LC-MS- and 1H NMR Spectroscopy-Guided Identification of Antifungal Diterpenoids from Sagittaria latifolia

Article abstract of DOI:10.1021/acs.jnatprod.5b00470

Antifungal screening of small-molecule natural product libraries showed that a column fraction (CF) derived from the plant extract of Sagittaria latifolia was active against the fungal pathogen Cryptococcus neoformans. Dereplication analysis by liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (¹H NMR) indicated the presence of new compounds in this CF. Subsequent fractionation of the plant extract resulted in the identification of two new isopimaradiene-type diterpenoids, 1 and 2. The structures of 1 and 2 were determined by chemical methods and spectroscopic analysis as isopimara-7,15-dien-19-ol 19-O-α-l-arabinofuranoside and isopimara-7,15-dien-19-ol 19-O-α-l-(5′-acetoxy)arabinofuranoside, respectively. Compound 1 exhibited IC₅₀ values of 3.7 and 1.8 μg/mL, respectively, against C. neoformans and C. gattii. Its aglycone, isopimara-7,15-dien-19-ol (3), resulting from acid hydrolysis of 1, was also active against the two fungal pathogens, with IC₅₀ values of 9.2 and 6.8 μg/mL, respectively. This study demonstrates that utilization of the combined LC-MS and ¹H NMR analytical tools is an improved chemical screening approach for hit prioritization in natural product drug discovery.

Full text of DOI:10.1021/acs.jnatprod.5b00470

Article
pubs.acs.org/jnp
1
LC-MS- and H NMR Spectroscopy-Guided Identification of
Antifungal Diterpenoids from Sagittaria latifolia
†
†
§
§
†,‡
Ranga Rao Ravu, Melissa R. Jacob, Cynthia Jeffries, Ying Tu, Shabana I. Khan,
Ameeta K. Agarwal,†,‡ R. Kiplin Guy, Larry A. Walker, Alice M. Clark, and Xing-Cong Li*,†,‡
§
†,‡
†,‡
†
‡
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and Department of BioMolecular
Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States
§
Department of Chemical Biology and Therapeutics, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, United
States
*
S Supporting Information
ABSTRACT: Antifungal screening of small-molecule natural product libraries showed that a column fraction (CF) derived from
the plant extract of Sagittaria latifolia was active against the fungal pathogen Cryptococcus neoformans. Dereplication analysis by
1
liquid chromatography−mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy ( H NMR) indicated
the presence of new compounds in this CF. Subsequent fractionation of the plant extract resulted in the identification of two new
isopimaradiene-type diterpenoids, 1 and 2. The structures of 1 and 2 were determined by chemical methods and spectroscopic
analysis as isopimara-7,15-dien-19-ol 19-O-α-L-arabinofuranoside and isopimara-7,15-dien-19-ol 19-O-α-L-(5′-acetoxy)-
arabinofuranoside, respectively. Compound 1 exhibited IC50 values of 3.7 and 1.8 μg/mL, respectively, against C. neoformans
and C. gattii. Its aglycone, isopimara-7,15-dien-19-ol (3), resulting from acid hydrolysis of 1, was also active against the two fungal
pathogens, with IC values of 9.2 and 6.8 μg/mL, respectively. This study demonstrates that utilization of the combined LC-MS
5
0
1
and H NMR analytical tools is an improved chemical screening approach for hit prioritization in natural product drug discovery.
ur previous work has shown that small-molecule natural
product libraries consisting of chromatographically
tractable column fractions (CFs) are a valuable resource for
natural product drug discovery and that UPLC-MS-ELSD-PDA
extraction of the flowers and leaves of S. latifolia, followed by
isolation and structure elucidation of bioactive components,
was conducted, leading to the identification of two new
isopimaradienes (1 and 2). Using this as an example, the study
reported here further illustrates the advantage of the combined
O
(
abbreviated as LC-MS below) is a powerful dereplication tool
1
1
to facilitate compound identification. The present study
further demonstrates the utility of this resource for antifungal
natural products discovery. The in vitro antifungal screening of
greater than 14 000 CFs was conducted against the
opportunistic fungal pathogen Cryptococcus neoformans, which
causes life-threatening systemic cryptococcosis in immunocom-
LC-MS and H NMR spectroscopic analytical tools for hit
prioritization in natural products drug discovery.
