Steroidal Saponins in Oat Bran

Abstract

Saponins are one type of wide-spread defense compounds in plant kingdom and have been exploited for production of lead compounds with diverse pharmacological properties in drug discovery. Oats contain two unique steroidal saponins avenacosides A, 1, and B, 2. However, the chemical composition, the levels of these saponins in commercial oat products, and their health effects are still largely unknown. In this study, we directly purified five steroidal saponins (1-5) from a methanol extract of oat bran, characterized their structures by analyzing their MS and NMR spectra, and also tentatively identified 11 steroidal saponins (6-16) based on their tandem mass spectra (MSn: n = 2-3). Among the five purified saponins, 5 is a new compound and 4 is purified from oats for the first time. Using HPLC-MS techniques, a complete profile of oat steroidal saponins was determined, and the contents of the two primary steroidal saponins 1 and 2 were quantitated in fifteen different commercial oat products. The total levels of these two saponins vary from 49.6-443.0 mg/kg, and oat bran or oatmeal has higher levels of these two saponins than cold oat cereal. Furthermore, our results on the inhibitory effects of 1 and 2 against the growth of human colon cancer cells HCT-116 and HT-29 showed that both had weak activity, with 2 being more active than 1.

Full text

Steroidal Saponins in Oat Bran
Junli Yang, Pei Wang, Wenbin Wu, Yantao Zhao, Emmanuel Idehen, and Shengmin Sang*
Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina
Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081,
United States
ABSTRACT: Saponins are one type of widespread defense compound in the plant kingdom and have been exploited for the
production of lead compounds with diverse pharmacological properties in drug discovery. Oats contain two unique steroidal
saponins, avenacoside A, 1, and avenacoside B, 2. However, the chemical composition, the levels of these saponins in commercial
oat products, and their health effects are still largely unknown. In this study, we directly purified 5 steroidal saponins (1−5) from
a methanol extract of oat bran, characterized their structures by analyzing their MS and NMR spectra, and also tentatively
identified 11 steroidal saponins (6−16) on the basis of their tandem mass spectra (MS
n
, n =2−3). Among the five purified
saponins, 5 is a new compound and 4 is purified from oats for the fi rst time. Using HPLC-MS techniques, a complete profile of
oat steroidal saponins was determined, and the contents of the two primary steroidal saponins, 1 and 2, were quantitated in
15 different commercial oat products. The total levels of these two saponins vary from 49.6 to 443.0 mg/kg, and oat bran or
oatmeal has higher levels of these two saponins than cold oat cereal. Furthermore, our results on the inhibitory effects of 1 and 2
against the growth of human colon cancer cells HCT-116 and HT-29 showed that both had weak activity, with 2 being more
active than 1.
KEYWORDS: oat bran, steroidal saponin profile, avenocoside D, cytotoxic effect
■
INTRODUCTION
Oats (Avena sativa L.) have been considered as one of the
healthiest foods worldwide.
1,2
Oat grains are able to thrive in poor
soil conditions.
3
Most oat products are made from a hulled grain
without stripping their bran and germ, and these parts retain large
amounts of dietary fiber and bioactive phytochemicals, which
display a serum cholesterol lowering effect and reduce the risk of
heart disease and cardiovascular disease.
4−7
Besides, consumption
of oat products showed other health benefits, such as anticancer
and antidiabetic effects,
8
enhancing human immunity,
9
and
reducing the risk of high blood pressure.
10
Oats produce a series of phytochemicals contributing to their
health-related effects including steroidal saponins,
11
avenan-
thramides,
12
phenolic acids,
13,14
tocols,
15
and flavanoids.
11
As the
only saponin-accumulating cereal,
16
oats contain two different
saponin forms, avenasides and avenacosides, synthesized via two
different biosynthetic pathways.
17
Avenasides are triterpenoid
saponins mainly stored in roots for inhibiting pathogens such as
Gaeumannomyces graminis,
17
whereas avenacosides belong to
steroid glycosides and are mainly accumulated in oat leaves
and grains.
18,19
Avenacosides A and B, the two primary and also
unique avenacosides in oats, are glycosylated at C-3 with a
trisaccharide (one rhamnose and two glucose units) in the case
of avenacoside A or a tetrasaccharide (one rhamnose and three
glucose units) in the case of avenacoside B, and at C-26 with
a glucose unit (Figure 1). Upon tissue disruption, the O-β-
glucosidic bond at C-26 is immediately hydrolyzed by a special
β-glucosidase, named avenacosidase, to yield the bioactive
26-deglucoavenacosides,
20
which poss ess strong antifungal
activity.
