Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants

Article abstract of DOI:10.1021/acs.jnatprod.5b00874

Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.

Full text of DOI:10.1021/acs.jnatprod.5b00874

Article
pubs.acs.org/jnp
Identification of Compounds with Efficacy against Malaria Parasites
from Common North American Plants
Shengxin Cai,†,‡,# April L. Risinger,§,⊥,# Shalini Nair,∥ Jiangnan Peng,§ Timothy J. C. Anderson,∥
Lin Du,†,‡ Douglas R. Powell,† Susan L. Mooberry,*,§,⊥ and Robert H. Cichewicz*,†,‡
‡
†Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, and Natural Products Discovery Group,
and Institute for Natural Products Applications and Research Technologies, University of Oklahoma, Norman, Oklahoma 73019,
United States
§Department of Pharmacology and ⊥Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio,
Texas 78229, United States
∥Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas 78227, United States
S
* Supporting Information
ABSTRACT: Some of the most valuable antimalarial compounds,
including quinine and artemisinin, originated from plants. While these
drugs have served important roles over many years for the treatment of
malaria, drug resistance has become a widespread problem. Therefore, a
critical need exists to identify new compounds that have efficacy against
drug-resistant malaria strains. In the current study, extracts prepared from
plants readily obtained from local sources were screened for activity
against Plasmodium falciparum. Bioassay-guided fractionation was used to
identify 18 compounds from five plant species. These compounds
included eight lupane triterpenes (1−8), four kaempferol 3-O-rhamno-
sides (10−13), four kaempferol 3-O-glucosides (14−17), and the known
compounds amentoflavone and knipholone. These compounds were
tested for their efficacy against multi-drug-resistant malaria parasites and
counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3)
isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive
and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as
compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains
can be identified from native and introduced plant species in the United States, which traditionally have received scant
investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.
n 2013, malaria infections impacted an estimated 198 million
people across 97 different countries, resulting in an
infectious organism. While several promising new antimalarial
compounds are being explored,5,6 the high rate of attrition for
candidate molecules under clinical investigation suggests that
additional efforts are needed to develop alternative therapeutic
strategies.
The majority of approved treatment options for malaria are
either plant-derived natural products or synthetic analogues of
plant-derived compounds. Quinine, originally obtained from
the bark of the South American cinchona tree, was introduced
to Europe in the 17th century as an antimalarial treatment by
Jesuit priests returning from Peru.7 Chloroquine has been
widely used for over 50 years before resistance became
widespread. Artemisinin, derived from sweet wormwood
(Artemisia annua), clears parasites from the blood of patients
I
estimated 584 000 deaths.1 While several related Plasmodium
spp. are implicated in the disease, Plasmodium falciparum is
responsible for the majority of deaths. No effective vaccine has
been developed for malaria, which has meant that small-
molecule therapeutics must continue to fulfill the treatment
needs of infected patients and at-risk individuals. There are four
major classes of compounds that are used clinically to treat
malaria: (1) quinolines (e.g., chloroquine, quinine, meflo-
quine), (2) antifolates (e.g., sulfadoxine, pyrimethamine), (3)
artemisinin derivatives (e.g., artesunate, artemether), and (4)
antimicrobials (e.g., doxycycline); however, resistance to the
most widely used agents from the first three major drug classes
is now widespread.2 Specifically, resistance to artemisinin
derivatives, which form the basis of widely used combination
therapies for malaria, is now common across Southeast Asia.3
The further spread of artemisinin resistance is anticipated to be
disastrous for malaria control efforts.4 It is therefore imperative
that new classes of drugs be developed for the treatment of this
Special Issue: Special Issue in Honor of John Blunt and Murray
Munro
Received: October 1, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00874
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
more rapidly than any other known antimalarial.8 Artemisinin
derivatives are now widely used as part of combination
therapies with other drugs such as lumefantrine or mefloquine.
Even atovaquone, which is combined with proguanil in the drug
Malarone, is an analogue of the plant-derived compound
lapachol from Tabebuia sp. (Bignoniaceae). The recent
identification of the alkaloid tazopsine from a Madagascan
plant (Strychnopsis thousarsii), which kills the liver stage of
malaria, further supports the continued exploration of plants for
antimalarial leads.9 Despite these successes, only a small
fraction of the world’s plant species have been screened for
antimalarial activity. Furthermore, the plants that have been
tested tend to have been obtained solely from the flora of
malaria-endemic countries or have been selected based on a
history of medicinal use against the disease.10,11
The intent of this study was to expand the diversity of plants
tested for bioactive compounds that are active against malaria
by focusing on readily available botanical resources from our
local environment in the south-central United States.
Specifically, we have focused exclusively on plants that grow
well in the sometimes harsh environment of southern Texas.12
Our studies have yielded several potent and selective inhibitors
of drug-resistant P. falciparum, which highlight the value of local
North American plants as a resource for bioactive natural
product leads for malaria.
guided fractionation based on their capacity to specifically
inhibit the growth of the malaria parasite with minimal or no
toxicity toward human cells. It is worth noting the important
role that the extraction methods played in liberating the active
compounds from the freeze-dried plant material; among the
hits, only a single extract from a given plant yielded a bioactive
hit. In other words, the different extraction methods performed
on a given plant captured unique sets of metabolites that were
distinct enough from one another so as to preclude the
possibility that a single extraction method would have sufficed
to produce the hits detected in this project.