RESULTS AND DISCUSSION
■
The present dereplication analysis is based on comparison of
the molecular weight (MW), mass spectrometry (MS)
fragmentation patterns, UV absorptions, and characteristic H
2
1
promised patients. Using LC-MS and H NMR spectroscopic
analytical tools for hit dereplication, an active CF, coded 80680-
c4, from the ethanol extract of Sagittaria latifolia Willd.
1
NMR signals with previously reported data in databases such as
SciFinder Scholar (Chemical Abstracts Service, Columbus, OH,
USA) and the Dictionary of Natural Products (CRC Press, www.
dnp.chemnetbase.com), as well as chemotaxonomic consid-
erations that structurally similar compounds are present in the
same species, genus, or even family of the plant.
(
Alismataceae), was predicted to possess new compounds.
S. latifolia, an herbaceous aquatic plant, has long been an
important food source to indigenous peoples of the Americas.
This genus consists of nearly 25 species distributed through the
3
tropical regions of North and South America. Previous
CF80680-c4 showed promising antifungal activity against C.
neoformans ATCC 90113 with an IC50 of 12.7 μg/mL and was
phytochemical investigations of other Sagittaria species
revealed the presence of various chemotypes of diterpe-
3
−12
noids.
Several ent-rosane and ent-kaurane diterpenoids
showed potent antimicrobial activities against oral patho-
Received: May 26, 2015
1
0,11
gens.
To identify the antifungal compound(s), a scale-up
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00470
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Journal of Natural Products
Article
1
Figure 1. LC-MS and H NMR analysis of the antifungal chromatographic fraction CF80680-c4 from Sagittaria latifolia. LC-MS: Acquity UPLC
BEH C18 column (2.1 × 50 mm, 1.7 μm); gradient elution starting at 15%, ramping to 20% in 0.2 min, then to 95% CH CN in water with 0.1%
3
HCOOH in 2.65 min at a flow rate of 1.0 mL/min. Ionization and detection of natural products were carried out on a Waters SQ mass spectrometer
1
1
using both the positive and negative ESI modes. H NMR: Recorded on a Bruker DRX NMR spectrometer operating at 400 ( H) using TMS as an
internal standard. (a) ELSD chromatogram showing compounds 1 and 2 with retention times of 1.93 and 2.06 min, respectively; (b and c) positive
and negative ESIMS total-ion chromatograms (TIC) of compound 1, respectively; (d and e) positive and negative ESIMS TIC of compound 2,
1
respectively; and (f) 400 MHz H NMR spectrum of CF80680-c4 indicating the structural information on compounds 1 and 2.
the most active of all CFs generated from the plant extract of S.
latifolia by an automated high-throughput fractionation
indicated a MW of 462. The difference of 42 mass units
between 1 and 2 suggested that an acetyl group was likely
present in 2, explaining the slightly longer retention time of 2.
Since several diterpenoids have been isolated from other
Sagittaria species, the MW ranges and the non-UV absorption
nature of compounds 1 and 2 suggested they could be
diterpenoid derivatives. Using the MW and taxonomic
information to search the aforementioned databases, it was
determined that one diterpenoid named sagittine B from S.
