Phytochemical Studies on Two Australian Anigozanthos Plant Species

Article abstract of DOI:10.1021/acs.jnatprod.7b00063

Phytochemical studies of two Australian Anigozanthos (kangaroo paw) species, A. rufus and A. pulcherrimus, resulted in the identification of 13 secondary metabolites. 2-Amino-6-O-p-coumarylheptanedioic acid (3) and chalcone-5′-O-(4-O-p-coumaryl)-O-β-d-glucopyranoside (12) are reported as new compounds and are accompanied by nine flavonoids (2, 5-11, 13) and two anthocyanins (1, 4). Compounds 1 and 4 were isolated as red solids from A. rufus and are likely responsible for the coloration of the flowers. Compounds 1, 3, and 6 showed weak antimicrobial activities against Acinetobacter baumannii ATCC 19606 at concentrations of 52.4, 94.9, and 53.9 μM, respectively.

Full text of DOI:10.1021/acs.jnatprod.7b00063

Note
pubs.acs.org/jnp
Phytochemical Studies on Two Australian Anigozanthos Plant
Species
Rudi Hendra and Paul A. Keller*
School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia
S
* Supporting Information
ABSTRACT: Phytochemical studies of two Australian Anigozanthos (kangaroo paw) species, A. rufus and A. pulcherrimus,
resulted in the identification of 13 secondary metabolites. 2-Amino-6-O-p-coumarylheptanedioic acid (3) and chalcone-5′-O-(4-
O-p-coumaryl)-O-β-D-glucopyranoside (12) are reported as new compounds and are accompanied by nine flavonoids (2, 5−11,
13) and two anthocyanins (1, 4). Compounds 1 and 4 were isolated as red solids from A. rufus and are likely responsible for the
coloration of the flowers. Compounds 1, 3, and 6 showed weak antimicrobial activities against Acinetobacter baumannii ATCC
19606 at concentrations of 52.4, 94.9, and 53.9 μM, respectively.
he Anigozanthos genus is a member of the Hemodoraceae
family with examples more commonly known as kangaroo
despite the similar name, the black kangaroo paw belongs to a
different genus from the rest of the kangaroo paw species.
We have an ongoing interest in colors from Nature,
concentrating particularly on flowers that are endemic to
Australia,9 as well as any relationship between the isolated
compounds and indigenous uses. Although the aforementioned
studies investigated the roots of Anigozanthos sp. and its family,
the flowers from these species have not yet undergone
phytochemical investigation. Here, we report the extraction,
isolation, and identification of compounds from the flowers of
the red (A. rufus) and yellow (A. pulcherrimus) kangaroo paw and
document the compounds responsible for the intense coloration
present in the flowers. Further, we extrapolate correlations
between the biological activities of some of the isolated
compounds and known traditional medicinal uses within the
Hemodoraceae family.
