2.1. Identification of Compounds 1–10
Compound
1 was obtained as a white amorphous powder, and its molecular formula was analyzed as C
27H
38O
12 by HRESIMS (
m/z 577.2260 [M + Na]
+, calculated 577.2261 for C
27H
38NaO
12). The NMR spectra of
1 showed two stereoisomers:
1a and
1b (5:3). In the
1H NMR spectrum of
1a (
Table 1), the following signals were observed: (1) a 4-substituted phenyl at
δH 6.77, 7.43 (2H each, d,
J = 8.4 Hz); (2) two trans double bonds at
δH 6.33, 7.63 (1H each, d,
J = 15.6 Hz) and 5.36, 5.42 (1H each, dt,
J = 17.4, 6.6 Hz); (3) two anomeric protons at
δH 4.31 (1H, d,
J = 8.4 Hz) and 5.18 (1H, d,
J = 1.8 Hz); (4) a methylene linking with oxygen at
δH 3.55, 3.80 (1H each, m), two methylene groups at
δH 2.05, 2.37 (2H each, m), and two methyl groups at
δH 0.93 (2H, t,
J = 7.2 Hz, 6a), 0.97 (1H, t,
J = 7.2 Hz, 6b) and 1.25 (3H, d,
J = 6.0 Hz). In the
13C NMR spectrum of
1a (
Table 2), the following signals were observed: a carbonyl at
δC 169.2, a phenyl at
δC 117.4–163.0, two double bonds at
δC 114.1–147.1, two anomeric carbons at
δC 102.7 and 104.4, nine sugar carbons at
δC 64.6–84.0, a methylene linking with oxygen at
δC 70.8, two methylene groups at
δC 21.5 and 28.9, and two methyl groups at
δC 14.6 and 17.9. The above
1H and
13C NMR data suggested
1a should be a glycoside, including a trans-
p-coumaroyl and two monosaccharide moieties. The
1H-
1H COSY experiment of
1a (
Figure 2) showed correlations between
δH 2.37 (H-2 of aglycone) and
δH 3.80 (H-1b of aglycone); 5.36 (H-3 of aglycone) between
δH 5.36 (H-3 of aglycone) and
δH 5.42 (H-4 of aglycone); between
δH 2.05 (H-5 of aglycone) and
δH 5.42 (H-4 of aglycone), 0.93 (H-6a of aglycone). Together with the HMBC experiment on
1a (
Figure 2), the aglycone of
1a was affirmed as (
E)-3-hexen-1-ol. The acid hydrolysis experiment of
1 resulted in
d-glucose and
l-rhamnose, affirmed by TLC and a comparison of its NMR data with those of ligurobustoside E [
12]. The HMBC experiment on
1a (
Figure 2) displayed the following long-distance correlations: between
δH 4.31 (H-1′ of glucosyl) and
δC 70.8 (C-1 of aglycone), between
δH 5.18 (H-1″ of rhamnosyl) and
δC 84.0 (C-3′ of glucosyl), and between
δH 4.35 (H-6′a of glucosyl), 4.48 (H-6′b of glucosyl), and
δC 169.2 (carbonyl of coumaroyl). The
1H and
13C NMR signals of
1 were assigned by
1H-
1H COSY, HSQC, and HMBC experiments (
Figure S1). Based on above evidence,
1a was identified as (
E)-3-hexen-1-yl 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
trans-
p-coumaroyl)-
O-β-
d-glucopyranoside. It is a novel hexenol glycoside, named ligurobustoside X.
The NMR data of
1b (
Table 1 and
Table 2) were similar to those of
1a, except the
trans-
p-coumaroyl in
1a was replaced by the
cis-
p-coumaroyl (
δH 5.79, 6.88 (1H each, d,
J = 13.2 Hz, H-8′″, H-7′″)) in
1b. The HMBC experiment on
1b (
Figure 2) displayed long-distance correlations between
δH 4.27 (H-1′ of glucosyl) and
δC 70.7 (C-1 of aglycone), between
δH 5.16 (H-1″ of rhamnosyl) and
δC 84.0 (C-3′ of glucosyl), and between
δH 4.34 (H-6′a of glucosyl), 4.46 (H-6′b of glucosyl), and
δC 168.1 (carbonyl of coumaroyl). Therefore, the structure of compound
1b was identified as (
E)-3-hexen-1-yl 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
cis-
p-coumaroyl)-
O-β-
d-glucopyranoside. It is a novel hexenol glycoside, named ligurobustoside X
1. In conclusion, compound
1 is a mixture of ligurobustosides X and X
1.
