Top-down targeted metabolomics reveals a sulfur-containing metabolite with inhibitory activity against angiotensin-converting enzyme in asparagus officinalis

Article abstract of DOI:10.1021/acs.jnatprod.5b00092

The discovery of bioactive natural compounds containing sulfur, which is crucial for inhibitory activity against angiotensin-converting enzyme (ACE), is a challenging task in metabolomics. Herein, a new S-containing metabolite, asparaptine (1), was discovered in the spears of Asparagus officinalis by targeted metabolomics using mass spectrometry for S-containing metabolites. The contribution ratio (2.2%) to the IC₅₀ value in the crude extract showed that asparaptine (1) is a new ACE inhibitor.

Full text of DOI:10.1021/acs.jnatprod.5b00092

Note
pubs.acs.org/jnp
Top-down Targeted Metabolomics Reveals a Sulfur-Containing
Metabolite with Inhibitory Activity against Angiotensin-Converting
Enzyme in Asparagus officinalis
Ryo Nakabayashi,*,† Zhigang Yang,†,§ Tomoko Nishizawa,† Tetsuya Mori,† and Kazuki Saito*,†,‡
†RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
‡Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
S
* Supporting Information
ABSTRACT: The discovery of bioactive natural compounds
containing sulfur, which is crucial for inhibitory activity against
angiotensin-converting enzyme (ACE), is a challenging task in
metabolomics. Herein, a new S-containing metabolite,
asparaptine (1), was discovered in the spears of Asparagus
officinalis by targeted metabolomics using mass spectrometry
for S-containing metabolites. The contribution ratio (2.2%) to
the IC50 value in the crude extract showed that asparaptine (1) is a new ACE inhibitor.
ulfur-containing metabolites (S-metabolites) are common
across the plant kingdom and constitute a family of natural
approach based on the targeted analysis for S-metabolites (S-
omics) has been established using Fourier transform ion
cyclotron resonance mass spectrometry (FTICRMS), which
offers the best performance with respect to ultrahigh mass
accuracy (<1 ppm) and peak resolution (>1 000 000 fwhm).10
S-Omics using edible parts of three crops, i.e., onion (Allium
cepa L.), green onion (Allium fistulosum L.), and garlic (Allium
sativum L.), revealed the distribution of S-metabolites.11 The
tagged information on S-metabolites by FTICRMS coupled
with liquid chromatography (LC) may be expected to
streamline the isolation and chemical assignment of S-
metabolites.
S-Omics was used to seek a S-ion from a new S-metabolite,
asparaptine (1), which is a conjugate of arginine and
asparagusic acid, in 47 plant samples, including asparagus
(Table S1, Supporting Information). Aqueous methanol extract
solutions of the plants were analyzed using LC−FTICRMS to
understand the S-ion distribution in all detectable metabolite
peaks. On the basis of the differences in the exact mass and
relative signal intensity of S-ions and their 34S-substituted
counterparts, 733 putative S-ions were extracted from the
metabolome matrix, which included 4537 peaks. Then, the S-
ions were filtered using their relative signal intensity value
(>1.0) to isolate asparaptine (1). Ultimately, elemental
composition was assigned for 13 S-ions using their exact
mass and isotope pattern (Figure 1A and Table S2, Supporting
Information). The S-ion derived from asparaptine (1) had the
highest relative signal intensity in asparagus. The isotopic
pattern was nearly identical to the theoretical one of
C10H19N4O3S2 (Figure 1B).
S
products with a wide range of beneficial biological activities for
humans, such as anti-inflammatory, antioxidative, and anti-
cancer effects. The presence of a sulfur atom is crucial for the
inhibitory activity against angiotensin-converting enzyme
(ACE), which catalyzes the reaction from angiotensin I to
angiotensin II in the rennin−angiotensin system and plays a
major role in hypertension.1,2 Interestingly, a saturated five-
membered sulfide ring (1,2-dithiolane ring) in synthetic
peptides has a prominent role in the inhibitory activity of ACE.3
The inhibitory activity of plant extracts against ACE is
broadly investigated.4 Asparagus officinalis L. (Asparagaceae)
has a positive effect in the prevention of hypertension by
inhibiting the activity of ACE.5 Reports show that N-containing
metabolites (e.g., nicotianamine and 2″-hydroxynicotianamine)
are possible inhibitors in asparagus; however, the existence of
other molecules has been suggested. Asparagus is compared
with Allium species (Alliaceae),6 which accumulate a variety of
S-metabolites.7,8 Therefore, it is hypothesized that asparagus
might contain unknown S-metabolites exhibiting inhibitory
activity against ACE.
