioactivity. We therefore developed a detection method to screen and differentiate cucurbitacins via high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS). In order to confirm the identification, the fragmentation patterns of two cucurbitacins and one 23,24-dihydrocucurbitacin were also investigated. Six characteristic fragments were identified and three of them were employed for the identification of cucurbitacins and 23,24-dihydrocucurbitacins in the extract. As a result, in addition to eight previously reported cucurbitacins from this plant four distinct 23,24-dihydrocucurbitacins (B, D, E, and I) were putatively identified and newly found in the ethyl acetate extract of the tuber of C. naudinianus. The established methodology enables rapid and efficient LC-MS-based analysis and identification of cucurbitacins and 23,24-dihydrocucurbitacins in plant extracts.

Keywords: 23,24-dihydrocucurbitacins; Citrullus naudinianus; HPLC; MS; MS/MS; QTOF; cucurbitacins; orbitrap.

Figure 1

An unearthed tuber of C. naudinianus.

Figure 2

Cucurbitane [19(10 → 9β)abeo-10α-lanost-5-en] skeleton.

Figure 3

MS/MS spectrum of the hemslecin A standard [M + FA − H]⁻ adduct (flow injection, negative mode, 40 eV collision energy).

Figure 4

Putative fragment structures with their theoretical m/z value resulting from CID (40 eV) of hemslecin A.

Figure 5

Section of the total ion current (TIC) chromatogram of the HPLC-QTOF-MS analysis of the ethyl acetate extract of the tuber of C. naudinianus with numbered peaks (see Figure 6 and Table 2). HPLC settings: ZORBAX Eclipse Plus C18 RRHD Column (50 × 3 mm; 1.8 µm); solvent gradient of water + 0.1% FA (A) and acetonitrile:isopropanol (9:1) + 0.1% FA (B); column temperature, 40 °C; flow rate 0.6 mL/min. MS settings: Negative mode, capillary voltage, 3000 V; fragmentor voltage, 135 V; skimmer voltage, 65 V.

Figure 6

Extracted compound chromatograms (ECCs) of cucurbitacin G/H (orange, 1 and 2), J/K (yellow, 3 and 4), D (purple, 5), I (red, 7), B (green, 9), and E (blue, 11) and of 23,24-dihydrocucurbitacin D (light purple, 6), I (light red, 8), B (light green, 10), and E (light blue, 12) of the HPLC-QTOF-MS analysis of the ethyl acetate extract of the tuber of C. naudinianus (see Table 2). HPLC settings: ZORBAX Eclipse Plus C18 RRHD Column (50 × 3 mm; 1.8 µm); solvent gradient of water + 0.1% FA (A) and acetonitrile:isopropanol (9:1) + 0.1% FA (B); column temperature, 40 °C; flow rate 0.6 mL/min. MS settings: Negative mode; capillary voltage, 3000 V; fragmentor voltage, 135 V; skimmer voltage, 65 V.

Figure 7

Known cucurbitacins and newly found 23,24-dihydrocucurbitacins B (green), D (purple), E (blue), and I (red) putatively identified in the tuber of C. naudinianus.

Figure 8

Section of the total ion current (TIC) chromatogram of the HPLC-Orbitrap-MS analysis of the ethyl acetate extract of the tuber of C. naudinianus with numbered peaks of the found cucurbitacins (encircled in blue) or 23,24-dihydrocucurbitacins (encircled in green). HPLC settings: ZORBAX Eclipse Plus C18 RRHD Column (50 × 3 mm; 1.8 µm); solvent gradient of water + 0.1% FA (A) and acetonitrile:isopropanol (9:1) + 0.1% FA (B); column temperature, 40 °C; flow rate 0.6 mL/min. Orbitrap-MS settings: Negative mode, source voltage, 4.5 kV; source current, 100 µA; capillary voltage, −48 V.