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Steroidal Saponins in Oat Bran

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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.
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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 eects are still largely unknown. In this study, we directly puried 5 steroidal saponins (15) from
a methanol extract of oat bran, characterized their structures by analyzing their MS and NMR spectra, and also tentatively
identied 11 steroidal saponins (616) on the basis of their tandem mass spectra (MS
n
, n =23). Among the ve puried
saponins, 5 is a new compound and 4 is puried from oats for the rst time. Using HPLC-MS techniques, a complete prole of
oat steroidal saponins was determined, and the contents of the two primary steroidal saponins, 1 and 2, were quantitated in
15 dierent 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 eects 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 prole, avenocoside D, cytotoxic eect
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 ber and bioactive phytochemicals, which
display a serum cholesterol lowering eect and reduce the risk of
heart disease and cardiovascular disease.
47
Besides, consumption
of oat products showed other health benets, such as anticancer
and antidiabetic eects,
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 eects including steroidal saponins,
11
avenan-
thramides,
12
phenolic acids,
13,14
tocols,
15
and avanoids.
11
As the
only saponin-accumulating cereal,
16
oats contain two dierent
saponin forms, avenasides and avenacosides, synthesized via two
dierent 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 eects of 26-deglucoavenacosides,
21
and these saponins
can be detoxied 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 puried
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 eects 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 eects.
2831
The chemical prole and the anticancer eects 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
prole of steroidal saponins in oats, quantitate their levels in
commercial oat products, and evaluate their inhibitory eects on
the growth of human colon cancer cells.
MATERIALS AND METHODS
Materials. Silica gel (230400 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, 225 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, XXXXXX
MO, USA). All analytical and HPLC-MS grade solvents were obtained
from Thermo Fisher Scientic (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
ow rate of 0.2 mL/min. A gradient eluting system was applied: 40% B
from 0 to 3 min; 4052% B from 3 to 35 min; 52100% 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 puried compound in methanol solution
(10 μg/mL) was infused in ESI source and analyzed in negative ion
Figure 1. Structures of compounds 116 identied from oat bran. ∗∗, new compound; , rst purication from oat.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b06071
J. Agric. Food Chem. XXXX, XXX, XXXXXX
B
mode to give the following optimized parameters: spray voltage, 3.6 kV;
sheath gas (nitrogen) ow rate, 34 (arbitrary units); capillary voltage,
13 V; capillary temperature, 300 °C; tube lens oset, 60 V. For the
quantication 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 identication
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 ltration 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 aord four fractions,
F1F4, 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.
Purication of Steroidal Saponins 15. Repeated purication
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) aorded ve 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 Eects 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 nal concentrations (25, 50, 75, 100,
150, and 200 μM, the nal DMSO concentrations for control and
treatments were 0.1%, n =816). 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 reected 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 McCoys 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 15. As
part of our eorts to complete the chemical prole of oat bran,
ve steroidal saponins 15 (Figure 1) were isolated by means
of chromatographic methods, including silica gel and Diaion
HP-20 chromatography. Compounds 13 were identied 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 rst 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 dierence 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 ve
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, XXXXXX
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 rst time.
This is the rst 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 ve 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
olenic 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 congurations 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 GlcRha 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 =23) 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, XXXXXX
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 =23) Fragment Ions of Compounds 116 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 116 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, XXXXXX
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).
Identication of Steroidal Saponins 616 in Oats by
LC-ESI/MS
n
Spectra. On the basis of the ionization and
fragmentation patterns of the known compounds 15,we
searched the minor components of steroidal saponins from oat
extract. Eleven compounds (616) were identied, 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 identied 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
dierent linkage of sugars between the glucose and rhamnose
units at C-3 (Figure 1).
Compounds 1114 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 rst (Table 1). On the basis of
the fragmentation patterns that we observed from 13 and 5,
we hypothesized that 13 and 14 had unique side chains with
GlcGlcGlcRha or GlcGlc GlcGlcRha 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 quantication 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 dierent 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.
Quantication of the Contents of the Major Steroidal
Saponins 1 and 2 in 15 Commercial Oat Products by
HPLC-MS. The content of avenacosides is inuenced 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 dierent 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 dierent
commercial oat products including three oat brans, six oatmeals,
and six cold oat cereals. The contents of 1 and 2 in 15 dierent
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, 13; oatmeal, 49; and cold oat cereal, 1015.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. XXXX, XXX, XXXXXX
F
products, the contents of 1 were 36 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 eects 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 (116) were charac-
terized by NMR experiments or HPLC-ESI/MS
n
techniques.
