Elderberry flavonoids bind to and prevent H1N1 infection in vitro

Article abstract of DOI:10.1016/j.phytochem.2009.06.003

A ionization technique in mass spectrometry called Direct Analysis in Real Time Mass Spectrometry (DART TOF-MS) coupled with a Direct Binding Assay was used to identify and characterize anti-viral components of an elderberry fruit (Sambucus nigra L.) extr

Full text of DOI:10.1016/j.phytochem.2009.06.003

Phytochemistry 70 (2009) 1255–1261
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Elderberry flavonoids bind to and prevent H1N1 infection in vitro
a
b
a
c
a,*
Bill Roschek Jr. , Ryan C. Fink , Matthew D. McMichael , Dan Li , Randall S. Alberte
a
HerbalScience Group LLC, 1004 Collier Center Way, Suite 200, Naples, FL 34110, USA
Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33136, USA
HerbalScience Singapore, Pte. Ltd., 1 Science Park Road, Capricorn, Science Park II, Singapore 117528, Singapore
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A ionization technique in mass spectrometry called Direct Analysis in Real Time Mass Spectrometry
DART TOF-MS) coupled with a Direct Binding Assay was used to identify and characterize anti-viral com-
ponents of an elderberry fruit (Sambucus nigra L.) extract without either derivatization or separation by
Received 9 September 2007
Received in revised form 18 May 2009
Available online 12 August 2009
(
standard chromatographic techniques. The elderberry extract inhibited Human Influenza A (H1N1) infec-
tion in vitro with an IC50 value of 252 ± 34
from the elderberry extract bind to H1N1 virions and, when bound, block the ability of the viruses to
infect host cells. Two compounds were identified, 5,7,3 ,4 -tetra-O-methylquercetin (1) and 5,7-dihy-
droxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate (2), as
H1N1-bound chemical species. Compound 1 and dihydromyricetin (3), the corresponding 3-hydroxyfl-
avonone of 2, were synthesized and shown to inhibit H1N1 infection in vitro by binding to H1N1 virions,
lg/mL. The Direct Binding Assay established that flavonoids
Keywords:
Sambucus nigra L. Caprifoliaceae
Elderberry
Anti-viral flavonoids
DART TOF-MS
Flavonoids
0
0
Influenza
blocking host cell entry and/or recognition. Compound 1 gave an IC50 of 0.13
infection inhibition, while dihydromyricetin (3) achieved an IC50 of 2.8 g/mL (8.7
bition activities of the elderberry flavonoids compare favorably to the known anti-influenza activities of
l
g/mL (0.36
lM) for H1N1
l
lM). The H1N1 inhi-
Ò
Oseltamivir (Tamiflu ; 0.32 lM) and Amantadine (27 lM).
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
The chemical complexity of botanical extracts has made mass
ingatmosphericpressurechemicalionization (Sciex, 1992) andelec-
trospray ionization (Pramanik et al., 2002) mass spectrometric
techniques. Fragmentation of the samples during DART ionization
can be induced by adjusting the mass spectrometer voltages, allow-
ing for more detailed structural information (Cody et al., 2005). Re-
cently, DART TOF-MS was used to determine the molecular
formulae and structures of toxoid compounds in cell cultures of
Taxus wallichiana (Banerjee et al., 2008), and alkaloids expressed in
the hairy roots of Rauvolfia serpentine (Madhusudanan et al., 2008).
spectrometric characterization of whole extracts difficult due to
the lack of reliable extraction methodologies that yield optimized
extracts with dose-to-dose reliable chemical compositions
(Schmidt et al., 2007). A relatively new ionization source in mass
spectrometry, termed DART (Direct Analysis in Real Time) (Cody
et al., 2005), is coupled to a time-of-flight mass spectrometer, mak-
ing it possible to rapidly and accurately identify the chemical com-
ponents in botanicals and extracts at atmospheric pressure,
typically with no sample preparation or processing requirements.
The DART ion source utilizes electronic excited-state species,
suchasmetastableheliumandnitrogenatoms, asplasmas. Theseex-
cited atoms ionize samples directly for mass spectrometric analysis.
The most common ions produced during DART analysis are the
2
The combination of enhanced super critical CO extraction tech-
nologies and affinity chromatography has enabled the production
of optimized and dose-reliable botanical extracts from variable
feedstocks that possess a defined bioactive profile (Alberte et al.,
2007). These extraction technologies were employed herein to
generate reproducible extracts of elderberry (Sambucus nigra L.)
fruits for both chemical characterization and assessment of biolog-
ical activity. Elderberries are known to be rich in phenolic com-
pounds, including phenolic acids, flavonoids, catechins, and
proanthocyanidins (de Pascual-Teresa et al., 2000; Hakkinen
et al., 1999), as well as possessing a variety of anti-oxidant proper-
ties (Abuja et al., 1998; Rice-Evans et al., 1996; Seeram and Nair,
+
+
[
4
M+H] cations and the [M+NH ] adducts (observed if ammonium
hydroxide is present near the DART source); however metal, cation
adducts are never observed (Cody et al., 2005). DART is capable of
analyzing surface materials without direct exposure of the samples
to elevated temperatures and/or electrical potentials as occurs dur-
2
002; Wang et al., 1997), and enhancing the immune response
(
Barak et al., 2001; Zakay-Rones et al., 1995). In addition, elder-
*
Corresponding author. Tel.: +1 239 597 8822; fax: +1 239 597 8001.