T
paws. The genus contains nine species and is endemic to
southwest Australia, where it is the floral emblem of the State of
Western Australia.1 The flowers have a variety of colors such as
red, yellow, black, and red-green depending on the species, and
their unique shape resembles a tubular structure coated with
dense hairs that opens at the apex with six claw-like structures.2,3
Apart from the unique and attractive flowers, kangaroo paws
possess tuberous roots that contain significant levels of starch
and form part of the diet of the indigenous people in the
Yellagonga region of Western Australia.4 While there are no
documented reports of members of the Anigozanthos genus as
medicinal plants, four phenylpropanoids were isolated and their
structures elucidated from the red root of Anigozanthos rufus in
1975; extracts from these roots were used as a dye by the
indigenous people in the Northern Territory, Australia.5,6 This
characteristic class of secondary metabolites occurs in plant
families such as Hemodoraceae, Ponederaiaceae, Strelitziaceae,
and Musaceae;7 for example, six phenylphenalenones were
isolated from Hemodorum simplex (Hemodoraceae) and found to
possess moderate cytotoxicity against the P388 murine leukemia
cell line, with an IC50 value of >26−39 μM.8 In a separate study,
Hidalgo et al. reported the biosynthesis of phenylphenalenone
from in vitro root cultures of A. preissii and isolated four
phenylpropanoids, including 4′-methoxyanigorufone.7 Similar
compounds were isolated from the flowers of the black kangaroo
paw (Macropidia fuliginosa) and showed low antibacterial activity
against Staphylococcus aureus ATCC 25923.7 Interestingly,
RESULTS AND DISCUSSION
■
Separate MeOH extractions of the flowers of A. rufus and
A. pulcherrimus were concentrated and sequentially solvent-
partitioned into n-hexane/MeOH, resulting in polar and
nonpolar fractions. The chemical constituents from the nonpolar
fraction were analyzed using GC-EIMS (Figure S42, Supporting
Information). The polar fractions from both species were
subjected to analytical RP-HPLC followed by semipreparative
Received: January 20, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00063
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Journal of Natural Products
Note
RP-HPLC, which resulted in the isolation of 13 compounds
(Figure 1), 11 of which were known. The structures were
Table 1. NMR Spectroscopic Data (500 MHz, Methanol-d4)
for Compound 3
position
δC, type
δH (J in Hz)
gCOSY
gHMBC
1
171.9, C
a
2
53.9, CH
31.9, CH2
22.2, CH2
31.3, CH2
72.8, CH
173.8, C
3.99, dd (8.8, 7.5)
2.10−2.00, m
1.75−1.50, m
1.95−1.93, m
5.10, dd (7.4, 4.9)
3
4, 1
3
2, 4
3, 5
4, 6
1, 5
2, 6
3, 7
4
5
a
6
4, 7, 1′
7
1′
2′
3′
1″
2″
3″
4″
5″
6″
168.6, C
a
114.5, CH
145.6, CH
127.4, C
6.39, d (15.9)
7.68, d (15.9)
3′
2′
1′ , 1″
1′, 2″, 6″
131.1, CH
116.9, CH
161.4, C
7.48, d (8.3)
6.82, d (8.2)
3″
2″
3′, 4″, 6″
1″, 5″
116.9, CH
131.1, CH
6.82, d (8.2)
7.48, d (8.3)
6″
5″
1″, 3″
3′, 2″,4″
a
Indicates weak signal.
Figure 1. Molecular structures of compounds isolated from the flowers
of Anigozanthos sp. (A. rufus and A. pulcherrimus). Compounds 2, 3, and
5−12 were isolated from both species. Compounds 1 and 4 were
isolated from A. rufus, and compound 13 was isolated from
A. pulcherrimus.
Figure 2. Key HMBC and COSY correlations for compound 3.
elucidated through extensive NMR analyses and by literature
comparisons. The known compounds were identified as
cyanidin-3-rutinoside (1),10 quercetin-3-rutinoside (2),11 cyani-
din-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside (4),12
kaempferol (5),11 kaempferol-3-O-rutinoside (6),11 apigenin-7-
O-β-D-glucopyranoside (7),13 kaempferol-3-O-(6-O-p-coumar-
yl)-O-β-D-glucopyranoside (8),14 quercetin-3-O-(6-O-p-cou-
maryl)-O-β-D-glucopyranoside (9),15 quercetin-3-O-β-D-gluco-
pyranoside (10),11 luteolin-7-O-β-D-glucopyranoside (11),16
H-2, H-3, H-5, and H-4, respectively. Through gHMBC analysis,
the carbonyl resonance at δC 171.9 (C-7) correlated with H-5,
and the carbonyl resonance at δC 173.8 (C-1) correlated with H-
3. Moreover, gHMBC spectroscopic analysis revealed a
correlation between H-5 and carbon resonances at δC 168.6
and 22.2, assigned as C-1′ and C-4, respectively. Furthermore, a
three-bond proton−carbon correlation between H-2 and the
carbon resonance at δC 22.2 allowed its assignment to C-4.
