Nature Methods

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Supplemental Data: Figures
Direct isolation of flavonoids from plants using ultra-small anatase
TiO2 nanoparticles
Jasmina Kurepa, Ryo Nakabayashi, Tatjana Paunesku, Makoto Suzuki, Kazuki Saito, Gayle E.
Woloschak and Jan A. Smalle
Figure S1
List and nomenclature of cyanidin derivatives and flavonoid
glycosides detected in Col-0 plants grown on MS/2 media with 4%
sucrose.
Figure S2
UPLC-PDA chromatograms of aqueous acid methanol extracts of
Col-0 plants grown on 1% or 4% sucrose media.
Figure S3
Figure S4
Nanoharvesting flavonoids from rosettes and roots.
Effects of nanoparticle concentration and incubation temperature
on nanoharvesting yield.
Figure S5
Figure S6
Figure S7
Effects of pH on nanoharvesting.
Efficiency of nanoharvesting at different pH values.
Representative ion chromatograms of total plant extracts and
anthocyanins released from nanoparticle coronas.
Cellular damage and superoxide radical production in leaves
treated with nanoparticles.
Figure S8
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Supplementary Figure S1. List and nomenclature of cyanidin derivatives and flavonoid
glycosides detected in Col-0 plants grown on MS/2 media with 4% sucrose.
Total flavonoids were isolated and analyzed using LC-ESI-Q-TOF-MS/MS as described
(Matsuda et al. 2009).
(a) Isolated antocyanin derivatives (A1-A11)
A1: cyanidin 3-O-[2-O-(-D-xylopyranosyl)--D-glucopyranoside]-5-O--D-glucopyranoside
A2:
cyanidin
glucopyranoside]
3-O-[2-O-(-D-xylopyranosyl)--D-glucopyranoside]-5-O-[6-O-(malonyl)--D-
A3: cyanidin 3-O-[2-O-(-D-xylopyranosyl)-6-O-(E-p-coumaroyl)--D-glucopyranoside]-5-O--Dglucopyranoside
A4: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)--D-glucopyranoside-5-O--Dglucopyranoside
A5: cyanidin 3-O-[2-O-(-D-xylopyranosyl) -6-O-(E-p-coumaroyl)--D-glucopyranoside]-5-O-[6O-(malonyl)--D-glucopyranoside]
A6: cyanidin 3-O-[2-O-(-D-xylopyranosyl) -6-O-(4-O-(-D-glucopyranosyl)-E-p-coumaroyl)--Dglucopyranoside]-5-O--D-glucopyranoside
2
A7: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-E-p-coumaroyl)--Dglucopyranoside]-5-O--D-glucopyranoside
A8: cyanidin 3-O-[2-O--D-xylopyranosyl-6-O-(4-O-E-p-coumaroyl)--D-glucopyranoside]-5-O[6-O-(malonyl)--D-glucopyranoside]
A9:
cyanidin-3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-E-p-coumaroyl)--Dglucopyranoside]-5-O-[6-O-(malonyl)--D-glucopyranoside]
A10: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-Ep-coumaroyl)--D-glucopyranoside]-5-O--D-glucopyranoside
A11: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-(Ep-coumaroyl)--D-glucopyranoside)-5-O-[6-O-(malonyl)--D-glucopyranoside]
(b) Isolated flavonols (f1-f8).
f1: kaempferol 3-O--L-rhamnopyranoside-7-O--L-rhamnopyranoside
f2: kaempferol 3-O--D-glucopyranoside-7-O--L-rhamnopyranoside
f3:
kaempferol
rhamnopyranoside
3-O--L-rhamnopyranosyl
(1
→
2)--D-glucopyranoside-7-O--L-
f5: quercetin 3-O--D-glucopyranoside-7-O--L-rhamnopyranoside
f6: quercetin 3-O--L-rhamnopyranoside-7-O--L-rhamnopyranoside
f8: quercetin 3-O--L-rhamnopyranosyl (1→2)--D-glucopyranoside-7-O--L-rhamnopyranoside
3
Supplementary Figure S2. UPLC-PDA chromatograms of aqueous acid methanol extracts of
Col-0 plants grown on 1% or 4% sucrose media.
