Antioxidant glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, solanum viarum

Article abstract of DOI:10.1021/np300553t

Eggplant and related Solanum species contain abundant caffeoylquinic acid (CQA) derivatives. Fruit of the invasive species Solanum viarum Dunal contain numerous complex CQA derivatives, but only a few have been identified. The structures of two new compounds isolated from methanolic extracts of S. viarum fruit by C₁₈-HPLC-DAD were determined using 2D NMR and MS data. Both include two 5-CQA molecules joined by glucose via ester and glycosidic linkages. The structures of compounds 1 and 2 (viarumacids A and B) are, respectively, 5-caffeoyl-and 3-malonyl-5-caffeoyl-[4-(1β-[6-(5-caffeoyl)quinate] glucopyranosyl)]quinic acid. The antioxidant activities determined by ABTS ^?+ and DPPH^? assays were in the order 1 > 2 > 5-CQA.

Full text of DOI:10.1021/np300553t

Note
pubs.acs.org/jnp
Antioxidant Glucosylated Caffeoylquinic Acid Derivatives in the
Invasive Tropical Soda Apple, Solanum viarum
†
‡,§
,⊥
§
†,‡
Shi-Biao Wu, Rachel S. Meyer, Bruce D. Whitaker,* Amy Litt, and Edward J. Kennelly
†
Department of Biological Sciences, Lehman College, 250 Bedford Park Boulevard West, Bronx, New York 10468, United States
The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, New York 10016, United States
The New York Botanical Garden, 2900 Southern Boulevard, Bronx, New York 10458, United States
‡
§
⊥
Food Quality Laboratory, Building 002, Room 117, Beltsville Agricultural Research Center-West, Agricultural Research Service,
USDA, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States
*
S Supporting Information
ABSTRACT: Eggplant and related Solanum species contain
abundant caffeoylquinic acid (CQA) derivatives. Fruit of the
invasive species Solanum viarum Dunal contain numerous com-
plex CQA derivatives, but only a few have been identified. The
structures of two new compounds isolated from methanolic
extracts of S. viarum fruit by C -HPLC-DAD were determined
1
8
using 2D NMR and MS data. Both include two 5-CQA molec-
ules joined by glucose via ester and glycosidic linkages. The
structures of compounds 1 and 2 (viarumacids A and B) are,
respectively, 5-caffeoyl- and 3-malonyl-5-caffeoyl-[4-(1β-[6-(5-
caffeoyl)quinate]glucopyranosyl)]quinic acid. The antioxidant
activities determined by ABTS• and DPPH assays were in
+
•
the order 1 > 2 > 5-CQA.
Solanum viarum Dunal is an aggressive pan-tropical invasive
their structural elucidation using 2D NMR and MS data. Com-
pounds 1 and 2, new complex 5-CQA derivatives, were tested
for radical scavenging activity relative to Trolox and 5-CQA in
ABTS and DPPH assays.
Profiling of hydroxycinnamic acid (HCA) conjugates in a
weed that has recently become problematic in the southern
1
2,3
United States. It is toxic to mammals, inducing neuronal lesions.
•+
•
However, this species has also been reported to contain unusual
phenolic compounds with therapeutic potential that may be useful
4
for the natural products industry. Toxic compounds reported
methanolic extract of S. viarum Meyer 311 fruit tissue by C -
1
8
in S. viarum include steroidal glycoalkaloids such as solasodine,
HPLC-DAD revealed two unknown compounds with similar
UV spectra showing maxima at 324 and 296 nm. Compounds 1
and 2 eluted after 5-CQA (21.6 min) at 25.6 and 26.9 min and
composed 10.2% and 11.3%, respectively, of the total HCA
conjugate peak area determined at 325 nm. Isolation of the two
compounds from a concentrated methanolic extract of powdered
freeze-dried tissue from mature unripe fruit was achieved by
solid-phase extraction to obtain an enriched fraction followed
by C -HPLC-DAD separation and peak collection. Two rounds of
which coincidentally are useful precursors for the synthesis of
5
cortisone and other therapeutic steroids. Four recently identi-
fied new phenolic compounds are all 6-sinapoylglucosyl deriva-
4
tives of 5-O-(E)-caffeoylquinic acid (5-CQA). Caffeoylquinic
acids such as 5-CQA are the most potent antioxidants in the
6
genus Solanum, and anticarcinogenic activity has also been
7
−9
demonstrated.