Therefore, compound 3 was assigned as the novel 2-amino-6-O-
p-coumarylheptanedioic acid. Its specific rotation was deter-
mined as [α]2D5 0 (c 0.13, MeOH), indicating the likely presence
of a racemate.
and dihydroquercetin (13).17 Compounds 3 and 12 (tR
=
10.36, and 16.99 min) are new and were isolated from both
plants.
Compound 3 was isolated as a bright yellow solid with
HRESIMS analysis indicating a peak at m/z 338.1249 ([M +
1
H]+), assigned to the molecular formula C16H20NO7. The H
Compound 12 was isolated as a yellow solid with HRESIMS
analysis indicating a peak at m/z 603.1489 ([M + Na]+), assigned
to the molecular formula C30H28O12Na. The 1H and 13C NMR
spectroscopic analysis is collated in Table 2.
and 13C NMR spectroscopic data are collated in Table 1.
The 13C NMR spectroscopic analysis of 3 showed resonances
at δC 168.5 (C-6), 171.9 (C-7), and 173.8 (C-1) assigned to three
carbonyls, suggesting the presence of an ester and two hydroxy
Analysis of the 1H NMR spectra of 12 showed resonances at
δH 6.03 (s) and 6.26 (s) assigned to H-4′ and H-6′ of ring A,
respectively, which, via gHMBC analysis, correlated to a
quaternary carbon resonance at δC 127.2, assigned to C-1′, and
three oxygenated tertiary carbon resonances at δC 165.9, 161.4,
and 161.1, assigned to C-3′, C-5′, and C-2′, respectively.
Furthermore, the 1H NMR spectrum revealed resonances at δH
7.62 (2H, d, J = 8.0 Hz) and 6.87 (2H, d, J = 8.0 Hz), assigned to
H-2/6 and H-3/5 of ring B, respectively. This 1,4-disubstituted
benzene ring contained a quaternary carbon C-1 and an
oxygenated tertiary carbon C-4, which were assigned using
gHMBC analysis to resonances at δC 128.3 and 161.8,
respectively. A pair of trans olefinic proton resonances at δ
8.02 (1H, d, J = 15.0 Hz) and 7.68 (1H, d, J = 15.0 Hz) were
assigned to H-α and H-β, respectively, and through gHMBC
analysis and showed a proton−carbon correlation to the signals
1
carbonyl functional groups. Analysis of the H NMR data
revealed two sets of doublets at δH 7.48 (H-2″/6″) and 6.82 (H-
3″/5″) with coupling constants of 8.3 Hz, assigned as aromatic
ortho hydrogen atoms, with supporting evidence from gCOSY
analysis indicating a correlation between these protons. Two
further doublets at δH 7.68 (H-3′) and 6.39 (H-2′), together with
carbon resonances at δC 114.5 (C-2′) and 145.6 (C-3′), indicated
the presence of a pair of E-olefinic protons with a coupling
constant of 15.9 Hz. From the analysis of gHMBC data, a doublet
at δH 7.48 (H-2″/6″) showed both a long-range correlation with
the oxygenated aromatic carbon resonance at δC 161.4 (C-4″)
and a correlation with the resonance at δC 145.6, which was
assigned to the E-olefinic C-3′ (Figure 2).
The two shielded resonances at δH 5.10 and 3.99 and three
methylene groups at δH 2.02, 1.93, and 1.69 were assigned as H-6,
B
DOI: 10.1021/acs.jnatprod.7b00063
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Journal of Natural Products
Note
further doublets at δH 7.49 (1H, d, J = 15.0 Hz) and 6.83 (1H, d, J
= 15.0 Hz) were assigned to H-α′ and H-β′, respectively, and
showed proton−carbon correlations to resonances at δC 125.8,
131.4, and 168.7, assigned to C-1‴, C-2‴/6‴, and the ester
carbonyl carbon, respectively (Figure 3).
