2.1. Structure Elucidation of Compounds
Compound
1 was obtained as a white amorphous powder, with a molecular formula determined as C
25H
38O
5 on the basis of HREIMS which indicated a molecular ion peak at
m/
z 418.2722 M
+ (calcd. for C
25H
38O
5, 418,2719). The IR spectrum revealed absorption bands of hydroxyl (3431 cm
−1) and carbonyl (1711 cm
−1) groups. In the
1H-NMR spectrum (
Table 1), the downfield olefinic proton at δ
H 6.02 (1H, q,
J = 7.0 Hz) and two methyl signals at δ
H 1.82 (3H, s) and 1.92 (3H, d,
J = 7.0 Hz), indicated the presence of an angeloyloxy group in
1 [
8].
Apart from five carbon signals assigned to the angeloyloxy group (δ
C 167.9, 128.1, 138.6, 20.9, and 16.0), the
13C-NMR (DEPT) spectrum of
1 (
Table 1) also exhibited 20 carbons composed of three methyls, eight methylenes, four methines (one oxygenated), and five quaternary carbons, which were consistent with a skeleton of an
ent-kauranoid [
9]. In particular, the NMR spectroscopic features of
1 are similar to those of
8 (16α-hydroxy-
ent-kauran-19-oic acid), and only differed in the appearance of an angeloyloxy group at C-3 in
1. It was also confirmed by the chemical shift value of C-3 (δ
C 78.9, CH), C-9 (δ
C 56.1, CH) and the HMBC correlations (
Figure 2) from H-3 (δ
H 4.50, dd,
J = 12.2, 4.7 Hz) to C-1′ (δ
C 167.9, C), C-1 (δ
C 38.9, CH
2), and C-18 (δ
C 24.7, CH
3) as well as the correlations from Me-20, H-12, and H-15 to C-9, and from the methyl at C-4 (Me-18) to a downfield quaternary carbon (C-19) at δ
C 178.1. The ROESY correlations of H-3 with H-5 and H
3-18 suggested that the angeloyloxy was α-orientated, and the hydroxy at C-16 was also assigned as α-orientated by the ROESY correlations of H
3-17 with H
2-11 and H-14β along with the ROESY correlations of H
3-20 with H
2-15. Consequently, the structure of
1 was finally determined as 3α-angeloyloxy-16α-hydroxy-
ent-kauran-19-oic acid.
Compound
2 had the molecular formula C
25H
38O
6 as determined by the HREIMS, with 16 mass units more than
1. The
1H- and
13C-NMR data similarities between
2 and
1 (
Table 1 and
Table 2) suggested that they were structural analogues. As compared with compound
1, the main differences were due to the presence of a hydroxymethyl group (δ
C 66.8) and the absence of a methyl group in
2. The hydroxymethyl group was assigned to C-17 by the HMBC correlations of H
2-17 to C-14, C-15, and C-16. Therefore, the structure of
2 was established as shown.
Compounds
3 and
4 showed the same mass units as those of
1 and
2, respectively, on the basis of the HREIMS. The 1D NMR data of
3 and
4 (
Table 1 and
Table 2) also closely resembled those of
1 and
2, respectively, except for the presence of the tigloyloxy group at C-3 of
3 and
4 instead of the angeloyloxy group. These conclusions were verified by the HMBC correlations from H-3′ (δ
H 6.80 in
3, δ
H 6.89 in
4) and H-3 (δ
H 4.50 in
3, δ
H 4.50 in
4) to C-1′ (δ
C 167.9 in
3, δ
C 169.4 in
4). The NMR data suggested that compounds
3 and
4 possessed the same relative configuration as those of
1 and
2, respectively. Thus, compounds
3 and
4 were determined as 3α-tigloyloxy-16α-hydroxy-
ent-kauran-19-oic acid and 3α-tigloyloxy-16α, 17-dihydroxy-
ent-kauran-19-oic acid, respectively.
Compound
5, a white powder, possessed the molecular formula C
29H
38O
4, as determined by the HREIMS,
13C-NMR (
Table 2) and DEPT data. Comparison of the 1D- and 2D-NMR spectroscopic data of
5 with those of 3
α-cinnamoyloxy-
ent-kaur-16-en-19-oic acid (
15) revealed that their structures were closely similar to each other. The only difference between them was that the double bond of the cinnamoyloxy group at C-3 in
15 was reduced in
5, which was supported by the molecular weights of
5, showing two mass units more than those of
15. This was further confirmed by the HMBC cross-peaks of H-2′ and H-3′ with C-1′ and C-4′. The α-orientation of the 3-dihydrocinnamoyloxy group was apparent from the ROESY correlations of H-3β with H-5β and H
3-18β. Thus, compound
5 was determined as 3α-dihydrocinnamoyloxy-
ent-kaur-16-en-19-oic acid.