It has become increasingly important to find
industrial applications of invasive species. The problematic and
uncontrollable spread of S. viarum warrants further investiga-
tion of the therapeutic and economic potential of its abundant
phytochemicals. The marked invasivity of this species may in
fact partly result from its unique phenolic composition, as sug-
gested by the finding that increased phenolic levels in eggplant
1
8
HPLC isolation yielded 1.83 mg of compound 1 and 1.74 mg of
compound 2 at 87% and 85% purity (by HPLC-DAD, 325 nm),
respectively.
Compound 1 was obtained as a colorless oil. Its HRESIMS
+
showed the protonated molecular ion [M + H] (Table 1) corre-
(
S. melongena) hybrids of S. viarum correlated with greater
10
sponding to the formula C H O . The UV spectrum of 1 showed
38
44 22
resistance to the fruit and shoot borer, Leucinodes orbonalis.
absorption maxima at 324 and 296 nm. Two sets of aromatic ABX
In a recent survey of phenolics in fruits of Asian eggplant and
related species, it was noted that the HPLC profile of fruit from
a Chinese accession of S. viarum (Meyer 311) included two
prominent peaks not seen in the profile of fruit from S. viarum
1
protons were indicated by the H NMR signals at δ 7.04, 6.79, and
4,11
1
6
.95 and those at δ 7.17, 7.12, and 7.10 (Table 2). The H NMR
4
accession PI319855 studied by Ma et al. Herein we report
Received: August 14, 2012
the isolation of these two compounds by C -HPLC-DAD and
Published: December 13, 2012
1
8
©
2012 American Chemical Society and
American Society of Pharmacognosy
2246
dx.doi.org/10.1021/np300553t | J. Nat. Prod. 2012, 75, 2246−2250

Journal of Natural Products
Note
and 38.5 (C-6‴). These assignments were determined from
HSQC data. Moreover, there were two oxygenated qua-
ternary carbons at δ 76.3 (C-1″) and 76.5 (C-1‴) and two
carboxy resonances at δ 175.0 (C-7″) and 177.7 (C-7‴) in the
13
C NMR spectrum (Table 2), which confirm the presence of
two quinic acid moieties. In the HMBC spectra, correlations
between δ 5.36 (H-5″) and 168.5 (C-9), and between δ 5.32
(
H-5‴) and 168.6 (C-9′), indicated that the two caffeoyl groups
are linked at C-5″ and C-5‴ of the two quinic acid moieties
(
Figure 1).
Chemical shifts of the six carbons at δ 103.4 (C-1⁗), 74.8
(
6
C-2⁗), 77.6 (C-3⁗), 71.9 (C-4⁗), 75.8 (C-5⁗), and
13
6.0 (C-6‴′) in the C NMR spectrum (Table 2) indicated
a 1⁗,6⁗-disubstituted hexose moiety. Acid hydrolysis of 1
with 2 M HCl afforded β-D-glucose, identified by HPLC-TOF-
MS analysis and co-elution with an authentic β-D-glucose
standard (t 7.12 min). A 7.8 Hz coupling constant for the
R
anomeric proton confirmed the β-anomeric configuration.