18
The sugar moieties at C-3 are essential for the anti-
microbial effects of 26-deglucoavenacosides,
21
and these saponins
can be detoxified via sequential hydrolysis of the sugar units at
C-3 by α-rhamnosidase and β-glucosidase secreted by pathogenic
fungi.
21
To date there are only six steroidal saponins purified
from oat brans, including avenacoside A, 1,
22
avenacoside B, 2,
23
and avenacoside C, 3,
24
and 26-desglucoavenacosides A and
B
11,25,26
as well as one sulfated saponin.
19
Saponins are one type of widespread defense compound in
the plant kingdom,
27
and they are mainly characterized for their
antimicrobial effects and less frequent ly for insecticidal
properties.
28
Apart from their important role in plant defense
systems, more and more saponins have been utilized for the
production of lead compounds with diverse pharmacological
properties; one such property is their anticancer effects.
28−31
The chemical profile and the anticancer effects of oat steroidal
saponins are still unknown. In addition, the levels of these
saponins in commercial oat products have not been reported.
In this regard, a systematic investigation on oat steroidal saponins
was conducted here. The objective of the present study was to
explore more avenacoside-type components from oats, give a
profile of steroidal saponins in oats, quantitate their levels in
commercial oat products, and evaluate their inhibitory effects on
the growth of human colon cancer cells.
■
MATERIALS AND METHODS
Materials. Silica gel (230−400 mesh) (Sorbent Technologies Inc.,
Atlanta, GA, USA) and Diaion HP-20 (Mitsubishi Chemical, Japan)
were used for open column chromatography (CC). Chromatographic
separations were monitored by analytical thin-layer chromatography
(TLC) on 250 μm thick, 2−25 μm particle size, glass-backed silica gel
plates, which were purchased from Sigma (Sigma-Aldrich, St. Louis,
Received: December 22, 2015
Revised: February 4, 2016
Accepted: February 7, 2016
Article
pubs.acs.org/JAFC
© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

MO, USA). All analytical and HPLC-MS grade solvents were obtained
from Thermo Fisher Scientific (Waltham, MA, USA). All of the oat
products were purchased online at Walmart and from a local super-
market, Harris Teeter (Kannapolis, NC, USA).
HPLC-MS Analysis. HPLC-MS was performed with a Thermo-
Finnigan Spectra System consisting of an Ultimate 3000 degasser,
an Ultimate 3000 RS pump, an Ultimate 3000 RS autosampler, an
Ultimate 3000 RS column compartment, and an LTQ Velos Pro ion
trap mass spectrometer (Thermo Electron, San Jose, CA, USA) in-
corporated with an electrospray ionization (ESI) interface. The column
used was a 150 mm × 3.0 mm i.d., 5 μm, Gemini RP-18 (Phenomenex,
Torrance, CA, USA). The mobile phase consisted of water containing
0.2% formic acid (mobile phase A) and methanol with 0.2% formic acid
(mobile phase B). The gradient elution was carried out for 60 min at a
flow rate of 0.2 mL/min. A gradient eluting system was applied: 40% B
from 0 to 3 min; 40−52% B from 3 to 35 min; 52−100% B from
35 to 45 min; 100% B from 45 to 50 min, and then to 40% B from
50 to 55 min. The column was then re-equilibrated with 0% B for
5 min. The injection volume was 10 μL for each sample. The HPLC
eluent was introduced into the ESI interface. For mass spectrometric
parameter optimization, the purified compound in methanol solution
(10 μg/mL) was infused in ESI source and analyzed in negative ion
Figure 1. Structures of compounds 1−16 identified from oat bran. ∗∗, new compound; ∗, first purification from oat.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
B

mode to give the following optimized parameters: spray voltage, 3.6 kV;
sheath gas (nitrogen) flow rate, 34 (arbitrary units); capillary voltage,
−13 V; capillary temperature, 300 °C; tube lens offset, −60 V. For the
quantification of the two major saponins, target ions at m/z 1061.7
[M − H]
−
for 1 and at m/z 1223.9 [M − H]
−
for 2 were monitored
using the selected ion monitoring (SIM) mode. For the identification
of steroidal saponins, the collision-induced dissociation (CID) was con-
ducted with an isolation width of 1.2 Da, and the normalized collision
energy was set to 35% for MS
n
analysis. The mass range was measured
from m/z 50 to 1400. Data acquisition and analysis were performed
with Xcalibur 2.0 version (Thermo Electron).
Nuclear Magnetic Resonance (NMR) Analysis.
