Clausena lansium, (Lour.) Skeels (Lour.) Skeels

Tang, Xuemei, Zhao, Meiyan, Chen, Zhiting, Huang, Jianxiang, Chen, Yan, Wang, Fuhua & Wan, Kai, 2021, Visualizing the spatial distribution of metabolites in Clausena lansium (Lour.) skeels using matrix-assisted laser desorption / ionization mass spectrometry imaging, Phytochemistry (112930) 192, pp. 1-7 : 3-4

2.2. Identification of metabolites in C. lansium extract

Over past decades, many compounds including flavonoids, coumarins and alkaloids, have been isolated from Clausena plants. In this study, MALDI MS and ultra-performance liquid chromatography-electrospray ionization (UPLC-ESI) analysis of the C. lansium fruit showed the presence of prominent metabolites in the range of m/z 100–1000 ( Fig. 3 and Fig. S2 ). Alkaloids, coumarins, sugars and organic acids were identified by MALDI-MS analysis. The characterization of the main compounds was achieved by both accurate mass measurement and mass fragmentation analysis. Table 1 displays the identified compounds, including compound details and associated literature. All the compounds presented in this study were relatively high content with most being reported previously in Clausena plants.

The identification of alkaloid compounds has been pivotal in understanding the medicinal properties of Clausena plants. MALDI-MSI was adopted to study the composition of C. lansium , which revealed the existence of different types of alkaloids. A m/z 182.06 ion was assigned to the potassium adduct of DL-Stachydrine and confirmed with a standard. One of the main active components of the medicinal plant Leonurus heterophyllus , is DL-Stachydrine, the simplest pyrrole alkaloid which is known to elicit multiple pharmacological effects ( Yin et al., 2010). We report here for the first time the existence of this compound in C. lansium , together with its potassium dimer ion of m/z 325.15 and neutral loss CO 2 ion of m/z 243.21. Other literature has also indicated that C. lansium possesses numerous carbazole alkaloids ( Peng et al., 2019). We also performed a manual search for the m/z values of known alkaloids in the average mass spectrum obtained by MALDI MS analysis of the C. lansium fruit extract. By means of combining literature, analysis of standards, and secondary data, a series of carbazole alkaloids including simple carbazole alkaloids and furancazole alkaloids were identified ( Table 1 ). The MS/MS spectrum of the m/z 182.09 ion contained an intense ion peak at m/z 167.07, which corresponds to carbazole and the neutral loss of a methyl group (15.02 Da), therefore identifying this as the [M+H] + ion of 3-methylcarbazole. Ion peaks at m/z 226.08, 252.14, and 264.14 were assigned to protonated murrayanine, heptaphylline, and girinimbine, respectively( Fig. S3 ). The m/z 280.12 ion was identified as murrastinine B, and the isomers murrayamine A and clauraila B were also found in C. lansium ( Huang et al., 2017) . The fragmentation of m/z 626.19 and m/z 688.21 produced a characteristic ion peak at m/z 264.14. Therefore, those two ions tentatively identified as alkaloid derivatives with a similar structure to giriminbine. Secondary data and additional structures of alkaloids are presented in the Supplementary materials ( Fig. S5 ).

Clausena plants also contain a series of coumarin derivatives. Coumarins are widely present and distributed in all parts of Clausena rutaceae , mainly including simple coumarins, furanocoumarins and monoterpenoid coumarins ( Deng et al., 2014; Liu et al., 2014). Coumarins produce a characteristic fragment ion at m/z 203.03 in MALDI-MS/MS analysis. Notably in the C. lansium fruit extract we observed a very strong ion peak at m/z 203.03 which was identified as furocoumarin 8-hydroxylpsoralen. The difference in mass-to-charge ratio between precursor ions and furocoumarin peak revealed the difference in substituent groups. The MS/MS spectrum of the m/z 367.11 ion, which corresponded to wampetin, contained intense ions at m/z 203.03 and 165.09. Similarly, the MS/MS spectra of the m/z 383.11 and 407.09 also produced characteristic fragment ion at m/z 203.03, those two ions were identified as the [M+H] + ions of 2 ′,3 ′ -epoxyindicolactone and [M+K] + ions of dihydroindicolactone, respectively. Because the MS/MS spectra of the m/z 771.18 ion contained only an intense ion at m/z 405.07 and its neutral loss was 366.11 Da ( Fig. S4 ), the ion was identified as the [M+K] + ion of coumarin dimer. Dimerized coumarins are relatively rare in plants of the genus Clausena , but, the existence of coumarin dimer has been previously reported in Clausena lenis ( Hong et al., 2003) . Presently six different forms of dimerized coumarins have been reported from C. excavate and C. lenis . ( Peng et al., 2017).

Data from previous studies indicate the main saccharides in C. lansium are glucose, fructose and sucrose while the main organic acid compounds are citric and malic acid. In MALDI-MS, these substances usually exist in the form of sodium or potassium salts. In this study, the [M+K] + ions of hexoses (glucose and fructose), sucrose and citric acid were detected at m/z 219.03, 381.08 and 230.94, respectively, while ions corresponding to malic acids were not observed.

Figures

Fig. 3. MALDI-MS spectrum of C. lansium fruit extract in positive ion mode.

Fig. 2. Comparison of sample pretreatment methods for MALDI-MSI analysis. (A) Intensities of ion peaks corresponding to organic acids, sugars, and alkaloids in the three different sections using airbrush, iMLayer or combined methods for matrix application. Data represent the mean ± SE of intensities of ions at m/z 230.9, 381.0, 264.1 and 367.1 (n = 3), respectively. Photographs of DHB matrix material prepared by different methods: (B) Spray by airbrush, (C) Sublimation by iMLayer, (D) Spray after sublimation. Films and crystals observation were recorded under the light microscope (× 40).

Fig. 5. Distribution of the main coumarins in diverse tissue parts in the plant of C. lansium. All the MSI were acquired in positive ion mode. The number of pixels in x and y axis was 243 × 248 for the fruit, and 100 × 70 for the stem and 65 × 37 for the leaf parts. The distributions are displayed as heat maps, with the color code between black (low) and red (high). Images were exported from the Shimadzu Imaging software. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Distribution of the main alkaloids in diverse tissue parts in the plant of C. lansium. All the MSI were acquired in positive ion mode. The number of pixels in x and y axis was 243 × 248 for the fruit, and 100 × 70 for the stem and 65 × 37 for the leaf parts. The distributions are displayed as heat maps, with the color code between black (low) and red (high). Images were exported from the Shimadzu Imaging software. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)