E-mail addresses: ralberte@herbalsciencegroup.com, rsalbertehs@aol.com (R.S.
berry extracts have shown anti-influenza activity in human clinical
trials (Zakay-Rones et al., 2004).
Alberte).
0
031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.06.003

1
256
B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
We utilized an optimized elderberry extract as well as a newly
the H1N1 particles. It was also determined, based upon the molec-
ular formulae for each of these compounds, that many of the abun-
dant compounds bound to H1N1 were DART-generated fragments
of the parent ions. The structures of compounds 1 and 2 were
determined by: (1) measuring the accurate isotopic abundance ra-
tios; (2) determining the precise molecular formula based on these
isotopic ratios; (3) monitoring the DART-generated fragmentation
of these compounds bound to H1N1 virions; and (4) molecular
modeling of various proposed structures based on the determined
molecular formulae.
developed Direct Binding Assay to identify key bioactive flavonoids
in elderberry fruits that contribute to the reported anti-influenza
activities. The identified flavonoids bind to Human Influenza A
(
H1N1) viruses and block viral infection in vitro.
2
. Results and discussion
2
.1. Anti-viral activity of elderberry fruit extracts
+
The first flavonoid (1) at m/z [M+H] = 359.325 amu (with corre-
A viral focus reduction assay was used to characterize the
+
sponding DART-generated fragments at m/z [M+H] = 341.310,
in vitro anti-influenza activity of the elderberry extract. Human
influenza A (H1N1) virus particles were used to infect Madin-Dar-
by canine kidney NBL-2 (MDCK) cells. The elderberry extract
showed clear dose-dependent inhibition of H1N1 virus infection
3
31.289, 313.275, and 285.205 amu, representing the loss of water
and/or CO from the parent ion) (Cuyckens and Claeys, 2004) was
0
0
identified as 5,7,3 ,4 -tetra-O-methylquercetin (1) (Fig. 3). The sec-
+
ond flavonoid identified (2) at m/z [M+H] = 479.232 amu was
(
Fig. 1). The 50% inhibition concentration (IC50) of the extract for
H1N1 was 252 (±34) g/mL, while 100% inhibition of H1N1 infec-
tion was achieved at 1000 g/mL, the highest concentration tested.
esterified with 3,4,5-trihydroxy-cyclohexanecarboxylic acid
l
(
2
Fig. 3) on the 3-OH of the 3-hydroxyflavonone C-ring. Compound
contains multiple stereogenic centers, and as such, we have pre-
sented one of several possible diastereomers. However, free-en-
ergy minimizations of compound indicate that the ester
l
To address possible extract-induced cellular toxicity, and to
confirm that the viral inhibition effects of the elderberry extract
were not due to non-specific effects of the extract on target cells,
the elderberry extracts were evaluated in a MTT cell viability assay
using the target MDCK cells. No non-specific effects on cell viability
2
functionality of compound 2 is not likely a requirement for the
anti-influenza activity observed here (see below) and therefore,
racemic dihydromyricetin (3), the free 3-hydroxyflavonone of 2,
was pursued for synthesis.
To validate the entry inhibition mechanism-of-action of the
elderberry extract, the H1N1 virus particles were subjected to
the Direct Binding Assay. After incubation of the H1N1 viruses in
the elderberry extract, unbound chemicals were removed,
and the H1N1 virus particles with bound compounds were allowed
to infect MDCK cells. The identified flavonoids were found to bind
to H1N1 virions in a ratio (2.9:1; 1:2) different from the ratio of
these compounds in the elderberry extract (1.5:1) indicating that
binding of these flavonoids to H1N1 is not non-specific.
or cellular toxicity were observed up to 2000
data not shown), well above the IC100 concentration for viral
infection.
lg/mL of the extract
(
2
.2. Identification of elderberry extract compounds that bind to H1N1
virions
Fig. 2 shows the DART TOF-MS fingerprint of the compounds
that are bound to H1N1 virions (Fig. 2A) and those compounds that
do not bind to the virus particles (Fig. 2B). This was accomplished
by incubating the H1N1 virus in the elderberry extract and remov-
ing the unbound components by washing through a membrane fil-
When H1N1 virions were incubated at the IC50 and IC100 con-
centrations determined for the elderberry extract (252 and
ter. One of the extremely abundant compounds (m/z
+
1
108 lg/mL, respectively), 58% and 95% inhibition of H1N1 infec-
[
M+H] = 369.353) was identified as cholestadiene. This compound
tion was achieved. This level of inhibition indicates that the active
chemicals in the elderberry extract bind stoichiometrically to
H1N1 virions, and when bound, block viral infection in vitro.
To verify the proposed mode-of-action established with the
elderberry extract, and to confirm the proposed structures as well
as the in vitro anti-influenza activity of these compounds, 5,7,3 ,4 -
tetra-O-methyl quercetin (1) and dihydromyricetin (3) were syn-
thesized. Compound 1 was synthesized in two steps from Rutin
was present in the washing medium and, from our control exper-
iments, did not possess any anti-influenza activity.