Linkages of D-glucose within the molecular structure were
established by an HMBC experiment. The H-6⁗ proton at δ
4.32 correlated with the carbonyl carbon signal δ 175.0 (C-7″)
of one of the quinic acid moieties, and the signal of the
anomeric proton of glucose (H-1⁗) at δ 4.86 showed a
doublets at δ 7.53, 6.22, 7.68, and 6.38 (Table 2), along with
1
1
H− H COSY cross-peaks H-7/H-8 and H-7′/H-8′ (Figure 1),
3
indicated four trans-oriented olefinic protons ( J = 15.9 Hz).
HMBC experiments showed the correlations H-7/C-1,
1
13
/
C-4, /C-6, and/C-9 and H-8/C-1 and/C-9, in addition
H− C long-range correlation with a signal of one of the
to the correlations H-7′/C-1′, /C-4′, /C-6′, and/C-9′ and
H-8′/C-1′ and/C-9′ (Figure 1). Taken together, the above
NMR data indicated that two caffeoyl groups exist in the
structure.
caffeoyl moieties at δ 148.8 (C-4′), indicating that C-6⁗ of
glucose is linked to C-7″ of one quinic acid moiety by an ester
bond and that C-1⁗ of glucose is ether-linked to C-4′ of one
caffeoyl group (Figure 1).
The presence of two quinic acid moieties was indicated by
The HRESIMS fragmentation analysis data for 1 verified the
structure assigned by NMR analysis (Table 1). When the aperture
1 voltage of the MS detector was set at 60 V, TOF-TOF MS/MS
yielded the fragments m/z 679.1874 (C H O , [M + H −
1
H NMR resonances of six oxymethine protons at δ 4.18, 3.73,
5
.36, 4.17, 3.74, and 5.32, together with four pairs of sp3
methylene protons at δ 2.10−2.22, 2.01−2.19, 2.12−2.22, and
3
1
35 17
1
3
+
+
2
.01−2.18 (Table 2). By inspection of the C NMR and DEPT
quinic acid] ), 691.2039 (C H O , [M + H − caffeic acid] ),
2
9
39 19
+
spectra, these resonances were in agreement with six oxymethine
517.1567 (C H O , [M + H − quinic acid − caffeic acid] ), and
2
2
29 14
resonances at δ 71.8 (C-3″), 73.9 (C-4″), 72.5 (C-5″), 71.7
355.1025 (C H O , [M + H − quinic acid − caffeic acid −
1
6
19
9
3
+
(
C-3‴), 74.0 (C-4‴), and 72.4 (C-5‴), as well as with four sp
glucosyl group] ), which provided further proof of caffeic acids,
methylene carbons at δ 39.2 (C-2″), 38.5 (C-6″), 39.1 (C-2‴),
quinic acids, and a sugar moiety in the structure. Thus, compound
Table 1. HRESIMS Fragmental Analysis of Viarumacids A (1) and B (2) (Positive Mode)
compound
fragmental ions (m/z)
ion formulas
C H O Na
mDa difference
ppm difference
calcd mass
structures of fragments
1
875.2228
0.6
−1.1
−0.2
−4.7
1.0
0.7
−1.3
−0.3
−6.8
1.9
875.2222
853.2402
679.1874
691.2086
517.1557
499.1452
355.1029
