HIV-1 integrase and neuraminidase inhibitors from alpinia zerumbet

Article abstract of DOI:10.1021/jf104813k

AIDS and influenza are viral pandemics and remain one of the leading causes of human deaths worldwide. The increasing resistance of these diseases to synthetic drugs demands the search for novel compounds from plant-based sources. In this regard, the leav

Full text of DOI:10.1021/jf104813k

ARTICLE
pubs.acs.org/JAFC
HIV-1 Integrase and Neuraminidase Inhibitors from Alpinia zerumbet†
Atul Upadhyay,§ Jamnian Chompoo,§ Wataru Kishimoto,^ Tadahirio Makise,# and Shinkichi Tawata*,^
§Department of Biochemistry and Applied Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University,
Kagoshima 890-0065, Japan
#Makise Clinic, 750 Minamiuehara, Nakagusuku, Okinawa 901-2424, Japan
^Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan
ABSTRACT: AIDS and influenza are viral pandemics and remain one of the leading causes of human deaths worldwide. The
increasing resistance of these diseases to synthetic drugs demands the search for novel compounds from plant-based sources. In this
regard, the leaves and rhizomes of Alpinia zerumbet, a traditionally important economic plant in Okinawa, were investigated for
activity against HIV-1 integrase (IN) and neuraminidase (NA). The aqueous extracts of leaves and rhizomes had IN inhibitory
activity with IC50 values of 30 and 188 μg/mL, whereas against NA they showed 50% inhibition at concentrations of 43 and 57 μg/
mL, respectively. 5,6-Dehydrokawain (DK), dihydro-5,6-dehydrokawain (DDK), and 8(17),12-labdadiene-15,16-dial (labdadiene)
were isolated from the rhizomes and were tested for enzyme inhibitions. DK and DDK strongly inhibited IN with IC50 of 4.4 and 3.6
μg/mL, respectively. Against NA, DK, DDK, and labdadiene exhibited mixed type of inhibition with respective IC50 values of 25.5,
24.6, and 36.6 μM and Ki values ranging from 0.3 to 2.8 μM. It was found that DDK is a slow and time-dependent reversible inhibitor
of NA, probably with a methoxy group as its functionally active site. These results suggest that alpinia could be used as a source of
bioactive compounds against IN and NA and that DK and DDK may have possibilities in the design of drugs against these viral
diseases.
KEYWORDS: Alpinia zerumbet, 5,6-dehydrokawain, dihydro-5,6-dehydrokawain, 8(17),12-labdadiene-15,16-dial, anti-HIV-1
integrase, antineuraminidase
’ INTRODUCTION
Alpinia [Alpinia zerumbet (family Zingiberaceae)] is a per-
ennial ginger growing widely in the subtropics and tropics. It is
used in folk medicine for its anti-inflammatory, bacteriostatic,
and fungistatic properties.7 The essential oil extracted from its
leaves possessed both relaxant and antispasmodic actions in rat
ileum.8 Early reports have shown the R-pyrones, dihydro-5,6-
dehydrokawain (DDK), and 5,6-dehydrokawain (DK), are major
compounds in alpinia leaves, and they have shown plant growth
inhibition against lettuce seeds,9 insecticidal activity against
Coptotermes formosanus, and antifungal activity against Pythium
sp. and Corticium rolfsii.10 The aqueous extract of its leaves has
demonstrated hypotensive activity,11 mainly due to flavonoids
and kava pyrones.12 Furthermore, DK and DDK are reported to
inhibit the aggregation of ATP release from rabbit platelets.13 DK
and DDK are described to have antiulcerogenic and antithrom-
botic activities.12 The inhibitory properties of DK on human
platelet aggregation, anti-inflammatory, and cancer chemopre-
ventive therapeutic properties are reported.14 Labdadiene was
traditionally used as a medicine against inflammatory diseases.15
The cardiovascular effects induced by labdadiene were evaluated
in male Wistar rats.16 Moreover, labdadiene has also been
reported to inhibit lipid peroxidation, cyclooxygenase enzymes,
and human tumor cell proliferation.17 Our laboratory has
Acquired immunodeficiency syndrome (AIDS) represents