Because the AccuTOF mass spectrometer accurately determines
isotopic abundances (JEOL, 2007) and yields high resolution mass
measurements from the time-of-flight mass spectrometer, it was
possible to determine the candidate molecular formulae for the
compounds at m/z = 359.325 and 479.232 amu found to bind to
0
0
(4; Fig. 4), while racemic dihydromyricetin (3) was synthesized
1
1
20
00
in five steps by coupling acetophenone (6) and benzaldehyde (7;
Fig. 5). The anti-influenza mode-of-action of compounds 1 and 3
was confirmed by utilizing the Direct Binding Assay. Fig. 6 shows
the DART TOF-MS fingerprints of the compounds bound to the
H1N1 virions. The binding of dihydromyricetin (3) and compound
8
0
0
0
0
1
to H1N1 viruses (Fig. 4A and B, respectively) confirms the entry
inhibition mode-of-action of these flavonoids.
The synthesized flavonoids were also subjected to focus-form-
ing inhibition assays against the H1N1 virus. Dihydromyricetin
6
4
2
(
3) achieved 50% inhibition of H1N1 infection at a concentration
of 2.8 g/mL (8.7 M) (Table 1), which is approximately 100 times
lower than the IC50 of the elderberry extract (252 g/mL). Based on
l
l
l
the results of the Direct Binding Assay for the elderberry extract,
we expected compound 1 to have an IC50 7–10ꢀ lower (more ac-
tive) than dihydromyricetin (3). In fact, compound 1 achieved an
0
0
200
400
600
800
1000
1200
IC50 of 0.13
l
g/mL (0.36
l
M) (Table 1), which is 20ꢀ lower than
[
Elder berry extract] ( µµ g /mL)
the IC50 determined for (±)-dihydromyricetin, and 3-orders-of-
magnitude (1000ꢀ) lower than the elderberry extract. Of particular
Fig. 1. The dose-dependent inhibition curve of influenza A (H1N1) virus infection of
0
0
interest, 5,7,3 ,4 -tetra-O-methylquercetin (1) achieved an IC50
MDCK cells incubated with an elderberry fruit extract. The IC50 and IC100 values
2
Ò
were determined using the line-of-best-fit (R = 0.92; n = 22).
against H1N1 similar to that of Oseltamivir (Tamiflu ; 0.32 lM),

B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
1257
5
4
3
2
1
000
000
000
000
000
359.325
3
59.33
B
2
14.089
3
369.353
69.36
214.09
A
3
41.310
3
41.31
331.289
3
31.29
3
13.276
3
13.28
370.36
6
07.570
285.205
285.29
371.33
607.57
140.11
55 77 99 . .55 44 2
575.89
1
1
58.03
60.03
2
21 15 5. 0. 09 9257.26
16.08
479.232
15.24 449.09 479.51
4
372.40
2
0
A
6
4
2
000
129 0. 7
B
000
000
130.05
2
09.10
1
55 0. 9
81.13
1
211 1. 4
259.14
60 1. 4
1
15.09
2
320 .1 8
366.19 391.21
455.28
356 2. 2
438.28
483 2. 8
535 .3 2
0
1
00
200
300
400
500
600
700
800
m/z (amu)
m/z (a mu
Fig. 2. DART TOF-MS fingerprints of the compounds present in the elder berry extract that are bound (A) and not bound (B) to H1N1 virus particles after 1-h incubations.
0
0
Fig. 3. (A) The structures of 5,7,3 ,4 -tetra-O-methylquercetin (1), 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate
2), and (±)-dihydromyricetin (3). (B) The region most likely to bind to the hemagglutinin proteins of Influenza A is bracketed in the free-energy minimized three-dimensional
structure of 1 and 2.
(
0
0
2 3
Fig. 4. The synthesis of 5,7,3 ,4 -tetra-O-methylquercetin (1) from Rutin (4). (a) DMS, K CO , acetone, reflux, 70 h; (b) 20% HCl, reflux 2 h.
a known influenza neuraminidase (N1) inhibitor that prevents the
release of progeny from infected cells, and 27ꢀ lower than that of
the known influenza M2 proton channel inhibitor Amantadine
(27 lM) (Pinto and Lamb, 2006). Dihydromyricetin (3) had an
IC50 value 3ꢀ higher (less active) than Oseltamivir and ca. 3ꢀ lower
than Amantadine (Table 1).

1
258
B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
Fig. 5. The synthesis of (±)-dihydromyricetin (3) from 6 and 7. (a) NaH, MOM-Cl, DMF, 0 °C; (b) K
MeOH, 20 °C; and (e) 20% HCl, MeOH, 45 °C.
2
CO
3 2 2
, MOM-Cl, acetone, 10 °C; (c) 40% KOH, EtOH, 20 °C; (d) H O , 2 N NaOH,
6
5
4
3
2
1
00
00
00
00
00
A
(±)-dihydromyricetin
00
0
1000
5
,7, 3' 4'-tetra
O
-methylquercetin
8
00
00
00
00
0
B
6
4
2
310
320
330
340
350
360
370
m/z (amu)
0
0
Fig. 6. DART TOF-MS fingerprints of (A) (±)-dihydromyricetin (3) (m/z = 321.0674), and (B) 5,7,3 ,4 -tetra-O-methylquercetin (1) (m/z = 359.2825) bound to H1N1 virions.