Compound
2 was obtained as a white amorphous powder, and its molecular formula was determined as C
25H
34O
12 by HRESIMS (
m/z 549.1941 [M + Na]
+, calculated 549.1948 for C
25H
34NaO
12). The NMR spectra of
2 showed two stereoisomers:
2a and
2b (2:1). In the
1H NMR spectrum of
2a (
Table 1), the following signals were revealed: (1) a 4-substituted phenyl at
δH 6.80 and 7.47 (2H each, d,
J = 8.4 Hz); (2) a trans double bond at
δH 6.37 and 7.65 (1H each, d,
J = 16.2 Hz); (3) two olefinic proton signals at
δH 4.88 and 5.02 (1H each, br. s); (4) two anomeric protons at
δH 4.30 (1H, d,
J = 7.2 Hz) and 5.18 (1H, d,
J = 1.8 Hz); (5) a methylene linking with oxygen at
δH 4.07 and 4.20 (1H each, d,
J = 12.6 Hz); and two methyl groups at
δH 1.75 (3H, s) and 1.25 (3H, d,
J = 6.6 Hz). In the
13C NMR spectrum of
2a (
Table 2), the following signals were shown: a carbonyl at
δC 169.1, a phenyl at
δC 116.9–161.6, two double bonds at
δC 113.4–146.9, two anomeric carbons at
δC 102.8 and 103.0, nine sugar carbons at
δC 64.6–84.0, a methylene linking with oxygen at
δC 74.0, and two methyl groups at
δC 17.9 and 19.7. The above
1H and
13C NMR data indicated that
2a should be a glycoside, including a
trans-
p-coumaroyl and two monosaccharide moieties. In the HMBC experiment on
2a (
Figure 2), the following long-distance correlations were displayed: between
δH 4.07 (H-1a of aglycone) and 4.20 (H-1b of aglycone) and
δC 143.1 (C-2 of aglycone), 113.4 (C-3 of aglycone), and 19.7 (C-4 of aglycone); between
δH 4.88 (H-3a of aglycone), 5.02 (H-3b of aglycone), and
δC 19.7 (C-4 of aglycone). Together with the HSQC experiment on
2a (
Figure S2), the aglycone of
2a was affirmed as 2-methyl-2-propen-1-ol. The acid hydrolysis experiment on
2 afforded
d-glucose and
l-rhamnose, confirmed by TLC and a comparison of its NMR data with those of ligurobustoside E [
12]. Furthermore, the HMBC experiment on
2a (
Figure 2) displayed the following long-distance correlations: between
δH 4.30 (H-1′ of glucosyl) and
δC 74.0 (C-1 of aglycone), between
δH 5.18 (H-1″ of rhamnosyl) and
δC 84.0 (C-3′ of glucosyl), and between
δH 4.36 (H-6′a of glucosyl), 4.48 (H-6′b of glucosyl), and
δC 169.1 (carbonyl of coumaroyl). The
1H and
13C NMR signals of
2 were assigned by
1H-
1H COSY, HSQC, and HMBC experiments (
Figure S2). Thus, the structure of
2a was elucidated as 2-methyl-2-propen-1-yl 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
trans-
p-coumaroyl)-
O-β-
d-glucopyranoside. It is a novel butenol glycoside, named ligurobustoside Y.
The NMR data of
2b (
Table 1 and
Table 2) were similar to those of
2a, except the
trans-
p-coumaroyl in
2a was replaced by the
cis-
p-coumaroyl (
δH 5.80, 6.89 (1H each, d,
J = 12.6 Hz, H-8′″, H-7′″)) in
2b. In the HMBC experiment on
2b (
Figure 2), the following long-distance correlations were observed: between
δH 4.26 (H-1′ of glucosyl) and
δC 73.8 (C-1 of aglycone), between
δH 5.16 (H-1″ of rhamnosyl) and
δC 84.0 (C-3′ of glucosyl), and between
δH 4.36 (H-6′a of glucosyl), 4.46 (H-6′b of glucosyl), and
δC 168.1 (carbonyl of coumaroyl). Therefore, the structure of
2b was identified as 2-methyl-2-propen-1-yl 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
cis-
p-coumaroyl)-
O-β-
d-glucopyranoside. It is a novel butenol glycoside, named ligurobustoside Y
1. In summary, compound
2 is a mixture of ligurobustosides Y and Y
1.