one of the most important modern epidemics, with over 40
million people infected worldwide. The replication of HIV
requires three enzymes: protease, integrase (IN), and reverses
transcriptase. Insertion of a viral genome inside the infected cell is
mediated through the encoded enzyme IN, which has been a
rational target for treating HIV inhibition.1 Synthetic IN inhibi-
tors such as raltegravir (also known as isentress or MK-0518) are
being used in reducing HIV load.2 On the other hand, the
impingement of influenza on human health is undeniably esca-
lating, its impact being more serious during the winter season.3
Human cases of avian influenza and, more recently, the outbreak
of the aggressive porcine A/H1N1 strain in 2009 have heigh-
tened awareness of the threat of pandemic. The disease is
associated with a RNA virus that contains hemagglutinin and
neuraminidase (NA) as surface antigens. NA is involved in the
release of progeny virus from infected cells, by cleaving sugars
that bind the mature viral particles.4 Specifically, NA cleaves the
R-ketosidic bond that links a terminal neuraminic acid residue to
the adjacent oligosaccharide moiety. NA is therefore essential for
the movement of the virus to and from sites of infection in the
respiratory tract.5,6 With the ever-present threat of a pandemic
derived from the HIV and influenza viruses and the emergence of
strains resistant to synthetic drugs, the importance of the search
for novel compounds from plant-based source intensifies.
Special Issue: Casida Symposium
Received: May 11, 2010
† Part of the Symposium on Pesticide Toxicology in Honor of Professor John
Casida.
Accepted: December 27, 2010
Published: February 09, 2011
r
2011 American Chemical Society
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Figure 2. Chemical structures of (a) DK, (b) DDK, (c) DDK-OH, (d)
labdadiene, and (e) 4MHP.
Figure 1. Mu-chi, a traditional food of Okinawa. A rice ball or paste is
wrapped in alpinia leaves and served after steaming.
recorded on a JEOL JNM-ECA600 (JEOL, Japan) in CDCl3. Chemical
shifts are expressed in parts per million (δ) relative to TMS. 2D NMR
experiments (H, C-COSY, HMQC, HMBC) were obtained using
standard pulse sequences. DDK: electron ionization mass spectrometry
(EIMS), m/z 230 [M]þ (30), 202 (8), 125 (30), 111 (28), 91 (100), 69
reported DDK and phenolic compounds and their antioxidant
activities in the leaves and rhizomes of the plant.18
1
(12); H (CDCl3), δ 2.73-2.76 (m, 2H, CH2), 2.96-2.97 (m, 2H,
In Okinawa, alpinia leaves have been used to prepare a
traditional food, mu-chi (see Figure 1), and it is common folklore
that it prevents the common cold.19 In this study, we primarily
focused on investigating the IN and NA inhibitory activities of
aqueous extracts of alpinia leaves and rhizomes and three
compounds isolated from rhizomes. To the best of our knowl-
edge this is the first report on IN and NA inhibitory activities of
alpinia and the associated secondary metabolites. Furthermore,
in this study, we also exhibited the probable mechanism of NA
inhibition by DDK.
CH2), 3.77 (s, 3H, CH3), 5.42 (s, 1H, CH), 5.72 (s, 1H, CH), 7.18-7.29
(m, 5H, aromatic); 13C NMR (CDCl3), δ 32.80 (CH2), 35.42 (CH2),
55.80 (OCH3), 87.68 (CH), 100.25 (CH), 126.41, 128.57, 128.57,
128.27, 128.27, 139.82 (aromatic), 164.32 (CH), 164.93 (CH), 170.0
(C).21 DK was characterized by the following data: EIMS, m/z 228
[M]þ (60), 200 (20), 157 (35), 129 (20), 69 (20), 44 (35), 40 (100); 1H
(CDCl3), δ 3.79 (s, 3H, CH3), 5.51 (d, 1H, CH), 5.97 (s, 1H, CH),
6.81 (d, 1H, CH), 7.31 (d, 1H, CH), 7.32 (m, 5H, aromatic); 13C NMR
(CDCl3), δ 163.28 (C), 88.65 (CH), 171.21 (C), 100.95 (CH), 160.3
(C), 118.53 (CH), 135.36 (C), 131.88, 128.27, 129.75, 129.27, 127.98
(aromatic), 56.24 (OCH3).22 Labdadiene was isolated by another group
in our laboratory from the rhizomes of alpinia (unpublished results).