Minimum free-energy modeling analysis revealed that the A
and B rings of compounds 1 and 2 form an axis with inter-phenolic
ring distances of 10.5 Å and 10.9 Å, respectively (Fig. 3). This dis-
tance is well within the size constraints of the hemagglutinin
Proposed mechanisms for anti-H1N1 activity in vitro by com-
monly studied polyphenols (e.g. catechin, quercetin, cyanidin) in-
clude the prevention of endosome acidification (Imanishi et al.,
2002), the inhibition of membrane fusion (Nagai et al., 1995), the
inhibition of progeny virion release (Knox et al., 2001), the inhibi-
tion of neuraminidase activity (Knox et al., 2001; Macdonald et al.,
2004; Song et al., 2005), and the inhibition of intercellular replica-
tion (Serkedjieva et al., 1990). These previously described mecha-
nisms are different from the proposed molecular mode-of-action
of compounds 1 and 2. We show that the synthesized compounds,
as well as the elderberry extract, inhibit H1N1 infection by binding
to the viral envelope, most likely the HA domains involved is host
cell binding and recognition. This binding mechanism subse-
(
HA) binding domain pocket (14–15 Å) of influenza viruses, which
is responsible for host cell receptor binding and viral entry (Ste-
vens et al., 2004). The phenolic regions of dihydromyricetin (3),
as well as compound 1, most likely bind to the viral mannose-rich
HA binding domains and, as such, the proposed bound orientation
of dihydromyricetin (3) would leave the esterified functionality of
2
free to interact with the immune system, potentially increasing
an immune response to the viral particles in vivo (Vigerust and
Shepherd, 2007).

B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
1259
Table 1
(250 ꢀ 4.6 mm I.D., 5
l
, 300 Å; Phenomenex, Torrance, CA) with a
0
0
The IC50 values determined for 5,7,3 ,4 -tetra-O-methylquercetin (1), the (±)-dihydr-
omyricetin (3), Oseltamivir, and Amantadine.
flow rate of 1 mL/min. The column temperature was held at
2
2
5 °C. The mobile phase consisted of 5% (v/v) HCO H (solvent A)
Compound
IC50
(
lg/mL)
IC50
(
lM)
and MeOH (solvent B). The following linear gradient was used:
0–2 min, 5% B; 2–10 min, 5–24% B (hold 5 min); 15–30 min, 24–
35% B (hold 5 min); 35–50 min, 45% B (hold 5 min); 55–65 min,
0
0
5
,7,3 ,4 -Tetra-O-methyl quercetin (1)
0.15
2.8
0.1
0.36
8.7
0.32
27
(
±)-Dihydromyricetin (3)
Oseltamivir
Amantadine
5
% B (hold 3 min).
4.1
DART TOF-MS analysis of elderberry extracts, synthetic flavonoids
TM
and viral-bound compounds: The JEOL DART AccuTOF mass spec-
trometer (JMS-T100LC; Jeol USA, Peabody, MA) was used for chem-
ical analysis of the elderberry fruit extracts and was executed in
quently blocks the ability of the H1N1 virions to bind to, and con-
sequently enter, host cells.
+
positive ion mode [M+H] . The needle voltage was set to 3500 V,
heating element to 300 °C, electrode 1–150 V, electrode 2–250 V,
and He gas flow to 3.98 L/min. For the mass spectrometer, the fol-
lowing settings were loaded: orifice 1 set to 20 V, ring lens voltage
set to 5 V, and orifice 2 set to 5 V. The peak voltage was set to
3
. Conclusions
Through the use of the Direct Binding Assay and DART TOF-MS
analysis, it was possible to identify and characterize the molecular
mode-of-action of two anti-influenza flavonoids in an optimized
elderberry fruit extract. The identified compounds were 5,7-
dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-tri-
hydroxycyclohexanecarboxylate (2) and 5,7,3 ,4 -tetra-O-meth-
ylquercetin (1). These flavonoids are the major contributors to
the anti-influenza activity of the elderberry extract. The molecular
mode-of-action of these flavonoids was determined by demon-
strating their direct binding to H1N1 virus particles resulting in
the inability of the H1N1 viruses to enter host cells, effectively pre-
venting H1N1 infection in vitro. This mode-of-action was further
verified using synthesized 5,7,3 ,4 -tetra-O-methylquercetin (1)
and racemic dihydromyricetin (3) which bind to H1N1 virions
and, when bound, blocked H1N1 infection in vitro.
DART TOF-MS analysis of botanical extracts is a highly accurate
and efficient method for identifying compounds in complex mix-
tures. DART TOF-MS coupled with traditional analytical techniques
will dramatically broaden and greatly enhance the understanding
of the chemical complexity of botanical extracts.
1
000 V in order to give peak resolution beginning at 100 m/z. The
microchannel plate detector (MCP) voltage was set to 2550 V. Cal-
ibrations were performed internally with each sample using a 10%
(
w/v) solution of PEG 600 (Ultra Chemical, North Kingston, RI) that
provided mass markers throughout the required mass range of
00–1000 amu. Calibration tolerances were held to 5 mmu. Sam-
0
0
1
ples (as dry powders for the extracts and synthetic flavonoids,
and fixed virions for the Direct Binding Assays) were introduced
into the DART He plasma using the closed end of a borosilicate
glass melting point capillary tube until a signal was achieved in
the total-ion chromatogram (TIC). The next sample was introduced
when the TIC returned to baseline levels.