Our investigation of the compounds responsible for the
antimalarial activities of the active extracts was led by an HPLC-
based microtiter plate fractionation process13 in which
approximately 5 mg of extract was separated into 92 wells of
a 96-well plate. The fractions were split with half the material
used for bioassay testing, and the other half was retained for
LCESIMS analysis of the active wells. This approach enabled us
to use the LCESIMS data to rapidly guide the follow-up
metabolite purification process. We used this tactic to purify a
total of 18 compounds that exhibited varying degrees of
antimalarial activity, including knipholone,14 amentoflavone,15
eight lupane triterpenes [1−8; compounds 3−5 and 7−8 are
new, whereas 1 (23-O-(Z)-p-coumaroyl-23-hydroxybetulin), 2
(23-O-(E)-p-coumaroyl-23-hydroxybetulin), and 6 (3-O-(E)-p-
coumaroyl-23-hydroxybetulin) were reported earlier this year
by the Kinghorn group],16 four known kaempferol 3-O-
rhamnosides (10−13),17 and four known kaempferol 3-O-
glucosides (14−17).18−20
RESULTS AND DISCUSSION
■
A collection of 1281 plant extracts was generated from 463
species of plants that grow in Texas, USA. The extracts were
generated using supercritical fluid (CO2) extraction with or
without methanol, as well as traditional aqueous extractions to
capture as broad a range of metabolites as possible from each
sample. For the initial screening, extracts were tested for their
abilities to inhibit the growth of the drug-sensitive HB3 strain
of P. falciparum at a concentration of 20 μg/mL. Using this
delimiting parameter, five extracts were prioritized for bioassay-
Eight lupane triterpenes (1−8, of which 3−5 and 7−8 are
new analogues) were purified from the methanolic supercritical
extract of the common boxwood, Buxus sempervirens L.
(Buxaceae), after their detection using our microtiter plate
fractionation process. Compound 1 was obtained as colorless,
block-shaped crystals, and its molecular formula was established
as C39H56O5 based on HRESIMS data. The planar structure of
B
DOI: 10.1021/acs.jnatprod.5b00874
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Spectroscopic Data for Compounds 1, 3−5, 7, and 8 (δ in ppm, J in Hz)
ab
,
cd
,
cd
,
ad
,
cd
,
cd
,
no.
1
3
4
5
7
8
1
2
1.50, m
1.74, m
1.00, m
1.67, m
1.63, m
0.83, m
1.58, m
1.60, m
0.88, m
1.67, m
1.59, m
1.73, m
1.73, m
0.75, m
1.45, m
1.02, m
1.76, m
1.02, m
1.76, m
3
5
6
3.29, dd (9.6, 6.0)
0.86, m
3.61, dd (4.9, 11.2)
1.20, m
3.46, dd (5.2, 11.8)
0.95, m
4.78, dd (5.0, 11.8)
1.21, m
4.95, m
1.32, m
1.48, m
4.90, m
1.32, m
1.48, m
1.25, m
1.46, m
1.35, m
1.40, m
1.28, m
7
1.12, m
1.41, m
1.45, m
1.20, m
1.23, m
1.40, m
1.42, m
1.60, m
1.38, m
1.44, m
1.45, m
1.60, m
1.38, m
1.44, m
1.45, m
1.06, m
1.27, m
9
1.12, m
1.40, m
1.46, m
1.31, m
11
1.29, m
1.35, m
1.12, m
1.16, m
12
1.55, m
1.70, m
1.65, m
1.60, m
1.69, m
1.69, m
0.95, m
13
15
1.54, m
1.70, m
1.66, m
1.60, m
1.68, m
1.78, m
1.05, m
1.96, m
1.19, m
1.61, m
2.42, m
1.95, m
1.35, m
1.91, m
0.99, m
1.68, m
1.78, m
1.05, m
1.96, m
1.19, m
1.61, m
2.42, m
1.95, m
1.35 m,
1.91, m
0.99, m
1.55, m
1.76, m
1.58, m
1.68, m
0.78, m
1.01, m
0.91, m
0.91, m
16
1.85, m
1.92, m
1.92, m
1.90, m
0.98, m
1.14, m
1.12, m
1.05, m
18
19
21
1.42, m
1.58, t (11.7)
2.41, m
1.57, t (11.5)
2.41, m
1.48, t (11.8)
2.39 m
2.33, m
1.83, m
1.95, m
1.92, m
1.85, m
1.23, m
1.37, m
1.33, m
1.25, m
22
23
1.83, m
1.89, m
1.90, m
1.84, m
0.85, m
0.99, m
0.98, m
0.88, m
e
e
3.91, d (11.5)
3.87, d (11.5)
0.60, s
4.09, d (11.4)
4.04, d (11.4)
0.77, s
4.08, d (11.4)
3.99, d (11.4)
0.73, s
3.11, d (10.9)
2.93, d (10.9)
0.57, s
3.32
3.28
3.14, d (12.0)
0.80, s
3.10, d (11.5)
0.66, s
24
25
26
27
28
0.75, s
0.92, s
0.87, s
0.82, s
0.95, s
0.92, s
0.91, s
1.08, s
1.03, s
0.98, s
1.09, s
1.08, s
0.74, s
0.97, s
0.83, s
0.94, s
1.04, s
1.04, s
3.47, d (10.7)
3.04, d (10.7)
4.63, d (2.3)
4.51, brs
1.60, s
3.73, d (10.4)
3.27, d (11.1)
4.68, d (2.2)
4.57, brs
1.68, s
3.72, d (10.8)
3.27, d (10.8)
4.68, d (2.2)
4.57, brs
1.69, s
3.51, d (10.8)
3.07, d (10.8)
4.66, d (2.0)
4.53, brs
3.74, d (11.5)
3.74, d (11.5)
e
e
3.29
3.29
29
4.69, brs
4.57, brs
1.70, s
4.69, brs
4.57, brs
1.70, s
30
2′
3′
5′
6′
7′
8′
1.64, s
7.57, d (8.7)
6.72, d (8.7)
6.72, d (8.7)
7.57, d (8.7)
6.82, d (12.9)
5.75, d (12.9)
9.82, s
7.21, d (1.9)
7.66, d (2.0)
7.62, d (8.7)
6.73, d (8.7)
6.73, d (8.7)
7.62, d (8.7)
6.80, d (13.0)
5.71, d (12.8)
9.34, brs
7.18, d (1.8)
7.73, d (1.9)
6.83, d (8.2)
6.77, d (8.2)
6.80, d (8.2)
6.75, d (8.2)
7.08, dd (8.3, 1.9)
7.63, d (15.9)
6.38, d (15.9)
7.08, dd (2.0, 8.3)
6.89, d (12.9)
5.81, d (12.8)
7.05, dd (8.3, 1.8)
7.57, d (15.9)
6.33, d (15.9)
7.08, dd (8.2, 1.9)
6.84, d (12.8)
5.75, d (12.9)
OH-4′
OMe-3′
3.91, s
3.88, s
3.89, s
3.87, s
a
b
c
d
Solvent was DMSO-d6. Performed on a 400 MHz Varian instrument. Solvent was MeOH-d4. Performed on a 500 MHz Varian instrument.