337.1557
961.2226
939.2406
921.2310
853.2402
777.2089
679.1874
603.1561
585.1456
517.1557
499.1452
441.1033
423.0927
[M+Na]+
[M+H]+
3
8
44 22
8
6
6
5
4
3
3
53.2391
79.1874
91.2039
17.1567
99.1469
55.1025
37.0990
C H O
38 45 22
a
+
C H O
35 17
[M+H−Q ]
b
3
1
+
C H O
39 19
[M+H−C ]
[M+H−Q−C]
[M+H−Q−C−H O]
2
9
+
C H O
29 14
2
2
+
C H O
1.7
3.4
2
2
27 13
2
c
+
C H O
−0.4
6.7
−1.1
19.9
1.5
[M+H−Q−C−G ]
1
6
19
9
8
+
C H O
[M+H−Q−C−G−H O]
1
6
17
2
2
961.2240
C H O Na
1.4
[M+Na]+
4
1
46 25
9
9
8
7
6
6
5
5
4
4
4
39.2422
21.2309
53.2338
77.2108
79.1967
03.1555
85.1502
17.1563
99.1438
41.1133
23.0929
C H O
47 25
1.6
1.7
[M+H]+
4
1
+
C H O
0.8
0.9
[M+H−H O]
[M+H−P ]
[M+H−C]
4
1
45 24
2
d
+
C H O
45 22
−6.4
1.9
−7.5
2.4
3
8
+
C H O
41 22
3
2
+
C H O
35 17
9.3
13.7
−1.0
7.9
[M+H − Q − P]
[M+H-Q − C]
3
1
+
C H O
31 17
−0.6
4.6
2
5
+
C H O
[M+H−Q−C−H O]
[M+H−Q−C−P]
2
5
29 16
2
+
C H O
29 14
0.6
1.2
2
2
+
C H O
−1.4
−0.2
0.2
−2.8
−0.5
0.5
[M+H-Q−C−P−H O]
[M+H−Q−C−G]
2
2
29 14
2
+
C H O
21 12
1
9
+
C H O
[M+H−Q−C−G−H O]
1
9
19 11
2
a
b
c
d
Q: quinic acid group. C:caffeoyl group. G: glucosyl group. P:propanedioic acid group.
2
247
dx.doi.org/10.1021/np300553t | J. Nat. Prod. 2012, 75, 2246−2250

Journal of Natural Products
Note
Table 2. H and 13C NMR Data for Viarumacids A (1) and B (2)
1
a
1
2
1
2
position
δH (J in Hz)
δC
δH (J in Hz)
δC
position
δH (J in Hz)
δC
δH (J in Hz)
δC
Caffeoyl Group A
127.8, s
Quinic Acid Group A
72.5, d 5.37, 1H, m
1
2
3
4
5
6
7
8
9
127.8, s
115.6, d
147.5, s
149.8, s
116.7, d
5″
6″
5.36, 1H, m
2.01−2.19, 2H, m
overlapped)
72.5, d
38.4, t
7.04, 1H, d (2.1)
6.79, 1H, d (8.4)
115.6, d 7.04, 1H, d (2.1)
147.3, s
38.5, t 2.01−2.18, 2H, m
(
(overlapped)
7″
175.0, s
175.0, s
149.7, s
Quinic Acid Group B
116.7, d 6.80, 1H, d (8.1)
1
‴
‴
76.5, s
76.3, s
35.7, t
6.95, 1H, dd (8.4, 2.1) 123.2, d 6.96, 1H, dd (8.1, 2.1) 123.3, d
2
2.12−2.22, 2H, m
overlapped)
39.1, t 2.10−2.23, 2H, m
7.53, 1H, d (15.9)
6.22, 1H, d (15.9)
146.9, d 7.47, 1H, d (15.8)
115.5, d 6.23, 1H, d (15.8)
168.5, s
146.8, d
115.2, d
168.4, s
(
(overlapped)
3‴
4‴
5‴
6‴
4.17, 1H, m
71.7, d 5.30, 1H, m
73.6, d
3.74, 1H, dd (8.8, 4.2) 74.0, d 3.94, 1H, dd (8.0, 3.8) 70.0, d
Caffeoyl Group B
131.5, s
5.32, 1H, m
72.4, d 5.40, 1H, m
72.3, d
38.5, t
1′
2′
3′
4′
5′
6′
7′
8′
9′
131.4, s
117.8, d
148.7, s
148.8, s
116.1, d
2.01−2.18, 2H, m
38.5, t 2.01−2.18, 2H, m
7.17, 1H, d (2.1)
7.12, 1H, d (8.4)
117.9, d 7.18, 1H, d (2.1)
148.7, s
(overlapped)
(overlapped)
7‴
177.7, s
177.4, s
148.8, s
Glucose Moiety