Candidate molecular formulae were identified using elemental
composition and isotope matching programs in the Jeol MassCen-
terMain Suite software (JEOL USA, Peabody, MA). The candidate
molecular formulae were assigned with a confidence level greater
than 90%. The candidate molecular formulae were then used to
determine plausible chemical structures (JEOL, 2007).
MassCenterMain was also used to determine candidate molec-
ular formulae for the compounds that bind to the H1N1 virus.
The DART-generated fragments were confirmed using the deter-
mined molecular formulae for each of the masses identified as
bound to the H1N1 virions.
0
0
4
. Experimental
All solvents were purchased from Thermo Fisher Scientific (Fair-
lawn, NJ) unless specified below.
Three-dimensional free-energy minimizations: Chem3D Ultra
(Cambridgesoft, Cambridge, MA) molecular modeling package
was employed for the free-energy minimizations of the identified
compounds using the molecular mechanics two level of theory.
Viral focus reduction infection assays: The viral focus reduction
infection assays were modified from the procedure described by
Okuno et al. (1990). The specific procedure used is described
below.
The virus for this study was Influenza A (H1N1) virus strain A/
PR/8/34 (ATCC, Manassas, VA; ATCC No. VR-1469). The elderberry
extract and synthesized compounds were dissolved in a minimal
volume of EtOH (USP grade) prior to dilution in DMEM (pH 7.4).
Approximately 100 focus-forming units (FFU) of influenza virus
were incubated with dilutions of the elder berry extract solution
in DMEM for 1 h at room temperature and then allowed to infect
confluent MDCK cells for 1 h at room temperature. After infection,
cells were fixed with Formalde-fresh then permeabilized with
EtOH (USP). The FFU’s were visualized using goat anti-influenza
A virus IgG polyclonal antibody, rabbit Anti-Goat IgG (H&L) horse-
radish peroxidase conjugated affinity purified antibody (Chemicon,
Temecula, CA) and AEC chromogen substrate (Dako, Carpinteria,
CA). These same methods were employed for the re-infection as-
says with viral-bound compounds.
Elderberry extract preparation: Wild crafted elder berries (Sam-
bucus nigra L., Caprifoliaceae) from Hungary were purchased from
Blessed Herbs, Inc. (Oakham, MA; Product No. 724, Lot No.
L10379w). The polymer adsorbent extract was obtained by extract-
ing 20 g of ground elderberries using supercritical CO
00 bar for 2 h, followed by two extractions using EtOH:H
100 mL, 4:1, v/v) EtOH for 2 h each. The combined extracted slurry
was filtered through Fisherbrand P4 filter paper and centrifuged at
2
at 60 °C and
3
(
2
O
5
37ꢀg for 20 min. The supernatant was vacuum distilled to re-
move EtOH, and the final solution concentration was ꢁ35 mg/mL
for polymer adsorbent loading.
Adsorption experiments were carried out at room temperature
in an open batch system. The ADS5 polymer adsorbent (Nankai
University, China) was washed with EtOH to remove monomers
and impurities and soaked in distilled H
ing. The column was loaded with 60 mL of the above prepared
solution and washed with two column volumes of distilled H O.
The column was eluted using EtOH/H O (40 mL, 4:1, v/v). The col-
lected fraction was dried at 50 °C overnight to yield a dark purple
crystalline powder. This procedure was repeated multiple times to
ensure reproducibility of the extract.
HPLC analysis of elderberry extracts: The extracts were character-
ized by HPLC–UV on a Shimadzu LC-10AVP system (Shimadzu, Sin-
gapore) with a LC10ADVP pump and a SPD-M 10AVP diode array
2
O overnight before pack-
2
2
Extract non-specific and cytotoxicity assessments: The possible
non-specific effects of the elderberry extract on viral infection
(e.g. positive control) as well as the potential toxicity of extracts
was measured by monitoring mitochondrial reductase activity in
detector at 280 and 350 nm by injecting 10
solution in EtOH onto a reversed phase Jupiter C18 column
lL of a diluted extract

1
260
B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
TM
MDCK cells using the TACS MTT cell proliferation assay (R&D Sys-
tems, Inc., Minneapolis, MN) according to the manufacturer’s
instructions.
layer was concentrated under vacuum and the resultant oily resi-
due was purified by silica gel column chromatography by eluting
with hexanes:EtOAc (9:1) followed by hexanes:EtOAc (8.5:15) to
1
Direct Binding Assay: A Direct Binding Assay was developed to
determine which compounds in the elderberry extract bind to
H1N1 virions. The H1N1 virus particles were incubated in the
elderberry extract or the synthesized flavonoids, and were then
washed 3 times on an Amicon 100 kDa filter (Ultracel PL-100; Mil-
ipore Corp., Billerica, MA) with PBS to remove unbound com-
pounds. The virus particles were then collected and a portion
was fixed in 100% (USP) EtOH for DART TOF-MS analysis. In addi-
tion, the washed fractions containing the unbound chemicals were
collected and analyzed directly by DART TOF-MS for comparison.