sagittifolia had a MW of 420, and two diterpenoids named
sagittine C and sagittine D from S. sagittifolia had the same MW
of 462. Since the same MW (or molecular formula) generally
corresponds to multiple chemical structures, especially for small
MWs less than 500, it was undetermined as to whether 1 and 2
were the known compounds mentioned above, and the LC-MS
data were unable to address this without available authentic
1
3
system. It is worth noting that the parent ethanol extract of
S. latifolia had an IC50 of >50 μg/mL against this fungal
pathogen and was not considered as a hit, highlighting the
importance of activity enrichment by this fractionation
approach. The LC-MS data of CF80680-c4 are shown in
Figure 1. The evaporative light scanning detected (ELSD)
chromatogram indicated that it contained two major
compounds (1 and 2) in a ratio of approximately 1:1 at
retention times (t ) of 1.93 and 2.06 min, both of which lacked
R
UV absorptions (data not shown). Compound 1 gave a
10
+
protonated molecular ion at m/z 421.2 [M + H] in the
−
positive ESIMS and a [M + HCOO] ion at m/z 465.3 in the
negative ESIMS, indicating a MW of 420. For compound 2, the
+
ionized molecular ions at m/z 463.5 [M + H] and 507.2 [M +
HCOO]− in the positive and negative ESIMS, respectively,
B
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a
Table 1. NMR Spectroscopic Data for Compounds 1−3 in CDCl (δ, ppm; J, Hz)
3
1
2
3
H/C
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1.03 α/1.84 β
1.44
39.8
18.7
36.0
37.0
51.7
23.3
121.5
135.8
52.3
35.5
20.5
36.3
37.0
46.2
150.4
0.96 α/1.78 β
1.38
39.7
18.5
36.0
36.8
51.5
23.2
121.5
135.4
52.1
35.3
20.3
36.1
36.8
46.0
150.2
1.04 α/1.88 β
1.48/1.36
39.9
18.6
35.5
38.0
51.6
23.1
121.7
135.7
52.3
35.4
20.5
36.3
37.0
46.2
150.5
1.82 α/0.96 β
1.76 α/0.91 β
1.91 α/0.99 β
1.26 dd (12.3, 4.2)
1.83 α/1.98 β
5.33 br s
1.20 dd (12.1, 4.1)
1.79 α/1.92 β
5.28 br s
1.27 dd (12.2, 4.3)
1.89 α/2.00 β
5.34 br s
1.66
1.60
1.66
0
1
2
3
4
5
6
1.55 α/1.33 β
1.32 α/1.47 β
1.48 α/1.25 β
1.27 α/1.41 β
1.55 α/1.34 β
1.32 α/1.46 β
1.96 α/1.88 β
1.90 α/1.83 β
5.72 dd (17.4, 10.8)
4.85 dd (17.2, 1.4)
4.79 dd (10.6, 1.4)
0.79 s
1.98 α/1.83 β
5.79 dd (17.5, 10.8)
4.91 d (17.2)
4.85 d (10.7)
0.85 s
5.79 dd (17.5, 10.7)
4.91 dd (17.5, 1.4)
4
.85 dd (10.8, 1.4)
109.4
21.6
28.1
70.8
109.2
21.5
27.9
70.8
109.3
21.6
27.1
65.3
1
1
1
7
8
9
0.84 s
0.92 s
0.88 s
0.96 s
3.65 d (9.3)
3.60 d (9.4)
3.53 d (9.0)
0.78 s
3.89 d (10.7)
3.47 d (10.9)
0.83 s
3
.62 d (9.3)
20
0.83 s
16.3
16.1
171.4
20.9
16.3
CO
COCH3
2.05 s
4.86 br s
4.02
1′
2′
3′
4′
5′
4.96 br s
4.01
108.9
78.8
78.1
87.2
62.0
108.1
80.7
3.98
3.79
77.9
4.13
4.06
82.3
3.88 br d (11.7)
4.20
64.3
3
.80 br d (11.5)
a
1
13
1
13
Data obtained at 500 ( H) and 125 ( C) MHz for compound 1 and at 400 ( H) and 100 ( C) MHz for compounds 2 and 3. Assignments were
1
based on DEPT and 2D NMR including DQF-COSY, HMQC, HMBC, and ROESY. Well-resolved H NMR couplings are expressed with coupling
patterns and constants in Hz in parentheses. Some geminal protons were assigned as α- or β-oriented on the ring system.
compounds for direct comparison of retention times and MS
data.
The advantage of using H NMR spectroscopy for hit
of these CFs by reversed-phase C18 column chromatography
resulted in the purified compounds 1 and 2.
