Cytotoxicity Effect of Constituents of Pinus taiwanensis Hayata Twigs on B16-F10 Melanoma Cells

Abstract

Pinus taiwanensis Hayata (Pinaceae) is an endemic plant in Taiwan. According to the Chinese Materia Medica Grand Dictionary, the Pinus species is mainly used to relieve pain, and eliminate pus and toxicity. In this study, nineteen compounds were isolated from the ethyl acetate layer of the ethanolic extract of P. taiwanensis Hayata twigs using bioassay-guided fractionation, and their anti-melanoma effects were investigated through a B16-F10 mouse melanoma cell model. The structures of the purified compounds were identified by 2D-NMR, MS, and IR, including 1 triterpenoid, 9 diterpenoids, 2 lignans, 4 phenolics, 1 phenylpropanoid, 1 flavonoid, and 1 steroid. Among them, compound 3 was found to be a new diterpene. Some of the compounds (2, 5, 6, 17, 18) showed moderate cytotoxicity effects. On the other hand, the anti-melanoma effect was no better than that from the original ethyl acetate layer. We presumed it resulted from the synergistic effect, although further experimentation needs to be performed.

Keywords: B16-F10 melanoma cells; Pinaceae; Pinus taiwanensis Hayata; UHPLC-MS/MS.

Figure 1

Mass spectrum of crude extract of twigs from Pinus taiwanensis Hayata. (A) Partial spectrum from 100 to 400 m/z and 400 to 800 m/z, in positive ion mode. (B) Partial spectrum from 100 to 400 m/z and 400 to 800 m/z, in negative ion mode.

Figure 2

Cytotoxicity of crude extracts on B16-F10 melanoma cells. Cell toxicity of different parts of the extract were evaluated by the MTT assay on B16-10 cells. The EA layer, n-BuOH layer, and H₂O layer were partitioned from the crude extract and tested in the concentration of 50 μg/mL. Positive control was 5 μM (0.65 μg/mL) of fluorouracil. Results were expressed as % of control cells and mean ± standard deviation (SD). The value was compared with different concentrations in each group (n = 3). B, control cell; PC, positive control; CE, crude extract; EA, EA layer; BU, n-BuOH layer; H, H₂O layer. p-values were derived from one-way ANOVA with Dunnett’s multiple comparison tests. * p-value < 0.01, ** p-value < 0.001.

Figure 3

Flow diagram of bioassay-guided procedure. Bioassay-guided fractionation and isolation of the EA extract of Pinus taiwanensis Hayata twigs resulted in 19 isolated compounds. * is the new compound.

Figure 4

The structures of 1–19 from the twigs of Pinus taiwanensis Hayata.

Figure 5

Selected 2D NMR (Chloroform-d) correlations for compound 3. (A) COSY: ―; HMBC: . (B) NOESY.

Figure 6

Cytotoxicity of pure compounds to B16-F10 melanoma cells. Results of cytotoxicity of each sample were expressed as % of control cells and mean $\pm$ SD (n = 3). B, control cell; PC, positive control (5-FU, 5 μM). Compounds 1–19 (50 μM), expect 3. p-values were derived from one-way ANOVA with Dunnett’s multiple comparison tests. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.