The closed end of a borosilicate glass capillary tube was immersed
in the virion solution (in EtOH) and passed through the DART He
plasma to obtain mass spectra of virion surface-bound compounds
as described in Section 4.2.
give compound 8 (0.78 g, 48%). H NMR (CDCl
3
; 400 MHz) d 6.51
–, s), 3.47 (3H, –OCH , s),
, s). C NMR (CDCl
400 MHz) d 204.1 (C@O), 163.0 (C-4), 160.2 (C-2 and C-6), 116.1
), 33.0
(calcd. for
(2H, s, H-3 and H-5), 5.14 (6H, –CH
3.45 (6H, –OCH , s), 2.50 (3H, C(O)CH
2
3
1
3
3
3
3
;
(C-1), 95.1 (3ꢀ –CH
2
–), 93.8 (C-3, C-5), 55.7 (3ꢀ –OCH
3
(C(O)CH
3
).
HRMS
(positive ion) = 301.1281
C H
14 21
O
7
= 301.1287).
4.3. 3,4,5-Tris(methoxymethoxy)benzaldehyde (9)
A
mixture of 3,4,5-trihydroxy benzaldehyde (7, 0.5 g,
2.9 mmol), K CO (4 g, 29 mmol), and dry acetone (100 mL) were
placed in a 2-necked RB flask under N and the mixture was cooled
2
3
2
to 10–15 °C. Chloromethyl methyl ether (1.44 g, 18 mmol) was
added slowly over a period of 30 min at 10–15 °C, and the reaction
mass was allowed to reflux slowly over a period of 6 h. After reflux-
ing, the reaction mixture was filtered, washed with acetone
(50 mL), concentrated under vacuum and extracted with EtOAc
(2 ꢀ 25 mL). The combined organic layer was washed with distilled
0
0
Compound synthesis: The synthesis of the 5,7,3 ,4 -tetra-O-meth-
ylquercetin (1) and racemic dihydromyricetin (3) have been
adapted from previous reports (Koeppen et al., 1962; Li et al.,
1
990; Rao and Weisner, 1981). Specific methodologies are de-
scribed below.
2 2 4
H O (25 mL), brine (25 mL) and dried (Na SO ). The filtered organic
0
0
4
.1. Synthesis of 5,7,3 ,4 -tetra-O-methylquercetin (1)
layer was concentrated and the resultant oily residue was purified
by silica gel column chromatography by eluting with hex-
1
Dimethyl sulphate (138 g, 109 mmol) was added slowly to a
mixture of Rutin monohydrate (50 g, 82 mmol) and powdered
CO (210 g, 152 mmol) in acetone (1 L) at RT over a period of
anes:EtOAc (4:1) to give compound 9 (0.6 g, 72%). H NMR (CDCl
3
;
400 MHz) d 9.87 (1H, s, CH(O)), 7.40 (2H, s, H-2 and H-6), 5.29 (6H,
1
3
K
2
3
s, –CH
400 MHz) d 191.9 (C@O), 151.2 (C-3 and C-5), 140.3 (C-4), 132.2 (C-
1), 106.2 (C-2 and C-6), 98.0 (–CH
2 3 3 3
–), 3.66 (3H, s, –OCH ), 3.54 (6H, s, –OCH ). C NMR (CDCl ;
3
8
0 min. The reaction was heated to reflux and maintained for
0 h. The reaction was cooled to RT, filtered through Celite and
2
–), 94.9 (2ꢀ –CH
2
–), 55.9 (3ꢀ
washed with acetone (250 mL). The combined acetone layer was
concentrated under vacuum to give a pale yellow gummy solid
3
–OCH ).
HRMS
(positive
ion) = 287.1138
(calcd. for
C H
13 19
O
7
= 287.1131).
(
(
4, 48 g). The gummy solid was dissolved in 20% (v/v) HCl in H
500 mL), heated to 100 °C and maintained for 3 h. The reaction
Cl
(4 ꢀ 500 mL). The
combined organic layer was washed with H O (1 L), brine (1 L)
and dried (Na SO ). The organic layer was filtered and concen-
2
O
0
0
0
4.4. 2 ,4 ,6 ,3,4,5,-Hexakis(methoxymethoxy)chalcone (10)
mixture was cooled and extracted with CH
2
2
2
A solution of 40% KOH in EtOH (20 mL) was added to a mixture
of 8 (1 g, 3.33 mmol) in EtOH (5 mL) cooled to less than 20 °C. After
stirring for 15 min, a solution of 9 (1 g, 3.5 mmol) in EtOH (5 mL)
was added slowly over a period of 10 min and allowed to stir over-
2
4
trated under vacuum to give a dark solid (21 g) which was purified
by silica gel column chromatography and eluted with EtOAc:hex-
anes (1:1) followed by CH
2
Cl
2
:MeOH (4:1). The CH
2
Cl
2
:MeOH frac-
night at RT. The reaction was quenched with distilled H
and extracted with EtOAc (2 ꢀ 50 mL). The combined organic layer
was washed with distilled H O (50 mL), brine (50 mL) and dried
(Na SO ). The organic layer was concentrated under vacuum to
2
O (50 mL)
tions were concentrated under vacuum and yielded a brown
residue which was triturated with neat iso-PrOH (100 mL) and stir-
red for 1 h. The resulting pale green solid was filtered, washed with
cold iso-PrOH (25 mL) and dried at 60 °C under vacuum for 12 h
resulting in an off-white powder (9.0 g, 31% overall yield).
