Elderberry flavonoids bind to and prevent H1N1 infection in vitro
Bill Roschek Jr.a, Ryan C. Finkb, Matthew D. McMichaela, Dan Lic, Randall S. Albertea,*
aHerbalScience Group LLC, 1004 Collier Center Way, Suite 200, Naples, FL 34110, USA
bLeonard M. Miller School of Medicine, University of Miami, Miami, FL 33136, USA
cHerbalScience Singapore, Pte. Ltd., 1 Science Park Road, Capricorn, Science Park II, Singapore 117528, Singapore
a r t i c l e i n f o
Received 9 September 2007
Received in revised form 18 May 2009
Available online 12 August 2009
Sambucus nigra L. Caprifoliaceae
a b s t r a c t
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
standard chromatographic techniques. The elderberry extract inhibited Human Influenza A (H1N1) infec-
tion in vitro with an IC50value of 252 ± 34 lg/mL. The Direct Binding Assay established that flavonoids
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, 5,7,30,40-tetra-O-methylquercetin (1) and 5,7-dihy-
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,
blocking host cell entry and/or recognition. Compound 1 gave an IC50of 0.13 lg/mL (0.36 lM) for H1N1
infection inhibition, while dihydromyricetin (3) achieved an IC50of 2.8 lg/mL (8.7 lM). The H1N1 inhi-
bition activities of the elderberry flavonoids compare favorably to the known anti-influenza activities of
Oseltamivir (Tamiflu?; 0.32 lM) and Amantadine (27 lM).
? 2009 Elsevier Ltd. All rights reserved.
The chemical complexity of botanical extracts has made mass
spectrometric characterization of whole extracts difficult due to
the lack of reliable extraction methodologies that yield optimized
extracts with dose-to-dosereliable
(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,
The most common ions produced during DART analysis are the
[M+H]+cations and the [M+NH4]+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-
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).
The combination of enhanced super critical CO2extraction 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,
2002; Wang et al., 1997), and enhancing the immune response
(Barak et al., 2001; Zakay-Rones et al., 1995). In addition, elder-
berry extracts have shown anti-influenza activity in human clinical
trials (Zakay-Rones et al., 2004).
0031-9422/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +1 239 597 8822; fax: +1 239 597 8001.
E-mail addresses: email@example.com, firstname.lastname@example.org (R.S.
Phytochemistry 70 (2009) 1255–1261
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/phytochem
We utilized an optimized elderberry extract as well as a newly
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
A viral focus reduction assay was used to characterize the
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
(Fig. 1). The 50% inhibition concentration (IC50) of the extract for
H1N1 was 252 (±34) lg/mL, while 100% inhibition of H1N1 infec-
tion was achieved at 1000 lg/mL, the highest concentration tested.
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
or cellular toxicity were observed up to 2000 lg/mL of the extract
(data not shown), well above the IC100 concentration for viral
2.2. Identification of elderberry extract compounds that bind to H1N1
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-
ter. Oneof theextremely
[M+H]+= 369.353) was identified as cholestadiene. This compound
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
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
The first flavonoid (1) at m/z [M+H]+= 359.325 amu (with corre-
sponding DART-generated fragments at m/z [M+H]+= 341.310,
331.289, 313.275, and 285.205 amu, representing the loss of water
and/or CO from the parent ion) (Cuyckens and Claeys, 2004) was
identified as 5,7,30,40-tetra-O-methylquercetin (1) (Fig. 3). The sec-
ond flavonoid identified (2) at m/z [M+H]+= 479.232 amu was
esterified with 3,4,5-trihydroxy-cyclohexanecarboxylic
(Fig. 3) on the 3-OH of the 3-hydroxyflavonone C-ring. Compound
2 contains multiple stereogenic centers, and as such, we have pre-
sented one of several possible diastereomers. However, free-en-
ergy minimizations of compound 2 indicate that the ester
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
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.