Table 2. NMR Spectroscopic Data (500 MHz, Methanol-d4)
for Compound 12
a
position
δC, type
δH (J in Hz)
gCOSY gHMBC
1
128.3, C
131.9, CH
117.1, CH
161.8, C
117.1, CH
131.8, CH
127.2, C
161.1, C
165.9, C
98.7, CH
161.4, C
96.0, CH
1H NMR spectroscopic analysis showed the presence of sugar
moieties with resonances at δH 5.23 (1H, d, J = 7.8 Hz), 5.02 (1H,
dd, J = 9.8, 5.3 Hz), 3.90 (1H, t, J = 5.9 Hz), 3.82 (1H, dd, J = 9.8,
9.3 Hz), 3.72/3.62 (2H, dd, J = 12.6, 5.9 Hz), and 3.69 (1H, dd, J
= 10.5, 8.6 Hz), assigned to H-1″, H-4″, H-5″, H-3″, H-6″, and
H-2″, respectively. The proton sequences were confirmed by
gCOSY and TOCSY through bond analysis and suggested a
glucose moiety. In addition, acid hydrolysis of 12 indicated the
presence of a β-D-glucopyranose moiety via HPLC comparison
to authentic standards at a retention time of 8.33 min (Figure
S35, Supporting Information). Further gHMBC analysis
indicated a proton−carbon correlation from H-1″ to C-5′ and
from H-4″ to the ester carbonyl carbon, confirming the link
between ring A and glucose and from glucose to the cinnamoyl
moiety, respectively (Figure 3). Therefore, 12 was proposed as
the novel chalcone 5′-O-(4‴-O-p-coumaryl)-O-β-D-glucopyra-
nose.
2
7.62, d (8.0)
3
2
4, β, 6
3
6.87, d (8.0)
1, 5
4
5
6.87, d (8.0)
7.62, d (8.0)
6
5
1, 3
6
4, β, 2
1′
2′
3′
4′
5′
6′
6.03, s
6.26, s
2′, 6′
4′, 2′,
194.8
α1
125.6, CH
144.3, CH
194.8, CO
8.02, d (15.0)
7.68, d (15.0)
β1
α1
1, 1″
2, 194.8
β1
carbonyl
Glucosyl
1″
2″
3″
4″
5″
6″
101.7, CH
75.3, CH
76.3, CH
72.1, CH
76.,7 CH
62.3, CH2
5.23, d (7.8)
2″
5′, 3″, 5″
3.69, dd (8.9, 7.8)
3.82, dd (9.5, 9.0)
5.02, dd (9.5, 8.5)
3.90, ddd (8.9, 6.5, 12.0)
3.72, dd (6.5, 1.2)
3.62, dd (12.0, 6.5)
1″, 3″ 4″
2″, 4″ 1″, 5″
3″, 5″ 168.7
4″, 6″
The HPLC chromatogram profile of flowers from both species
showed the presence of similar compounds (2, 3, 5−12).
Compound 2 (tR = 10.07 min) was found be the major
constituent from the polar extract of both flowers followed by
compound 8 (tR = 13.00 min). Furthermore, compound 2 was
also found in the flowers of the black kangaroo paw
(M. fuliginosa).7 Compound 13 was found to be present only
in A. pulcherrimus, with 1 and 4 (tR = 9.62 and 10.88 min) found
only in A. rufus. Compounds 1 and 4 were dark red solids, and
analysis of their NMR data indicated the presence of
anthocyanins, supported by ESIMS data analysis. Although
phenalenone pigments are well known as red coloration in
several species of the plant family Hemodoraceae,6,18,19
anthocyanins 1 and 4 are reported here for the first time in
this species.
Antibacterial Activity Studies. The MeOH extracts from
both species and the isolated compounds (1−13) were screened
for their antibacterial activities against five bacteria and two fungi
at 32 μg/mL. Their activities were analyzed based on the ability
of the sample to inhibit the growth of microorganisms equal to or
more than 80% with a Z-score ≥ 2.5 (data in Supporting
Information). All samples tested were inactive against all the
tested microorganisms based on the above criteria. However,
based on the percentage of growth inhibition, compounds 1, 3,
and 6 showed weak inhibitory activity to Acinetobacter baumannii
ATCC 19606 at concentrations of 52.4, 94.9, and 53.9 μM,
respectively.