1
H (600 MHz),
13
C (150 MHz), heteronuclear single-quantum correlation (HSQC),
and heteronuclear multiple-bond correlation (HMBC) NMR spectra
were recorded on a Bruker 600 MHz NMR instrument. All samples
were dissolved in methanol-d
4
containing tetramethylsilane (TMS) as
the internal standard.
Extraction and Enrichment of Steroidal Saponins. Oat bran
(50 kg) purchased from Kalyx (www.kalyx.com) was continuously
extracted by 100% methanol (V
oat
:V
methanol
= 1:5) at room temperature
three times for 4 days each time. After filtration using cotton, the
methanol extract was concentrated under reduced pressure to yield a
crude residue (2136 g). This residue was reconstituted in water and
partitioned against n-hexane, ethyl acetate (EtOAc), and n-butanol
(n-BuOH). After concentration in vacuo, the n-BuOH fraction (242.8 g)
was suspended in water and applied to a Diaion HP-20 column (7.5 cm
i.d. × 60 cm) eluted with water, 30% ethanol in water, 70% ethanol in
water, and ethanol successively (5 L each) to afford four fractions,
F1−F4, respectively. Fraction F3 (70% ethanol elution) was evaporated
in vacuo and kept as the steroidal-saponin enriched sample (8.9 g)
at −80 °C. Fraction monitoring was by TLC (chloroform/methanol/
water, 70:35:5.5, v/v/v). The spots on TLC were visualized by spraying
with a H
2
SO
4
/ethanol (5:95, v/v) solution followed by heating.
Purification of Steroidal Saponins 1−5. Repeated purification
of fraction F3 by silica gel open column (5.0 cm i.d. × 30 cm) eluted
with a chloroform/methanol/water system (70:30:5.5 to 70:35:5.5,
v/v/v, 2 L each) afforded five steroidal saponins: 1 (1.2 g), AA; 2
(609 mg), AB; 3 (10.5 mg), AC; 4 (10.9 mg), chamaedroside E
2
; and a
new saponin, avenacoside D, 5 (5.8 mg).
Chamaedroside E
2
(4): yellow amorphous powder;
1
H NMR
(600 MHz, in methanol-d
4
) δ
H
5.37 (1H, br s, H-6), 4.45 (1H, m,
H-16), 4.41 (1H, d, J = 7.8 Hz, H-1″), 4.40 (1H, d, J = 7.8 Hz, H-1‴),
4.29 (1H, d, J = 7.7 Hz, H-1′), 3.80 (1H, d, J = 12.0 Hz, H-26a), 3.50
(1H, dd, J = 11.8, 2.6 Hz, H-3), 3.47 (1H, d, J = 11.2 Hz, H-26b), 1.22
(3H, s, H-27), 1.04 (3H, s, H-19), 0.98 (3H, d, J = 6.8 Hz, H-21), 0.81
(3H, s, H-18);
13
C NMR (150 MHz, in methanol-d
4
) δ
C
142.0 (C-5),
122.6 (C-6), 121.7 (C-22), 105.0 (C-1′), 104.6 (C-1‴), 102.3 (C-1″),
63.2/62.8/61.9 (C-6′/6″/6‴), 84.2 (C-25), 81.1 (C-16), 78.9 (C-3),
77.9 (C-26), 61.4 (C-17), 56.7 (C-14), 50.7 (C-9), 41.5 (C-20), 40.9
(C-12), 39.7 (C-4), 39.4 (C-13), 38.5 (C-1), 37.0 (C-10), 32.7 (C-24),
32.6 (C-8), 32.6 (C-7), 32.2 (C-15), 31.8 (C-23), 30.0 (C-2), 23.3
(C-27), 21.0 (C-11), 16.9 (C-19), 15.6 (C-18), 14.1 (C-21); negative
ESI/MS, m /z 915.7 [M − H]
−
and 961.7 [M + HCOOH − H]
−
.
