Three new compounds from the twigs and leaves of Nageia fleuryi Hickel

Abstract Two new diterpenoids, 12,15-di-O-acetylhypargenin B (1) and taiwanin F-12-O-β-D-glucopyranoside (2), one new monoterpenoid, (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate (3), together with eight known compounds (4−11), were obtained from the twigs and leaves of Nageia fleuryi Hickel. The structures of the new compounds were elucidated by extensive spectroscopic techniques including HR-ESI-MS and 1 D and 2 D NMR experiments. Spectroscopic data of the known compound 4 were provided for the first time. Compounds 1 and 11 exhibited strong inhibitory activity on LPS-stimulated production of NO in RAW 264.7 murine macrophages, while compounds 1, 3, and 5 showed significant quinone reductase inducing activity in Hepa 1c1c7 murine hepatoma cells. Moreover, compounds 7 and 8 showed inhibitory activity against the proliferation of the human prostate carcinoma DU145 cells. Graphical Abstract


Introduction
The genus Negeia (Family Podocarpaceae) includes seven species, which are divided into two groups: Nageia Sect. Nageia and Nageia Sect. Dammaroideae R.R.Mill. This genus is mainly distributed in South China, Japan, Indonesia, and Malay Peninsula (Sun and Wang 2005;Liu et al. 2017). Some of these plants have been shown to have pharmacological activities for the treatment of cancer (Shan et al. 2019), pulmonary fibrosis (Li et al. 2020), traumatic bleeding, fracture, and lumbar muscle degeneration (Xu and Xie 2007). Nageia fleuryi Hickel (taxonomic synonym Podocarpus fleuryi Hickel) is an evergreen tree widely distributed in Southwest China (Du et al. 2009). Previous studies on this plant proved that it contained diterpene, diterpene lactone, and other chemical components, some of which showed anti-cancer Xu et al. 1990;Xu and Fang 1991) and anti-proliferative activities (Zhang et al. 2013). In addition, studies showed that the methanol extract of its branches and leaves had good antibacterial activity (Wu et al. 2016), and its volatile oil had a certain anti-asthmatic effect (He et al. 2005). In the present study, a systemic isolation was carried out on the methanolic extract of this plant and three new compounds (1À3) as well as eight known ones (4À11) were obtained. Compounds 1À11 ( Figure 1) were evaluated for their NO production inhibiting activity and quinone reductase inducing activity. In the cell viability assay, some of them exhibited significant cytotoxicity and therefore, we further tested their anti-proliferation activity. Herein, we report the isolation, structure elucidation, and bioassay of these compounds.  discerned as three carbonyl carbons (d C 198.5, 169.6, and 169.0), six sp 2 carbons (d C 156.6, 152.1, 134.7, 128.7, 126.8, and 119.9), seven methyls (d C 32.6, 27.9, 27.7, 23.4, 22.0, 21.4, and 21.4), four methylenes (d C 41,4, 37.9, 36.3, and 18.9), two tertiary (including one oxygenated carbon at d C 80.2), and two quaternary carbons with the aid of the DEPT spectrum ( Figure S10). The 1 H NMR spectroscopic data (Table S1) of 1 resembled those of hypargenin B (Ayhan et al. 1988) closely, except for two more methyl signals (d H 2.33 and 1.97). The HMBC correlations ( Figure S2) of H 3 -20/C-1, C-5, C-9, and C-10; H 3 -18/C-3, C-4, C-5, and C-19; H 2 -6/C-5 and C-7; H-11/C-8 and C-13; H-14/C-7, C-12, and C-15; H 3 -16/C-13, C-15, and C-17 and the 1 H-1 H COSY ( Figure S2) correlation of H 2 -2/H 2 -1 and H 2 -3 proved that the skeleton of compound 1 was similar to that of hypargenin B. Moreover, the HMBC correlations of H 3 -2 0 /C-1 0 and H 3 -4 0 /C-3 0 together with the NOESY correlations ( Figure S3) of H 3 -2 0 /H-11 and H 3 -4 0 /H 3 -17 indicated that two acetyl groups were located at C-12 and C-15, respectively. The relative configuration of compound 1 was determined by analysis of the NOESY spectrum and comparison of the 1 H NMR data with literature values (Ayhan et al. 1988 , and six oxygenated methines with the aid of the DEPT spectrum ( Figure S19). A b-glucopyranosyl could also be determined by the chemical shifts and coupling constants of the anomeric carbon from the 1 H NMR (d H 4.47, d, J ¼ 7.9 Hz) and 13 C NMR (d C 107.6) spectra. Furthermore, acid hydrolysis of compound 2 revealed D-glucose (Takashi et al. 2007). The NMR data (Table S1) demonstrated that it was similar to szemaoenoid H (Pu et al. 2018), and the only difference was dehydroxylation of the latter to form a methyl of C-16 (d C 23.9). The HMBC correlations ( Figure S2) of H 3 -16/C-13, C-15, and C-17; H 3 -17/C-13, C-15, and C-16; and H-14/C-9, C-12, and C-15 and the 1 H-1 H COSY ( Figure S2) correlation of H 3 -16/ H-15 verified the above conclusion. The NOE correlations ( Figure S4) were similar to those of compound 1, suggesting that the stereochemistry of compound 2 was the same as that of compound 1. Moreover, the b-orientation of OH-3 was confirmed by the NOE correlation of H-3/H-5. Therefore, this structure was established as taiwanin F-12-O-b-D-glucopyranoside.  (Table S2) spectrum showed signals for four methyl singlets (d H 2.10, 2.05, 1.69, and 1.61), three olefinic protons (d H 5.11, 5.09, and 5.00), one oxygenated methylene (d H 4.26, dd, J ¼ 11.9, 3.2 Hz and 4.11, dd, J ¼ 11.9, 7.9 Hz), one oxygenated methine (d H 5.40, dd, J ¼ 7.9, 3.2 Hz), and two aliphatic methylenes (d H 2.16 and 2.08). The 13 C NMR spectrum of 3 revealed 14 resonances including four methyls (d C 25.8, 21.2, 20.9, and 17.9), four olefinic carbons (d C 144.5, 132.4, 123.6, and 112.9), two carbonyls (d C 170.8 and 170.1), two oxygenated carbons (d C 74.1 and 64.7), and two methylenes (d C 33.0 and 26.5). The NMR spectroscopic data (Table S2) of 3 resembled those of 4 closely, except for the presence of signals for two additional acetoxyls (d C 170.8, 170.1, 21.2, and 20.9). The HMBC correlations ( Figure S2) of H 3 -2 0 /C-1 0 and H 3 -4 0 /C-3 0 supported the above conclusion and the HMBC correlations of H 2 -1/C-2 and C-1 0 ; H-2/C-1 and C-3 0 further verified that the two acetyls were located at C-1 and C-2, respectively. The absolute configuration of compound 3 was determined by CD spectrum ( Figure S1). The CD spectrum of 3 showed a positive Cotton effect at around 220 nm, which meets the calculated CD curve for the 2S-isomer well. Thus, compound 3 was identified as (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate.