116.2, d 7.14, 1H, d (8.3)
1‴′ 4.86, 1H, d (7.8)
103.4, d 4.89, 1H, d (7.5)
103.2, d
7.10, 1H, dd (8.4, 2.1) 122.8, d 7.12, 1H, dd (8.3, 2.1) 122.5, d
2‴′ 3.52, 1H, dd (9.3, 7.2) 74.8, d 3.55, 1H, dd (8.0, 7.5) 74.8, d
3‴′ 3.46, 1H, dd (9.3, 7.8) 77.6, d 3.48, 1H, dd (8.0, 7.9) 77.4, d
‴′ 3.39, 1H, dd (7.8, 7.7) 71.9, d 3.38, 1H, dd (7.9, 7.7) 71.7, d
‴′ 3.74, 1H, m 75.8, d 3.75, 1H, m 75.7, d
a⁗ 4.32, 1H, dd (11.7, 6.9) 66.0, t 4.34, 1H, dd (12.3, 7.0) 66.2, t
b⁗ 4.50, 1H, dd (11.7, 1.8) 4.49, 1H, dd (12.3, 1.8)
Propanedioic Acid Group
7.68, 1H, d (15.9)
6.38, 1H, d (15.9)
146.3, d 7.61, 1H, d (15.8)
117.9, d 6.40, 1H, d (15.8)
168.6, s
146.3, d
117.8, d
168.6, s
4
5
6
6
Quinic Acid Group A
1
″
″
76.3, s
76.4, s
39.0, t
2
2.10−2.22, 2H, m
39.2, t 2.10−2.22, 2H, m
(overlapped)
(overlapped)
1a
2a
3a
168.0, s
50.2, t
3
″
″
4.18, 1H, m
71.8, d 4.18, 1H, m
71.7, d
3.36, 2H, s
4
3.73, 1H, dd (9.0, 4.1) 73.9, d 3.72, 1H, dd (9.0, 4.1) 73.7, d
170.5, s
a
Assignments were based on H− H COSY, HMQC, and HMBC experiments. Samples were in methanol-d . H and 13C scans were at 300 and
1
1
1
4
7
5 MHz, respectively.
they share the same skeleton. However, compound 2 includes a
propanedioic acid moiety linked with quinic acid, which is
supported by its 13C NMR data and molecular formula. More-
over, from the TOF-TOF MS/MS data (Table 1) obtained
under identical conditions, the fragment ions from 2 were
similar to those from 1. However, the presence of the m/z
+
8
6
53.2338 (C H O , [M + H − propanedioic acid] ),
3
8
45 22
79.1967 (C H O , [M + H − quinic acid − propanedioic
3
1
35 17
+
acid] ), and 517.1563 (C H O , [M + H − quinic acid −
2
2
29 14
+
caffeic acid − propanedioic acid] ) ions indicated compound 2
includes a propanedioic acid group. Additionally, the ions at
+
m/z 777.2108 (C H O , [M + H − caffeic acid] ), 603.1555
32
41 22
+
(
4
C H O , [M + H − quinic acid − caffeic acid] ), and
25
31 17
41.1133 (C H O , [M + H − quinic acid − caffeic acid −
19
21 12
+
glucosyl group] ) further confirmed that 2 has quinic acid, caffeic
acid, and a glucosyl group. Linkage of the propanedioic acid
moiety in the molecular structure was established via the HMBC
correlation between H-3‴ (δ 3.46) and C-1a (δ 168.0), indi-
cating that the propanedioic acid moiety is linked at C-3‴ by an
ester bond (Figure 1). Therefore, compound 2 was identified as
1
1
Figure 1. Key H− H COSY () and HMBC (→) NMR correlations
used in structural elucidation of the new glucosylated caffeoylquinic
acid derivatives.