Signal was overlapped with solvent peak.
e
compound 1 was determined by de novo analysis of its NMR
data (Tables 1 and 2 and Figure 1) and, later, by comparing its
NMR data to structurally related metabolites.16 The relative
configuration of the compound was determined based on
ROESY experimental data (Figure 1), which were consistent
with reported lupane triterpene analogues.16 During the course
of the NMR studies, we obtained crystals of compound 1 that
were suitable for X-ray crystallography, which served to confirm
the compound’s planar structure, as well as its relative
configuration (Figure 2). The absolute configuration of 1 was
determined by refinement of the Hooft parameter.21 To further
substantiate the absolute configuration of the metabolite,
compound 1 was hydrolyzed overnight in 1 M NaOH at
room temperature, and the triterpene portion of the molecule
(23-hydroxybetulin [9]) was purified from the hydrolysate.
NMR (Figures S45 and S46, Supporting Information) and
optical rotation ([α]20 +16) data obtained for the hydrolysis
D
product were consistent with that reported for 9 ([α]20
D
+22).22 The proposed absolute configuration is consistent
with an in silico derived specific rotation value for 9 (calcd
+68).23,24
Compound 2 was obtained as a white, amorphous powder,
and the planar structure was determined by comparing its
HRESIMS and NMR data with published data.16 The relative
C
DOI: 10.1021/acs.jnatprod.5b00874
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Spectroscopic Data for Compounds 1, 3−5, 7, and 8
ab
,
bc
,
bc
,
ab
,
cd
,
cd
,
no.
1
3
4
5
7
8
1
38.4 CH2
27.1 CH2
70.4 CH
41.9 C
38.4 CH2
26.1 CH2
71.3 CH
41.9 C
38.2 CH2
26.0 CH2
71.1 CH
41.7 C
38.1 CH2
23.1 CH2
74.2 CH
41.5 C
37.9 CH2
22.9 CH2
74.4 CH
41.6 C
37.9 CH2
22.9 CH2
74.2 CH
41.6 C
2
3
4
5
47.7 CH
18.2 CH2
33.9 CH2
40.9 C
47.8 CH
17.9 CH2
33.7 CH2
40.7 C
47.2 CH
17.9 CH2
33.7 CH2
40.6 C
46.6 CH
17.6 CH2
33.7 CH2
40.8 C
46.5 CH
17.3 CH2
33.3 CH2
40.6 C
46.5 CH
17.3 CH2
33.3 CH2
40.6 C
6
7
8
9
50.5 CH
36.9 C
50.7 CH
36.7 C
50.4 CH
36.6 C
50.1 CH
36.7 C
50.3 CH
36.5 C
50.3 CH
36.5 C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
3′
4′
5′
6′
7′
8′
9′
20.8 CH2
25.3 CH2
37.2 CH
42.5 C
20.6 CH2
25.2 CH2
37.3 CH
42.4 C
20.5 CH2
25.1 CH2
37.2 CH
42.3 C
20.8 CH2
25.2 CH2
37.1 CH
42.7 C
20.6 CH2
25.1 CH2
37.2 CH
42.4 C
20.6 CH2
25.1 CH2
37.2 CH
42.4 C
26.9 CH2
29.5 CH2
47.9 C
26.7 CH2
28.9 CH2
47.5 C
26.5 CH2
28.9 CH2
47.5 C
27.1 CH2
29.5 CH2
47.8 C
26.7 CH2
28.9 CH2
47.8 C
26.7 CH2
28.9 CH2
47.8 C
48.6 CH
47.8 CH
150.9 C
48.6 CH
47.7 CH
150.4 C
48.5 CH
47.7 CH
150.4 C
29.4 CH2
33.6 CH2
48.6 CH
47.7 CH
150.8 C
48.5 CH
47.9 CH
150.3 C
48.5 CH
47.9 CH
150.3 C
29.8 CH2
34.3 CH2
65.6 CH2
12.6 CH3
16.7 CH3
16.2 CH3
14.7 CH3
58.4 CH2
110.1 CH2
19.2 CH3
126.0 C
29.4 CH2
33.6 CH2
65.3 CH2
11.1 CH3
15.6 CH3
15.1 CH3
13.8 CH3
58.9 CH2
108.8 CH2
17.8 CH3
126.2 C
29.7 CH2
34.3 CH2
63.1 CH2
13.6 CH3
16.7 CH3
16.1 CH3
14.9 CH3
58.3 CH2
110.1 CH2
19.2 CH3
125.9 C
29.4 CH2
33.7 CH2
63.1 CH2
12.3 CH3
15.6 CH3
15.1 CH3
13.8 CH3
58.9 CH2
108.8 CH2
17.9 CH3
126.2 C
29.4 CH2
33.7 CH2
63.1 CH2
12.1 CH3
15.6 CH3
15.1 CH3
13.8 CH3
58.9 CH2
108.8 CH2
17.9 CH3
126.3 C
65 CH2
11.1 CH3
15.5 CH3
15.0 CH3
13.6 CH3
58.9 CH2
108.8 CH2
17.7 CH3
126.8 C
132.7 CH
115.4 CH
159.3 C
110.2 CH
148.0 C
113.1 CH
147.0 C
132.9 CH
115.3 CH
159.2 C
110.0 CH
148.0 C
113.3 CH
147.0 C
149.3 C
148.1 C
149.0 C
148.0 C
115.4 CH
132.7 CH
142.9 CH
116.2 CH
166.7 C
115.1 CH
122.8 CH
145.4 CH
114.1 CH
167.7 C
114.4 CH
124.7 CH
142.9 CH
115.6 CH
167.1 C
115.3 CH
132.9 CH
143.1 CH
116.6 CH
166.1 C
115.0 CH
122.7 CH
145.0 CH
114.3 CH
167.7 C
114.0 CH
124.9 CH
144.1 CH
115.8 CH
166.6 C
OMe-3′
55.0 CH3
54.9 CH3
c
55.0 CH3
55.0 CH3
a
b
d
Solvent was DMSO-d6. Performed on a 100 MHz Varian instrument. Solvent was MeOH-d4. Carbon data were determined by a combination of
13C (100 MHz), HSQC (500 MHz), and HMBC (500 MHz) experiments.