Avenacoside D (5): yellow amorphous powder;
1
H NMR (600 MHz,
in methanol-d
4
) δ
H
5.33 (1H, br s, H-6), 5.20 (1H, br s, H-1″″), 4.51
(1H, d, J =7.8Hz,H-1″″″), 4.47 (1H, d, J = 7.8 Hz, H-1″), 4.42 (1H, d,
J = 7.8 Hz, H-1″″′), 4.40 (1H, overlap, H-16), 4.35 (1H, d, J = 7.8 Hz,
H-1‴), 4.25 (1H, d, J = 7.7 Hz, H-1′), 3.81 (1H, d, J = 11.2 Hz, H-26a),
3.59 (1H, br d, J = 11.6 Hz, H-3), 3.50 (1H, d, J = 11.2 Hz, H-26b), 1.19
(3H, d, J =7.6Hz,H-6″″), 1.18 (3H, s, H-27), 1.00 (3H, s, H-19), 0.90
(3H, d, J = 6.8 Hz, H-21), 0.76 (3H, s, H-18);
13
C NMR (150 MHz, in
methanol-d
4
) δ
C
140.9 (C-5), 121.6 (C-6), 120.7 (C-22), 104.2 (C-1′),
104.0 (C-1″″″), 103.6 (C-1″″′), 103.2 (C-1‴), 101.0 (C-1″″), 99.4
(C-1″), 84.2 (C-25), 81.1 (C-16), 78.4 (C-3), 77.1 (C-26), 62.2/61.7/
61.6/61.5/60.9 (C-6′/6″/6‴/6″″′/6″″″), 61.4 (C-17), 56.7 (C-14),
50.7 (C-9), 40.5 (C-20), 40.5 (C-12), 39.9 (C-4), 39.8 (C-13), 38.5
(C-1), 37.0 (C-10), 32.7 (C-24), 32.7 (C-7), 32.6 (C-8), 32.2 (C-15),
31.8 (C-23), 29.7 (C-2), 23.3 (C-27), 20.9 (C-11), 18.8 (C-6″″),
16.9 (C-19), 15.6 (C-18), 14.1 (C-21); negative ESI/MS, m/z 1385.8
[M − H]
−
and 1431.8 [M + HCOOH − H]
−
.
Preparation of Standards of 1 and 2 and the Extracts of
Commercial Oat Products. The stock solutions (0.1 mg/mL) of
1 and 2 were prepared in 50% (v/v) aqueous methanol solution. Stock
solutions were stored at −20 °C before use. The above stock solutions
were diluted with 50% methanol to prepare 0.5, 0.75, 1.5, 2.5, 5.0,
and 10.0 μg/mL 1 and 0.25, 0.375, 0.75, 1.25, 2.5, and 5.0 μg/mL 2,
respectively. All of the samples were freshly prepared before use.
Three independent samples of each oat product were used in this
study. One gram of each oat product was accurately weighed and
extracted three times with 50 mL of methanol under sonication for
30 min and then cooled and centrifuged at 8000 rpm for 10 min.
Supernatants from the three extractions were combined and concentrated
to dryness under vacuum at 35 °C. The residue was reconstituted in
2.0 mL of 50% methanol and centrifuged for 10 min at 16000 rpm.
Before injection, the supernatant of each sample was diluted 10 times (for
oat cereal) or 20 times (for oat bran and oatmeal) with 50% methanol.
Each sample was analyzed in triplicate.
Growth Inhibitory Effects of 1 and 2 on Human Colon
Cancer Cells. Cell growth inhibition was determined by a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colori-
metric assay. Human colon cancer cells HCT-116 and HT-29 were
plated in 96-well microplates with 5000 cells/well and allowed to
attach for 24 h at 37 °C. The test compounds (in DMSO) were added
to cell culture medium to desired final concentrations (25, 50, 75, 100,
150, and 200 μM, the final DMSO concentrations for control and
treatments were 0.1%, n =8−16). After the cells had been cultured for
72 h, the medium was aspirated, and cells were treated with 200 μLof
fresh medium containing 2.41 mmol/L MTT. After incubation for 3 h
at 37 °C, the medium containing MTT was aspirated, 100 μLof
DMSO was added to solubilize the formazan precipitate, and the plates
were shaken gently for 1 h at room temperature. Absorbance values
were derived from the plate reading at 550 nm on a Biotek microtiter
plate reader. The reading reflected the number of viable cells and was
expressed as a percentage of viable cells in the control. Both HCT-116
and HT-29 cells were cultured in McCoy’s 5A medium. All of the
above media were supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin, and 1% glutamine, and the cells were kept
in a 37 °C incubator with 95% humidity and 5% CO
2
.IC
50
values
were obtained using GraphPad Prism (GraphPad Software, San Diego,
CA, USA).
■
RESULTS AND DISCUSSION
Structural Elucidation of Steroidal Saponins 1−5. As
part of our efforts to complete the chemical profile of oat bran,
five steroidal saponins 1−5 (Figure 1) were isolated by means
of chromatographic methods, including silica gel and Diaion
HP-20 chromatography. Compounds 1−3 were identified as
avenacosides A, B, and C, respectively, according to literature
data.