5″
Coumaryl
1‴
125.8, C
2‴
3‴
4‴
131.4, CH
117.0, CH
161.2, C
7.49, d (8.0)
6.83, d (8.0)
3‴
2‴
5‴
6‴
α2
117.0, CH
131.4, CH
114.8, CH
147.6, CH
168.7, CO
6.83, d (8.0)
7.49, d (8.0)
6.36, d (15.0)
7.70, d (15.0)
6‴
5‴
β2
α2
β2
carbonyl
a
1H and 13C NMR experiments were performed in methanol-d4 at 500
and 125 MHz, respectively.
at δC 127.2, 128.3, and 194.8, assigned as C-1′, C-1, and the
ketocarbonyl carbon, respectively (Figure 3).
Plants from the family Hemodoraceae, which are found
predominantly in Australia, have been reportedly used by the
indigenous people for several purposes. Extracts of the bulbs
from the genus Hemodorum have been used as a purgative, an
ointment for snake bites, and coloring agents.8 Recent studies
reported that the MeOH and CH2Cl2 extracts from flowers of
black kangaroo paw (M. fuliginosa) showed potential as
antimicrobial agents against Pseudomonas aeruginosa and
Streptococcus pyogenes.18 Despite the lack of reports regarding
medicinal usage from the genus Anigozanthos, especially from the
flowers, it was found that the isolated compounds from both
species possess various biological activities from previous studies.
Flavonoid compounds were the major compounds isolated from
both flowers, and these are well known to possess antimicrobial
Figure 3. Key HMBC and COSY correlations for compound 12.
A set of ortho-coupled aromatic resonances of ring C protons
at δH 7.49 (2H, d, J = 8.0 Hz) and 6.83 (2H, d, J = 8.0 Hz) were
assigned to H-2‴/H6‴ and H-3‴/H5‴, respectively, with
gHMBC analysis showing correlations between these protons
and the quaternary carbon resonance at δC 125.8 (C-1‴) and the
oxygenated tertiary carbon resonance at δC 161.2 (C-4‴). Two
C
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Journal of Natural Products
Note
(A. pulcherrimus). The residues were liquid−liquid extracted with n-
hexane and MeOH, resulting in polar extracts (3.154 and 2.832 g,
respectively). The nonpolar extracts were analyzed for their chemical
constituents using GC-EIMS analysis. The MeOH fraction from A. rufus
(400 mg) in MeOH (50 mL) was filtered through a Whatman syringe
filter 0.45 μm, and the volume was reduced to 20 mL. The solution was
injected (10 × 2.0 mL/injections) onto preparative HPLC with a
gradient elution from 90% of solvent A (0.1% trifluoroacetic acid (TFA)
in H2O) to 50% of solvent B (0.1% TFA in MeCN) within 35 min. All
isolates were manually trapped based on the chromatographic profile
with the following retention times (min) for each compound (Figure S1,
Supporting Information): 1 (22 mg, tR = 9.62 min), 2 (90 mg, tR = 10.07
min), 3 (17 mg, tR = 10.36 min), 4 (21 mg, tR = 10.88 min), 5 (5.5 mg, tR
= 12.09 min), 6 (10 mg, tR = 12.28 min), 7 (12 mg, tR = 12.54 min), 8 (14
mg, tR = 13.00 min), 9 (8.5 mg, tR = 13.59 min), 10 (11 mg, tR = 14.47
min), 11 (8.0 mg, tR = 14.47 min), and 12 (6.5 mg, tR = 16.99 min). In an
identical HPLC procedure, the MeOH fraction from A. pulcherrimus
(400 mg) resulted in the isolation of compounds 2 (83 mg), 3 (12.5
mg), 5 (6.2 mg), 6 (8.3 mg), 7 (10.3 mg), 8 (11.5 mg), 9 (7.2 mg), 10
(10.4 mg), 11 (7.8 mg), 12 (7.5 mg), and 13 (4.0 mg, tR = 14.47 min).
