Chemical investigation on the liquid and solid fermented products of N. aurantiaca, an endophytic fungi from M. leucadendra, afforded one new compound together with five known compounds. By comparison with literature data, the known compounds were identified as xyloketal K (2) (Sun et al. 2016), bostrycin (3) (Stevens et al. 1979; Chen et al. 2012), deoxybostrycin (4) (Chen et al. 2012; Wang et al. 2013), xylanthraquinone (5) (Sommart et al. 2008), and ergosterol (6) (Kawai et al. 2018).
Compound 1, a colorless oil, was determined to have a molecular formula of C11H18O2, ([M + H]+ m/z 183.1381, calcd 183.1380) by HRESIMS analysis and evidenced by 13C NMR spectrum. The IR spectrum confirmed the presence of a hydroxy and an olefinic functionalities at 3334 and 1646 cm− 1, respectively. The 1H NMR data (MeOH-d4, 600 MHz) spectrum shows two methyl groups at δH 1.67 (3H, d, J = 6.2 Hz, H3-1) and δH 1.77 (3H, dd, J = 6.7, 1.2 Hz, H3-9); six methine signals at δH 3.00 (1H, q, J = 7.0 Hz, H-4), δH 5.43 (1H, dd, J = 16.4, 7.0 Hz, H-3), δH 5.52 (1H, dq, J = 16.4, 6.2 Hz, H-2), δH 5.70 (1H, dq, J = 14.8, 6.7 Hz, H-8), δH 5.94 (1H, d, J = 11.0 Hz, H-6), and δH 6.43 (1H, ddq, J = 14.8, 11.0, 1.2 Hz, H-7); and two oxygenated methylene signals at δH 3.55 and 3.63 (each 1H, dd, J = 10.7, 7.0 Hz, H2-11) and δH 4.14 and 4.18 (each 1H, d, J = 12.0 Hz, H2-10). The DEPT 13C NMR in combination with the 13C NMR (MeOH-d4) and HSQC spectrum of 1 contained 11 carbon signals corresponding to two methyls at δC 16.8 (C-1) and 17.00 (C-9); six methines at δC 50.7 (C-4), 126.2 (C-2), 126.9 (C-7), 129.2 (C-6), 129.6 (C-8), and 130.7 (C-3); and two methylenes at δC 58.1 (C-10) and 64.4 (C-11). The COSY spectrum (Fig. 2) revealed contiguous protons of H-9/H-8/H-7/H-6 and H-1/H-2/H-3 /H-4 /H-11. Key cross-peaks of HMBC spectrum (Fig. 2) including δH 4.18 (H2-10)/δC 137.6 (C-5), 50.4 (C-4), and 129.2 (C-6); δH 3.63 (H2-11)/δC 137.6 (C-5), 50.4 (C-4), and 130.7 (C-3); δH 3.00 (H-4)/δC 130.7 (C-3), 137.6 (C-5), 129.2 (C-6), and 126.2 (C-2) were observed. The structure of 1 was thus determined to be as shown in Fig. 1, and it was named nigaurdiol. The chemical skeleton of 1 has not been reported previously, and it could be a recemate since the optical rorational value of 1 was close to zero.
All six isolates were evaluated for nitric oxide (NO) production inhibitory activity assays in LPS-activated microglial BV-2 cell and also its cytotoxic activity to these cells. For positive control, curcumin was used with an IC50 value of 6.0 ± 0.3 µM. Compounds 3, 4, and 6 showed potently inhibitory activities with IC50 value of 2.3 ± 0.3, 2.5 ± 0.5, and 7.2 ± 1.4, respectively; however, compounds 3 and 4 exhibited significant cytotoxicity against microglial BV-2 cell with viabillity of 10.7 ± 0.8 and 11.3 ± 1.3% (10 µM), respectively. Furthermore, compounds 6 showed no cytotoxic activity with the survival of cells at concentration 10 µM of 90.8 ± 6.7%. Compounds 1, 2 and 5 showed weak inhibitory activities and no cytotoxic activity (see Table 2). Ergosterol (6) is the major sterol endogenous produced by fungi and protozoa with diverse bioactivity including anti-inflammatory, anti-cancer, and immune-modulatory (Lee et al. 2017; Papoutsis et al. 2020).
Table 1 NMR data of compound 1 in CD3OD.
Position
|
δC
|
δH (J, in Hz)
|
1
|
16.8
|
1.67 (d, 3H, J = 6.2 Hz)
|
2
|
126.2
|
5.52 (dq, 1H, J = 16.4, 6.2 Hz)
|
3
|
130.7
|
5.43 (dd, 1H, J = 16.4, 7.0 Hz)
|
4
|
50.4
|
3.00 (q, 1H, J = 7.0 Hz)
|
5
|
137.6
|
|
6
|
129.2
|
5.94 (d, 1H, J = 11.0 Hz)
|
7
|
126.9
|
6.43 (ddq,, 1H, J = 14.8, 11.0, 1.2 Hz)
|
8
|
129.6
|
5.70 (dq, 1H, J = 14.8, 6.7 Hz)
|
9
|
17.0
|
1.77 (dd, 3H, J = 6.7, 1.2 Hz)
|
10a
|
58.1
|
4.18 (d, 1H, J = 12.0 Hz)
|
10b
|
|
4.14 (d, 1H, J = 12.0 Hz)
|
11a
|
64.4
|
3.63 (dd, 1H, J = 10.7, 7.0 Hz)
|
11b
|
|
3.55 (dd, 1H, J = 10.7, 7.0 Hz)
|
Table 2 IC50 and cell viabillity values of compounds in BV-2 microgial cells. Data are as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the stimulation (V); ###p < 0.001 compared with the resting (R)
Compounds
|
IC50 (µM)
|
Cell viabillity (%)
|
1
|
32.2 ± 3.3
|
102.6 ± 8.8
|
2
|
30.1 ± 3.0*
|
98.3 ± 7.6
|
3
|
2.3 ± 0.3***
|
10.7 ± 0.8***
|
4
|
2.5 ± 0.5***
|
11.3 ± 1.3***
|
5
|
32.1 ± 6.7
|
102.8 ± 6.9
|
6
|
7.2 ± 1.4***
|
90.8 ± 6.7
|
R
|
1.4 ± 0.8
|
100.0 ± 0
|
V
|
38.2 ± 4.7###
|
-
|
Curcumin
|
6.0 ± 0.3
|
-
|