The exact mass and natural abundance of stable isotopes in
Nature are reflected in their mass spectrometry (MS) spectra.
Sulfur has two major stable isotopes, 32S and 34S, representing
their natural abundances of 95.02% and 4.21%, respectively.
The theoretical differences in the exact mass and relative signal
intensity between 32S-containing monoisotopic ions (S-ions)
and their 34S-substituted counterparts can be used for the
chemical assignment of S-ions. Since the clear separation of 34S-
ions and other ions substituted by 18O and 13C2 at the region of
the monoisotope (M) + 2 requires high mass accuracy (<1
mDa) and peak resolution [ca. 250 000 full width at half-
maximum (fwhm)] in small molecules,9 these ions have not
been used for chemical assignment. Recently, a metabolomic
Received: January 29, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Targeted metabolomics for S-containing metabolites to elucidate the new compound asparaptine (1) in Asparagus officinalis. (A)
Hierarchical cluster analysis of S-containing monoisotopic ions (S-ions). The elemental compositions of S-ions were determined using their exact
mass and isotope pattern. The color bar indicates the strength of the relative signal intensity. The gray color indicates less than 1.0 or not detected.
(B) Isotope pattern at the region of the monoisotope (M) + 2. The upper diagram indicates 34S-, 18O-, and 13C2-substituted ions of the
monoisotopic ion (m/z 307.08931). The lower diagram shows the theoretical pattern of C10H19N4O3S2. The resolution power of these ions was
approximately 400 000 fwhm. (C) MS- and UV-based chromatograms. The peak range of asparaptine (1) is colored in blue. BPC, base peak
chromatogram. (D) Product and difference ion analysis. The diamond symbol indicates the protonated molecular ion. (E) Chiral high-performance
liquid chromatography analysis of the hydrolysate of asparaptine (1) and D,L-arginine. The hydrolysate was identified as L-arginine.
(Figure 1C). The peak of asparaptine (1) ([M + H]+, m/z
307.0893) was confirmed in a base peak chromatogram (BPC),
while there were no redundant visible peaks of asparaptine (1)
in the UV-based chromatograms at 220, 254, 340, and 540 nm.
These findings suggested that the structure of asparaptine (1)
contains no chromophores. Therefore, asparaptine (1) was
isolated by preparative LC−PDA−MS after open column
chromatography. By tracking the ion with m/z 307 in the
positive ion mode, asparaptine (1) (61.0 mg) was finally
isolated from lyophilized asparagus powder (57.5 g dry weight).
Asparaptine (1) was obtained as a white powder, and its
elemental composition was determined by FTICRMS to be
C10H18N4O3S2. By MS/MS analysis, the product ion (m/z
248.040, C9H14NO3S2) and the difference ion (m/z 59.047,
Subsequently, tandem MS (MS/MS) analysis was performed
for chemically assigning the structure of S-metabolites
according to the guidelines of the Metabolomics Standards
Initiative.12 Using elemental compositions assigned by the
isotope analysis, corresponding S-metabolites were searched in
the in-house, ReSpect, and KNApSAcK databases.13,14 The
CH5N3) suggested the desorption of the guanidine moiety
(Figure 1D). The product ion (m/z 150.003, C4H8NOS2) and
the difference ion (m/z 157.084, C6H11N3O2) suggested the
analysis of product and difference ions (between protonated
molecular and product ions) characterized and annotated eight
S-ions from known S-metabolites in the databases, such as
glucosinolates, glutathione derivatives, and S-alkenylcysteines in
Arabidopsis thaliana L. Heynh, onion, garlic, Glycine max L.
Merr., and Wasabia japonica (Miq.) Matsum. (Table S2 and
Figure S1, Supporting Information). However, the structure of
three ions in asparagus could not be assigned in this analysis.