When H1N1 virions were incubated at the IC50and IC100con-
centrations determined for the elderberry extract (252 and
1108 lg/mL, respectively), 58% and 95% inhibition of H1N1 infec-
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, 5,7,30,40-
tetra-O-methyl quercetin (1) and dihydromyricetin (3) were syn-
thesized. Compound 1 was synthesized in two steps from Rutin
(4; Fig. 4), while racemic dihydromyricetin (3) was synthesized
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
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
(3) achieved 50% inhibition of H1N1 infection at a concentration
of 2.8 lg/mL (8.7 lM) (Table 1), which is approximately 100 times
lower than the IC50of the elderberry extract (252 lg/mL). Based on
the results of the Direct Binding Assay for the elderberry extract,
we expected compound 1 to have an IC507–10? lower (more ac-
tive) than dihydromyricetin (3). In fact, compound 1 achieved an
IC50of 0.13 lg/mL (0.36 lM) (Table 1), which is 20? lower than
the IC50 determined for (±)-dihydromyricetin, and 3-orders-of-
magnitude (1000?) lower than the elderberry extract. Of particular
interest, 5,7,30,40-tetra-O-methylquercetin (1) achieved an IC50
against H1N1 similar to that of Oseltamivir (Tamiflu?; 0.32 lM),
[Elder berry extract] (µ µg /mL)
Percent inhibition of infection
Fig. 1. The dose-dependent inhibition curve of influenza A (H1N1) virus infection of
MDCK cells incubated with an elderberry fruit extract. The IC50and IC100values
were determined using the line-of-best-fit (R2= 0.92; n = 22).
B. Roschek Jr et al./Phytochemistry 70 (2009) 1255–1261
a known influenza neuraminidase (N1) inhibitor that prevents the
release of progeny from infected cells, and 27? lower than that of
the known influenza M2 proton channel inhibitor Amantadine
(27 lM) (Pinto and Lamb, 2006). Dihydromyricetin (3) had an
IC50value 3? higher (less active) than Oseltamivir and ca. 3? lower
than Amantadine (Table 1).
129. 07129. 07
155. 09155. 09
211. 14211. 14
356. 22 356. 22
320. 18 320. 18
483. 28483. 28
535. 32535. 32
331.2 9 331.2 9
371.3 3 371.3 3
313.2 8313.2 8
140.1 1 140.1 1
216.0 8 216.0 8
479.5 1 479.5 1 415.24415.24
575.8 9575.8 9
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.
Fig. 3. (A) The structures of 5,7,30,40-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.
Fig. 4. The synthesis of 5,7,30,40-tetra-O-methylquercetin (1) from Rutin (4). (a) DMS, K2CO3, acetone, reflux, 70 h; (b) 20% HCl, reflux 2 h.
B. Roschek Jr et al./Phytochemistry 70 (2009) 1255–1261
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
(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
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-
Fig. 5. The synthesis of (±)-dihydromyricetin (3) from 6 and 7. (a) NaH, MOM-Cl, DMF, 0 ?C; (b) K2CO3, MOM-Cl, acetone, 10 ?C; (c) 40% KOH, EtOH, 20 ?C; (d) H2O2, 2 N NaOH,
MeOH, 20 ?C; and (e) 20% HCl, MeOH, 45 ?C.
310 320 330 340 350 360 370
5,7,3' 4'-tetra O-methylquercetin
Fig. 6. DART TOF-MS fingerprints of (A) (±)-dihydromyricetin (3) (m/z = 321.0674), and (B) 5,7,30,40-tetra-O-methylquercetin (1) (m/z = 359.2825) bound to H1N1 virions.
B. Roschek Jr et al./Phytochemistry 70 (2009) 1255–1261
quently blocks the ability of the H1N1 virions to bind to, and con-
sequently enter, host cells.
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-
hydroxycyclohexanecarboxylate (2) and 5,7,30,40-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 5,7,30,40-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.
All solvents were purchased from Thermo Fisher Scientific (Fair-
lawn, NJ) unless specified below.
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 CO2at 60 ?C and
300 bar for 2 h, followed by two extractions using EtOH:H2O
(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
537?g 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 H2O overnight before pack-
ing. The column was loaded with 60 mL of the above prepared
solution and washed with two column volumes of distilled H2O.
The column was eluted using EtOH/H2O (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
detector at 280 and 350 nm by injecting 10 lL of a diluted extract
solution in EtOH onto a reversed phase Jupiter C18 column
(250 ? 4.6 mm I.D., 5 l, 300 Å; Phenomenex, Torrance, CA) with a
flow rate of 1 mL/min. The column temperature was held at
25 ?C. The mobile phase consisted of 5% (v/v) HCO2H (solvent A)
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,
5% B (hold 3 min).
DART TOF-MS analysis of elderberry extracts, synthetic flavonoids
and viral-bound compounds: The JEOL DART
trometer (JMS-T100LC; Jeol USA, Peabody, MA) was used for chem-
ical analysis of the elderberry fruit extracts and was executed in
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
1000 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
100–1000 amu. Calibration tolerances were held to 5 mmu. Sam-
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.