(a) Accumulation of anthocyanins in plants grown on 4% sucrose media. Anthocyanins were
extracted by aqueous acid methanol followed by chloroform partitioning (Kubasek et al. 1992).
Data are mean ± standard error of mean of three measurements.
(b) Peak labels correspond to compounds shown in Supplementary Figure 2. s1 and s2 are
sinapoyl derivatives, and s2i is an isomer of s2 (Yonekura-Sakakibara et al. 2008).
The analyses show that in addition to anthocyanin species such as A11 (cyanidin 3-O-[2-O-(2O-(E-sinapoyl--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-(E-p-coumaroyl)--Dglucopyranoside)-5-O-[6-O-(malonyl)--D-glucopyranoside]), sucrose led to an increase in
flavonols such as f3 and f1 (kaempferol 3-O--L-rhamnopyranosyl (1→2)--D-glucopyranoside7-O--L-rhamnopyranoside
and
kaempferol
3-O--L-rhamnopyranoside-7-O--Lrhamnopyranoside, respetively) and sinapoyl derivates. The zoomed chromatogram and relative
abundances of flavonols in 1% and 4% sucrose-treated plants are shown in the Figure 3.
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Supplementary Figure S3. Nanoharvesting flavonoids from rosettes and roots.
Methods: Col-0 plants grown on MS/2 media with 1% sucrose for three weeks were dissected,
and an equal mass (~20 mg) of shoot or root tissue was mixed with TiO 2 nanoparticles (pH 5.0,
76.7 mg/L). Harvesting was done for 24 hours at 22°C in the dark. Plant tissues were removed
from the tubes prior to photography. Nanoconjugates (NC) where pelleted for 1 min at 1000 g,
resuspended in a 10 mM phosphate buffer (pH 5.7), vortexed and sonicated for 1 min. 1 µl of
NC suspension was used to determine the absorption spectra using NanoDrop 2000.
(a) Nanoharvesting from shoots and roots.
(b) The UV-Vis light absorption spectra of nanoconjugates isolated from rosettes and roots. NP,
phosphorylated TiO2 nanoparticles.
Conclusions: The most prominent flavonoid species in roots are quercetin and kaempherol
derivates (Yonekura-Sakakibara et al. 2008). Both quercetin and kaempherol upon conjugation
to anatase nanoparticles form yellow-orange nanoconjugates (Figure 1). The UV-Vis absorption
spectrum of root NCs has a distinct peak at 420 nm, which corresponds to the conjugated
flavonols, and the spectrum of shoot NC has a peak at 600 nm that corresponds to the
conjugated cyanidin derivatives.
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Supplementary Figure S4. Effects of nanoparticle concentration and incubation temperature
on nanoharvesting yield.
(a) and (b) Equal amounts of leaf tissue (~20 mg) from plants grown on 4% sucrose were
incubated in 10 mM sodium phosphate buffer (pH 6.0) containing the denoted concentration of
ultra-small TiO2 nanoparticles (NP). After 4 hours of co-incubation at 22°C in the dark, the
tissues were removed, the incubation vessels were photographed (a). The nanoconjugates
were pelleted (1 min, 1000 g, room temperature) and resuspended in 10 mM sodium phosphate
buffer (pH 6.0). One µl was used to determine the absorption at 590 nm (peak absorption for
anthocyanin-TiO2 nanoconjugates (b).
(c) Equal amounts of leaf tissue (~20 mg) from plants grown on 4% sucrose were incubated in a
nanoparticle suspension in the dark at 4°C or 24°C for 12 hours. The leaf tissues were removed
prior to photography.
Supplementary Figure S5. Effects of pH on nanoharvesting.