Figure 1. Key H−1H COSY, HMBC, and ROESY correlations for
compound 1.
1
Figure 2. ORTEP structure generated from the X-ray diffraction data
for a single crystal of 1.
D
DOI: 10.1021/acs.jnatprod.5b00874
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Journal of Natural Products
Article
Table 3. Bioassay Data of Amentoflavone, Knipholone, and Compounds 1−17
HB3 IC
SE NHP1337 IC50 SE
relative resistance
(NHP1337/HB3)
HeLa IC
SE
fold selectivity
(HeLa/HB3)
fold selectivity
(HeLa/NHP1337)
50 a
a
50b
(μM)
compound
(μM)
(μM)
amentoflavone
25
2
19
5
7
2
0.8
1.0
1.9
0.9
0.5
0.3
0.7
0.7
0.8
1.5
12
46.4 0.4
32.3 0.3
1.9
6.6
2.4
6.5
knipholone
4.9 0.6
0.8 0.4
0.9 0.1
0.5 0.2
1.0 0.1
2.3 0.1
1.9 0.2
3.0 0.3
3.4 0.5
0.6 0.2
2.0 0.6
0.50 0.03
1.8 0.4
0.6 0.1
0.9 0.2
0.8 0.1
2.1 0.9
1
1.53 0.04
0.85 0.05
0.27 0.03
0.31 0.05
1.6 0.1
7
28
1
3
8.8
4.6
2
31
33
3
>20
20
>40
20
>74
65
4 and 3 (3:1)
4
1
5 and 6 (4:1)
22
9.6
14
6
1.4 0.2
>20
>20
>20
>11
>6.7
>5.9
34
>14
>8.7
>3.8
2.9
7 and 8 (2:1)
2.3 0.4
9
5.2 0.3
10
11
12
13
14
15
16
17
7
4
1
1
20.6 0.5
11.9 0.7
9.3 0.2
2.0
8.2
3.9
3.5
5.6
5.0
1.8
6.0
3.0
4.1 0.5
19
2.3
7
1
16
<3
<3
<3
<3
1
8.9
2.3
2.1 0.6
<5.0
<3.3
<3.8
<1.4
<1.4
<0.6
<0.8
<0.8
5
4
1
1
3.8 0.6
a
b
Inhibition of malaria parasites was evaluated using a SYBR green-based assay. Inhibition of HeLa cells was evaluated using the sulforhodamine B
assay.
configuration was confirmed using data from a ROESY
experiment (Figure S1, Supporting Information). On the
basis of these results, as well as biosynthetic considerations,
the shared 23-hydroxybetulin scaffolds in 1 and 2 are presumed
to possess the same absolute configurations.
Compound 3 was purified as a white, amorphous powder.
HRESIMS indicated that this compound had the molecular
formula C40H58O6 ([M − H]− ion at m/z 633.4178, calcd
633.4161). NMR analysis revealed that 3 exhibited 1H and 13C
NMR data (Tables 1 and 2) that were similar to 2 with the
addition of proton (δ 3.91, 3H, s) and carbon (δ 55.0, CH3)
signals indicative of a new methoxy group. Further 2D NMR
analysis confirmed the placement of the methoxy group on the
aromatic ester moiety, thus establishing that 3 is a new trans-
feruloyl-containing analogue of 2.
Compound 4 was obtained as a mixture with compound 3
(ca. ∼3:1 ratio of 4:3). It was observed by LCESIMS that 4
readily interconverted to 3 within several hours of its
purification by HPLC. Therefore, structure determination of
the metabolite proceeded using the compound mixture. Both
compounds were found to bear the same molecular formula
based on HRESIMS data. The 1D NMR data (Tables 1 and 2)
indicated that the two components in the mixture were nearly
identical, with the major difference being that the olefinic trans-
coupled protons in 3 (J = 15.9 Hz) appeared to be cis
configured (J = 12.9 Hz) in 4. Subsequent 2D NMR analysis of
4 confirmed that the remainder of the compound was
structurally identical to 3. On the basis of these data, as well
as their shared biogenic origin, the absolute configuration of the
triterpene portions of both molecules were presumed to be
identical. The cis−trans isomerization of 4 to 3 has precedence
among other feruloyl-containing triterpene metabolites.25,26
Both natural and laboratory-related factors are likely involved in
the interconversion between these two geometric isomers.
While our testing of the plant material remains incomplete, the
available data suggest that both isomers might be present in the
original plant tissues.
Compound 6, which was very recently reported by the
Kinghorn group and named 3-O-(E)-p-coumaroyl-23-hydrox-
ybetulin, was isolated as a white, amorphous powder, and its
structure determined by NMR spectroscopy (Table S1,
Supporting Information) and HRESIMS, as well as by
comparing its experimentally derived data to those published
for structurally related metabolites.16 The assignment of the
position of the trans-p-coumaroyl group was based on an
HMBC correlation from H-3 (δ 4.83 dd, 5.0, 11.2) to C-9′ (δ
166.6 C). Compound 5 was isolated as a mixture with 6 (ratio
of 5:6, ∼4:1). An analysis of its 1D and 2D NMR data (Tables
1 and 2 and Figure S3, Supporting Information) indicated that
the difference between compounds 5 and 6 was also due to
isomerization of an olefinic bond. The trans-configured
coumaroyl group in compound 6 was replaced by a cis-
configured coumaroyl group in 5 based on J-based analysis of
the olefinic protons (J = 16.0 Hz in 6 versus J = 12.9 Hz in 5).
The cis-p-coumaroyl group was determined to be appended to
the C-3 oxygen atom based on an HMBC correlation from H-3
(δ 4.78 dd, 5.0, 11.8) to C-9′ (δ 166.1 C).
Compounds 7 and 8 were also obtained as a mixture in a
∼2:1 ratio. The HRESIMS data revealed that these two
compounds shared the same molecular formula (C40H58O6).