Results and discussion
Compound 4 was obtained as a white amorphous solid and its molecular formula was determined as C 10 H 18 O 2 . This compound was elucidated to be the known compound 1,2-dihydroxymyrcene (Yang et al. 2014). As no NMR data were reported for this compound previously, we assigned the 1 H and 13 C NMR data (Table S2) of it with the aid of 2 D NMR experiments. The CD spectrum of 4 ( Figure S1) showed a positive Cotton effect at around 220 nm, which was similar to that of compound 3. Thus, the absolute configuration of compound 4 can also be determined as 2S.
As many terpenoids were reported to have anti-inflammatory activity (Toshio et al. 2017), we tested compounds 1-11 for their inhibitory effects on the inflammatory response by detecting the nitric oxide (NO) formation in lipopolysaccharide (LPS)stimulated RAW 264.7 murine macrophages. Compounds 1 and 11 dose-dependently inhibited LPS-induced overproduction of NO with the IC 50 of 20.4 lM and 39.3 lM, while cell viability was also assayed and they did not exhibit cytotoxicity at concentrations up to 100 lM. In the cell viability assay, compounds 7 and 8 showed significant cytotoxicity at low concentration ( Figure S5) and therefore, we further tested their anti-proliferation activity against the human prostate carcinoma DU145 cells. Compounds 7 and 8 exhibited strong inhibitory activity with IC 50 values of 0.917 and 8.135 lM, respectively. ( Figure S6) The quinone reductase (QR) inducing activity in Hepa 1c1c7 murine hepatoma cells was used to estimate the inhibitory effect of chemicals against oxidative stress. The induction of QR activity was expressed by the maximum folds of QR induction (MQI) compared with the non-treatment group. Compound 3 showed a strong QR-inducing effect (MQI ! 2). Compounds 1 and 5 moderately induced QR activity (2 > MQI ! 1.5). Compounds 4, 6, and 9 showed weak inhibitory effects (1.5 > MQI ! 1.2) ( Figure S7). Other compounds showed no obvious activities in the above assay.
For the isolated compounds, the anti-proliferation activity against the human prostate carcinoma of 7 and 8 were carried out for the first time, which further proved the anti-cancer activity of nagilactones. The anti-inflammatory and anti-oxidative activities of monoterpenoids 3 and 4 were also evaluated for the first time. Based on the present findings, some compounds could serve as potential anti-inflammatory agents worthy of further investigations.

Conclusion
Two new diterpenoids, 12,15-di-O-acetylhypargenin B (1) and taiwanin F-12-O-b-D-glucopyranoside (2), one new monoterpenoid, (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate (3), together with eight known compounds (4À11), were obtained from the N. fleuryi Hickel. The structures of the new compounds were elucidated by extensive spectroscopic techniques including HR-ESIMS and NMR experiments. Compounds 1 and 11 exhibited a strong anti-inflammatory activity by inhibiting LPS-induced formation of nitric oxide in RAW 264.7 cells, while compounds 1, 3, and 5 showed significant anti-oxidative activity by inducing the QR activity in Hepa 1c1c7 cells. Moreover, compounds 7 and 8 showed inhibitory activity against the proliferation of the human prostate carcinoma DU145 cells. These data suggested the potential of terpenoids as preventive agents for inflammation, oxidation and proliferation related diseases.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support from the National Natural Science Foundation of China (No. 21877071) is gratefully acknowledged.