2
2
4
1
give compound 10 as a pale yellow solid (1.5 g, 78%). H NMR
1
0
H
(CDCl
3
; 400 MHz) d 7.22 (1H, d, J = 12 Hz, H-
a
), 7.04 (2H, s, H-3
0
0
0
NMR (CDCl
3
; 400 MHz) d 7.82 (1H, s, H-2 ), 7.41 (1H, s, H-3 ),
and H-5 ), 6.87 (1H, d, J = 12 Hz, H-b), 6.57 (2H, s, H-2 and H-6),
0
6
3
4
1
1
.99 (1H, s, H-6 ), 6.55 (1H, s, H-8), 6.35 (1H, s, H-6), 3.99 (6H, s),
.96 (3H, s), 3.92 (3H, s). 13C NMR (CDCl
; 400 MHz) d 172.0 (C-
), 164.5 (C-7), 160.7 (C-5), 159.0 (C-9), 150.5 (C-3 and C-4 ),
5.20 (6H, s), 5.17 (2H, s), 5.11 (4H, s), 3.61 (3H, s), 3.51 (3H, s),
13
3
3
3.49 (6H, s), 3.39 (6H, s). C NMR (CDCl ; 400 MHz) d 192.9
0
0
0
0
0
(C@O), 164.2 (C-4 ), 163.6 (C-2 , C-6 ), 151.0 (C-3, C-5), 145.3 (C-
0
0
0
0
42.2 (C-2), 137.7 (C-3), 124.0 (C-1 ), 120.8 (C-6 ), 111.3 (C-2 ),
b), 133.1 (C-4), 127.2 (C-
C-6), 95.3 (3ꢀ –CH –), 94.7 (3ꢀ –CH
37
(positive ion) = 569.2251 (calcd. for C27H O
a
), 126.5 (C-1), 112.9 (C-1 ), 103.6 (C-2,
0
10.9 (C-5 ), 106.3 (C-10), 95.8 (C-6), 92.6 (C-8), 56.2, 55.9. ESI-
2
2
–), 55.9 (6ꢀ –OCH
3
). HRMS
13 = 569.2234).
+
+
+
MS (positive): [M] = 358.2; [M+H] = 359.3; [M+HꢂCH
3
] = 344.4;
+
[
M+Hꢂ2ꢀCH
3
] = 329.3.
0
0
0
4
.5. 2 ,4 ,6 -Tris(methoxymethoxy)phenyl(3-(3,4,5-
4.2. 2,4,6-Tris(methoxymethoxy)acetophenone (8)
tris(methoxymethoxy)phenyl)oxiran-2-yl)methanone (11)
A mixture of 2,4,6-trihydroxyacetophenone (6, 1 g, 5.4 mmol) in
dry DMF (20 mL) was added to a slurry of NaH (60% in mineral oil,
2 2
H O (50% [v/v], 1 mL, 17.35 mmol) was added to a mixture of
chalcone 10 (1 g, 1.8 mmol) and 2 N NaOH (3 mL), and stirred for
overnight at RT in MeOH (30 mL). The MeOH was concentrated un-
der vacuum and the resultant residue was extracted with EtOAc
(2ꢀ 50 mL). The combined organic layer was washed with distilled
0
3
.9 g, 20 mmol) in dry DMF (10 mL) at 0–5 °C over a period of
0 min under N and stirred for 1 h at RT. The reaction mixture
2
was cooled to 0–5 °C; a solution of chloromethyl methylether
1.75 g, 22 mmol) in dry DMF was added slowly over a period of
5 min. The reaction mixture was stirred at RT for 4 h, poured in
to ice-cold H
O (100 mL), and extracted with EtOAc (2 ꢀ 50 mL).
The combined organic layer was washed with distilled H
50 mL), brine (50 mL) and dried (Na SO ). The filtered organic
(
1
2 2 4
H O (50 mL), brine (50 mL) and dried (Na SO ). The organic layer
was concentrated under vacuum to give compound 11 as a thick
1
2
pale yellow oil (0.72 g, 70%). H NMR (CDCl
3
; 400 MHz) d 6.77
0
0
2
O
(2H, s, H-2 and H-6), 6.49 (2H, s, H-3 and H-5 ), 5.13 (12H, s,
(
2
4
2
–CH –), 3.91 (1H, d, J = 2 Hz, H-a), 3.83 (1H, d, J = 2 Hz, H-b), 3.58

B. Roschek Jr et al. / Phytochemistry 70 (2009) 1255–1261
1261
1
3
(
(
1
3H, s), 3.44 (9H, s), 3.37 (6H, s). C NMR (CDCl
3
; 400 MHz) d 196.5
Imanishi, N., Tuji, Y., Katada, Y., Maruhashi, M., Konosu, S., Mantani, N., Terasawa, K.,
Ochiai, H., 2002. Additional inhibitory effect of tea extract on the growth of
influenza A and B viruses in MDCK cells. Microbiol. Immunol. 46, 491–494.
0
0
0
C@O), 163.5 (C-4 ), 162.2 (C-2 and C-6 ), 150.9 (C-3 and C-5),
0
34.1 (C-4), 131.6 (C-1), 103.4 (C-1 ), 99.7 (C-2 and C-6), 95.2
TM
JEOL, 2007. AccuTOF DART Applications Notebook. JEOL USA, Peabody, MA.
(
–
C
3ꢀ –CH
2
–), 94.9 (3ꢀ –CH
HRMS (positive
14 = 585.2183).