22,32,33
Avenacoside A, 1, and avenacoside B, 2, have been
reported as the primary saponins in oat bran and analyzed by
HPLC-TQ-MS and HPLC with UV detection.
32,33
Compound
3 was first discovered from fresh bulbs of Lilium brownii
34
and
subsequently isolated from oat bran and named avenacoside C
by Lu et al.
24
Compound 4 gave a deprotonated io n at m/z 915.7
[M − H]
−
in an HPLC-MS spectrum. The MS/MS spectrum
of the precursor ion at m/z 915.7 displayed a fragment ion at
m/z 753.5, generated by the loss of a glucose unit.
1
H and
13
C
NMR spectra of this compound showed glycone signals similar
to those of AA, 1, and AB, 2. The difference lies in the number
of sugar units. There are three sugar units found in the NMR
spectra of 4 [δ
H
4.41 (1H, d, J = 7.9 Hz), 4.40 (1H, d, J =
7.9 Hz), 4.29 (1H, d, J = 7.7 Hz); δ
C
105.0 (CH), 104.6 (CH),
102.3 (CH)], whereas AA, 1, and AB, 2, have four and five
sugar units, respectively. On the basis of HSQC and HMBC
data, 4 was determined as chamaedroside E
2
, which has been
reported as a chemical component from Veronica chamaedrys L.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
C

However, the literature did not give its full NMR assignment.
Here we report the full
1
H and
13
C NMR data for the first time.
This is the first report of this compound from oats.
Compound 5 showed a molecular formula of C
63
H
102
O
33
by
analyzing its deprotonated ions at m/z 1385.8 [M − H]
−
and
1431.8 [M + HCOOH − H]
−
. In the MS/MS spectrum, the
precursor ion at m/z 1385.8 [M − H]
−
gave fragment ions
at m/z 1223.8, 1061.7, 899.6, 753.5, and 591.4, which were
generated by the loss of the sugar units in its structure in
sequence.
1
H and
13
C NMR data of 5 demonstrated the
existence of five glucose units [δ
H
4.51 (1H, d, J = 7.8 Hz), 4.47
(1H, d, J = 7.8 Hz), 4.42 (1H, d, J = 7.8 Hz), 4.35 (1H, d, J =
7.8 Hz), and 4.25 (1H, d, J = 7.7 Hz); δ
C
104.2, 104.0, 103.6,
103.2, and 99.4] and one rhamnose unit [δ
H
5.20 (1H, br s)
and 1.19 (3H, d, J = 6.5 Hz); δ
C
101.0 and 18.8]. The NMR
spectra also showed diagnostic signals for one trisubstituted
olefinic bond [δ
H
5.33 (1H, br s); δ
C
140.9 and 121.6], one
methyl doublet [δ
H
0.91 (3H, d, J = 6.8 Hz)], and three methyl
singlets [δ
H
1.18 (3H, s), 1.00 (3H, s), and 0.76 (3H, s)]. Finally,
the linkage patterns of sugars and the whole planar structure of 5
were constructed by HSQC and HMBC techniques (Figure 2).
The similarity of NMR chemical shifts between 5 and AA and
AB was used to determine relative configurations of 5 as shown
in Figure 1.Compound5 is a new steroidal saponin and was
named here avenacoside D.
Fragmentation Patterns of Steroidal Saponins. When
there is one −Glc−Rha unit at C-3 and one −Glc unit at C-26,
the fragmentation priority of the sugar units is the loss of the
Figure 2. HMBC correlations (from H to C) of compound 5.
Figure 3. ESI/MS
n
(n =2−3) spectra and fragmentation pattern of (A) compound 3 and (B) compound 1.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
D

-Rha unit instead of the −Glc unit at C-26. This is supported by
the observation of m/z 753.5 [M − Rha − H]
−
as the base ion
of the tandem mass of 3 (m /z 899.7 [M − H]
−
)(Figure 3A).