The isolation of asparaptine (1) was performed using
standard chromatographic approaches, and the MS- and UV-
based chromatograms were analyzed to elucidate its structure
1
presence of arginine and asparagusic acid moieties. The H
NMR, COSY, and HMQC spectra (Figures S2, S3, and S4,
Supporting Information) indicated 12 proton signals for 10
protons on five methylene carbons and two methine protons,
suggesting that the molecule contains arginine and asparagusic
acid moieties. The 13C NMR spectrum (Figure S2, Supporting
Information) showed 10 carbon signals for five methylenes (δ
27.2, 31.5, 43.3, 44.6, 45.4), two methines (δ 54.0, 57.6), and
B
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three quaternary carbons (δ 159.5, 176.7, 181.2). In the HMBC
spectrum, the α-proton signal of arginine at δ 4.21 (H-8)
revealed a strong correlation with the signal of the amide
carbonyl carbon at δ 176.7 (C-6) (Figure S5, Supporting
Information). The acid hydrolysate was detected at the same
retention time of L-arginine by chiral high-performance liquid
chromatography (HPLC) (Figure 1E). Consequently, the
chirality of C-8 was determined to be S.
activity is much weaker than that of asparaptine (1).17 The total
flavonoid glycosides in the crude extracts seemed to have a
partial role in the inhibitory activity.
The amount of asparaptine (1) in the green asparagus spears
was 251 mg/kg fresh weight (Table 2). It has been reported
Table 2. Quantification of Specialized Metabolites in
Asparagus
Asparaptine (1) was assayed to evaluate its inhibitory activity
against ACE. Captopril, nicotianamine, and N-succinyl-L-
proline were used as positive controls, because they possess
potent inhibitory activity against ACE. L-Arginine, D-arginine, L-
aspartic acid, and asparagusic acid glucose ester15 were
investigated to gain an in-depth insight into the structure−
activity relationship. Consequently, the IC50 values of captopril,
nicotianamine, N-succinyl-L-proline, asparaptine (1), and
asparagusic acid glucose ester were 1.6 nM, 18.7 μM, 14.5
μM, 113 μM, and 347 μM, respectively (Table 1). The
amount (mg/kg fresh weight)
compound
asparaptine (1)
green
purple
white
251 17
98.6 11
289 23
482 48
185 26
84.6 8.4
301 24
460 48
298 22
106 13
324 20
11 1.3
asparagusic acid
asparagusic acid glucose ester
rutin
that the amount of 2″-hydroxynicotianamine in green asparagus
is 47.0−54.0 mg/kg fresh weight, and that of nicotianamine is
less than or equal to one-tenth of that in green asparagus.5 The
amount of asparaptine (1) was approximately 5-fold higher
than that of 2″-hydroxynicotianamine. The amount of
asparaptine (1) tends to be higher in white asparagus than in
green or purple asparagus as well as in asparagusic acid and its
glucose ester (Table 2). Conversely, the amount of rutin was
the lowest in the white asparagus. Rutin is a light-inducible
flavonol glycoside,18 and white asparagus is generally grown
under sunlight-shielded conditions. These facts suggest that the
biosyntheses of asparaptine (1) and the other S-metabolites are
unaffected by sunlight irradiation.
The biosynthetic pathway of asparaptine (1) remains to be
elucidated, but its structure implies the occurrence of a specific
arginine N-acyltransferase, which catalyzes the conjugation of
asparagusic acid with arginine. Comparative analysis using non-
and accumulating organs in the integrated analysis of
transcriptomics and metabolomics should enable streamlining
the identification of the gene. The biosynthetic pathway of
asparagusic acid is under investigation, and stable isotope
labeling experiments suggest that asparagusic acid and its
analogues are derived from valine through isobutyric acid,
methacrylic acid, 2-methyl-3-mercaptopropionic acid, and S-(2-
carboxy-n-propyl)cysteine.19 Along with the biosynthetic path-
way and enzymes of asparaptine (1), the investigation of the
biological role of asparaptine (1) in asparagus is also intriguing
with regard to the general function of S-metabolites in
monocotyledons.