All the isolates were freeze-dried, producing solid compounds for
spectroscopic analysis.
Cyanidin-3-rutinoside (1): UV-active red solid; UV (MeOH) λmax
280 (3.87); 519 (3.95) nm; LREIMS m/z 595 [M + 1]+; HRESIMS m/z
595.1781 (calcd for C27H31O15 595.1752). All NMR data were identical
to reported data.10
Quercetin-3-rutinoside (2): UV-active, pale yellow solid; LREIMS
m/z: 633 [M + Na]+, 609 [M + H]−. All NMR data were identical to
reported data.11
2-Amino-6-O-p-coumarylheptanedioic acid (3): UV-active, pale
yellow, oily solid; [α]2D5 0 (c 0.13, MeOH); IR [cm−1] 3365 (br, m), 2944
(s), 831 (s), 1717 (s); 1H NMR (500 MHz, methanol-d4) and 13C NMR
(125 MHz, methanol-d4) data, see Table 1; HRESIMS m/z 338.1240
(calcd for C16H20NO7, 338.1240).
Cyanidin-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside (4): UV-
active, red solid; UV (MeOH) λmax 282 (3.92); 313 (3.85); 523 (3.84)
nm; LREIMS m/z 595 [M + 1]+. All NMR data were identical to
reported data.12
activities. Quercetin and quercetin glycosides (2, 9, and 10) have
been reported to exhibit antioxidative, anticarcinogenic, anti-
inflammatory, and vasodilating effects.20
Many studies report the benefit of kaempferol and its glucoside
to reduce the risk of chronic disease. Kaempferols (5−8) have
the ability to modulate a number of key elements in cellular signal
transduction pathways linked to apoptosis, angiogenesis,
inflammation, and metastasis.21 Additionally, taxifolin (13) is
believed to have therapeutic promise in treating cancer and
cardiovascular and liver disease;22 this study is the first to report
its isolation from the Aningozanthos genus.
In summary, phytochemical studies led to the successful
isolation of the major constituents from the flowers of two
species from the Aningozanthos genus. Anthocyanins 1 and 4
from A. rufus flowers are reported for the first time, and
additionally, the new compounds 3 and 12 were isolated and
identified. An antimicrobial assay of the isolated compounds
indicated they were inactive against the tested microorganisms.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Jasco P-2000 polarimeter. UV−visible spectra of samples
diluted in MeOH were obtained using a Shimadzu UV-1601 UV−vis
spectrophotometer. ECD spectra were recorded on a JASCO J-810
spectropolarimeter with a path length of 0.1 cm and concentration
between 50 and 100 μM in MeOH. IR spectra were recorded with a
Shimadzu IR Affinity-1 FT-IR spectrometer fitted with a 1.5 round
diamond crystal. NMR spectra were recorded at 500 and 125.7 MHz,
respectively, on a Varian Unity Inova-500 MHz spectrometer,
controlled by Varian VNMR software (version 6.1 revision C). NMR
spectra were acquired in methanol-d4 with chemical shifts (δ) reported
in parts per million (ppm) relative to methanol-d4 (1H: δ = 3.31; 13C: δ =
49.2) (unless otherwise specified). Coupling constants (J) are reported
1
in hertz (Hz). J values listed in H NMR spectroscopic data refer to
coupling between hydrogen nuclei. ESI mass spectra were obtained on
an LCMS-2010 EV (Shimadzu). Samples were injected as a solution in
methanol HPLC grade. HRESIMS were acquired on a Micromass
QTOF2 Ultima spectrometer.
Kaempferol (5): UV-active, yellow solid; UV (MeOH) λmax 228
(3.26); 267 (4.08); 366 (4.02) nm; LREIMS m/z 310 [M + Na]+. All
NMR data were identical to reported data.11
The HPLC profiles from both species were obtained using a Waters
system (Waters 1525 pump, Waters 2487 detector, controlled by Breeze
software v3.30) with a Symmetry C18 column (5 μm, 4.9 × 150 mm) and
a Wakosil C-18 RS column (5 μm, 4.6 × 250 mm). All compounds were
isolated by Preparative HPLC using a Waters prep-LC system (LC-600
controller, 2489 detector, LC150 pump, PD1 degasser) with a Waters
reversed-phase OBD Sunfire C18 column (5 μm, 19 × 150 mm)
protected with a Waters Sunfire C18 guard column (5 μm, 19 × 10 mm).
