H.N. Ngoc, et al.
Fitoterapia 137 (2019) 104252
Table 3
(Fig.S4.7, Supporting Information) can be assumed [9]. Therefore, the
structure of 19 was elucidated as (2R, 3R)-taxifolin 3-O-β-D-quinovo-
pyranoside.
Half maximal inhibitory concentration (IC50) of compounds regarding the
prevention of intracellular ROS formation in AAPH treated BEAS-2B cells.
2
Additionally, fifteen known compounds (3, 4, and 6–18) were iso-
lated and identified as follows: quercetin 3-O-β-D-glucopyranoside (3)
Compounds
IC50 (μM)
(lower CI
–
upper CI)
R
1
3
4
5
6
7
8
9
59.9
24.1
18.8
–
15.1
19.2
18.0
–
–
–
–
237.4
30.3
19.7
–
0.9009
0.9916
0.9995
–
[
10], quercetin 3-O-α-L-rhamnopyranoside (4) [11], quercetin 3-O-β-D-
apiofuranosyl-(1 → 2)-α-L-rhamnopyranoside (6) [12], quercetin 3,3´-
O-di-α-L-rhamnopyranoside (7) [13], quercetin 3-O-β-D-xylopyranosyl-
(1 → 2)-α-L-rhamnopyranoside (8) [14], epicatechin (9) [15], catechin
3-O-α-L-rhamnopyranoside (10) [16], (2R,3R)-taxifolin 3-O-β-D-gluco-
pyranoside (11) [17], (2R,3R)-taxifolin 3-O-β-D-galactopyranoside (12)
38.5
–
25.2
–
–
48.9
–
0.9835
–
35.3
47.0
47.9
61.4
63.5
31.2
–
14.0
29.7
28.7
32.1
38.3
11.9
–
–
–
–
–
–
89.5
74.3
79.8
117.5
105.4
82.1
–
0.9212
0.9842
0.9809
0.9758
0.9854
0.9044
–
[
17,18], 5-hydroxy-6,7,8-trimethoxyflavanone (13) [19], p-hydro-
1
1
1
1
1
1
0
1
2
4
5
6
xyphenethyl-trans-ferulate (14) [20], piperolactam C (15) [21], piper-
olactam A (16) [22], aristololactam AII (17) [23], and 4,5-dioxodehy-
droasimilobine (18) [24]. NMR and MS data of the known compounds
are provided as supplementary information in Tables S1 to S6.
–
–
–
–
Compounds that were available in sufficient amount (≥ 5.0 mg)
were tested for their antioxidant capacity against the human bronchial
epithelial cells BEAS-2B. They were treated with the peroxyl radical
generator AAPH to increase ROS levels. The well-known antioxidant
quercetin was able to reduce the intracellular ROS formation by ap-
proximately 60% at a concentration of 10 μM. For some compounds,
e.g. compounds 3, 4, 6, 8–12, and 14, a significant and dose-dependent
inhibition of ROS formation was found, whereby compounds 3 and 4
were most active, with half maximal inhibitory concentrations (IC50) of
hydroxyl (3306 cm−1) functionalities, while the 1H and C NMR
13
spectra indicated a quercetin backbone. Specifically, an ABX spin
system at δ 7.74 (1H, d, J = 1.8 Hz), 7.64 (1H, dd, J = 1.8, 8.4 Hz),
H
and 7.00 (1H, d, J = 1.8 Hz), two diagnostic aromatic protons of the A
ring at δ 6.20 (1H, d, J = 1.8 Hz) and 6.49 (1H, d, J = 1.8 Hz), as well
as a conjugated ketone at δ 177.9 (s). A methoxylation at position C-3
3.80) to C-3 (δ
H
C
was deduced by a HMBC correlation from OMe (δ
H
C
2
4.1 and 18.8 μM. However, some substances showed pro-oxidative
1
37.7). As for the glycone part, two sugar moieties were observed by
properties, for example compounds 5 and 16. The antioxidant activities
of all tested compounds are summarized in Fig.S5 (Supporting
Information), and the IC50 values including lower and upper confidence
intervals are given in Table 3. When studying a possible structure-ac-
tivity-relationship of the investigated flavonoids the following trends
were noticed: ROS inhibition correlated with the number of OH groups
the co-appearance of anomeric protons at δ
H
5.12 (1H, d, J = 7.2 Hz)
and 5.19 (1H, brs) (Table 2). The COSY spectrum showed two coupling
networks (Fig. 2) typical for two hexopyranose-type sugars, with one
belonging to the deoxy type. The two sugars were determined to be β-D-
glucopyranose and α-L-rhamnopyranose by analysis of NOESY data
1
3
(
Fig.S3.6, Supporting Information), by comparison with the C NMR
(
see 4 and 7), flavones were more active than flavanones (3 and 11),
data of quercetin 3,7-dimethylether-3´-O-α-L-rhamnopyranosyl-(1 →
and the substitution of C3 with a disaccharide instead of a mono-
saccharide slightly reduced activity (4 and 6); all these observations
were according to literature [25]. More unexpected were similar results
for the two catechin derivatives 9 and 10, because a free OH group in
position C3 is usually associated with a stronger antioxidant capacity; a
different stereochemical arrangement at this position could be a pos-
sible explanation [25]. Cell viability was strongly affected by com-
2
)-β-D-glucopyranoside [7], as well as confirmed by GC–MS analysis of
the hydrolysis product, compared with authentic sugars (L-rhamnose
and D-glucose) (Fig.S3.8, Supporting Information). Linkage between
the two sugars was confirmed by HMBC correlations from H-1˝´ (δ
H
5
.19) to C-2˝ (δ
C
76.9) and from H-2˝ (δ
H
3.58) to C-1˝´ (δ
C
100.5),
whereas the position of the glycone moiety became obvious by an
HMBC crosspeak between H-1˝ (δ
H
5.12) and C-3˝ (δ
C
144.8) (Fig. 2).