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
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.
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
TMAccuTOF mass spec-
The IC50values determined for 5,7,30,40-tetra-O-methylquercetin (1), the (±)-dihydr-
omyricetin (3), Oseltamivir, and Amantadine.
5,7,30,40-Tetra-O-methyl quercetin (1)
B. Roschek Jr et al./Phytochemistry 70 (2009) 1255–1261
MDCK cells using the TACSTMMTT cell proliferation assay (R&D Sys-
tems, Inc., Minneapolis, MN) according to the manufacturer’s
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.
Compound synthesis: The synthesis of the 5,7,30,40-tetra-O-meth-
ylquercetin (1) and racemic dihydromyricetin (3) have been
adapted from previous reports (Koeppen et al., 1962; Li et al.,
1990; Rao and Weisner, 1981). Specific methodologies are de-
4.1. Synthesis of 5,7,30,40-tetra-O-methylquercetin (1)
Dimethyl sulphate (138 g, 109 mmol) was added slowly to a
mixture of Rutin monohydrate (50 g, 82 mmol) and powdered
K2CO3(210 g, 152 mmol) in acetone (1 L) at RT over a period of
30 min. The reaction was heated to reflux and maintained for
80 h. The reaction was cooled to RT, filtered through Celite and
washed with acetone (250 mL). The combined acetone layer was
concentrated under vacuum to give a pale yellow gummy solid
(4, 48 g). The gummy solid was dissolved in 20% (v/v) HCl in H2O
(500 mL), heated to 100 ?C and maintained for 3 h. The reaction
mixture was cooled and extracted with CH2Cl2(4 ? 500 mL). The
combined organic layer was washed with H2O (1 L), brine (1 L)
and dried (Na2SO4). The organic layer was filtered and concen-
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 CH2Cl2:MeOH (4:1). The CH2Cl2:MeOH frac-
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).
NMR (CDCl3; 400 MHz) d 7.82 (1H, s, H-20), 7.41 (1H, s, H-30),
6.99 (1H, s, H-60), 6.55 (1H, s, H-8), 6.35 (1H, s, H-6), 3.99 (6H, s),
3.96 (3H, s), 3.92 (3H, s).13C NMR (CDCl3; 400 MHz) d 172.0 (C-
4), 164.5 (C-7), 160.7 (C-5), 159.0 (C-9), 150.5 (C-30and C-40),
142.2 (C-2), 137.7 (C-3), 124.0 (C-10), 120.8 (C-60), 111.3 (C-20),
110.9 (C-50), 106.3 (C-10), 95.8 (C-6), 92.6 (C-8), 56.2, 55.9. ESI-
MS (positive): [M]+= 358.2; [M+H]+= 359.3; [M+H?CH3]+= 344.4;
4.2. 2,4,6-Tris(methoxymethoxy)acetophenone (8)
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,
0.9 g, 20 mmol) in dry DMF (10 mL) at 0–5 ?C over a period of
30 min under N2and stirred for 1 h at RT. The reaction mixture
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
15 min. The reaction mixture was stirred at RT for 4 h, poured in
to ice-cold H2O (100 mL), and extracted with EtOAc (2 ? 50 mL).
The combined organic layer was washed with distilled H2O
(50 mL), brine (50 mL) and dried (Na2SO4). The filtered organic
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
give compound 8 (0.78 g, 48%).1H NMR (CDCl3; 400 MHz) d 6.51
(2H, s, H-3 and H-5), 5.14 (6H, –CH2–, s), 3.47 (3H, –OCH3, s),
3.45 (6H, –OCH3, s), 2.50 (3H, C(O)CH3, s).
400 MHz) d 204.1 (C@O), 163.0 (C-4), 160.2 (C-2 and C-6), 116.1
(C-1), 95.1 (3? –CH2–), 93.8 (C-3, C-5), 55.7 (3? –OCH3), 33.0
13C NMR (CDCl3;
ion) = 301.1281 (calcd.for
4.3. 3,4,5-Tris(methoxymethoxy)benzaldehyde (9)
Amixture of3,4,5-trihydroxybenzaldehyde(7,0.5 g,
2.9 mmol), K2CO3(4 g, 29 mmol), and dry acetone (100 mL) were
placed in a 2-necked RB flask under N2and the mixture was cooled
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
H2O (25 mL), brine (25 mL) and dried (Na2SO4). The filtered organic
layer was concentrated and the resultant oily residue was purified
by silica gel column chromatography by eluting with hex-
anes:EtOAc (4:1) to give compound 9 (0.6 g, 72%).1H NMR (CDCl3;
400 MHz) d 9.87 (1H, s, CH(O)), 7.40 (2H, s, H-2 and H-6), 5.29 (6H,
s, –CH2–), 3.66 (3H, s, –OCH3), 3.54 (6H, s, –OCH3).13C NMR (CDCl3;
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 (–CH2–), 94.9 (2? –CH2–), 55.9 (3?