DDK-OH was synthesized by stirring DDK in concentrated HCl for
18 h followed by overnight crystallization at 8 ꢀC.10
’ MATERIALS AND METHODS
Preparation of Plant Extracts and Isolation of Com-
pounds. The leaves and rhizomes of alpinia were collected from the
University of the Ryukyus campus, Okinawa. Twenty grams of fresh
leaves or rhizomes was separately boiled in 1 L of water for 30 min, and
the cooled extract was filtered and dried under vacuum at 40 ꢀC. All of
the residues were weighed and dissolved in 50% DMSO to make a stock
solution of 1 mg/mL before the IN and NA inhibition of crude extracts
was tested. To isolate the active compounds, we used the rhizomes
of alpinia. Our laboratory has been working with two pyrone com-
pounds10,18-20 (see Figure 2), and thus we chose first to investigate the
anti-IN and -NA properties of these compounds. For the isolation of
these compounds, 2 kg of fresh alpinia rhizomes was boiled in 10 L of
water for 20 min. The cooled extract was filtered and reduced to 1 L
under vacuum at 40 ꢀC. Further extraction with hexane (500 mL ꢀ 3)
was done, and the hexane fraction was evaporated to complete dryness
under vacuum. The dried extract was boiled in water and filtered hot.
The residue obtained was purified by preparative HPLC to obtain DK
using a TSK gel ODS-100Z column (Tosoh Corp., Japan) (15 ꢀ 0.46
cm i.d.; 5 μm particle size) monitored continuously at 280 nm. The
mobile phase consisted of 0.1% acetic acid and MeOH, which was
increased from 50 to 100% in 20 min at a flow rate of 0.8 mL/min. For
DDK, the filtrate was crystallized at 4 ꢀC, which was further purified
using preparative HPLC as above. Compounds were identified using
NMR and GC-MS. The amounts of the purified compounds were
weighed and expressed as milligrams per 100 g of fresh rhizomes. DDK
and DK were determined to be 24.1 and 18.8 mg/100 g fresh rhizomes.
HIV-1 Integrase Assays. IN inhibition was determined using a
multiplate integration assay.23 Fifty microliters of biotinylated-LTR
donor DNA was added to a streptavidin-coated 96-well microtiter plate
and incubated for 60 min at room temperature. After discarding the
solution, IN buffer (12 μL), target DNA (5 pmol), sterilized water (32
μL), inhibitors (6 μL), and IN (180 ng) were added to wells and
incubated at 37 ꢀC for 80 min. After the completion of reaction,
unbound DNA was removed by washing with PBS containing 0.5%
Tween 20 and PBS alone. The relative activity of bound DNA was
determined using an alkaline phosphatase labeled anti-digoxigenin
antibody (50 mU) after incubation at 37 ꢀC for 1 h. The resulting
yellow color was measured using a microplate reader at 405 nm. A
control contained enzyme without sample, whereas the blank did not
contain enzyme.
The percent inhibition against IN was calculated as
% inhibition ¼½ODcontrol - ODsampleꢁ=ODcontrol ꢀ 100
where OD = absorbance detected from each well.
NA (Clostridium perfringens) Inhibition Assay. The enzyme
assay was performed as reported with slight modifications.24 Briefly,
4-methylumbellifery-l-R-D-N-acetylneuramic acid sodium salt hydrate
(Sigma, M8639), 0.1 mM in 50 mM sodium acetate buffer (pH 5.0), was
used as substrate. NA (Sigma, N2876), 0.1 U/mL in acetate buffer, was
used as enzyme source. All of the inhibitors were dissolved in MeOH and
diluted to the appropriate concentration in acetate buffer. Fifty micro-
liters of enzyme was added to 20 μL of inhibitor mixed with 80 μL of
1
The H NMR (600 MHz) and 13C NMR (150 MHz) spectra were
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Table 1. IN and NA Inhibitory Activities of Different Extracts
rhizomes of alpinia. The IC50 values of the extract and the
isolated compounds are shown in Table 1. When the inhibition
by isolated compounds was checked, DDK (IC50 = 24.6 ( 0.4
μM) and DK (IC50 = 25.5 ( 0.7) both had more potent activity
than the positive control quercetin25 with an IC50 of 34.7 ( 0.9
and Isolated Compoundsa
NA inhibition
IN inhibition
μM under these conditions (see Table 1). Labdadiene (IC50
=
test sample
leaf
IC50
IC50
type of inhibition Ki
36.6 ( 1.0) had activity similar to that of quercetin. The kinetic
studies of individual compounds showed mixed type of inhibition
(see Figure 3) with estimated Ki values ranging from 0.2 to
2.8 μM.