Compound
3 was obtained as a white amorphous powder, and its molecular formula was determined as C
21H
28O
12 by HRESIMS (
m/z 495.1474 [M + Na]
+, calculated 495.1478 for C
25H
34NaO
12). The NMR spectra of
3 exhibited two stereoisomers:
3a and
3b (4:1). The
1H and
13C NMR spectra of
3a (
Table 3 and
Table 4) showed a
trans-
p-coumaroyl (
δH 7.63, 6.33 (1H each, d,
J = 16.2 Hz, H-7″, H-8″), 7.45 and 6.80 (2H each, d,
J = 8.4 Hz, H-2″, H-3″, H-5″, H-6″);
δC 126.9 (C-1″), 161.6 (C-4″), 169.2 (CO)], an α-rhamnosyl (
δH 5.18 (1H, d,
J = 1.8 Hz, H-1′), 1.26 (3H, d,
J = 6.0 Hz, H-6′);
δC 102.7 (C-1′), 17.9 (C-6′)), and a substituted glucose, which kept balance between the β and α configurations in CD
3OD (β-configuration:
δH 4.52 (1H, d,
J = 7.8 Hz, H-1),
δC 98.1 (C-1); α-configuration:
δH 5.08 (1H, d,
J = 3.6 Hz, H-1),
δC 94.0 (C-1)). The acid hydrolysis experiment on
3 offered
d-glucose and
l-rhamnose confirmed by TLC and a comparison of its NMR data with those of ligurobustoside E [
12]. The HMBC experiment on
3a (β,
Figure 2) displayed the following long-distance correlations: between
δH 5.18 (H-1′ of rhamnosyl) and
δC 84.1 (C-3 of glucose) and between
δH 4.36 (H-6a of glucose), 4.45 (H-6b of glucose) and
δC 169.2 (carbonyl of coumaroyl). The
1H and
13C NMR signals of
3 were assigned by
1H-
1H COSY, HSQC and HMBC experiment (
Figure S3). Based on the above evidence, the structure of compound
3a was identified to be 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
trans-
p-coumaroyl)-
d-glucopyranose. It is a new sugar ester, named ligurobustate A.
The NMR data of
3b (
Table 3 and
Table 4) were close to those of
3a. The main difference was that the
trans-
p-coumaroyl in
3a was replaced by the
cis-
p-coumaroyl (
δH 6.86, 5.76 (1H each, d,
J = 13.2 Hz, H-7″, H-8″)) in
3b. The HMBC experiment on
3b (β,
Figure 2) displayed the following long-distance correlations: between
δH 5.15 (H-1′ of rhamnosyl) and
δC 84.2 (C-3 of glucose) and between
δH 4.26 (H-6a of glucose), 4.39 (H-6b of glucose), and
δC 168.2 (carbonyl of coumaroyl). Therefore, the structure of compound
3b was identified to be 3-
O-(α-
l-rhamnopyranosyl)-6-
O-(
cis-
p-coumaroyl)-
d-glucopyranose. It is a new sugar ester, named ligurobustate B. In summary, compound
3 is a mixture of ligurobustates A and B.
Compound
4, a white amorphous powder, was determined as C
21H
28O
12 by HRESIMS (
m/z 495.1476 [M + Na]
+, calculated 495.1478 for C
21H
28NaO
12). The NMR spectra of
4 exhibited two stereoisomers:
4a and
4b (3:1). The
1H and
13C NMR data of
4a (
Supplementary Materials Section S2) was in accordance with those of 3-
O-(α-
l-rhamnopyranosyl)-4-
O-(
trans-
p-coumaroyl)-
d-glucopyranose (cistanoside I) [
20]. The NMR data of
4b (
Table 3 and
Table 4) were similar to those of
4a, except the
trans-
p-coumaroyl (
δH 7.67, 6.35 (1H each, d,
J = 16.0 Hz, H-7″, H-8″)) in
4a was replaced by the
cis-
p-coumaroyl (
δH 6.94, 5.81 (1H each, d,
J = 12.8 Hz, H-7″, H-8″)) in
4b. The acid hydrolysis experiment on
4 resulted in
d-glucose and
l-rhamnose, confirmed by TLC. The HMBC experiment on
4b (β,
Figure 2) showed the following long-distance correlations: between
δH 5.12 (H-1′ of rhamnosyl) and
δC 81.9 (C-3 of glucose), and between
δH 4.85 (H-4 of glucose) and
δC 167.0 (carbonyl of coumaroyl). The
1H and
13C NMR signals of
4 were assigned by
1H-
1H COSY, HSQC, and HMBC experiments (
Figure S4). Thus,
4b was identified as 3-
O-(α-
l-rhamnopyranosyl)-4-
O-(
cis-
p-coumaroyl)-
d-glucopyranose. It is a new sugar ester, named ligurobustate C. To sum up, compound
4 is a mixture of cistanoside I and ligurobustate C.