In summary, solid evidence of the existence of a new S-
containing inhibitor, asparaptine (1), of ACE has been
provided in asparagus using S-omics, a targeted metabolomic
approach. So far, the number of compounds from plants
beneficial to humans discovered by the top-down metabolomics
approach is limited.20 To our knowledge, this study is the first
example showing that S-omics could be used for efficient
screening of unknown S-metabolites in plants. By developing
databases storing metabolic information such as compound
name, structure, elemental composition, and MS/MS spectrum
details, it may be understood whether target metabolites are
known or unknown. Given that newly approved drugs are
developed on the basis of the structures of natural products
containing heteroatoms (N, O, S, and halogens),21 this type of
approach could be used for the screening of seed compounds
containing heteroatoms for drug development and for func-
tional food health benefit. It is recognized that S-omics is
limited to redundant S-metabolites showing 34S isotopic ions in
Table 1. Compound ACE Inhibitory Activity
compound
IC50 (μM)
a
captopril
0.001 61 0.000 43
14.5 5.3
a
N-succinyl-L-proline
a
nicotianamine
18.7 0.54
113 11
a
asparaptine (1)
a
asparagusic acid glucose ester
347 22
a
c
D-arginine
N.D.
a
c
L-arginine
N.D.
a
c
L-aspartic acid
N.D.
b
c
asparagusic acid
N.D.
a
b
Compound was dissolved in water. Compound was dissolved in 1%
c
dimethyl sulfoxide. N.D., not determined.
inhibitory activity of asparaptine (1) was 6-fold lower than that
of nicotianamine, for which the inhibitory activity was nearly
equal to that of 2″-hydroxynicotianamine.16 The IC50 values of
L-arginine, D-arginine, and L-aspartic acid could not be
determined. These results suggested that the conjugation of
the asparaptine (1) or asparagusic acid moiety alone is a key
aspect of the inhibitory activity. Next, asparagusic acid was
assayed to evaluate its contribution to the inhibitory activity.
The IC50 value of asparagusic acid could not be determined
(Table 1), indicating that the 1,2-dithiolane ring in conjugated
form is important for the inhibitory activity. Arginine is more
suitable than glucose as the counterpart of the asparagusic acid
moiety in the conjugate to exhibit the activity.
The contribution ratio of asparaptine (1) to the ACE
inhibitory activity was calculated in the edible parts of green
asparagus spears. The IC50 value of aqueous methanol extracts
of the green asparagus spears was 157 μg/mL. Quantitative
analysis using liquid chromatography−quadrupole time-of-
flight−mass spectrometry (LC−QTOF−MS) showed that the
amount of asparaptine (1) (0.479%) was in the crude extract.
Nicotianamine and 2″-hydroxynicotianamine were not detected
in this analysis. The contribution of asparaptine (1) to the total
ACE inhibitory activity of asparagus accounted for 2.2%,
indicating that asparaptine (1) is another potent inhibitor of
ACE in asparagus in addition to previously characterized
compounds such as nicotianamine derivatives. In this analysis,
flavonoids such as rutin [quercetin 3-O-rhamnosyl-(1→6)-
glucoside] were also detected. Flavonoid constituents also have
an inhibitory activity against ACE; however, their inhibitory
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extraction and evaporation, the water phase (approximately 50 mL)
was subjected to open-column chromatography [column diameter 7.3
cm, length 15 cm, Cosmosil 75C18-OPN materials, (Nacalai Tesque,
Kyoto, Japan)] and eluted with MeOH−H2O (0:100 → 100:0 v/v) to
afford 13 fractions (Frs. 1 and 2, 0% MeOH; Frs. 3 and 4, 5% MeOH;