1
1
13
The H and C NMR data of compound 1 (Table 1) due to
the aglycone moiety differed from those of any diterpenoid
previously isolated from Sagittaria spp., while its sugar moiety
appeared to be a single β-arabinofuranose that was present in
dereplication lies in its fingerprinting nature providing excellent
structural information that can significantly narrow down
possible hits when combined with LC-MS for this purpose. In
1
this study, a 400 MHz H NMR spectrum of CF80680-c4 was
several diterpenoid glycosides that have been isolated from
acquired in deuterated chloroform with a quantity of 0.4 mg
10,11
Sagittaria spp.
Acid hydrolysis of 1 afforded an aglycone
(
dried from 200 μL of the sample at 2 mg/mL in DMSO). This
(
7
3) and L-arabinose. The aglycone was identified as isopimara-
1
H NMR spectrum (Figure 1f) showed methyl groups of the
1
,15-dien-19-ol by comparison of its H NMR spectrum and
diterpenoid skeleton in the range δ 0.6−1.0, an acetyl group at
14,15
H
optical rotation data with those reported in the literature.
Its C NMR data were assigned by 2D NMR experiments and
δ
2.05, sugar moiety signals in the range δH 3.5−4.5, and
13
H
vinylic protons of the aglycone moiety and anomeric protons of
1
13
are reported for the first time in Table 1. The H and C NMR
assignments of compound 1 were facilitated by 2D NMR
experiments. The linkage of the arabinose to the C-19 hydroxy
group in 1 was confirmed by the HMBC correlations between
H -19 at δ 3.65 and 3.62 (1H, each, d, J = 9.3 Hz) and C-1′ at
the sugar moiety in the range δ 4.7−6.0. Comparison of the
H
characteristic methyl resonances with those of sagittines B−D
10
indicated distinct differences, suggesting that compounds 1
and 2 had a different diterpenoid skeleton and thus might be
new natural products.
2
H
δ 108.9 and between H-1′ at δ 4.96 (1H, br s) and C-19 at
δ 70.8, as well as the NOE correlation between H-19 and H-
To isolate compounds 1 and 2, a 95% ethanol extract of the
dried leaves and flowers of S. latifolia was fractionated into CFs
by normal-phase silica gel chromatography using a solvent
system of chloroform with an increasing percentage of
methanol. The CFs containing the two compounds were
C
H
C
1
5
′. In addition, the key NOE correlations, H -20 and H -19, H-
3
2
and H-9, and H -17 and H-11β, supported the isopimara-
3
7,15-dien-19-ol skeleton exhibiting a conformation of chair/
chair-like/chair for the tricyclic A/B/C ring system, as shown in
the optimized geometry by ChemBio3D Ultra 12.0 (Figure 2).
1
located readily by direct comparison of TLC behavior and H
NMR spectra with those of CF80680-c4. Subsequent isolation
C
DOI: 10.1021/acs.jnatprod.5b00470
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Journal of Natural Products
Article
efficiency of rapid isolation of active compounds was proven in
this study, whereas the conventional bioassay-guided fractiona-
tion often requires multiple steps of fractionation and bioassay
before isolation of the desired active compounds. The inclusion
1
of H NMR spectroscopic analysis for dereplication is
particularly powerful for identifying known compounds without
time-consuming isolation from CFs that contain structurally
similar compounds. Two such examples that illustrate rapid
dereplication of known antifungal compounds are further
shown in the Supporting Information. Thus, the application of
this improved dereplication tool will enhance discovery
potential in a natural product discovery program.
Figure 2. Key NOE correlations of aglycone 3.