Among them, 4 was puried from oats for the rst time, and 5
is a new compound. This is also the rst report of the complete
NMR data for 4. Saponins 616, tentatively identied by
HPLC-ESI/MS
n
analysis, are also reported from oats for the
rst time. As a result, we have outlined the comprehensive
prole of steroidal saponins in oat bran and have quantied the
levels of 1 and 2 in 15 commercial oat products. Furthermore,
we found 2 has a weak eect 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 reect 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 eects 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 nancial interest.
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Oat is classified as a whole grain and contains high contents of protein, lipids, carbohydrates, vitamins, minerals, and phytochemicals (such as polyphenols, flavonoids, and saponins). In recent years, studies have focused on the effects of oat consumption on reducing the risk of a variety of diseases. Reports have indicated that an oat diet exerts certain biological functions, such as preventing cardiovascular diseases, reducing blood glucose, and promoting intestinal health, along with antiallergy, antioxidation, and cancer preventive effects. At present, cancer is the second leading cause of death worldwide. The natural products of oat are an important breakthrough for developing new strategies of cancer prevention, and their ability to interact with multiple cellular targets helps to combat the complexity of cancer pathogenesis. In addition, the comprehensive study of the cancer prevention activity and potential mechanism of oat nutrients and phytochemicals has become a research hotspot. In this Review, we focused on the potential functions of peptides, dietary fiber, and phytochemicals in oats on cancer prevention and further revealed novel mechanisms and prospects for clinical application. These findings might provide a novel approach to deeply understand the functions and mechanisms for cancer prevention of oat consumption.
Article
There is an increasing trend today towards plant-based diets in western society, often resulting in milk restriction. In the case of very young children, the direct substitution of milk by other foods, without proper nutritional advice, may lead to a lack of nutrients and hence to growth and development alterations. This study focuses on the nutritional assessment of various commercially available plant-based drinks, to determine their adequacy as alternatives to ruminant milk, in relation to the nutritional requirements of toddlers (1–3 years old), and to establish whether other sources of nutrient supplementation may be needed, as well as any other possible positive and /or negative health effects associated to their consumption. A sample of 179 commercial plant-based drinks (almond, coconut, hemp, oat, rice, soy, tigernut) were chosen and their nutrient contents were compared to the EFSA nutrient reference values for toddlers. The scientific literature on the presence of bioactive and/or undesirable compounds was reviewed. None of the plant-based drinks studied should be considered as a milk substitute, since they are different food products with a different composition. However, from the results obtained, the best choice for toddlers who do not consume milk would be to consume at least 250 mL/day of fortified soy drink (for its higher amount and quality of protein, polyunsaturated fatty acids and phytosterols), and always in the context of a carefully-balanced diet. Almond, hemp or oat drinks are other alternatives that can be used in combination or for soy-allergic toddlers. The key nutrients that should be fortified in plant-based drinks are: vitamins A and B12, calcium, zinc and iodine, as they represent the most significant nutritional differences with milk; vitamin D would also be desirable. Of these, vitamins A, B12, D and calcium, are easily found in many commercial plant-based drinks on the Spanish market (most frequently in soy drinks), unlike iodine and zinc, which were not added to any. Given the fish restriction in vegetarians/vegans and the fact that plant-based drinks provide high amounts of phytates and tannins, which act as antinutrients, a good strategy for the industry would be to fortify plant-based drinks with iodine and zinc to improve the nutritional value of products aimed to vegetarians/vegans.
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Control methods alternative to synthetic pesticides are among the priorities for both organic and conventional farming systems. Plants are potential sources of compounds with antimicrobial properties. In this study, the antifungal potentialities of saponins derived from Medicago species and oat grains and of brassica sprouts have been explored for the control of Verticillium dahliae, a widely distributed fungal pathogen that causes vascular wilt disease on over 200 plant species. All the tested plant extracts showed antifungal properties. Such compounds, able to reduce mycelium growth and conidia formation, deserve deeper in vivo evaluation, even in combination with a delivery system.
Article
Cellular senescence is representing a potential anticancer therapeutic arsenal. Avenanthramide C (AVN C), as signature compounds of oats, exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and anti-tumor activities. However, the relationship between AVN C and cellular senescence in tumors remains largely unclear. Here, we elucidated that AVN C treatment predisposed colorectal cancer cells to senescent phenotype confirmed by flattened and enlarged shape characteristics, elevated senescence-associated β-galactosidase (SA-β-Gal) activity, and G1 phase arrest. Furthermore, AVN C triggered cellular senescence via transcriptionally repressing miR-183/96/182 cluster and subsequently reduced the levels of mature miR-183, -96, and -182. Mechanistically, AVN C exerted its senescence induction by attenuating β-catenin-mediated transactivation of miR-183/96/182 cluster to unleash its common target FOXO1 and two other targets, FOXO3 and SMAD4, which subsequently foster the p21 and p16 expression. In addition, AVN C is also noted to facilitate p53-mediated p21 transactivation via suppressing β-catenin. Collectively, we identified a novel mechanism of β-catenin/miR-183/96/182 cluster/FOXO1 mediated-CRC cellular senescence that entails that AVN C serves as an auxiliary agent for CRC treatment.