pound
1 (IC50 = 36.1 μM), and somewhat by compounds 15
Thus, the structure of 5 could be assigned as quercetin 3-methoxy-3´–O-
α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside.
(
IC50 = 394.5 μM) and 16 (IC50 = 179.2 μM), while the other com-
pounds did not show any cytotoxicity within the tested concentration
range (3.1 μM to 25 μM).
Compound 19 was also isolated as a yellow gum, and its molecular
formula C21
H O11 established by an ion peak at m/z 473.1049
22
+
(
[M + Na] , calcd for C21
H O11Na 473.1054). The IR spectrum
22
−1
showed characteristic absorption bands for carbonyl (1634 cm ) and
−
1
1
13
4. Conclusions
hydroxyl (3272 cm ) groups, respectively. The H and C NMR
spectrum indicated the presence of an ABX spin system [δ 6.76 (d,
J = 7.8 Hz), 6.78 (dd, J = 7.8, 1.8 Hz), and 6.91 (d, J = 1.8 Hz)], two
meta-aromatic protons [δ 5.90 (s) and 5.90 (s)], two doublet signals
4.72 (d, J = 8.4 Hz) and 5.28 (d, J = 8.4 Hz)], and a ketone re-
sonance at δ 195.6, which are diagnostic signals for a flavanone ske-
leton (Table 2). Besides that, the proton resonances at δ 3.99 (1H, d,
H
The continuous phytochemical investigation of Fissistigma poly-
anthoides stems resulted in the isolation and characterization of four
new natural products (1, 2, 5, and 19), along with fifteen known (3, 4,
and 6–18), yet for F. polyanthoides new constituents. Concerning the
latter, it is noteworthy to say that lactams like 15–17 are typical for
species from the Annonaceae family and that these compounds are
nephrotoxic [26]. Compounds 1 and 2 represent new polymethoxylated
chalcones, while compounds 5 and 19 are previously undescribed fla-
vone glycosides, partly substituted with rare sugar moieties. When
evaluating the potential anti-oxidant activity of fourteen of the isolated
compounds, substances 3 and 4 turned out to be interesting candidates
for further investigations, because of their non-toxic nature and pro-
nounced ability of inhibiting ROS formation in a dose-dependent
manner. They therefore might explain, or at least significantly con-
tribute to, the activity of F. polyanthoides stems if used as an anti-in-
flammatory remedy.
H
[
δ
H
C
H
J = 7.8 Hz) and 1.17 (3H, d, J = 6.0 Hz) were typical for a deoxy-type
sugar. The latter was determined as β-D-quinovopyranose by the large
coupling constant (J = 7.8 Hz), NOESY correlations of H-3˝/ H-1˝, H-5˝
and H-4˝/ H-2″, H-6˝ (Fig.S4.6, Supporting Information), and in com-
parison with literature data [8]. A confirmation by hydrolysis/GC–MS
as described before was not possible due to the limited amount of
compound available. The binding position of β-D-quinovopyranose to
the flavanone aglycon was established by an HMBC correlation of H-1″
(
δ
H
3.99) to C-3 (δ 77.5) (Fig. 2). The large coupling constants of H-2
C
[
δ
H
5.28 (d, J = 8.4 Hz)] and H-3 [δ 4.72 (d, J = 8.4 Hz)] indicated
H
trans relationship [9], and the CD spectrum of 19 was identical to that
of (2R, 3R)-taxifolin, so that the (2R, 3R) configuration of C-2 and C-3
5