ion) = 287.1138(calcd.for
4.4. 20,40,60,3,4,5,-Hexakis(methoxymethoxy)chalcone (10)
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-
night at RT. The reaction was quenched with distilled H2O (50 mL)
and extracted with EtOAc (2 ? 50 mL). The combined organic layer
was washed with distilled H2O (50 mL), brine (50 mL) and dried
(Na2SO4). The organic layer was concentrated under vacuum to
give compound 10 as a pale yellow solid (1.5 g, 78%).
(CDCl3; 400 MHz) d 7.22 (1H, d, J = 12 Hz, H-a), 7.04 (2H, s, H-30
and H-50), 6.87 (1H, d, J = 12 Hz, H-b), 6.57 (2H, s, H-2 and H-6),
5.20 (6H, s), 5.17 (2H, s), 5.11 (4H, s), 3.61 (3H, s), 3.51 (3H, s),
3.49 (6H, s), 3.39 (6H, s).
(C@O), 164.2 (C-40), 163.6 (C-20, C-60), 151.0 (C-3, C-5), 145.3 (C-
b), 133.1 (C-4), 127.2 (C-a), 126.5 (C-1), 112.9 (C-10), 103.6 (C-2,
C-6), 95.3 (3? –CH2–), 94.7 (3? –CH2–), 55.9 (6? –OCH3). HRMS
(positive ion) = 569.2251 (calcd. for C27H37O13= 569.2234).
13C NMR (CDCl3; 400 MHz) d 192.9
H2O2(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
(2? 50 mL). The combined organic layer was washed with distilled
H2O (50 mL), brine (50 mL) and dried (Na2SO4). The organic layer
was concentrated under vacuum to give compound 11 as a thick
pale yellow oil (0.72 g, 70%).
(2H, s, H-2 and H-6), 6.49 (2H, s, H-30and H-50), 5.13 (12H, s,
–CH2–), 3.91 (1H, d, J = 2 Hz, H-a), 3.83 (1H, d, J = 2 Hz, H-b), 3.58
1H NMR (CDCl3; 400 MHz) d 6.77
B. Roschek Jr et al./Phytochemistry 70 (2009) 1255–1261
(3H, s), 3.44 (9H, s), 3.37 (6H, s).13C NMR (CDCl3; 400 MHz) d 196.5 Download full-text
(C@O), 163.5 (C-40), 162.2 (C-20and C-60), 150.9 (C-3 and C-5),
134.1 (C-4), 131.6 (C-1), 103.4 (C-10), 99.7 (C-2 and C-6), 95.2
(3? –CH2–), 94.9 (3? –CH2–), 69.1 (C-a), 58.2 (C-b), 55.5 (6?
(±)-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
30 min. The MeOH was concentrated under vacuum and the resul-
tant dark residue was purified by silica gel column chromatogra-
phy byeluting with EtOAc:hexanes
CH2Cl2:MeOH (9:1) to give compound 3 as an off-white powder
(0.70 g, 66%).1H NMR (CDCl3; 400 MHz) d 6.62 (2H, s, H-20and
H-60), 5.98 (1H, s, H-8), 5.94 (1H, s, H-6), 4.96 (1H, d, J = 12 Hz, H-
2), 4.57 (1H, d, J = 12 Hz, H-3).
197.9 (C-4), 167.6 (C-7), 164.8 (C-9), 164.0 (C-5), 146.1 (C-30),
134.0 (C-10), 128.9 (C-40), 107.9 (C-20and C-60), 101.4 (C-10), 96.8
(C-8),95.7(C-6), 84.4 (C-3),
ion) = 320.0541 (calcd. for C15H12O8= 320.0532).
ion) = 585.2193 (calcd.for
13C NMR (CDCl3; 400 MHz) d
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 5,7,30,40-tetra-O-methylquercetin, dihydromy-
ricetin, Oseltamivir and Amantadine. Support for this research
was provided by HerbalScience Singapore, Pte. Ltd.
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