Mangrove plants contain a variety of secondary metabolites, which are important for their survival and adaptation to the coastal environment, as well as for producing bioactive compounds. To reveal differences in mangrove volatiles, a GC-MS method was built up and used to analyze and identify five mangrove species' leaf, root, and stem. The relative content and type of volatile compounds were counted and compared, and their pathway enrichment analysis was performed. The results showed that 532 compounds were detected in the leaf, root, and stem parts of five mangrove species, which were grouped into 18 classes including alcohols, aldehydes, alkaloids, alkanes, etc. The number of compounds found was from 41 to 86 in each part of five mangrove species, A. corniculatum leaves and A. marina roots contained a maximum of 86 compounds. 247 compounds were found in A. corniculatum, 245 in K. candel, and 240 in A. marina. Roots, stems, and leaves each had 399, 342, and 339 compounds. There were 40 unique compounds in A. corniculatum leaves, and 39 in A. corniculatum stems. 71 common compounds occurring in more than two species or organ parts were analyzed by PLS-DA model. Compared with the contents and compositions of their compounds, A. ilicifolius and B. gymnorrhiza differed significantly from the other species, while the leaves differed significantly from the other parts. Unique compounds and common compounds were found to have a significant difference in composition and concentration between species and parts based on the results of one-way analysis of variance, principal component analysis, and hierarchical clustering analysis. VIP screening and pathway enrichment analysis were performed on 17 common compounds closely related to mangrove tree species or parts, and these compounds were involved in metabolic pathways of C10 isoprenoids, C15 isoprenoids, fatty alcohols, etc. These findings might help in the development of genetic varieties and medicinal utilization of mangrove plants.

Plant Material

The seedlings of five mangroves, A. ilicifolius, B. gymnorrhiza, A. corniculatum, K. candel, and A. marina were collected from Tongming river, which is located in Zhanjiang Mangrove National Nature Reserve, Guangdong (E110.1667°, N 20.9765°). The seedlings were 1.5 years old and 25–50 cm high. The morphological characteristics of the seedlings of the five mangrove species used in this study species were shown in Figure 1. Three plants were taken from each mangrove species' leaves, roots, and stems for biological duplication, and the combinations of species and organ parts from the leaves, roots, and stems were coded and named species_part. Leaves, roots, and stems of A. ilicifolius (AI), B. gymnorrhiza (BG), A. corniculatum (AC), K. candel (KC), and A. marina (AM) were simplified as AI_leaf, AI_root, AI_stem, BG_leaf, BG_root, BG_stem, AC_leaf, AC_root, AC_stem; KC_leaf, KC_root, KC_stem, AM_leaf, AM_root, AM_stem, respectively.

Sample preparation

In April 2020, healthy leaves, stems and roots from the whole plant of each mangrove species were picked, washed and dried in the shade and crushed. 5 g of leaves, roots, and stems, respectively, were weighed accurately and put into a triangular flask, 100 mL of 70% ethanol was added and extracted at 25℃ for 48 h (shook for 5 min every 12 h), and then centrifuged for 10 min at 4,000 R/min. The supernatant was filtered with quantitative filter paper and concentrated to dryness under reduced pressure, then 1 mL of ethyl was added to dissolve and prepared to test.

GC-MS analysis conditions

1.0 µL of the sample solution was used in a Shimadzu GC-2010 gas chromatograph (Shimadzu Scientific Instrument, Inc.).

The GC-MS system was equipped with an HP-5ms (30 m × 250 μm × 0.25 μm) chromatographic column, made of (5%-phenyl) -methylpolysiloxane.

In the heating procedure, the initial temperature was set at 40℃ for 2 minutes, increased at 5°C/minute to 230℃ and held at 230℃ for 2 minutes, then increased to 250℃ at 20℃/minute and held at 250℃ for another 2 minutes. Samples were injected in splitless mode; injection time was 1.00 min. The carrier gas was helium with a flow rate of 1 mL/min. The temperature of the GC injector was 250℃.

The GC MS-QP2010 SE mass spectrometer was operated in EI mode at 70 eV of electron energy; ion source temperature was equal to 230℃, and the interface temperature was equal to 250℃. The solvent delay time was 1.00 min; The Scan interval was 0.30 sec with a 2,000 amu/sec scan speed, and the scan Mas range was 50~550 m/z. The maximum length of retent time was 43.00 min.

All measurements were repeated three times.

Data Analysis

The mass spectral fragmentation patterns of the compounds detected by GC-MS were compared with those in the NIST 2014, Wiley (version 9) libraries, and those with a mass spectral similarity of over 90% were selected for identification. The relative content (%) of each compound was calculated by comparing the peak area, expressed as % from total peak areas, in GC-MS analysis.

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