The 1H and 13C NMR data of compound 2 (Table 1)
indicated that it had the same aglycone as 1, and an additional
acetyl group was confirmed to be attached to the C-5 hydroxy
group of the α-L-arabinofuranose by the HMBC correlation
between H-5′ and the carbonyl carbon of the acetyl group. This
acetylation resulted in a downfield shift for both C-5′ and H-5′
compared to those for compound 1. The chemical correlation
between the two compounds was achieved by converting 2 into
EXPERIMENTAL SECTION
■
General Experimental Procedures. Specific rotations were
1
13
measured on an Autopol IV polarimeter. H and C NMR spectra
were recorded on a 400 MHz Bruker DRX NMR spectrometer
1
13
operating at 400 ( H) and 100 ( C) MHz or a 500 MHz Bruker DRX
1
13
1
through a mild alkaline hydrolysis procedure. The NOE
NMR spectrometer operating at 500 ( H) and 125 ( C) MHz.
Chemical shifts are expressed in ppm relative to the residual solvent
signals. High-resolution ESIMS data were obtained on an Agilent
Series 1100 SL mass spectrometer. Column chromatography was
performed on silica gel (40 μm, J. T. Baker) and reversed-phase silica
gel (C , 40 μm, J. T. Baker). Silica gel 60 F TLC plates (Merck,
correlations of 2 obtained from a 2D ROESY experiment
showed exactly the same patterns as those for compound 1,
supporting the configurational and conformational assignment
for its aglycone (3).
18
254
In vitro antifungal testing showed that compound 1 had an
IC50 value of 3.7 μg/mL against C. neoformans ATCC 90113,
while compound 2 was inactive at the highest test
concentration of 20.0 μg/mL. The IC50 value of 1 was
approximately one-third of that observed for CF80680-c4. It
was thus concluded that compound 1 is responsible for the
antifungal activity of CF80680-c4. Compound 3, the aglycone
of compound 1, was also active against C. neoformans ATCC
Darmstadt, Germany) and reversed-phase TLC plates (C , Merck,
1
8
Darmstadt, Germany) were used for analytical TLC. The plates were
visualized by spraying 10% H SO followed by heating. UPLC-MS-
2
4
ELSD-PDA data of CFs were obtained with a Waters Acquity
UPLCMS system (Waters Corp., Milford, MA, USA). The general
conditions are shown in Figure 1, while the detailed instrumentation
1
protocol has been described previously.
Plant Material. The leaves and flowers of Sagittaria latifolia Willd.
were collected in Marinette, WI, USA, with coordinates of 45°05′12″
N 087°35′15″ W by Andrew Townesmith and Greg Gust on August
9
0113, with an IC50 of 9.2 μg/mL. Since the recent
cryptococcosis outbreak throughout the Pacific Northwest
was caused by Cryptococcus gattii, a highly virulent fungal
1
9, 2005, and identified by A. Townesmith. A voucher specimen
(collection no. 272) is deposited in the Herbarium of the Missouri
16,17
pathogen that generally affects immunocompetent hosts,
compounds 1 and 3 were tested further against C. gattii ATCC
2609, showing IC /MIC values of 1.8/20.0 and 6.8/20.0 μg/
Botanical Garden, St Louis, MO, USA.
Extraction and Isolation. The air-dried and powdered leaves and
flowers of S. latifolia (40 g) were extracted three times with 95% EtOH
3
5
0
(
360 mL) at 37 °C for 10 min using an accelerated solvent extractor to
mL, respectively. For comparison, the positive control
amphotericin B exhibited IC /MIC values of 0.2/0.6 and
yield a crude EtOH extract (9.8 g). The extract (9.8 g) was
5
0
chromatographed on silica gel using CHCl first and then a gradient
3
0.1/0.3 μg/mL against C. neoformans ATCC 90113 and C. gattii
elution of CHCl −MeOH (0−30% MeOH) to yield 29 fractions,
3
ATCC 32609, respectively. It was noted that acetylation of 1
resulted in the loss of activity for compound 2, while
deglycosylation of 1 retained the activity for compound 3.
The retention of activity for aglycone 3 implies that more
chemically tractable, structurally similar natural and synthetic
diterpenoids can be explored for anticryptococcal activity.