Methods: An equal mass (~10 mg) of true leaves from plants grown on MS/2 with 4% sucrose
or of roots from plants grown on vertically positioned MS/2 plates with 1% sucrose was
incubated in a 500 µl of TiO2 nanoparticle suspension in 10 mM Na phosphate buffer (final mass
concentration 76.7 mg/L) for 4 hours in the dark at 22°C. The pH of the nanoparticle suspension
was adjusted with HCl or NaOH.
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(a) pH dependence of nanoharvesting from leaves. The highest yield of anthocyanin
nanoconjugates (NCs) was observed at the pH 5–6 range. A similar trend was observed when
the pH of the nanoparticle suspensions was adjusted by H2PO4 or KOH (not shown). SN,
supernatant.
(b) Nanoconjugates shown in (a) were pelleted by centrifugation, and resuspended in a 0.2 V of
10 mM Na phosphate buffer at pH 6. These suspensions were used for the quantifications by
UV-Vis spectroscopy shown in Figure S6.
(c) pH dependence of nanoharvesting from roots. Similar to the nanoharvesting efficiency from
leaves, the maximal yield of nanoparticles with plant metabolite corona was obtained within the
pH 5-6 range.
Supplementary Figure S6. Efficiency of nanoharvesting at different pH values.
Methods: After nanoharvesting, the source plant tissue was saved, briefly washed in a 10 mM
sodium phosphate buffer (pH 6.0), and used for anthocyanin extraction by the acid aqueous
methanol method (Kubasek et al. 1992). Nanoconjugates (shown in Figure S6) were pelleted by
centrifugation (1 min at 1000 g) and the pellet was resuspended in 100 µl of a 10 mM sodium
phosphate buffer (pH 6.0). The supernatant was also saved for further analyses. The acid
aqueous methanol extracts of the source plant tissue, supernatants of the nanoharvesting
reactions and nanoconjugates suspensions in phosphate buffer were used for spectral analyses
using NanoDrop 2000.
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(a) Comparison of the UV-Vis spectra of nanoconjugates (NC) isolated at different pH values
(red line) and control, phosphorylated TiO2 nanoparticles (NP, blue line). The representative UVVis spectra are shown, and the arrowhead pointing at 590 nm (a peak unique for nanoparticles
with anthocyanin corona) is added for clarity.
(b) Relative levels of anthocyanins (measured at 530 nm, A530) and anthocyanin
nanoconjugates (measured at 590 nm, A590). The absorption was measured in the coincubation solutions separated from nanoconjugates by centrifugation (supernatant),
nanoconjugates (pellet) and in the acid methanol extracts of the plants used for nanoharvesting
(residual). Data are shown as mean ± SD (n=9) and are expressed in relative units (RU).
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Supplementary Figure S7. Representative ion chromatograms of total plant extracts and
anthocyanins released from in vivo-formed nanoconjugates (NC).
The identities of cyanidin glycosides (A) are listed in Supplemental Figure 2. Arrows point to the
peaks with the denoted m/z ratios.
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Supplementary Figure S8. Cellular damage and superoxide radical production in leaves
treated with nanoparticles.
(a) Axenically grown seedlings were incubated with a 76.7 mg/L nanoparticle suspension for 4
hours, rinsed in sterile water and stained with the vital stain SYTOX Green (Molecular Probes,
http://www.lifetechnologies.com) essentially as described (Truernit and Haseloff 2008). In brief,
seedlings were incubated in 0.2 μM SYTOX Green solution (pH 7) in the dark for 20 min and
then rinsed in distilled water. Seedlings were mounted in Fluoro-Gel solution (Electron
Microscopy Sciences, http://www.emsdiasum.com) and analyzed using an Olympus BX51
fluorescent microscope. Representative images (scale bar = 100 µm) are shown.
(b) The number of nuclei per leaf area (mm2) was quantified using ImageJ software. Data are an
average ± SD of 5 counts.
(c) Superoxide radical (O2.-) detection. Plants were grown and treated with nanoparticles as
described in (a). The nitro blue tetrazolium (NBT) staining of leaves was done as described
(Kurepa et al. 2010).
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