1
The H and 13C NMR data (Tables 1 and 2) indicated that
both components in the mixture were structurally similar and
contained the same lupane triterpene skeleton found in
compounds 5 and 6. The validity of this assessment was
confirmed by a hydrolysis experiment, which yielded triterpene
9 from both metabolites. Subsequent 1D and 2D NMR analysis
indicated that the major component, 7, exhibited resonances
characteristic for a trans-configured feruloyl group connected to
the C-3 oxygen [HMBC correlation from H-3 (δ 4.95 m) to C-
9′ (δ 167.7 C)]. Similarly, the less abundant component, 8,
contained a cis-configured feruloyl group connected to C-3.
The eight lupane triterpenes exhibited modest differences in
their respective potencies (IC50 values ranging from 0.5 to 3.0
μM) against the drug-sensitive HB3 parasites (Table 3). This is
consistent with a report published earlier this year, wherein it
E
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Journal of Natural Products
Article
was disclosed that compounds 1, 2, and 6 were active against
the drug-sensitive P. falciparum Dd2 strain at concentrations
that were similar to those used in our experiments.16 Notably,
the most potent metabolite in our assay was the new
compound 3, which contains a trans-feruloyl group attached
to the C-23 oxygen atom, with an IC50 of 0.5 μM against the
HB3 parasite. Compared to 3, compounds with structurally
related moieties attached to the C-23 oxygen atom, including
cis-feruloyl (4), cis-p-coumaroyl (1), and trans-p-coumaroyl (2)
groups, also inhibited the parasite, but at slightly higher (2-fold
greater) concentrations. In contrast, compounds containing
these substituents on the C-3 oxygen were 4- to 6-fold less
potent than 3, indicating that modifications at this position had
a less desirable influence on their biological profiles.
Interestingly, it was noted that the absence of any feruloyl or
coumaroyl groups resulting in the unadorned lupane triterpene
9 yielded a less potent inhibitor with an IC50 value of 3.4 μM.
The efficacies of the lupane triterpenes were also evaluated
against a multi-drug-resistant clone of P. falciparum that was
recently isolated from the Thailand−Myanmar border. Other
than compounds 1 and 9, the triterpenes were slightly more
potent against the drug-resistant NHP1337 parasites as
compared to the drug-sensitive HB3 strain (Table 3). Overall,
the triterpene derivatives showed selective activities against the
parasites versus human cells with compounds 2 and 3, which
contain trans-p-coumaroyl or trans-feruloyl groups, respectively,
at C-23, having greater than 30-fold selectivity for both the
drug-sensitive and drug-resistant parasites. The potency and
selectivity of 3 against both drug-sensitive and -resistant
parasites further supports that this class of compounds may
be an interesting lead for exploiting as a new class of
antimalarial agents that are chemically distinct from existing
therapeutics.
Four known kaempferol 3-O-rhamnosides were isolated from
the methanolic supercritical fluid extract of Platanus occidentalis
L. (Platanaceae), the American sycamore. The structures of
these metabolites were determined to be kaempferol 3-O-α-L-
(2″,3″-di-E-p-coumaroyl)rhamnoside (10), kaempferol 3-O-α-
L-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)rhamnoside (11),
kaempferol 3-O-α-L-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)-
rhamnoside (12), and kaempferol 3-O-α-L-(2″,3″-di-Z-p-
coumaroyl)rhamnoside (13) based on analysis of their NMR
and HRESIMS data and by comparing these values with data
reported for these compounds.17 The metabolites exhibited
inhibitory activities against the drug-sensitive parasites with
IC50 values ranging from 0.5 to 2.0 μM (Table 3). Among the
kaempferol 3-O-rhamnosides, compound 10 showed the best
selectivity for malaria compared to human cells, but the
compounds were less potent against the drug-resistant malaria
line (Table 3). This resulted in selectivity values of less than 4-
fold for the drug-resistant malaria strains compared to human
cells.
exhibited IC50 values in the range 0.6−2.1 μM against the drug-
sensitive parasite, but were 2 to 5 times less potent against the
drug-resistant strain and showed no selectivity for the parasite
compared to human cells (Table 3). While there has been some
effort to explore flavonoids as antimalarial agents,6 members of
this class have been reported to exhibit a wide variety of
biological activities due to their proclivities for nonspecific
inhibition of various biological targets.27,28 This is consistent
with their less than 5-fold selectivity for malaria as compared to
HeLa cells in our assay, making them less than ideal candidates
for further investigation.
The aqueous extract of Bulbine frutescens Willd. (Asphode-
laceae) yielded the known compound knipholone.14 The active
compound was identified by 96-well microtiter plate fractiona-
tion of the extract, which generated a single bioactive well.
Dereplication was performed based on taxonomic consid-
erations, as well as the metabolite’s UV-PDA profile and MS
data.14 Scale-up processing of the crude extract over silica gel,
HP20ss, and C18 HPLC yielded several milligrams of the pure
metabolite, which enabled the confirmation of its structure by
NMR spectroscopy. Knipholone exhibited an IC50 of 4.9 μM
against the drug-sensitive HB3 parasite and almost identical
potency against the NHP1337 drug-resistant parasite (Table 3).
This is consistent with the reported activity of knipholone
against both drug-sensitive and -resistant parasites.29 Addition-
ally, knipholone showed greater than 6-fold selective activity
against the parasite versus HeLa cells (Table 3), which is also
consistent with published results.29
Another known compound, amentoflavone,15 was obtained
from the methanolic supercritical fluid extract of the evergreen
sumac, Rhus virens Lindheim ex A. Gray (Anacardiaceae).
Amentoflavone was previously demonstrated to have a wide
range of biological activities and was identified in a large-scale
screen for inhibitors of the M1 family of alanyl aminopeptidases
in P. falciparum.30 In the current study, it was determined that
amentoflavone exhibited relatively weak activity against both
drug-sensitive and -resistant parasites and was only 2-fold more
selective toward P. falciparum compared to its activity against
HeLa cells (Table 3).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation data were
determined on a Rudolph Research AUTOPOL III automatic
polarimeter. IR data were collected on a Shimadzu IR Affinity FTIR.
UV data were collected on a Hewlett-Packard 8452A diode array
spectrophotometer. NMR data were collected on Varian 400 and 500
MHz NMR spectrometers. Accurate mass data were collected on an
Agilent 6538 HRESI QTOF MS coupled to an Agilent 1290 HPLC.
