Transcriptome and metabolome reveal the accumulation of secondary metabolites in different varieties of Cinnamomum longepaniculatum.

BACKGROUND: Cinnamomum longepaniculatum (Gamble) N. Chao ex H. W. Li, whose leaves produce essential oils, is a traditional Chinese medicine and economically important tree species. In our study, two C. longepaniculatum varieties that have significantly different essential oil contents and leaf phenotypes were selected as the materials to investigate secondary metabolism. RESULT: The essential oil content and leaf phenotypes were different between the two varieties. When the results of both transcriptome and metabolomic analyses were combined, it was found that the differences were related to phenylalanine metabolic pathways, particularly the metabolism of flavonoids and terpenoids. The transcriptome results based on KEGG pathway enrichment analysis showed that pathways involving phenylpropanoids, tryptophan biosynthesis and terpenoids significantly differed between the two varieties; 11 DEGs (2 upregulated and 9 downregulated) were associated with the biosynthesis of other secondary metabolites, and 12 DEGs (2 upregulated and 10 downregulated) were related to the metabolism of terpenoids and polyketides. Through further analysis of the leaves, we detected 196 metabolites in C. longepaniculatum. The abundance of 49 (26 downregulated and 23 upregulated) metabolites differed between the two varieties, which is likely related to the differences in the accumulation of these metabolites. We identified 12 flavonoids, 8 terpenoids and 8 alkaloids and identified 4 kinds of PMFs from the leaves of C. longepaniculatum. CONCLUSIONS: The combined results of transcriptome and metabolomic analyses revealed a strong correlation between metabolite contents and gene expression. We speculate that light leads to differences in the secondary metabolism and phenotypes of leaves of different varieties of C. longepaniculatum. This research provides data for secondary metabolite studies and lays a solid foundation for breeding ideal C. longepaniculatum plants.

Diversity and spatial distribution of endophytic fungi in Cinnamomum longepaniculatum of Yibin, China.

Cinnamomum longepaniculatum (Gamble) N. Chao is an important woody incense plant that contains volatile terpenoids and has been extensively cultivated in Yibin, China. However, the relationship between endophytic fungal diversity and C. longepaniculatum species remains unclear. Here, fungal taxa in different tissue samples were analyzed using Illumina-based sequencing of ITS1 region of fungal rDNA genes. Results showed that 476 OTUs were identified in all tissues of C. longepaniculatum, with 78 OTUs common among all tissues. Similarity cluster analysis indicated that these OTUs belong to 5 phyla and at least 18 genera, with a large number of OTUs remaining unidentified at family and genus levels. The fungal community in seeds exhibited the greatest richness and diversity, followed by those in branches, leaves, and roots, respectively. Unclassified Chaetosphaeriales (91.66%), Passalora (57.17%), and unclassified Ascomycota (58.79%) OTUs dominated in root, branch, and leaf communities, respectively, and other common groups in the branch community included unclassified Ascomycota (12.13%), Houjia (10.38%), and Pseudoveronaea (5.43%), whereas other common groups in leaf community included Passalora (11.43%) and Uwebraunia (8.58%). Meanwhile, the seed community was dominated by unclassified Ascomycota (16.98%), unclassified Pleosporaceae (15.46%), and Talaromyces (12.50%) and also included high proportions of unclassified Nectriaceae (7.68%), Aspergillus (6.95%), Pestalotiopsis (6.02%), and Paraconiothyrium (5.11%) and several seed-specific taxa, including Peniophora, Cryptodiscus, and Penicillium. These findings suggest that Yibin-native C. longepaniculatum harbors rich and diverse endophytic communities that may represent an underexplored reservoir of biological resources.

Efficient approach for the extraction of proanthocyanidins from Cinnamomum longepaniculatum leaves using ultrasonic irradiation and an evaluation of their inhibition activity on digestive enzymes and antioxidant activity in vitro.