Frs. 5 and 6, 10% MeOH; Frs. 7 and 8, 20% MeOH; Frs. 9 and 10,
40% MeOH; Frs. 11 and 12, 60% MeOH; Fr. 13, 100% MeOH; 500
mL solvent for each elution). Next, the fractions were analyzed with a
Shimadzu LC-MS-2020 system. The protonated molecular ion of
asparaptine (1) was observed at m/z 307 [M + H]+ and was mainly
found in fraction 6 (10% MeOH). After evaporation, fraction 6 (ca. 12
mL) was purified using a mass-directed fractionation system, the
preparative LC−PDA−MS system (Shimadzu), to yield 61 mg of
asparaptine (1). The purification was performed under the following
conditions: LC-20AP pump A and B; Unison UK-C18 column [150 ×
10 mm i.d.; 3 μm (Imtakt, Kyoto, Japan)]; column oven temperature
of 40 °C, linear gradient solvents at a flow of 3 mL/min [solvent A:
water (0.1% HCOOH); B: acetonitrile (0.1% HCOOH) 5% B (0−2
min), 5−8% B (2−3 min), 8−10% B (3−21 min), 10−100% B (21−
22 min), 100% B (22−27 min), 100−5% B (27−28 min), 5% B (28−
35 min)]; a makeup flow (LC-20AD pump C, solvent B, 0.2 mL/min)
was used to split the LC eluent into an LC-MS-2020 mass
spectrometer at a ratio of 1:150; electrospray ionization mass spectra
were recorded through a range of m/z 100−1000 in the positive-
ionization mode with a probe voltage of 4.5 kV, nebulizing gas flow of
1.5 L/min, “DL” temperature of 250 °C, heat block temperature of
the MS spectrum. However, profiling results of the redundant
S-metabolites could be good markers for isolation of target
metabolites. S-Omics and the discovery of asparaptine (1) will
present opportunities to find new S-metabolites with biological
activities potentially beneficial to humans.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotation was
measured using a JASCO P-1020 polarimeter. NMR spectra were
recorded using a Bruker 600 MHz spectrometer with a DCH
CryoProbe (Bruker BioSpin GmbH, Rheinstetten, Germany). MS and
MS/MS data were recorded using a Bruker solariX 7.0 T FTICRMS
instrument (Bruker Daltonik GmbH, Bremen, Germany). Purification
was performed on a preparative LC−PDA−MS system comprising
two LC-20AP binary gradient pumps, an LC-20AD makeup pump, an
SPD-M20A photodiode array detector, an SIL-10AP autosampler, a
CTO-20A column oven, an LC-MS-2020 single quadrupole mass
spectrometer, an FRC-10A fraction collector, and a CBM-20A module
(Shimadzu, Kyoto, Japan). N-Succinyl-L-proline was purchased from
Sigma-Aldrich Co., LLC (Saint Louis, MO, USA), and captopril,
nicotianamine, D-arginine, L-arginine, and L-aspartic acid were
purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). Asparagusic acid was purchased from Shanghai Haoyuan
Chemexpress Co., Ltd. (Shanghai, China). Isolated asparagusic acid
glucose ester was used in this study.
200 °C, and drying gas flow of 20.0 L/min.
MS and MS/MS spectra were recorded using Hystar 3.0 software
(Bruker Daltonik GmbH). Data were processed using DataAnalysis 4.0
(Bruker Daltonik GmbH). The raw files were converted to netCDF.
Peak picking of S-ions was performed using the theoretical mass
1
Asparaptine (1): white powder; [α]20 +6.5 (c 0.03, H2O); H
D
NMR (600 MHz, in D2O containing 0.01% DDS-d6, 25 °C) δ 4.21
(1H, dd, J = 8.2, 5.0 Hz, H-8), 3.46 (1H, m, H-5a), 3.44 (1H, m, H-4),
3.42 (1H, m, H-3a), 3.34 (1H, m, H-3b), 3.28 (1H, m, H-5b), 3.21
(2H, t, J = 6.9 Hz, H-11), 1.86 (1H, m, H-9a), 1.72 (1H, m, H-9b),
1.61 (2H, m, H-10); 13C NMR (150 MHz, in D2O containing 0.01%
DDS-d6, 25 °C) δ 181.2 (C, C-14), 176.7 (C, C-6), 159.5 (C, C-13),
57.6 (CH, C-8), 54.0 (CH, C-4), 45.4 (CH2, C-5), 44.6 (CH2, C-3),
43.3 (CH2, C-11), 31.5 (CH2, C-9), 27.2 (CH2, C-10); FTICRMS m/
z 307.08931 ([M + H]+, calcd for C10H19N4O3S2, 307.08930).