Compound
5, a white amorphous powder, was analyzed as C
27H
38O
16 by HRESIMS (
m/z 641.2057 [M + Na]
+, calculated 641.2058 for C
27H
38NaO
16). The NMR spectra of
5 showed two stereoisomers:
5a and
5b (5:1). The NMR data of
5a (
Table 3 and
Table 4) were close to those of
3a, except for another α-rhamnosyl (
δH 5.19 (1H, d,
J = 1.6 Hz, H-1′), 1.29 (3H, d,
J = 6.0 Hz, H-6′);
δC 102.4 (C-1′), 18.6 (C-6′)). The acid hydrolysis experiment on
5 afforded
d-glucose and
l-rhamnose, affirmed by TLC and a comparison of its NMR data with those of
3. The HMBC experiment on
5a (β,
Figure 2) revealed the following long-distance correlations: between
δH 5.19 (H-1′ of inner rhamnosyl) and
δC 83.6 (C-3 of glucose), between
δH 5.20 (H-1″ of outer rhamnosyl) and
δC 81.2 (C-4′ of inner rhamnosyl), and between
δH 4.33 (H-6a of glucose), 4.45 (H-6b of glucose), and
δC 169.2 (carbonyl of coumaroyl). The
1H and
13C NMR signals of
5 were assigned by
1H-
1H COSY, HSQC, and HMBC experiment s(
Figure S5). Based on the above evidence,
5a was identified to be 3-
O-[α-
l-rhamnopyranosyl-(1→4)-α-
l-rhamnopyranosyl]-6-
O-(
trans-
p-coumaroyl)-
d-glucopyranose. It is a new sugar ester, named ligurobustate D.
The NMR data of
5b (
Table 3 and
Table 4) were close to those of
5a; the main difference was that the
trans-
p-coumaroyl (
δH 7.64, 6.35 (1H each, d,
J = 16.0 Hz, H-7″′, H-8″′)) in
5a was replaced by the
cis-
p-coumaroyl (
δH 6.87, 5.79 (1H each, d,
J = 12.8 Hz, H-7′″, H-8′″)) in
5b. The HMBC experiment on
5b (β,
Figure 2) showed the following long-distance correlations: between
δH 5.17 (H-1′ of inner rhamnosyl) and
δC 83.6 (C-3 of glucose), between
δH 5.20 (H-1″ of outer rhamnosyl) and
δC 81.2 (C-4′ of inner rhamnosyl), and between
δH 4.33 (H-6a of glucose), 4.45 (H-6b of glucose), and
δC 168.2 (carbonyl of coumaroyl). Thus, the structure of
5b was elucidated to be 3-
O-[α-
l-rhamnopyranosyl-(1→4)-α-
l-rhamnopyranosyl]-6-
O-(
cis-
p-coumaroyl)-
d-glucopyranose. It is a new sugar ester, named ligurobustate E. In conclusion, compound
5 is a mixture of ligurobustates D and E.
Compounds
6–
10 (
1H,
13C NMR data see
Supplementary Materials Section S2) were identified as reported 3-
O-(α-
l-rhamnopyranosyl)-4-
O-(
trans-caffeoyl)-
d-glucopyranose (cistanoside F,
6) [
21]; kaempferol 3, 7-diglucoside (peonoside,
7) [
22]; (+)-cycloolivil 6-
O-β-
d-glucopyranoside (
8) [
23]; (
E)-methyl
p-hydroxycinnamate (
9a) [
24]; (
Z)-methyl
p-hydroxycinnamate (
9b) [
25]; and 4-hydroxyphenylethanol (
10) [
26]; by comparison with published NMR data and 2D-NMR experiments (
1H-
1H COSY, HSQC, and HMBC). Compounds
4a,
6,
7,
8,
9a,
9b, and
10 were isolated from this plant for the first time.