However, if there are additional glucose units on the -Glc-Rha
unit, the additional glucose units will have priority over the −Rha
unit to be cleavaged from the side chain at C-3. For example,
Table 1. ESI-MS and ESI-MS
n
(n =2−3) Fragment Ions of Compounds 1−16 in Oat Bran
t
R
(min) [M − H]
−
[M + HCOOH − H]
−
MS
2
MS
3
1 27.58 1061.7 1108.0 1061.7/899.6 [M − Glc − H]
−
(B), 753.5 [M − Glc − Rha − H]
−
899.6/881.6, 753.5 (B), 737.6, 591.5
753.5/591.5 (B), 573.5, 429.4
2 27.10 1223.9 1269.7 1223.9/1061.7 [M − Glc − H]
−
(B), 899.7 [M − 2Glc − H]
−
,
753.6 [M − 2Glc − Rha − H]
−
1061.7/899.7 (B), 881.6, 753.6, 591.6,
573.6
899.7/753.5(B), 737.7, 591.5, 573.4
753.5/591.6 (B), 429.4
3 28.17 899.7 945.9 899.7/753.5 [M − Rha − H]
−
(B) 753.5/591.4 (B), 573.5, 429.3
4 28.45 915.7 961.7 915.7/753.5 [M − Glc − H]
−
(B) 753.5/591.5 (B), 573.6, 429.4
5 26.93 1385.8 1431.8 1385.8/1223.8 [M − Glc − H]
−
(B), 1061.7 [M − 2Glc − H]
−
,
899.6 [M − 3Glc − H]
−
, 753.5[M − 3Glc − Rha − H]
−
1061.7/915.6, 899.6 (B), 753.5
899.6/753.5 (B), 737.6, 591.4
753.6/591.4 (B), 573.4, 429.4
6 30.87 899.7 945.9 899.7/737.5 [M − Glc − H]
−
(B) 737.5/591.5 (B), 429.3
7 32.78 899.7 945.6 899.7/737.5 [M − Glc − H]
−
(B) 737.5/591.5 (B)
8 22.74 915.6 961.7 915.6/753.5 [M − Glc − H]
−
(B) 753.5/591.4 (B)
9 27.62 915.6 961.7 915.6/753.5 [M − Glc − H]
−
(B) 753.5/591.5 (B), 573.5
10 21.10 1061.8 1107.8 1061.8/899.6 [M − Glc − H]
−
(B), 753.6 [M − Glc − Rha − H]
−
899.6/753.6 (B), 737.5, 591.5
753.6/591.5 (B), 429.4
11 20.15 1223.9 1269.9 1223.9/1061.7 [M − Glc − H]
−
, 899.6 [M − 2Glc − H]
−
(B),
753.5 [M − 2Glc − Rha − H]
−
1061.7/899.7 (B), 881.6, 753.6, 591.6,
573.6
899.6/753.5 (B), 737.6, 591.5, 573.4
753.5/591.6 (B), 573.5, 429.4
12 25.64 1223.9 1269.7 1223.9/1061.7 [M − Glc − H]
−
(B), 899.7 [M − 2Glc − H]
−
,
753.6 [M − 2Glc − Rha − H]
−
1061.7/899.7 (B), 881.6, 753.6, 591.6,
573.6 899.7/753.5 (B), 737.6, 591.5,
573.4
753.6/591.6 (B), 573.5, 429.4
13 25.64 1223.9 1269.8 1223.9/1077.7 [M − Rha − H]
−
(B), 915.7 [M − Glc − Rha −
H]
−
, 753.6 [M − 2Glc − Rha − H]
−
1077.7/915.7 (B), 573.6
915.7/753.6 (B), 591.5, 573.4
753.6/591.4 (B),
14 27.35 1223.9 1269.7 1223.9/1077.7 [M − Rha − H]
−
(B), 915.7 [M − Glc −
Rha − H]
−
1077.7/915.6 (B)
915.7/753.6 (B), 735.6, 591.5, 573.4
15 25.18 1385.8 1431.8 1385.8/1223.8 [M − Glc − H]
−
, 1061.7 [M − 2Glc − H]
−
(B),
899.6 [M − 3Glc − H]
−
, 753.5 [M − 3Glc − Rha − H]
−
1061.7/915.6, 899.6 (B), 753.5
899.6/753.5 (B), 737.6, 591.5
753.5/591.4 (B), 429.4
16 26.63 1385.8 1431.7 1385.8/1223.8 [M − Glc − H]
−
, 1061.7 [M − 2Glc − H]
−
(B),
899.6 [M − 3Glc − H]
−
, 753.5 [M − 3Glc − Rha − H]
−
1061.7/915.7, 899.6 (B), 753.5
899.6/753.5 (B), 737.6, 591.4
753.6/591.5 (B), 573.5, 429.4
Figure 4. Total ion chromatogram (TIC) of compounds 1−16 in oat bran extract generated from negative HPLC-ESI/MS
n
.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
E

compound 1 has one more glucose unit on the side chain at C-3
than 3 . The base ion of the MS
2
of 1 is the ion at m/z 899.6
[M − Glc − H]
−
and the MS
2
of this fragment ion (MS
3
,
899.6/1061.7) is almost identical to that of 3 (MS
2
, 899.7)
(Figure 3B). We observed the same fragmentation pattern from
the tandem mass of 2 and 5, which have two and three more
glucose units as 3, respectively (Table 1).