Hydrolysis of Asparaptine (1). Asparaptine (1) (1.9 mg) was
suspended in 6.0 N HCl (2.0 mL) and heated at 85 °C for 8 h. The
acid hydrolysate was dried under N2 and dissolved in H2O−MeOH−
HCOOH (30:70:0.02). The sample was then analyzed by HPLC
under the following conditions: LC-20AD pump A and B; Astec
CHIROBIOTIC T column (150 × 2.1 mm i.d., 1024AST, Sigma-
Aldrich); column oven temperature of 25 °C; solvent H2O−MeOH−
HCOOH (30:70:0.02) at a flow rate of 0.2 mL/min; UV detection at a
wavelength of 200 nm. D- and L-Arginine showed retention times at
11.1 and 8.1 min, respectively. L-Arginine in the hydrolysate was
identified by comparing its retention time with that of an authentic
sample.
Quantification of S-Metabolites in Asparagus Using LC−
QTOF−MS. The fresh samples were extracted with 5 μL of 80%
MeOH containing 2.5 μM lidocaine and 10-camphor sulfonic acid per
mg fresh weight using a mixer mill with zirconia beads for 7 min at 18
Hz and 4 °C. After centrifugation for 10 min, the supernatant was
filtered using an HLB μElution plate (Waters). Extract solutions (1
μL) were analyzed using LC−QTOF−MS (LC, Waters Acquity
UPLC system; MS, Waters Xevo G2 Q-TOF). Analytical conditions
were as follows: LC column, Acquity bridged ethyl hybrid C18 (1.7 μm,
2.1 mm × 100 mm, Waters); solvent system, solvent A (water
including 0.1% formic acid) and solvent B (acetonitrile including 0.1%
formic acid); gradient program, 99.5% A/0.5% B at 0 min, 99.5% A/
0.5% B at 0.1 min, 20% A/80% B at 10 min, 0.5% A/99.5% B at 10.1
min, 0.5% A/99.5% B at 12.0 min, 99.5% A/0.5% B at 12.1 min and
99.5% A/0.5% B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/
min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min, and
0.3 mL/min at 14.5 min; column temperature, 40 °C; MS detection:
capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature,
120 °C, desolvation temperature, 450 °C, cone gas flow, 50 L/h;
desolvation gas flow, 800 L/h; collision energy, 6 V; mass range, m/z
100−1500; scan duration, 0.1 s; interscan delay, 0.014 s; data
difference (1.995
0.001 Da) between 32S-monoisotopic ions and
their 34S-substituted counterparts and using the natural abundance of
34S (4.21 5%) with the appropriate Web-based analytical tool for
heteroatom-containing metabolites, which will be publicly available
after reaching the final step. S-Ions were extracted under the condition
that the retention time was 1−20 min. Peak alignment was conducted
using Progenesis CoMet (Nonlinear Dynamics, Durham, NC, USA).
Signal intensity values of all ions were divided by the signal intensity
value of the internal standard lidocaine for normalization. To obtain S-
ions with redundant signal intensity, ions with an intensity less than
1.0 were cut out. The software MeV 4.8 (http://www.tm4.org/mev.
html) was used for hierarchical cluster analysis. Pearson’s correlation
analysis was used in this study. The elemental composition was
calculated using SmartFormula (Bruker Daltonik GmbH) with the
following limiting conditions: <1 mDa; C0−50H0−100N0−5O0−50S0−5
charge, 1.
;
Plant Material. Commercially available spears of Asparagus
officinalis produced in Japan (Hokkaido for both S-omics using
LC−FTICRMS and quantification analysis using LC−QTOF−MS,
Tochigi for isolation) were used in this study. Voucher specimens
[green asparagus (Hokkaido), APGDo-FD; purple (Hokkaido),
APPDo-FD; white (Hokkaido), APWDo-FD; and green (Tochigi),
APGTo-FD] were deposited at RIKEN Freeze-dried Collection
(RIKEN CSRS). Vegetables were purchased at U-Takaraya
(Arakawa-ku, Tokyo, Japan). Arabidopsis thaliana and Oryza sativa
L. were grown in a plant growth room.22,23 All samples were
immediately frozen, lyophilized, and ground and then stored at room
temperature with silica gel until use.