2
–), 69.1 (C-
a
), 58.2 (C-b), 55.5 (6ꢀ
Knox, Y.M., Hayashi, K., Suzutani, T., Ogasawara, M., Yoshida, I., Shiina, R., Tsukui, A.,
Terahara, N., Azuma, M., 2001. Activity of anthocyanins from fruit extract of
Ribes nigrum L. against influenza A and B viruses. Acta Virol. 24, 209–215.
Koeppen, B.H., Smit, J.B., Roux, D.G., 1962. The flavone C-glycosides and flavonol O-
glycosides of Aspalathus acuminatus (rooibos tea). Biochem. J. 83, 507–511.
Li, S., Onda, M., Kawaga, H., Kawase, H., Iguchi, M., Hatigaya, Y., 1990. Sodium
borohydride reduction of flavanols. J. Heterocycl. Chem. 27, 2029–2035.
Macdonald, S.J., Watson, K.G., Cameron, R., Chalmers, D.K., Demaine, D.A., Fenton,
R.J., Gower, D., Hamblin, J.N., Hamilton, S., Hart, G.J., Inglis, G.G., Jin, B., Jones,
H.T., McConnell, D.B., Mason, A.M., Nguyen, V., Owens, I.J., Parry, N., Reece, P.A.,
Shanahan, S.E., Smith, D., Wu, W.Y., Tucker, S.P., 2004. Potent and long-acting
dimeric inhibitors of influenza virus neuraminidase are effective at a once-
weekly dosing regimen. Antimicrob. Agents Chemother. 48, 4542–4549.
Madhusudanan, K.P., Banerjee, S., Khanuja, S.P., Chattopadhyay, S.K., 2008. Analysis
of hairy root culture of Rauvolfia serpentina using direct analysis in real time
mass spectrometric technique. Biomed. Chromatogr. 22, 596–600.
OCH
3
).
ion) = 585.2193
(calcd. for
27
H
37
O
(
±)-Dihydromyricetin (3): A mixture of 11 (0.2 g) and HCl in
MeOH (1.25 M, 3.0 mL, 3.75 mmol) was stirred at 45 °C for
0 min. The MeOH was concentrated under vacuum and the resul-
3
tant dark residue was purified by silica gel column chromatogra-
phy by eluting with EtOAc:hexanes (1:1) followed by
CH Cl :MeOH (9:1) to give compound 3 as an off-white powder
2 2
1
0
(
0.70 g, 66%). H NMR (CDCl
3
; 400 MHz) d 6.62 (2H, s, H-2 and
0
H-6 ), 5.98 (1H, s, H-8), 5.94 (1H, s, H-6), 4.96 (1H, d, J = 12 Hz, H-
), 4.57 (1H, d, J = 12 Hz, H-3). 1 C NMR (CDCl
97.9 (C-4), 167.6 (C-7), 164.8 (C-9), 164.0 (C-5), 146.1 (C-3 ),
34.0 (C-1 ), 128.9 (C-4 ), 107.9 (C-2 and C-6 ), 101.4 (C-10), 96.8
3
2
1
1
3
; 400 MHz) d
0
Nagai, T., Moriguchi, O., Suzuki, Y., Tomimori, T., Yamada, H., 1995. Mode of action
of the anti-influenza virus activity of plant flavonoid 5,7,4-trihydroxy-8-
methoxyflavone, from the roots of Scutellaria baicalensis. Antiviral Res. 26, 11–
0
0
0
0
(
C-8), 95.7 (C-6), 84.4 (C-3), 72.9 (C-2). HRMS (positive
25.
12 8
ion) = 320.0541 (calcd. for C15H O = 320.0532).
Okuno, Y., Tanaka, K., Baba, K., Maeda, A., Kunita, N., Ueda, S., 1990. Rapid focus
reduction neutralization test of influenza A and B viruses in microtiter system. J.
Clin. Microbiol. 28, 1308–1313.
Pinto, L.H., Lamb, R.A., 2006. The M2 proton channels of Influenza A and B viruses. J.
Biol. Chem. 281, 8997–9000.
Acknowledgments
We acknowledge Dr. S. Puppali (NORAC Pharmaceuticals, Azu-
sa, CA) who conducted the flavonoid synthesis. We also acknowl-
edge Dr. L. Holland (IITRI, Chicago, IL) for conducting the viral
infection assays on 5,7,3 ,4 -tetra-O-methylquercetin, dihydromy-
ricetin, Oseltamivir and Amantadine. Support for this research
was provided by HerbalScience Singapore, Pte. Ltd.
Pramanik, B.N., Ganguly, A.K., Gross, M.L., 2002. Applied Electrospray Mass
Spectrometry. Marcel Dekker, New York.
Rao, K.V., Weisner, N.T., 1981. Microbial transformation of quercetin by Bacillus
cereus. Appl. Environ. Microbiol. 42, 450–452.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure–antioxidant activity
relationships of flavonoids and phenolic acids. Free Rad. Biol. Med. 20, 933–956.
Schmidt, B.M., Ribnicky, D.M., Lipsky, P.E., Raskin, I., 2007. Revisiting the ancient
concept of botanical therapeutics. Nat. Chem. Biol. 3, 360–366.
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0
0
Seeram, N.P., Nair, M.G., 2002. Inhibition of lipid peroxidation and structure–
activity-related studies of the dietary constituents anthocyanins,
anthocyanidins, and catechins. J. Agric. Food Chem. 50, 5308–5312.
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