The cytotoxicity of compounds 1 and 3 was evaluated against
two mammalian cell lines to determine their selectivity of
antifungal activity. The IC values of 1 were 30.0 and 21.5 μg/
which were subjected to TLC analysis by comparison with CF80680-
c4. Compound 2 was present in fractions 11 and 12, which were
combined (540 mg) and chromatographed on reversed-phase C18
silica gel using 80−100% MeOH in H O to yield compound 2 (159.1
2
mg). The combined fractions 16−18 (280 mg) contained compound
1, which was obtained in a quantity of 172.5 mg by reversed-phase C18
silica gel chromatography using 85−100% MeOH in H O. While
2
conducting the isolation work, antifungal testing of the 29 CFs against
C. neoformans ATCC 90113 was performed, indicating that compound
5
0
1
-containing fractions were most active, with IC50 values of ∼5 μg/mL,
mL against Vero (African green monkey kidney fibroblast) and
thereby excluding the possibility of missing any antifungal compounds
from other CFs.
LLC-PK (pig kidney epithelial) cells, respectively, while for
1
compound 3 the values were 37.5 and 42.5 μg/mL,
respectively. Both compounds were not cytotoxic at the
concentrations that were responsible for antifungal activity
and are significantly less cytotoxic than the positive control
doxorubicin (IC values of 5.4 and 0.4 μg/mL against the same
Isopimara-7,15-dien-19-ol 19-O-α-L-arabinofuranoside (1): white
powder; [α]25 −89.9 (c 0.1, CHCl ); NMR data, Table 1; HRESIMS
D
3
−
m/z 465.2860 (calcd for [C H O + HCOO] , 465.2858; Δ = −0.41
25
40
5
ppm).
Isopimara-7,15-dien-19-ol 19-O-α-L-(5′-acetoxy)arabinofurano-
50
side (2): colorless liquid; [α]25 −65.9 (c 0.1, CHCl ); NMR data,
D
3
O
two cell lines, respectively).
−
1
Table 1; HRESIMS m/z 507.2967 (calcd for [C27
H
42
6
+ HCOO] ,
In conclusion, the LC-MS and H NMR spectroscopic
analysis of the active CF80680-c4 from our small-molecule
natural product libraries has led to the identification of
5
07.2963; Δ = −0.68 ppm).
Acid Hydrolysis of Compound 1. A solution of 1 (25 mg) in 2
M HCl−1,4-dioxane (1:1, 2 mL) was stirred at 80 °C for 45 min. After
1
antifungal diterpenoids. The combination of LC-MS and H
cooling, the reaction mixture was diluted with H O (10 mL) and
2
NMR spectroscopy is an improved chemical dereplication tool
for structural analysis of compounds in semipurified CFs. The
extracted with CHCl (10 mL × 3). The combined organic layers were
3
washed with H O (5 mL × 3), dried over Na SO , and evaporated to
2
2
4
D
DOI: 10.1021/acs.jnatprod.5b00470
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Journal of Natural Products
Article
a
Table 2. In Vitro Antifungal Activity and Cytotoxicity of Compounds 1 and 3
b
c
antifungal activity (IC /MIC, μg/mL)
cytotoxicity (IC , μg/mL)
50
50
compound
C. neoformans ATCC 90113
C. gattii ATCC 32609
Vero
LLC-PK1
1
3
3.7 ± 0.3/>20.0
9.2 ± 1.1/>20.0
0.2 ± 0.02/0.7 ± 0
1.8 ± 0.6/20.0 ± 0
6.8 ± 0.6/20.0 ± 0
0.1 ± 0.06/0.3 ± 0.1
30.0 ± 0
37.5 ± 3.5
21.5 ± 4.9
42.5 ± 3.5
amphotericin B
doxorubicin
5.4 ± 0.2
0.4 ± 0.2
a
b
Both antifungal activity and cytotoxicity are expressed as mean values of three experimental values with standard deviations. IC : concentration
50
responsible for 50% growth inhibition of fungal cells; MIC: minimum inhibitory concentration (lowest concentration that allows no detectable
c
growth). The highest test concentration used for both compounds 1 and 3 was 20 μg/mL. IC : concentration responsible for 50% growth
50
inhibition of mammalian cells. The highest test concentration used for both compounds 1 and 3 was 50 μg/mL.
dryness to yield a residue (21 mg). The residue was subjected to C18
reversed-phase silica chromatography using MeOH−H O (98:2) to
Agricultural Research Service Specific Cooperative Agreement
No. 58-6408-2-0009.