LCESIMS data were obtained on a Shimadzu LC-MS 2020 system
(ESI quadrupole) coupled to a photodiode array detector, with a
Phenomenex Kintex column (2.6 μm C18 column, 100 Å, 75 × 3.0
mm). The preparative HPLC system utilized SCL-10A VP pumps and
system controller with a Luna 5 μm C18 column (110 Å, 250 × 21.2
mm, 10 mL/min), and the analytical and semipreparative HPLC
system utilized Waters 1525 binary pumps with Waters 2998
photodiode array detectors and Luna 5 μm C18 columns (110 Å,
250 × 4.6 mm, 1 mL/min and 110 Å, 250 × 10 mm, 4 mL/min). X-ray
data were collected using a diffractometer with a Bruker APEX CCD
area detector and graphite-monochromated Mo Kα radiation (λ =
0.710 73 Å). All solvents were of ACS grade or better.
Additionally, four known kaempferol 3-O-glycosides (14−
17) were purified from the methanolic supercritical fluid extract
of the Lacey oak (Quercus laceyi Small, Fagaceae). The
structures of the flavonoid glycosides were determined to be
kaempferol-3-O-(3″,4″-diacetyl-2″,6″-di-E-p-coumaroyl)-
glucoside (14),18 kaempferol 3-O-(2″-cis-p-coumaroyl-3″,4″-
diacetyl-6″-trans-p-coumaroyl)-β-D-glucopyranoside (15),19
kaempferol-3-O-(2″-trans-p-coumaroyl-3″, 4″-diacetyl-6″-cis-p-
coumaroyl)-β-D-glucopyranoside (16),20 and kaempferol-3-O-
(3″,4″-diacetyl-2″,6″-di-Z-p-coumaroyl)glucoside (17),18 based
on comparisons with published data. Compounds 14−17
For each experiment, a sample of 20 mg of crude plant extract was
dissolved in 2 mL of HPLC grade MeOH, and the suspension was
centrifuged. The supernatant was passed over HP20ss and C18 silica
gel columns (both eluted with MeOH), and the residue resuspended
to yield a 50 mg/mL solution in HPLC grade MeOH. A 100 μL
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Journal of Natural Products
Article
aliquot (5 mg of soluble plant extract material) was loaded onto a C18
semipreparative HPLC column, and fractions were collected in a deep-
well 96-well plate. HPLC separations were performed under gradient
conditions progressing from 30:70 MeOH−H2O to 100% MeOH in
30 min, followed by a column wash with 100% MeOH for another 10
min. The resulting microtiter plate contained 92 wells, which were
each filled with 1.7 mL of eluent. The contents of each well were split
into two plates, and the solvent was removed under vacuum. For
bioassay analysis, the contents in each well of one plate were
resuspended in DMSO for testing, whereas the contents of the second
matched plate were retained for follow-up LCESIMS analysis of the
bioactive components.
λmax (log ε) 206 (4.05), 326 (4.03) nm; IR (film) νmax 2935, 2866,
1
1739, 1689, 1541, 1514, 1454, 1265 cm−1; H and 13C NMR, see
Tables 1 and 2; HRESIMS [M − H]− m/z 633.4162 (calcd for
C40H57O6, 633.4161).
3-O-(cis)-p-Coumaroyl-23-hydroxybetulin and 3-O-(E)-p-cou-
maroyl-23-hydroxybetulin (mixture of 5 and 6, ∼4:1): white,
amorphous powder; [α]20 +27.4 (c 0.18, MeOH); UV (MeOH)
D
λmax (log ε) 208 (4.09), 312 (4.16) nm; IR (film) νmax 3415, 2943,
1
2870, 1641, 1512, 1392, 1165, 1016 cm−1; H and 13C NMR, see
Tables 1 and 2; HRESIMS [M − H]− m/z 603.4074 (calcd for
C39H55O5, 603.4055).
3-O-(E)-p-Coumaroyl-23-hydroxybetulin (6): white, amorphous
powder; [α]20 +24.8 (c 0.25, CHCl3−MeOH, 1:1); UV (MeOH)
Plant Material. The leaves and stems of Bulbine frutescens
(bulbine) and Buxus sempervirens (the common boxwood) were
collected from the San Antonio Botanical Garden (SABG) in June
2005. The leaves and stems of Rhus virens (the evergreen sumac) were
obtained from Natives of Texas Nursery in March 2007. The leaves
and stems of Platanus occidentalis (the American sycamore) and
Quercus laceyi (the Lacey oak) were collected from the SABG in April
2014. Additional re-collections of plant material were obtained from
SABG or local plant nurseries (Casa Verde, Hill Country African
Violets). Voucher specimens of Bulbine frutescens (SLM223), Buxus
sempervirens (SLM245), Rhus virens (SLM316), Platanus occidentalis
(SLM2047), and Quercus laceyi (SLM2046) were made and stored in
the Mooberry laboratory herbarium at UTHSCSA. The remaining
plant materials were rapidly frozen to preserve their chemical integrity
prior to freeze-drying. Half of the plant material was used to generate
two lipophilic extracts by supercritical fluid extraction. The first
lipophilic extract was generated using CO2 only, which was followed
by a second extraction with methanol and CO2 to yield a less
hydrophobic extract. The other half of the plant material was extracted
using 70% H2O and 30% EtOH to generate an aqueous extract.
Extraction and Isolation. The methanolic supercritical fluid
extract (2.5 g) of Buxus sempervirens was fractionated over silica gel
and eluted with hexane−CH2Cl2−MeOH (hexane, 50:50 hexane−
CH2Cl2, CH2Cl2, 90:10 CH2Cl2−MeOH, MeOH). Fractions 4 and 5
were combined, applied to an HP20ss column, and eluted with
MeOH−H2O. The fourth fraction (MeOH−H2O, 90:10) was further
separated over Sephadex LH20 (eluted with MeOH) to yield five
subfractions. Subfraction 3 was separated by C18 preparative HPLC
(gradient elution with MeOH−H2O from 30:70 to 100% organic
phase in 30 min) followed by C18 semipreparative HPLC (MeCN−
H2O, 75:25) to obtain 1 (15.0 mg), 2 (5.5 mg), 3 (0.9 mg), and 4 (1.2
mg, isomerized to a ∼3:1 mixture of 4 and 3). Compounds from the
Sephadex-LH20-derived subfraction 4 were further purified by C18
preparative HPLC (MeOH−H2O gradient from 30:70 to 100%
organic phase in 30 min) and C18 semipreparative HPLC (MeCN−
H2O, 75:25) to yield 5 (8.4 mg) (this isomerized into a 4:1 mixture of
5 and 6), 6 (15.4 mg) (this also isomerized over time into a mixture of
5 and 6), and a 2:1 mixture of 7 and 8 (1.4 mg).