30 ( 1 μg/mL
43 ( 1 μg/mL
57 ( 1 μg/mL
nt
nt
rhizomes 188 ( 2 μg/mL
nt
nt
DK
4.4 ( 0.5 μg/mL a 25.5 ( 0.7 μM a
3.6 ( 0.9 μg/mL a 24.6 ( 0.4 μM a
mixed
mixed
mixed
nt
0.3 μM
2.8 μM
0.6 μM
nt
DDK
’ DISCUSSION
labdadiene nt
DDK-OH nt
36.6 ( 1 μM b
525.6 ( 11.3 μM
822.9 ( 7.2 μM
nt
The present study examines the inhibitory properties of
aqueous extract of leaves and rhizomes of alpinia against IN
and NA. DK, DDK, and labdadiene were isolated from the
rhizomes. To determine the inhibitory activity against IN, we
carried out strand transfer inhibition assay using a microtiter
plate method. Strand transfer is the second step of the
integration reaction, which corresponds to the ligation of
the viral 30-OH cDNA ends to the 50-DNA phosphate of an
acceptor DNA.1 In our result we found the leaf aqueous
extract had higher activity than the rhizome extract. This was
also true with NA inhibition (Table 1). The leaves and
rhizomes of alpinia have been a source of a wide variety of
bioactive constituents. The content of phenolics and flavo-
noids in the leaves and rhizomes of alpinia have been
extensively investigated.9-12,19 Our previous work reported
the presence of phenolic compounds in the leaves and
rhizomes of alpinia.18 These groups of compounds have
always been a promising class of molecules against a variety
of diseases. Therefore, we have supposed that IN or NA
inhibitory activities of alpinia leaves and rhizomes may be
attributed to the phenolic compounds. Besides, several com-
pounds present in other species of alpinia have been reported
to have IN or NA inhibitory properties.26-28 Furthermore,
4MHP
nt
nt
nt
suramin
quercetin
2.3 ( 0.7 μg/mL a
nt
nt
nt
34.7 ( 0.9 μM b
nt
nt
a Different letters in the same column indicate the existence of significant
difference (Tukey test). nt, not tested.
acetate buffer in a microplate. Reaction was started by adding 50 μL of
substrate, and fluorescence was measured using a GENIOS fluorescence
meter, Wako, Japan. The excitation wavelength was set at 360 nm, and
the emission wavelength was set at 450 nm. For kinetic studies, we used a
time-driven protocol with initial velocity recorded over a range of
substrate concentrations for different inhibitor concentrations (0, 10,
15, and 20 μM). Dixon plots were obtained by plotting the slopes of the
obtained line (Km/Vm) against substrate concentrations. For time-
dependent studies, we obtained progress curves for 600 s at several
preincubation times using 25 μM DDK, and the slopes of lines obtained
were plotted against preincubation time. To obtain the effect of enzyme
concentrations, we used different enzyme concentrations over a range of
inhibitor concentrations. All of the data were analyzed using Microsoft
Excel Office, 2007.
The inhibition was calculated using
% inhibition ¼½1-ðS - S0Þ=ðC - C0Þꢁ ꢀ 100
isolation and identification of specific compounds active
against IN and/or NA from alpinia are being undertaken in
our laboratory. Hence, once it was confirmed that the aque-
ous extract of the leaves and rhizomes of alpinia had con-
siderable activity, we explored the secondary metabolites
where S and C represent relative fluorescence units (RFU) for sample
and control after reaction time and S0 and C0 are RFU at zero time.