Extraction and Isolation. Extraction of metabolites for LC−
FTICRMS analysis was performed as previously described.10 The
column was exchanged for Xselect CSH Phenyl-Hexyl (3.5 μm, 2.1
mm × 150 mm, Waters, Milford, MA, USA).
Fresh asparagus spears (970.7 g) were cut into 2−3 mm pieces with
a ceramic knife. After flash-freezing in liquid nitrogen, the pieces were
freeze-dried and crushed. The lyophilized asparagus powder (57.5 g)
was extracted three times with 80% methanol (3 L × 3) overnight at
room temperature. The solvent was evaporated until a small volume of
water solution was left (approximately 400 mL). The solution was
sequentially extracted with n-hexane (400 mL × 3) and chloroform
(400 mL) to remove lipid and chlorophyll. After the liquid−liquid
D
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acquisition, centroid mode; polarity, positive/negative; lockspray
(leucine enkephalin): scan duration, 1.0 s; interscan delay, 0.1 s.
MS/MS data were acquired in the ramp mode under the following
analytical conditions: mass range, m/z 50−1500; collision energy, 10
to 50 V; scan duration, 0.1 s; interscan delay, 0.014 s; data acquisition,
centroid mode; polarity, positive for asparaptine (1) and negative for
asparagusic acid and asparagusic acid glucose ester. The solvents of
asparaptine (1), asparagusic acid, and asparagusic acid glucose ester
(<100 μM) were analyzed to produce a standard curve. These
metabolites were then quantified using this standard curve. Differences
and standard deviations were calculated from the results of three
replicates.
ACE in Vitro Assay. The assays of the ACE inhibitory activity on
concentrations of the compounds (<1 mM) and crude extracts (<1000
μg/mL) were conducted using an ACE Kit-WST (Dojindo Molecular
Technologies, Kumamoto, Japan). The fresh samples were extracted
with 5 μL of 80% MeOH per mg fresh weight using a mixer mill with
zirconia beads for 7 min at 18 Hz and 4 °C. After centrifugation for 10
min, the supernatant was filtered using the HLB μElution plate. After
concentration, crude extracts were dissolved with water, and the
extracts were used for the assay. The IC50 values were determined
according to the manufacturer’s instructions. However, in the case
where 50% inhibition was not reached at the high concentrations, the
IC50 value was considered as N.D. (not determined). Differences and
standard deviations were calculated from the results of three replicates.
Calculation of Contribution Ratio of Asparaptine (1) to ACE
Inhibitory Activity of Asparagus Crude Extract. The contribution
ratio was calculated as follows:
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +81-45-503-9442. Fax: +81-45-503-9489. E-mail: ryo.
nakabayashi@riken.jp (R. Nakabayashi).
*Tel: +81-45-503-9488. Fax: +81-45-503-9489. E-mail: kazuki.
saito@riken.jp (K. Saito).
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M.; Yamada, Y.; Nishizawa, T.; Matsuda, F.; Kojima, M.; Sakakibara,
H.; Shinozaki, K.; Michael, A. J.; Tohge, T.; Yamazaki, M.; Saito, K.
Plant J. 2014, 77, 367−379.
(23) Yang, Z.; Nakabayashi, R.; Okazaki, Y.; Mori, T.; Takamatsu, S.;
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Present Address
§School of Pharmacy, Lanzhou University, 199 West Donggang
Road, Lanzhou, Gansu, People’s Republic of China 730020.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank D. Fine, D. Wheritt, and L. Sumner
(Noble Foundation, Ardmore, OK, USA) for useful advice on
structure elucidation, and M. Kitajima and H. Takayama (Chiba
University) for collecting data on asparaptine (1). We also
thank K. Takano and Y. Okazaki for providing freeze-dried
samples, Y. Yamada and T. Sakurai (RIKEN CSRS) for
technical help in mining S-ions, and T. Tohge (Max-Planck
Institute, Potsdam, Germany) for useful comments on the
manuscript. This work was partially supported by Strategic
International Collaborative Research Program (SICORP), JST,
and Japan Advanced Plant Science Network.
E
DOI: 10.1021/acs.jnatprod.5b00092
J. Nat. Prod. XXXX, XXX, XXX−XXX