Proanthocyanidins were separated for the first time from Cinnamomum longepaniculatum leaves. An experiment-based extraction strategy was used to research the efficiency of an ultrasound-assisted method for proanthocyanidins extraction. The Plackett-Burman design results revealed that the ultrasonication time, ultrasonic power and liquid/solid ratio were the most significant parameters among the six variables in the extraction process. Upon further optimization of the Box-Behnken design, the optimal conditions were obtained as follows: extraction temperature, 100°C; ethanol concentration, 70%; pH 5; ultrasonication power, 660 W; ultrasonication time, 44 min; liquid/solid ratio, 20 mL/g. Under the obtained conditions, the extraction yield of the proanthocyanidins using the ultrasonic-assisted method was 7.88 ± 0.21 mg/g, which is higher than that obtained using traditional methods. The phloroglucinolysis products of the proanthocyanidins, including the terminal units and derivatives from the extension units, were tentatively identified using a liquid chromatography with tandem mass spectrometry analysis. Cinnamomum longepaniculatum proanthocyanidins have promising antioxidant and anti-nutritional properties. In summary, an ultrasound-assisted method in combination with a response surface experimental design is an efficient methodology for the sufficient isolation of proanthocyanidins from Cinnamomum longepaniculatum leaves, and this method could be used for the separation of other bioactive compounds.

Anti-inflammatory activity of leaf essential oil from Cinnamomum longepaniculatum (Gamble) N. Chao.

The anti-inflammatory activity of the essential oil from C. longepaniculatum was evaluated by three experimental models including the dimethyl benzene-induced ear edema in mice, the carrageenan-induced paw edema in rat and the acetic acid-induced vascular permeability in mice. The influence of the essential oil on histological changes and prostaglandin E2 (PGE2), histamine and 5-hydroxytryptamine (5-HT) production associated with carrageenan-induced rat paw edema was also investigated. The essential oil (0.5, 0.25, 0.13 ml/kg b.w.) showed significantly inhibition of inflammation along with a dose-dependent manner in the three experimental models. The anti-inflammatory activity of essential oil was occurred both in early and late phase and peaked at 4 h after carrageenan injection. The essential oil resulted in a dose dependent reduction of the paw thickness, connective tissue injury and the infiltration of inflammatory cell. The essential oil also significantly reduced the production of PGE2, histamine and 5-HT in the exudates of edema paw induced by carrageenan. Both the essential oil and indomethacin resulted relative lower percentage inhibition of histamine and 5-HT than that of PGE2 at 4 h after carrageenan injection.

Anti-hepatoma effect of safrole from Cinnamomum longepaniculatum leaf essential oil in vitro.

The aim of this study was to study the anti-hepatoma effect of safrole and elucidate its molecular mechanism, the human hepatoma BEL-7402 cells were incubated with various concentrations (40, 80, 160, 320 and 640 µg/ml) of safrole and the cell proliferation and apoptosis were evaluated. The results showed that both the cell proliferation determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium brominde (MTT) assay and cell colony determined by soft agar assay were significantly suppressed by safrole in a dose-time-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis, including cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed when treated with safrole for 24 h and 48 h. Cell cycle changes evaluated by flow cytometry analysis showed that the safrole could induce accumulation of cells arrested at G1 and S phases of the cell cycle. These results demonstrated that safrole is potent anti-hepatoma agent and the underlying mechanism may be attributed to suppress tumor cell growth by inducing cell apoptosis.

Antibacterial activity of leaf essential oil and its constituents from Cinnamomum longepaniculatum.

Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella enteritidis CMCC (B) 50041, were used in the antibacterial tests of Cinnamomum longepaniculatum leaf essential oil and its five chemical constituents. The effect of 1, 8-cineole on the ultrastructural structure of the bacteria (S. aureus and E. coli) was also investigated by transmission electron microscopy. The C. longepaniculatum leaf essential oil and the five chemical constituents showed variable levels of inhibition. Their MIC ( minimum inhibitory concentration ) and MBC (minimal bacteriocidal concentration) values were all in the range of 0.781 µL/mL~6.25 µL/mL and 0.781 µL/mL~12.5 µL/mL respectively except γ-terpinene. The MIC values of γ-terpinene against E. coli and S. aureus were all higher than 50 µL/mL, but the MIC and MBC values of γ-terpinene against S. enteritidis was only 3.125 µL/mL. Among them, α-terpineol possessed the best antibacterial activity. Under the transmission electron microscope, cell size of treated E. coli decreased, cell wall and cell membrane ruptured, and nucleoplasm was reduced and gathered onto the side. After the S. aureus was treated with 1, 8-cineole, the cell size and shape were damaged and nucleus cytoplasm was concentrated or reduced or agglomerated on the side. These results suggest that C. longepaniculatum leaf essential oil and its constituents have excellent antibacterial activities, the antibacterial mechanism of 1, 8-cineole against E. coli and S. aureus might attributable to its hydrophobicity.