Statistical Treatment. All of the experiments were conducted in
triplicates and repeated twice. The data represent the mean ( the
standard deviation (SD) of six results. The IC50 value was determined
graphically as the concentration of each sample required to give 50%
inhibition activity. For kinetic studies of NA inhibition, all calculations
were performed in Excel, Microsoft Office 2007. For significance
analysis, the data were analyzed by one-way ANOVA and the means
were separated using Tukey's HSD range test at p = 0.01. All statistical
analyses were performed using SPSS version 16.0 for Windows Vista.
present in the plant.
Two compounds, DK and DDK, were tested against IN
activity. Both of these compounds had activities similar to that
of suramin (p = 0.01), which is widely used as a positive control
against IN inhibition assays in vitro.23 Our results indicated that
both DK and DDK could be used as possible candidates for IN
inhibition (Table 1). Furthermore, the simple structures and low
molecular weights of DK and DDK would certainly add to their
merits over complex synthetic inhibitors. Besides, we had pre-
viously shown that DK could be metabolized to hispidin in vitro
using CYP2C9.29 Because hispidin has been shown to have IN
inhibitory activity,10 DK seems to act as an anti-IN in two forms,
first as DK itself and second after it has been catalyzed to hispidin
by CYP2C9. The mechanism of IN inhibition by DK and DDK is
still unclear, whereas the activity of labdadiene against IN is yet to
be determined.
’ RESULTS
IN Inhibition Assays. The IN inhibitory activities of different
extracts and the isolated compounds are shown in Table 1. It was
found that the aqueous extract of the leaves of alpinia had
stronger activity than the rhizomes. When inhibition by the
isolated compounds was carried out, IC50 values of DK and DDK
against IN were 4.4 ( 0.5 and 3.6 ( 0.9 μg/mL, respectively.
Suramin had an IC50 of 2.3 ( 0.7 μg/mL under the stated
conditions.
NA Inhibition Activities. The water extracts of alpinia leaves
and rhizomes showed considerable NA activity, with IC50 values
of 42.7 ( 1.2 and 57.1 ( 1.1 μg/mL, respectively. In the case of
NA inhibition, we also used labdadiene isolated from the
The IC50 values of the investigated compounds against NA
indicate that DK and DDK have significantly better inhibition
properties, whereas labdadiene had similar activity against NA
when compared to quercetin (Table 1). Our results therefore
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Figure 3. Effect of DK (A), DDK (B), and labdadiene (C) on neuraminidase inhibition. (Left) Lineweaver-Burk plot in the presence of compounds at
concentrations of 0, 10, 15, and 20 μM. Neuraminidase inhibition was assayed as described in the text. (Right) Secondary plot of Lineweaver-Burk plot.
The slopes were plotted against respective compound concentrations. The intercept on the x-axis gives an estimate of Ki.
suggest that DK, DDK, and labdadiene could be used effectively
against NA.
Furthermore, in the case of NA inhibition by R-pyrone,
Grienke et al. have reported that of four diarylheptanoids isolated
from A. katsumadai, the most potent NA inhibitory compound
had an R-pyrone moiety along with an additional phenyl group.27
However, the authors have not discussed whether the inhibition
was due to the pyrone moiety or some other functional groups.
To investigate the active functional group of DDK, we examined
NA inhibition by the R-pyrone compound 4-methoxy-6-methyl-
2H-pyran-2-one (4MHP), a moiety present in DDK. Interest-
ingly, the activity of 4MHP against NA was very low, with an IC50
= 822.9 ( 7.2 μM. This made us think that, for DDK, the pyrone
group may not have an effective role in inhibiting NA. Our next
step was to determine whether the ethylbenzene group or the
methoxy group of DDK is active against NA. For this, we
synthesized a compound, DDK-OH (see Figure 2), and exam-
The role of pyrone in inhibiting IN and NA is reported.
Four naphtha-γ-pyrones belonging to the chaetochromin and
ustilaginoidin family were identified as IN inhibitors.30 On the
other hand, all compounds containing pyrone rings do not show
inhibitory properties. A structure-activity relationship study of
32 different flavones with γ-pyrone showed that the activity
depends on the charge of the γ-oxygen atom of the pyrone ring.31
However, in our study we used DK and DDK, which are R-pyrone
compounds. Tipranavir, an R-pyrone compounds, has been shown
to have potent HIV protease inhibition32 and is a drug approved by
the U.S. FDA. Although yet to be confirmed, this information
might suggest that the IN inhibition of DK and DDK may be
attributed to the presence of the R-pyrone group.