Identification of Steroidal Saponins 6−16 in Oats by
LC-ESI/MS
n
Spectra. On the basis of the ionization and
fragmentation patterns of the known compounds 1−5,we
searched the minor components of steroidal saponins from oat
extract. Eleven compounds (6−16) were identified, and their
structures were tentatively elucidated as shown in Figure 1,on
the basis of the analysis of their MS
n
spectra (Table 1). Figure 4
shows the total ion chromatogram (TIC) of these 16 steroidal
saponins from oats.
Compounds 6 and 7 had the same molecular ion m/z 899.7
[M − H]
−
as 3. However, both 6 and 7 possessed a base peak
ion (m/z 737.5 [M − Glc − H]
−
) with the loss of a glucose
unit in their MS
2
spectra in lieu of the loss of a rhamnose unit
in the MS
2
spectrum of 3 (Table 1), indicating there are two
glucoses and one rhamnose at C-3 and no −Glc at C-26.
Furthermore, the tandem mass of m/z 737.5 (MS
3
, 737.5/
899.7) had a fragment ion that lost one rhamnose unit (m/z
591.5 [M − Glc − Rha − H]
−
)(Table 1). All of these features
suggest that 6 and 7 have two glucoses and one rhamnose at
C-3 but no glucose at C-26 (Figure 1).
As analyzed by the negative LC-ESI/MS
n
, both 8 and 9 had
the same molecular ion as 4 (m/z 915.6 [M − H]
−
), indicating
they have three glucoses. However, the tandem mass spectra of
8 and 9 and their major fragment ions were almost identical to
those of 4. Therefore, we were unable to elucidate the linkages
of these three glucoses. The structures of 8 and 9 were
tentatively identified as shown in Figure 1.
Compound 10 possessed a base peak ion at m/z 899.6
[M − Glc − H]
−
in its MS
2
spectra, and the tandem mass of
this fragment ion (MS
3
, 899.6/1061.8) was similar to that of 3
(MS
2
, 899.7), which were similar to those of AA, 1 (Table 1).
Thus, 10 was tentatively suggested to be the isomer of 1 with a
different linkage of sugars between the glucose and rhamnose
units at C-3 (Figure 1).
Compounds 11−14 had the same deprotonated ion at m/z
1223.9 [M − H]
−
as AB, 2 (Table 1). The MS
n
spectra of 11
and 12 were almost identical to those of 2 (Table 1), suggesting
their structures were similar to that of 2, except for the linkage
between the glucose and rhamnose units (Figure 1). 13 and 14
had fragment ions at m/z 1077.7 [M − Rha − H]
−
and 915.7
[M − Rha − Glc − H]
−
in their MS
2
spectra, which showed
that a rhamnose moiety was lost first (Table 1). On the basis of
the fragmentation patterns that we observed from 1−3 and 5,
we hypothesized that 13 and 14 had unique side chains with
−Glc−Glc−Glc−Rha or −Glc−Glc −Glc−Glc−Rha at C-3,
respectively (Figure 1).
The MS
2
spectra of 15 and 16 had three major fragment ions
at m/z 1223.8 [M − Glc − H]
−
, 1061.7 [M − 2Glc − H]
−
, and
899.6 [M − 3Glc − H]
−
and a major fragment ion at m / z 753.5
[M − 3Glc − Rha − H]
−
in the tandem mass spectra of m/ z
899.6 (MS
3
, 899.6/1385.8), which were similar to those of 5
(Table 1). Therefore, the structures of 15 and 16 were similar
to that of 5 except for the linkage between the glucose and
rhamnose units of the side chain at C-3 (Figure 1).
Validation of the Quantitative HPLC-MS Method. The
quantitative HPLC-MS method was validated in terms of linearity,
precision, and accuracy. Calibration curves were constructed by
plotting the integrated peak areas (x) of chromatography versus
the corresponding concentrations of the injected standard
solutions (y). The calibration curves were obtained over the
concentration ranges from 0.5 to 10 μg/mL for 1 and from
0.25 to 5 μg/mL for 2 with good linearity (R
2
> 0.999). The
limit of quantification was 0.12 μg/mL for 1 and 0.1 μg/mL
for 2. The intraday variation was determined by analyzing the
known concentrations of 1 and 2 in six replicates during a single
day, whereas interday variation was determined in duplicate
on three consecutive days, respectively. The overall intra- and
interday variations were <2.13%, indicating satisfactory pre-
cision of the instrumentation and stability of the samples were
achieved. Recovery tests were performed to examine the accuracy
of the analytical method. Accurate amounts of authentic
standards with two different concentration levels (low and
high, n = 3) were added into the oat bran product (product 4)
before the samples were extracted and analyzed by the HPLC-
MS method. The mean extraction recoveries were 97.8% for 1
and 98.0% for 2, indicating that this method was consistent,
reproducible, and acceptable.