The molecular formula of compound
6 was deduced as C
29H
34O
4 on the basis of the positive HREIMS at
m/
z 446, 2463 [M]
+ (calcd. for 446,2457). The
1H- and
13C-NMR data of
6 (
Table 1 and
Table 2) showed many similarities to those of
20, indicating that they were structural analogues as
ent-kaura-9(11),16-dien-19-oic acid. As compared with compound
20, the obvious difference was due to the presence of one more cinnamoyloxy group at C-3 in
6. HMBC correlations from H-3, H-2′, and H-3′ to C-1′ further validated the conclusion above. The ROESY correlations of H-3 with H-5 and H
3-18 suggested that the cinnamoyloxy was α-orientated. Consequently, the structure of
6 was determined as 3α-cinnamoyloxy-
ent-kaura-9(11), 16-dien-19-oic acid.
Compound
7 was isolated as a white powder, and its molecular formula was determined as C
29H
36O
6 by HREIMS based on
m/
z 480.2508 [M]
+ (calcd. for 480,2512). The IR spectrum showed absorptions at 3441 (OH), 1701 (C = O), and 1639 and 1449 cm
−1 (aromatic C = C). The presence of a cinnamoyloxy moiety was deduced by comparison with the NMR data (
Table 1 and
Table 2) of compound
6. Besides this cinnamoyloxy moiety, the remaining twenty C-atoms included a trisubstituted double bond (δ
H 5.58 (br. s); δ
C 134.2 and 149.2), a carboxyl group (δ
C 176.0), an O-bearing methylene group (δ
H 4.10 (d,
J = 14.3 Hz) and 4.14 (d,
J = 14.3 Hz); δ
C 60.9), and an O-bearing quaternary carbon (δ
C 75.6). Further analyses demonstrated that compound
7 showed a closely similar NMR pattern to that of
6, indicating that compound
7 was a structural analogue of
ent-kaurane-19-oic acid. The double bond was located between C-15 and C-16 by the HMBC cross-peaks of H-15 with C-8, C-14, C-16 and C-17. Meanwhile, the O-bearing methylene group was only connected to C-17 by the HMBC correlations from H
2-17 to C-14, C-15, and C-16. At last, the O-bearing quaternary carbon could be attributed to C-9 due to the HMBC correlations of H
2-7, H
2-11, and H
3-20 to C-9. The relative configuration of
7 was shown to be identical with that of 6 by NMR analysis. Thus, compound
7 was determined as 3α-cinnamoyloxy-9β, 17-dihydroxy-
ent-kaur-15-en-19-oic acid.
Nineteen known
ent-kaurane derivatives, namely 16α-hydroxy-
ent-kauran-19-oic acid (
8) [
10], 16α-methoxy-17-hydroxy-
ent-kauran-19-oic acid (
9) [
11], 16α-17-dihydroxy-
ent-kauran-19-oic acid (
10) [
12], 16α, 18-dihydroxy-
ent-kaurane (
11) [
13], 3α-tigloyloxypterokaurene L3 (
12) [
14], 3α-angeloyloxy-9β-hydroxy-
ent-kaur-16-en-19-oic acid (
13) [
15], 3α-cinnamoyloxy-9β-hydroxy-
ent-kaur-16-en-19-oic acid (
14) [
15], 3α-cinnamoyloxy-
ent-kaur-16-en-19-oic acid (
15) [
12], 3α-hydroxy-
ent-kaur-16-en-19-oic acid (
16) [
16], 3α-tiglinoyloxy-
ent-kaur-16-en-19-oic acid
ent-kaura-9 (
17) [
8],
ent-9α-hydroxy-16-kauren-19-oic acid (
18) [
17],
ent-15-oxokaur-16-en-19-oic acid (
19) [
18], (11),16-dien-19-oic acid (
20) [
19], 12α-hydroxy-
ent-kaur-9(11),16-dien-19-oic acid (
21) [
20], 12α-methoxy-
ent-kaur-9(11),16-dien-19-oic acid (
22) [
21], 3α-hydroxy-
ent-kaura-9(11), 16-dien-19-oic acid (
23) [
21],
ent-17-oxokaur-15-en-19-oic acid (
24) [
22], 15α,16α-epoxy-17-hydroxy-
ent-kauran-19-oic acid (
25) [
12], and wedeliaseccokaurenolide (seco) (
26) [
14], were also isolated. Their structures were identified on the basis of spectroscopic analysis and comparison with reported data.
The
in vitro antimicrobial activities of all
ent-kaurane derivatives isolated were tested against
Pseudomonas aeruginosa (ATCC 27853),
Staphyloccocus aureus (ATCC 25923),
Monilia albicans (ATCC Y0109) and
Escherichia coli (ATCC 25922) using an agar well diffusion method [
23]. Compounds
2,
4,
7,
10,
12, and
13 showed weak activities against
M. albicans (zone of inhibition > 10 mm at 1 mg/mL). The minimum inhibitory concentrations (MICs) of compounds above against
M. albicans, 1R (R: methicillin-resistant
M. albicans), 2R, 3R, 4R, 5R, and 535R were determined by the 2-fold dilution method (
Table 3). Fluconazole was used as standard drug for comparison.