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Figure 4. Effect of preincubation time on hydrolysis of substrate by NA:
(A) time-dependent inhibition of NA in the presence of 25 μM DDK;
(B) decrease in slopes of the lines of panel A as a function of time.
Figure 5. Effect of enzyme concentration on NA inhibition: (A) typical
plot of residual activity of NA at various concentrations (0-0.4 U/mL)
in the presence of DDK at 25 μM; (B) hydrolytic activity of NA as a
function of enzyme concentration at different concentrations of DDK.
ined its activity against NA. The IC50 of DDK-OH (525.6 ( 11.3
μM) was better than that of 4MHP but was >100-fold weaker
than DDK. These two results suggest that the active functional
group in DDK is probably the methoxy group present in the C-5.
Hence, it is likely that for DDK R-pyrone has very less function-
ality in inhibiting NA.
The plausible explanations of inhibition of NA by labdadiene are
still under investigation. However, it is noteworthy that this com-
pound contains double aldehyde groups in C-15 and 16 and that the
commercial influenza drug Tamiflu also has an aldehyde group.
Researchers have tried to develop active compounds against NA by
adding various aldehyde groups to the carboxylate and acetomido
moiety of Tamiflu to increase the inhibitory properties.33
The kinetics studies of individual compounds showed mixed
type of inhibition (see Figure 3). These types of inhibition are
quite common with natural compounds. The predominant in-
hibition mode shown by naturally occurring NA inhibitors is
noncompetitive. However, the compounds isolated from alpinia
exhibited a mixed type of inhibition, thereby indicating a different
class of compounds identified against NA. The estimated Ki
values for DK, DDK, and labdadiene are 0.2, 2.8, and 0.6,
respectively (Table 1). The low Ki of DK along with its low
IC50 value certainly makes it a potent compound against NA.
We further investigated the inhibitory mechanism of DDK at
its IC50 concentration. We explored the effect of preincubation
time on the inhibition of the hydrolysis of neuramic acid. Because
the decrease in residual activity was observed with increasing
preincubation time, DDK emerged as a slow-binding inhibitor at
low concentrations (see Figure 4A). Furthermore, increasing
preincubation time of DDK also led to a decrease in the slope,
thereby indicating reduction in both initial and steady state
velocity (see Figure 4B). This result along with low Ki indicates
that DDK is a slow binder to NA. DDK therefore is more like the
drug Tamiflu, which is also a slow and time-dependent inhibtior of
NA.34 Moreover, when the effect of enzyme concentration on the
inhibition was probed, it was found that the residual activity of NA
increased with the enzyme concentration at fixed substrate amount
(see Figure 5A). Plots of residual enzyme activity versus enzyme
concentration at different concentrations of DDK gave a family of
straight lines passing through the origin, indicating that DDK is a
reversible inhibitor (see Figure 5B). Hence, we may say that in our
study DDK acted as a time-dependent, slow, reversible inhibitor of NA.
To sum up, we found that the leaves and rhizomes of alpinia
had IN and NA inhibitory activities. We showed that compounds
isolated from alpinia had significant properties against these
enzymes and discussed the mechanism of inhibition of NA by
DDK. Our results indicate that DK and DDK could be used as
sources of IN and NA inhibitors; however, further research is
necessary to use them as lead candidates in drug design.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: b986097@agr.u-ryukyu.ac.jp.
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’ ACKNOWLEDGMENT
(19) Tawata, S.; Fukuta, M.; Xuan, T. D.; Deba, F. Total utilization
of tropical plants Leucaena leucocephala and Alpinia zerumbet. J. Pestic.
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(20) Elzaawely, A. A.; Xuan, T. D.; Tawata, S. Changes in essential
oil, kava pyrones and total phenolics of Alpinia zerumbet (Pers.) B.L.
Burtt. & R.M. Sm. leaves exposed to copper sulphate. Environ. Exp. Bot.
2007, 59, 347–353.
We express sincere thanks to Professor John E. Casida at the
University of California, Berkeley, for his useful comments and
suggestions.
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