Quantification of the Contents of the Major Steroidal
Saponins 1 and 2 in 15 Commercial Oat Products by
HPLC-MS. The content of avenacosides is influenced by plant
species and cultivar, the plant part, and physiological age, as well
as geographic environment.
17
Although there were two reports
on the variation of avenacoside contents among different oat
varieties,
33,35
the content of avenacosides in commercial oat
products remains unknown. Saponins 1 and 2 were revealed as
the primary saponins of oat bran in this study, which is in
accordance with published data.
33
Increasing interest in oat
products prompted us to analyze the contents of avenacosides
in commercial oat products. In this study, we developed an
HPLC-MS method to analyze the avenacosides in 15 different
commercial oat products including three oat brans, six oatmeals,
and six cold oat cereals. The contents of 1 and 2 in 15 different
commercial oat products are summarized in Table 2. In all oat
Table 2. Contents of Avenacoside A, 1, and Avenacoside B, 2, in 15 Commercial Oat Products
a
oat product
b
1 (mg/kg of product) 2 (mg/kg of product) oat product
b
1 (mg/kg of product) 2 (mg/kg of product)
1 377.5 ± 1.4 65.5 ± 1.7 9 169.5 ± 4.2 52.0 ± 1.1
2 224.1 ± 3.9 76.3 ± 4.9 10 37.9 ± 0.8 11.7 ± 1.2
3 224.8 ± 5.9 89.2 ± 7.1 11 65.4 ± 0.9 23.4 ± 2.1
4 163.2 ± 3.9 59.6 ± 3.7 12 72.2 ± 0.5 18.5 ± 1.8
5 76.1 ± 11.4 24.8 ± 6.7 13 57.2 ± 2.6 21.0 ± 2.8
6 193.1 ± 2.7 84.4 ± 6.3 14 62.9 ± 0.2 17.1 ± 1.3
7 221.9 ± 2.3 72.0 ± 1.3 15 61.7 ± 0.3 17.4 ± 0.6
8 156.5 ± 4.1 36.2 ± 2.1
a
Values expressed as the mean ± standard deviation.
b
Oat bran, 1−3; oatmeal, 4−9; and cold oat cereal, 10−15.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. XXXX, XXX, XXX−XXX
F

products, the contents of 1 were 3−6 times higher than those of
2. The total amount of 1 and 2 varies from 49.6 to 443.0 mg/kg
(Table 2). In general, the levels of these compounds in cold oat
cereal are much lower than those in oat bran or oatmeal.
Cell Growth Inhibition by 1 and 2. Saponins 1 and 2
were evaluated for growth inhibitory effects against human
colon cancer cells HCT-116 and HT-29 and showed weak
activity in both cell lines (Figure 5). 2 was more potent than 1 in
both cell lines, and the IC
50
of 2 in HCT-116 cells is 175.3 μM.
In conclusion, 16 steroidal saponins (1−16) were charac-
terized by NMR experiments or HPLC-ESI/MS
n
techniques.
Among them, 4 was purified from oats for the first time, and 5
is a new compound. This is also the first report of the complete
NMR data for 4. Saponins 6−16, tentatively identified by
HPLC-ESI/MS
n
analysis, are also reported from oats for the
first time. As a result, we have outlined the comprehensive
profile of steroidal saponins in oat bran and have quantified the
levels of 1 and 2 in 15 commercial oat products. Furthermore,
we found 2 has a weak effect against the growth of human
colon cancer cells. Saponins 1 and 2 are the unique and primary
saponins in oats. It is possible that they can be used as exposure
markers to reflect whole grain oat intake. However, there is no
study on the bioavailability of these two compounds. Therefore,
it is worthwhile to study the bioavailability and biotrasforma-
tion of 1 and 2. Understanding the health effects of these two
compounds is another topic of future study.
■
AUTHOR INFORMATION
Corresponding Author
*(S.S.) Phone: (704) 250-5710. Fax: (704) 250-5709. E-mail:
ssang@ncat.edu, shengminsang@yahoo.com.
Notes
The authors declare no competing financial interest.
■
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