1β,4β-dihydroxy-3β-carboxyacetoxy-5α-{[3-[4-[1β-(6-O-(5-(E)-
caffeoylquinic acid)-β-D-glucopyranosyl)oxy]-3-hydroxyphenyl]-
(E)-1-oxo-2-propenyl]oxy}cyclohexanecarboxylic acid, which
was named viarumacid B.
1
(
was determined to be 1β,3β,4β-trihydroxy-5α-{[3-[4-[1β-(6-O-
5-(E)-caffeoylquinic acid)-β-D-glucopyranosyl)oxy]-3-hydroxy-
phenyl]-(E)-1-oxo-2-propenyl]oxy}cyclohexanecarboxylic acid
(
IUPAC numbering), which was named viarumacid A.
Among the reported natural derivatives of 5-CQA, the 6-sinap-
oylglucosyl conjugates previously isolated from S. viarum fruit
are structurally the most similar to viarumacids A and B4.
Specifically, two of the four compounds identified by Ma et al.
are analogues of viarumacids A and B, wherein the 6-hydroxy
group of the glucose moiety is ester linked to the carboxy group
For compound 2, a prominent sodium adduct of the molec-
+
ular ion in the HRESIMS spectrum, [M + Na] , gave the
molecular formula C H O (Table 1). The UV spectrum of 2
showed maxima at 325 and 296 nm. The H and C NMR
spectra of 2 were similar to those of 1 (Table 2), indicating that
4
1
46 25
1
13
2
248
dx.doi.org/10.1021/np300553t | J. Nat. Prod. 2012, 75, 2246−2250

Journal of Natural Products
Note
of sinapic acid rather than to the carboxy group of quinic acid
in 5-CQA. Concerning the latter linkage, it appears that
esterification of quinic acid to glucose or other saccharides is
extremely rare; a 6-quinylglucoside of the flavonol herbacetin
from Ephedra alata is the only example we found in the
natural products literature.
Standards and Reagents. 5-O-(E)-Caffeoylquinic acid (5-CQA),
-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1-
6
diphenyl-2-picrylhydrazyl (DPPH), and HPLC grade MeOH were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Diammonium
2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was from
12
TCIAce (Tokyo, Japan). Ultrapure H O was prepared using a Milli-
2
RO 12 Plus system (Millipore Corp., Bedford, MA, USA).
Plant Material. Seeds of Solanum viarum accession 311, collected
Antioxidant activity was quantified for viarumacids A and B
•
•+
using the DPPH and ABTS radical scavenging assays. Because
viarumacids A and B both include two 5-CQA moieties linked by
glucose, 5-CQA was included in the assays as a reference com-
pound in addition to Trolox. The TEAC values ± SD from the
by one of the authors (R.S.M.) in China, were obtained from The New
1
3
York Botanical Garden DNA bank. Seeds were germinated indoors,
and two plants were transferred to an outdoor bed at the Calder
Research Center in Armonk, New York, where they were grown in
the summer of 2009. On the basis of morphological features, the
plants were confirmed to be S. viarum by Dr. Michael Nee, curator of
Solanaceae at The New York Botanical Garden. For further confirmation,
DNA was isolated and the nuclear ribosomal internal transcribed spacer
•
DPPH assay were 1.707 ± 0.013, 2.471 ± 0.052, and 2.158 ±
0
.043, respectively, for 5-CQA, viarumacid A, and viarumacid B. The
•+
•
ABTS assay (Table 3) gave similar results to the DPPH assay.
1
3
was sequenced according to Meyer et al., which showed Meyer 311
GenBank ID JQ638805) to be a 100% match to S. viarum accession
AY561275 used in the Solanum phylogenetic study by Levin et al.