2
25
give compound 3 (7.0 mg): colorless liquid: [α]
−13.9 (c 0.1,
D
14
20
D
CHCl ) {ref.: [α] −15 (CHCl )}.
3
3
REFERENCES
■
The H O layer was passed through an Amberlite MB-150 resin
2
(
1) Yang, J.; Liang, Q.; Wang, M.; Jeffries, C.; Smithson, D.; Tu, Y.;
column (8 g) eluting with H O (50 mL) to give L-arabinose (4.5 mg):
2
25
25
Boulos, N.; Jacob, M. R.; Shelat, A. A.; Wu, Y.; Ravu, R. R.; Gilbertson,
R.; Avery, M. A.; Khan, I. A.; Walker, L. A.; Guy, R. K.; Li, X.-C. J. Nat.
Prod. 2014, 77, 902−909.
[
0
α] +93.9 (c 0.1, H O), which was comparable to [α] +119.9 (c
D
2
D
.1, H O) for standard L-(+)-arabinose from Sigma-Aldrich. L-
2
Arabinose gave an R value of 0.7 on normal-phase silica gel TLC
f
(
2) Perfect, J. R. In Antifungal Therapy; Ghannoum, M. A., Perfect, J.
R., Eds.; Informa Healthcare: New York, 2010; Chapter 18, pp 308−
19.
3) Tanaka, C. M. A.; Radke, V. S. C. O.; da Silva, C. C.; Nakamura,
C. V.; de Oliveira, P. L.; Kato, L.; de Oliveira, C. M. A. Quim. Nova
010, 33, 30−32.
4) Sharma, S. C.; Tandon, J. S.; Dhar, M. M. Phytochemistry 1975,
4, 1055−1057.
5) Juneja, R. K.; Sharma, S. C.; Tandon, J. S. Indian J. Chem. Sect B:
Org. Chem. 1986, 25B, 748−749.
6) Yoshikawa, M.; Yamaguchi, S.; Murakami, T.; Matsuda, H.;
Yamahara, J.; Murakami, N. Chem. Pharm. Bull. 1993, 41, 1677−1679.
7) Yoshikawa, M.; Yoshizumi, S.; Murakami, T.; Matsuda, H.;
Yamahara, J.; Murakami, N. Chem. Pharm. Bull. 1996, 44, 492−499.
8) Radke, V. S. C. O.; Tanaka, C. M. A. Biochem. Syst. Ecol. 2004, 32,
29−531.
9) Radke, V. S. C. O.; Amaral, M. C. E.; Schuquel, I. T. A.; da Silva,
C. C.; de Oliveira, C. M. A.; Tanaka, C. M. A. J. Braz. Chem. Soc. 2007,
8, 444−447.
10) Liu, X.-T.; Pan, Q.; Shi, Y.; Williams, I. D.; Sung, H. H.-Y.;
Zhang, Q.; Liang, J.-Y.; Ip, N. Y.; Min, Z. D. J. Nat. Prod. 2006, 69,
55−260.
11) Liu, X.-T.; Shi, Y.; Yu, B.; Williams, I. D.; Sung, H. H.-Y.; Zhang,
Q.; Liang, J.-Y.; Ip, N. Y.; Min, Z. D. Planta Med. 2007, 73, 84−90.
12) Liu, X.-T.; Shi, Y.; Liang, J.-Y.; Min, Z.-D. Zhongguo Tianran
Yaowu 2009, 7, 341−345.
13) Tu, Y.; Jeffries, C.; Ruan, H.; Nelson, C.; Smithson, D.; Shelat,
developed with CHCl −MeOH−H O (6:4:1).