D
λmax (log ε) 208 (3.98), 310 (4.21) nm; IR (film) νmax 2935, 1687,
1
1539, 1514, 1419, 1394, 1157 cm−1; H and 13C NMR, see Table S1,
Supporting Information; HRESIMS [M − H]− m/z 603.4062 (calcd
for C39H55O5, 603.4055).
3-O-(trans)-Feruloyl-23-hydroxybetulin and 3-O-(cis)-feruloyl-23-
hydroxybetulin (mixture of 7 and 8, ∼2:1): white, amorphous
powder; [α]20D +22.9 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 206
(4.10), 326 (4.10) nm; IR (film) νmax 2937, 2870, 1678, 1539, 1454,
1
1269, 1178 cm−1; H and 13C NMR, see Tables 1 and 2; HRESIMS
[M − H]− m/z 633.4178 (calcd for C40H57O6, 633.4161).
23-Hydroxybetulin (9): A 0.5 mL amount of 1 M NaOH was added
to 23-O-(Z)-p-coumaroyl-23-hydroxybetulin (1) (5.0 mg), and the
system was stirred overnight at room temperature. A saturated
aqueous solution of NaCl (2 mL) was added to this mixture and was
further partitioned with EtOAc. The organic phase was evaporated
under reduced pressure. 23-Hydroxybetulin (9) (3 mg) was obtained
after further purification by semipreparative HPLC: white, amorphous
powder; [α]20 +16.0 (c 0.2, CHCl3). ESIMS [M − H2O + H]+ m/z
D
441, [M − 2H2O + H]+ m/z 423, [M + HCOOH − H]− m/z 503; 1H
and 13C NMR, see Figures S45 and S46, Supporting Information.
The methanolic supercritical extract (1.67 g) of Platanus occidentalis
was fractionated by HP20ss column chromatography (eluted with
MeOH−H2O). Fraction 4 was processed by C18 preparative HPLC
(MeOH−H2O gradient from 30:70 to 100% organic phase over 30
min) and C18 semipreparative HPLC (MeCN−H2O, 50:50) to yield
10 (3.0 mg), 11 (1.2 mg), 12 (1.0 mg), and 13 (0.8 mg). The
methanolic supercritical extract (0.95 g) of Quercus laceyi was
fractionated by HP20ss column chromatography (eluted with
MeOH−H2O). Fraction 4 (MeOH−H2O, 90:10) was processed by
C18 preparative HPLC (with a MeOH−H2O gradient from 30:70 to
100% organic over 30 min) and C18 semipreparative HPLC (MeCN−
H2O, 47.5:52.5) to yield 14 (2.0 mg), 15 (1.0 mg), 16 (1.0 mg), and
17 (0.5 mg). The aqueous extract (0.5 g) of Bulbine fruticosa was
fractionated over silica gel (eluted with hexane−CH2Cl2−MeOH).
Fraction 4 was passed over an HP20ss column and eluted with
MeOH−H2O. Subfraction 4 was further processed by C18 preparative
HPLC (MeOH−H2O gradient from 30:70 to 100% organic phase in
30 min) and C18 semipreparative HPLC (isocratic 50:50 MeOH−
H2O) to yield knipholone (3.0 mg). The supercritical/MeOH extract
(4.88 g) of Rhus virens was subjected to fractionation by silica flash
chromatography (gradient elution with CH2Cl2−MeOH). The
fraction eluting with 20:80 MeOH−H2O was further purified by
preparative HPLC using a Phenomenex C18 column with a MeCN−
H2O gradient from 20:80 to 80:20 in 50 min to yield amentoflavone.
The biological activities of amentoflavone were found to be identical
with a commercially obtained sample of amentoflavone from Sigma-
Aldrich (St. Louis, MO, USA).
23-O-(Z)-p-Coumaroyl-23-hydroxybetulin (1): colorless, block-
shaped crystals; [α]20 +80.9 (c 0.21, MeOH); UV (MeOH) λmax
D
(log ε) 208 (4.49), 310 (4.31) nm; IR (film) νmax 2931, 2864, 1739,
1
1689, 1541, 1514, 1454, 1161; H and 13C NMR, see Tables 1 and 2;
HRESIMS [M − H]− m/z 603.4066 (calcd for C39H55O5, 603.4055).
23-O-(E)-p-Coumaroyl-23-hydroxybetulin (2): white, amorphous
powder; [α]20 −3.6 (c 0.45, MeOH); UV (MeOH) λmax (log ε) 208
D
(4.05), 314 (4.25) nm; IR (film) νmax 2937, 2868, 1739, 1689, 1541,
1
1514, 1454, 1165 cm−1; H and 13C NMR, see Table S1, Supporting
Information; HRESIMS [M − H]− m/z 603.4078 (calcd for
C39H55O5, 603.4055).
X-ray Crystal Structure Analysis of 23-O-(Z)-p-Coumaroyl-
23-hydroxybetulin (1). A colorless, block-shaped crystal of
dimensions 0.420 × 0.220 × 0.090 mm was selected for structural
analysis. Intensity data for this compound were collected using a
diffractometer with a Bruker APEX CCD area detector31 and graphite-
monochromated Mo Kα radiation (λ = 0.710 73 Å). The sample was
cooled to 100(2) K. Cell parameters were determined from a
nonlinear least-squares fit of 8005 peaks in the range 2.45° < θ <
26.91°. A total of 27 638 data were measured in the range 1.832° < θ <
26.920° using φ and ω oscillation frames. The data were corrected for
23-O-(trans)-Feruloyl-23-hydroxybetulin (3): white, amorphous
powder; [α]20 +8.9 (c 0.045, MeOH); UV (MeOH) λmax (log ε)
D
206 (4.09), 326 (4.07) nm; IR (film) νmax 3437, 2941, 2868, 1641,
1
1514, 1454, 1392, 1263, 1159, 1018 cm−1; H and 13C NMR, see
Tables 1 and 2; HRESIMS [M − H]− m/z 633.4178 (calcd for
C40H57O6, 633.4161).