Table 3. ABTS• Results Reported in Trolox Equivalent
+
(
a
Antioxidant Capacity (TEAC)
1
4
(
2006). Two fruit from each plant of Meyer accession 311 were
5-CQA
viarumacid A (1)
viarumacid B (2)
harvested at the mature unripe stage, i.e., full size but mottled green
and white in color rather than yellow. The four fruit were pooled as a
time (min) TEAC
SD
TEAC
SD
TEAC
SD
0
5
1
1
2
2
3
3
4
1.44
1.67
1.68
1.60
1.72
1.65
1.71
1.72
1.73
±0.17
±0.12
±0.09
±0.05
±0.10
±0.07
±0.06
±0.05
±0.04
1.51
2.71
3.01
3.18
3.64
3.73
3.71
3.81
3.89
±0.26
±0.15
±0.22
±0.34
±0.17
±0.37
±0.16
±0.16
±0.16
−0.31
1.61
1.85
2.10
2.26
2.49
2.42
2.50
2.56
±0.25
±0.21
±0.21
±0.18
±0.22
±0.28
±0.18
±0.19
±0.20
single sample, frozen in liquid N , and stored at −80 °C. The frozen
2
tissue was lyophilized and powdered with a mortar and pestle prior to
extraction.
0
5
0
5
0
5
0
Fruit Tissue Extraction and Isolation of Compounds. Two 1.0 g
samples of the lyophilized, powdered tissue were each extracted twice
with 30 mL of MeOH−H O, 4:1, for 30 min using a magnetic stir bar
2
in a 50 mL screw-cap tube that was sealed after flushing with N . After
2
centrifugation for 5 min at 1500g, the supernatant was vacuum filtered
through a sintered glass funnel fitted with a glass fiber disk. The com-
bined extracts were reduced to 20 mL by N evaporation at 40 °C, and
2
a
Technical replicates: 7.
two 10 mL portions were fractionated on 500 mg Strata X polymeric
solid-phase extraction (SPE) tubes (Phenomenex, Torrance, CA,
USA) with a step gradient of 25%, 40%, and 60% aqueous MeOH
In both cases, viarumacid A was a better scavenger than
viarumacid B. Activity observed for viarumacid A in the ABTS
assay was roughly twice that of 5-CQA, suggesting that the caffeic
acid moieties contribute most or all of the activity (Table 3).
The roughly 1.5-fold greater values for viarumacid A compared
with B suggest that the malonyl group in B results in reduced
activity. Another factor that distinguished the viarumacids from
1
1
•
+
(10 mL each). Analysis of aliquots by C -HPLC-DAD showed the
18
4
0% MeOH SPE fraction was enriched in compounds 1 and 2. This
fraction was processed to yield two 1.2 mL samples in 10% aqueous
MeOH plus 0.02% H PO , which were injected in 80 μL portions onto
3
4
a Phenomenex Luna C18(2) column (5 μm particle size, 250 mm
long, 4.6 mm i.d.) for isolation of compounds 1 and 2 using an HP
1
100 Series HPLC system (Agilent Technologies) and a binary gra-
5
-CQA was the rate at which maximum activity was attained
dient consisting of 0.02% H PO in H O and MeOH as previously
3
4
2
•+
4
after initiation of incubation with the ABTS radical: 5-CQA
activity showed an immediate sharp increase and plateaued at
5
described by Ma et al.
DPPH Radical (DPPH ) Scavenging Activity. DPPH scaveng-
•
•
1
1
ing activity was quantified according to Ma et al. Five 1:2 serial
dilutions of each sample made from a stock solution of 250 μg/mL
were used to calculate the standard curve. Two technical replicates were
performed. In addition to Trolox, 5-CQA was included as a reference
standard. Activity was reported as Trolox equivalent antioxidant capacity
min, whereas activities of viarumacids A and B increased
asymptotically, rising rapidly from 0 to 5 min and reaching a
plateau by 20 to 25 min (Table 3). In summary, these two new
compounds that include two 5-CQA moieties as part of their
structure have antioxidant potentials exceeding those of 5-CQA
and Trolox. Moreover, the protracted radical scavenging activity of
the viarumacids compared with 5-CQA merits investigation into
whether these new 5-CQA derivatives exert different biological
activities potentially useful for the natural products industry.