The sugar moiety from compound 12 was identified by using an RP-
HPLC (Shimadzu HPLC) system, coupled with an automatic sampler
(Shimadzu SIL-10A XL) and a Sedere Sedex 60 LT evaporative light
scattering detector (ELSD) with a Prevail Carbohydrates ES column
(250 × 4.6 mm i.d.; Alltech). All analytical HPLC samples were filtered
through Grace syringe filter PTFE 0.45 μm, 4 mm, and preparative
HPLC samples were filtered through Grace syringe filter 0.45 μm, 30
Kaempferol-3-O-rutinoside (6): UV-active, yellow solid; UV
(MeOH) λmax 225 (4.08); 268 (4.03); 350 (3.99) nm; LREIMS m/z:
595 [M + H]+; HRESIMS m/z 617.1484 (calcd for C27H30O15Na,
617.1482). All NMR data were identical to reported data.11
Apigenin-7-O-β-D-glucopyranoside (7): UV-active, yellow solid;
LREIMS m/z 469 [M + Na]+. All NMR data were identical to reported
data.13
Kaempferol-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside (8):
UV-active, yellow solid; LREIMS m/z 617 [M + Na]+. All NMR data
were identical to reported data.14
Quercetin-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside (9): UV-
active, yellow solid; LREIMS m/z 609 [M − H]−, 633 [M + Na]+. All
NMR data were identical to reported data.15
Quercetin-3-O-β-D-glucopyranoside (10): UV-active, yellow solid;
UV (MeOH) λmax 228 (4.14); 259 (4.12); 359 (4.07) nm; LREIMS m/z
465 [M + 1]+. All NMR data were identical to reported data.11
Luteolin-7-O-β-D-glucoside (11): UV-active, yellow solid; LREIMS
m/z 449 [M + 1]+. All NMR data were identical to reported data.16
Chalcone-5′-O-(4-O-p-coumaryl)-O-β-D-glucoside (12): UV-active,
yellow solid; [α]2D2 +12.8 (c 0.15, MeOH); IR [cm−1] 3566 (br, m), 2360
(s), 1683 (s); 1H NMR (500 MHz, methanol-d4) and 13C NMR (125
MHz, methanol-d4) data, see Table 2; LREIMS, m/z 603 [M + Na]+;
HRESIMS m/z 603.1506 (calcd for C30H28O12Na, 603.1478).
Dihydroquercetin (13): UV-active, light yellow solid; [α]2D5 +46 (c
0.15, MeOH) [lit.23 [α]D25 +46.2 (c 0.12, MeOH)]; LREIMS m/z 304
[M − 1]−. All NMR data were identical to reported data.17
Acid Hydrolysis of Compound 12. A mixture of 12 (1.0 mg) in
aqueous HCl (15%, 2.5 mL) was heated with stirring at 50 °C for 6 h.
The solution was neutralized by the dropwise addition of NaOH (5%)
mm. A Buchi rotary evaporator (R-114/200) with a high vacuum pump
̈
was used for evaporation of solvents under reduced pressure at 40 °C.
Plant Material. The red (A. rufus, voucher specimen Woll11570)
and yellow (A. pulcherrimus, voucher specimen Woll11569) kangaroo
paw flowers were collected in January 2015 from the campus grounds of
the University of Wollongong, Australia (34°24′16.9″ S 150°52′38.2″
E). All the flowers were washed, freeze-dried, and stored in a refrigerator
until analysis. Voucher specimens of both species are stored in the Janet
Cosh Herbarium Reference Collection at the University of Wollongong.