23-O-(cis)-Feruloyl-23-hydroxybetulin and 23-O-(trans)-feruloyl-
23-hydroxybetulin (mixture of 4 and 3, ∼3:1 ratio): white,
amorphous powder; [α]20 +86.2 (c 0.065, MeOH); UV (MeOH)
D
G
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absorption by the empirical method,32 giving minimum and maximum
transmission factors of 0.969 and 0.993. The data were merged to form
a set of 7493 independent data with R(int) = 0.0325 and a coverage of
100.0%. The orthorhombic space group P212121 was determined by
systematic absences and statistical tests and verified by subsequent
refinement. The structure was solved by direct methods and refined by
full-matrix least-squares methods on F2.33 The positions of hydrogens
bonded to carbons were initially determined by geometry and were
refined using a riding model. Hydrogens bonded to oxygens were
located on a difference map, and their positions were refined
independently. Non-hydrogen atoms were refined with anisotropic
displacement parameters. Hydrogen atom displacement parameters
were set to 1.2 (1.5 for methyl) times the isotropic equivalent
displacement parameters of the bonded atoms. A total of 406
parameters were refined against 7493 data to give wR(F2) = 0.1081
and S = 0.994 for weights of w = 1/[σ2(F2) + (0.0600P)2 + 0.6000P],
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00874.
■
S
Summaries of 2D NMR correlation data, 1D and 2D
NMR spectra, and HRESIMS data (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Phone: 210-567-4788. Fax: 210-567-4300. E-mail:
mooberry@uthscsa.edu.
*Phone: 405-325-6969. Fax: 405-325-6111. E-mail:
rhcichewicz@ou.edu.
■
2
where P = [Fo + 2Fc2]/3. The final R(F) was 0.0421 for the 6343
Author Contributions
#S. Cai and A. L. Risinger contributed equally to this work.
observed, [F > 4σ(F)], data. The largest shift/s.u. was 0.000 in the final
refinement cycle. The final difference map had maxima and minima of
0.257 and −0.161 e/Å3, respectively. The absolute structure was
determined by refinement of the Hooft parameter21 [Hooft y = 0.3(4),
P2(true) = 0.831, P3(true) = 0.455, P3(false) = 0.093, P3(rac-twin) =
0.453]. The X-ray crystallographic data for 1 have been deposited with
the Cambridge Crystallographic Data Center under accession number
CCDC 1420620. The data can be accessed free of charge at http://
www.ccdc.cam.ac.uk.
Malarial Parasite Inhibition Assays. Samples were evaluated for
their abilities to inhibit the growth of malaria parasites using a SYBR
green-based assay.34 Briefly, an aliquot of plant extract was added to
100 μL of P. falciparum cell culture containing ring stage parasites at
0.5% parasitemia. Test plates were maintained at 37 °C under standard
parasite culture conditions for 72 h, at which time parasite growth was
measured. Each plate contained positive controls to measure
uninhibited parasite growth and negative controls consisting of
uninfected red blood cells to measure background fluorescence. The
percent inhibition was calculated as the reduction in SYBR
fluorescence measured in the presence of plant extract relative to
the positive control. Selective extracts were evaluated a second time to
verify their antimalarial activity and specificity. The relative potencies
of the purified compounds were determined by running full dose−
response curves over concentration ranges covering 3 orders of
magnitude. The IC50 values were calculated from the dose−response
curves.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this project was provided by a pilot studies grant
from the Texas Biomedical Forum and R21AI092235 awarded
to S.L.M. and T.J.C.A. from the National Institute for Allergy
and Infectious Disease, National Institutes of Health (NIH),
Bethesda, MD. This investigation was conducted in facilities
constructed with support from Research Facilities Improve-
ment Program grants C06 RR013556 and RR017515 from the
National Center for Research Resources of the NIH. Parasite
clone NHP1337 was isolated from an infected blood sample
collected on the Thailand−Myanmar border provided by F.
Nosten. T.J.C.A. and S.N. are supported by grant R37AI048071
from the National Institute of Allergy and Infectious Diseases.
The LC-MS instrument used for this project was provided in
part by a Challenge Grant from the Office of the Vice President
for Research, University of Oklahoma, Norman Campus, and
an award through the Shimadzu Equipment Grant Program
(R.H.C.). We thank A. Robles and L. Clark for their assistance
in plant collections and extractions and the San Antonio
Botanical Garden for access to plant samples.
Initial screening of crude plant extracts was conducted with a
laboratory parasite (HB3) that is sensitive to antimalarials (chlor-
oquine, antifolates, mefloquine, quinine, and artemisinin). Purified
compounds showing activity against HB3 were further tested using a
parasite clone recently isolated from the Thailand−Myanmar border.
This parasite is multi-drug-resistant, and its collection was approved by
the ethics review boards of the Faculty of Tropical Medicine, Mahidol
University, Thailand. The genome has mutations encoding chlor-
oquine resistance (chloroquine resistance transporter (pfcrt) K76T)
and artemisinin resistance (kelch-C580Y). Antifolate resistance was
also expected since parasites from this region are generally resistant to
antifolate drugs due to combinations of mutations in both the
dihydrofolate reductase (dhfr) and dihydropteroate synthase genes
(dhps).35 Furthermore, this parasite was isolated from a patient
showing slow parasite clearance following artemisinin combination
therapy, providing further confirmation of artemisinin resistance.
HeLa Cell Line Counterscreen. Each of the plant extracts and
purified compounds that exhibited antimalarial activity was evaluated
against the HeLa human cervical cancer cell line as a first step to
evaluate specificity for malaria versus nonspecific toxicity. HeLa cells
were obtained from the American Type Culture Collection (Manassas,
VA, USA) and cultured in Basal Medium Eagle with Earle’s salts
(Sigma-Aldrich) with 10% fetal bovine serum and 50 μg/mL
gentamicin. The sulforhodamine B assay was used to measure
antiproliferative and cytotoxic activities as previously described.36,37
DEDICATION
■
Dedicated to Professors John Blunt and Murray Munro, of the
University of Canterbury, for their pioneering work on
bioactive marine natural products.
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