3
2
Alkaline Hydrolysis of Compound 2. A solution of 2 (1.0 mg)
in MeOH−1% KOH (1:1, 200 μL) was kept at room temperature for
3
(
1
5 min. The reaction mixture was directly subjected to TLC analysis
using CHCl −MeOH (95:5) as a developing system. Compound 1
was detected with an R value of 0.3.
3
2
(
1
(
f
In Vitro Antifungal Assay. A modified version of the CLSI
formerly NCCLS) method was used for susceptibility testing.
(
Organisms (C. neoformans ATCC 90113 and C. gattii ATCC 32609)
were obtained from the American Type Culture Collection (ATCC)
(
(
Manassas, VA, USA). Amphotericin B (ICN Biomedicals, Aurora,
OH, USA) was used as a positive control. The detailed procedure has
been described previously.
18,19
(
In Vitro Cytotoxicity Assay. The two mammalian cell lines Vero
(
5
(
(
African green monkey kidney fibroblast) and LLC-PK (pig kidney
1
epithelial) used in this study were obtained from ATCC. Cytotoxicity
20
was determined by the neutral red method. Doxorubicin (ICN
Biomedicals) was used as a positive control. The detailed procedure
18
1
(
has been described in a previous paper.
ASSOCIATED CONTENT
Supporting Information
■
2
(
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00470.
(
(
Two additional examples for dereplication of known
1
A. A.; Brown, M. K.; Li, X.-C.; Hester, P. J.; Smillie, T.; Khan, I. A.;
antifungal compounds using LC-MS and H NMR;
Walker, L. A.; Guy, R. K.; Yan, B. J. Nat. Prod. 2010, 73, 751−754.
NMR and HRESIMS spectra of compounds 1−3 (PDF)
(14) Cagnoli, N.; Ceccherelli, P.; Curini, M. J. Chem. Res. (S) 1982,
2
54.
AUTHOR INFORMATION
Corresponding Author
Tel: 662-915-6742. Fax: 662-915-7989. E-mail: xcli7@olemiss.
(15) Passannanti, S.; Paternostro, M.; Piozzi, F. J. Nat. Prod. 1984,
7, 885−889.
■
4
(
16) Kidd, S. E.; Hagen, F.; Tscharke, R. L.; Huynh, M.; Bartlett, K.
*
H.; Fyfe, M.; MacDougall, L.; Boekhout, T.; Kwon-Chung, K. J.;
Meyer, W. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17258−17263.
(
edu.
Notes
17) Byrnes, E. J., 3rd; Li, W.; Lewit, Y.; Ma, H.; Voelz, K.; Ren, P.;
The authors declare no competing financial interest.
Carter, D. A.; Chaturvedi, V.; Bildfell, R. J.; May, R. C.; Heitman, J.
PLoS Pathog. 2010, 6, e1000850.
ACKNOWLEDGMENTS
(18) Yang, C.-R.; Zhang, Y.; Jacob, M. R.; Khan, S. I.; Zhang, Y.-J.; Li,
X.-C. Antimicrob. Agents Chemother. 2006, 50, 1710−1714.
■
The authors thank Ms. M. Bennett for extraction of the plant
material, Ms. M. Wright for in vitro antifungal testing, Mr. J.
Trott for cytotoxicity testing, Dr. B. Avula for high-resolution
mass spectrometry analyses, and Mr. F. T. Wiggers for
acquiring the 2D NMR spectra. This work was supported by
the NIH, NIAID, Grant No. AI27094, and the USDA
(19) Li, X.-C.; Jacob, M. R.; Khan, S. I.; Ashfaq, M. K.; Babu, K. S.;
Agarwal, A. K.; Elsohly, H. N.; Manly, S. P.; Clark, A. M. Antimicrob.
Agents Chemother. 2008, 52, 2442−2448.
(20) Borenfreund, E.; Puerner, J. Toxicol. Lett. 1985, 24, 119−124.
E
DOI: 10.1021/acs.jnatprod.5b00470
J. Nat. Prod. XXXX, XXX, XXX−XXX