Extraction and Isolation of Flowers. The freeze-dried flowers (70
g each) were crushed, suspended in MeOH (1.0 L), stirred for 24 h, and
then filtered, and the filtrate was extracted with MeOH (3 × 1.0 L). The
supernatants for each species were separately pooled and concentrated
in vacuo to produce residues, 12.371 g (A. rufus) and 11.946 g
D
DOI: 10.1021/acs.jnatprod.7b00063
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
with pH monitoring using universal indicator paper. This aqueous
mixture was partitioned with EtOAc (2 × 2.5 mL), and the aqueous
fraction evaporated under reduced pressure.24 The residue was
subjected to RP HPLC using a Carbohydrates ES column with ELSD
and an isocratic flow (H2O/MeCN, 20:80) for 20 min with a flow rate of
1.0 mL/min. The retention time of the fraction (8.3 min) corresponded
to β-D-glucopyranose, as evidenced by a co-injection with a standard.
Antimicrobial Assay. Antimicrobial activities were conducted by
the Community for Open Antimicrobial Drug Discovery, Queensland,
Australia. The MeOH extracts from both species and isolated
compounds were evaluated for their antimicrobial activities against
seven microorganisms (Staphylococcus aureus ATCC 43300 (MRSA),
Eschericia coli ATCC 25922 (FDA control strain), Klebsiella pneumoniae
ATCC 700603 (MDA), Pseudomonas aeruginosa ATCC 27853,
Acinetobacter baumannii ATCC 19606, Candida albicans ATCC
90028, and Cryptococcus neoformans ATCC 208821). All the samples
were dissolved in DMSO at a concentration of 10 mg/mL. Each sample
(5 μL) was plated in duplicate into a 384-well nonbinding surface plate
for each strain. All bacteria were cultured in cation-adjusted Mueller
Hinton broth at 37 °C overnight; then 45 μL was added to each well of
the compound-containing plates, giving a cell density of 5 × 105 CFU/
mL to give a final compound concentration of 32 μg/mL. All the plates
were covered and incubated at 37 °C for 18 h. For the fungi strains, all
the fungi were cultured for 3 days on yeast extract−peptone dextrose
agar at 30 °C, and these stock suspensions were diluted with yeast
nitrogen base broth to a final concentration of 2.5 × 105 CFU/mL.
Then, 45 μL was added to each well of the compound-containing plates,
giving a final compound concentration of 32 μg/mL, and incubated at 35
°C for 24 h. Inhibition of bacterial growth was determined by measuring
absorbance at 600 nm using a Tecan M1000 Pro monochromator plate
reader, while growth inhibition of the fungi was determined by
measuring absorbance at 530 nm after the addition of resazurin (0.001%
final concentration) and incubation at 35 °C for an additional 2 h. The
absorbance was measured using a Biotek Synergy HTX plate reader. The
percentage of growth inhibition was calculated for each well, using the
negative control (media only) and positive control (microorganisms
without inhibitors) on the same plate. The significance of the value was
determined by Z-scores, calculated using the average and standard
deviation of the sample wells (no controls). Samples with an inhibition
value above 80% and Z-score above 2.5 for either replicate were
classified as active.
Higher Education (DGHE) and UOW International Post-
graduate Tuition Fee Award 2016 for a scholarship.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00063.
(24) Dias, D. A.; Urban, S.; Roessner, U. Metabolites 2012, 2, 303−336.
General procedures, HPLC profiles, GCMS, antimicrobial
data, and NMR spectral data of compounds 1−13
including 2D NMR spectral data of compounds 3 and
12 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: keller@uow.edu.au. Tel: +61 2 4221 4692. Fax: +61 2
4221 4287.
■
ORCID
Paul A. Keller: 0000-0003-4868-845X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Antimicrobial screening was performed by CO-ADD (The
Community for Antimicrobial Drug Discovery), funded by the
Wellcome Trust (UK) and The University of Queensland
(Australia). R.H. thanks the Indonesian Directorate General
E
DOI: 10.1021/acs.jnatprod.7b00063
J. Nat. Prod. XXXX, XXX, XXX−XXX