(
TEAC, μM Trolox/μM compound) after incubation for 30 min.
•+
•+
ABTS Radical (ABTS ) Scavenging Activity. ABTS scaveng-
11
ing activity was quantified as described in Ma et al. Six 1:2 serial dilu-
tions were made from a stock solution of 500 μg/mL, and these were
used to calculate the standard curve. Seven technical replicates were
performed. Activity was reported in TEAC values at 5 min intervals
from 0 to 40 min.
EXPERIMENTAL SECTION
■
20
Viarumacid A (1): C H O , colorless oil; [α] −45.0 (c 0.020,
General Experimental Procedures. Optical rotations were
determined on an AUTOPOL III polarimeter (Rudolph Research
Analytical, Hackettstown, NJ, USA) equipped with a sodium lamp
38 44 22
D
−1
MeOH); IR νmax cm 3305, 2945, 2823, 1727, 1599, 1450, 1409,
106, 1025, and 604; UV (MeOH), λmax (log ε) 324 (4.26), 296
1
(
−
4.06); HRESIMS (negative) m/z 851.2282 ([M − H] calcd for
(
589 nm) and a 10 cm microcell. UV and IR spectra were obtained on
1
a UV-2450 spectrometer (Shimadzu, Japan) and a Nicolet iS10 spec-
C H O22, 851.2246), (positive) see Table 1; H NMR (methanol-d ,
300 MHz) and C NMR (methanol-d
3
8
4
3
4
1
3
trometer (Thermo Scientific, Waltham, MA, USA), respectively. A
Bruker Avance 300 NMR spectrometer, equipped with bbi (for H
, 75 MHz) data, see Table 2.
4
1
20
Viarumacid B (2): C38 22, colorless oil; [α]
H
44
O
−54.2 (c 0.048,
D
13
−1
and 2D) and bbo (for C) probes, was operated at 300.1312 MHz for
H and at 75.4753 MHz for 13C NMR experiments. HRESIMS was
performed using an LCT Premier XE TOF mass spectrometer
Waters, Milford, MA, USA) equipped with an ESI interface and con-
trolled by Masslynx V4.1 software.
MeOH); IR νmax cm 3313, 2945, 2823, 1726, 1598, 1446, 1409,
1
1115, 1017, and 620; UV (MeOH), λmax (log ε) 325 (4.19), 296 (4.00);
+
HRESIMS (negative) m/z 937.2253 ([M + H] calcd for C41
H
O ,
25
45
1
(
937.2250), positive please see Table 1; H NMR (methanol-d , 300
4
MHz) and 13C NMR (methanol-d , 75 MHz) data, see Table 2.
4
2
249
dx.doi.org/10.1021/np300553t | J. Nat. Prod. 2012, 75, 2246−2250

Journal of Natural Products
Note
ASSOCIATED CONTENT
■
*
S
Supporting Information
1
13
1
1
H and C NMR, H− H COSY, HSQC, HMBC, and HRESIMS
spectra of compounds 1 and 2, a diagram of HRTOFMS
fragmentation of compounds 1 and 2, and a C -HPLC-DAD
18
chromatogram of hydroxycinnamic acid conjugates in a methanolic
extract of S. viarum 311 fruit tissues are available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Tel: 301-504-6984. Fax: 301-504-5107. E-mail: bruce.
whitaker@ars.usda.gov.
■
*
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors wish to thank Dr. G. Subramaniam, Department
of Chemistry and Biochemistry, Queens College, CUNY, for
acquisition of the UV spectra and optical rotation data, and
Mr. S. Elhakem, Department of Chemistry, Lehman College,
CUNY, for acquisition of the IR spectra. The authors also thank
Mrs. M. Fredrick for her language assistance.
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