Article
Background/aims: Biomarkers can provide objective measures of dietary exposure, but their relationship with dietary intake in different populations needs to be characterized. This study aimed to determine the association between C14:0, C15:0 and C17:0 and children's dairy fat intake, and to ascertain whether these fatty acids can be used as biomarkers for detecting change in dairy fat intake. Methods: Data from a randomized controlled trial (114 healthy children of 4-13 years of age) was used. The intervention was a replacement of regular-fat dairy foods with reduced-fat or low-fat items. Serum fatty acid composition was measured and dairy intake was assessed via 3 × 24-hour diet recalls at baseline and at 12 weeks (the end of the intervention). Correlation analysis was used to evaluate the relationship between dietary intake and fatty acids at baseline and at week 12, and for the change in biomarkers and diet between these time points. Results: Total dairy fat intake correlated with C14:0, C15:0 and C17:0 at baseline (n = 114; r = 0.24; r = 0.42; r = 0.25 respectively, all p < 0.05), but not at week 12. The change in the total amount of dairy fat (g/day) after 12 weeks was associated with a change in serum C15:0 (n = 59; r = 0.27; p = 0.04). Conclusions: C15:0 is a useful biomarker of dairy fat intake in children and can detect short-term changes in the absolute intake of dairy fat.
Article
Background: Vitamin D is obtained from dietary sources and synthesized in the skin during exposure to ultraviolet B radiation in sunlight. During pregnancy, vitamin D is transported from mother to fetus through the placenta in the form of 25-hydroxyvitamin D [25(OH)D]. There is evidence that vitamin D influences neuronal differentiation, endocrine functions, and fetal brain growth. Animal studies indicate alterations in the offspring brain as a consequence of vitamin D deficiency during pregnancy. In humans, maternal vitamin D insufficiency has been linked to impaired child language development. Using data from a prebirth cohort with up to 22 years of follow-up, we examined the association of vitamin D status with proxies of offspring neurodevelopmental outcomes. During 1988-1989, pregnant women were recruited for the DaFO88 cohort (n = 965) in Aarhus, Denmark. Maternal concentrations of 25(OH)D were quantified in serum from week 30 of gestation via the LC-MS/MS method (n = 850). Offspring were followed up through national registries until the age of 22 years. We evaluated the association of the maternal concentration of 25(OH)D with offspring neurodevelopmental outcomes defined as first admission diagnosis or prescription of medication for (1) ADHD, (2) depression, and (3) scholastic achievement based on the mean grade on standardized written examinations in the 9th grade (final exams after 10 years of compulsory school in Denmark). Key messages: Maternal concentrations of 25(OH)D were higher compared to current levels (median 76 nmol/l; 5th to 95th percentiles 23-152). There was a direct association between maternal vitamin D status and offspring depression (p(trend) = 0.01); for ADHD there was no association. Scholastic achievement was slightly higher for offspring of mothers with a vitamin D status in the range of >50-125 nmol/l, but this nonlinear association was not statistically significant. Conclusions: Our analyses based on biomarker measurement of 25(OH)D from a cohort of 850 pregnant women combined with long-term follow-up showed no support for a beneficial fetal programming effect of vitamin D status with regard to behavioral and affective disorders and scholastic achievement.
Article
The stromacentre, a particular structure in the plastids of mostAvena species, was isolated from etioplasts ofAvena sativa and then characterized to determine its biological function. When comparing differentAvena species with or without stromacentre, it was shown that the stromacentre, a 63-kDa protein, and saponins (characteristic compounds ofAvena sativa) either occur together or not at all. This linkage was confirmed by demonstrating a transformation of saponins by the isolated stromacentre protein: avenacosides were hydrolyzed to 26-desgluco-avenacosides. Therefore, the stromacentre protein had to be regarded as aß-glucosidase. Enzyme assays usingp-nitrophenyl-ß-d-glucopyranoside as substrate showed that thisß-glucosidase has a pH optimum at pH 6.0. The calculatedKm value for this substrate was 2.2·10-3 M. Antibodies against the stromacentre protein inhibitedß-glucosidase activity. The determination of the molecular weight of theß-glucosidase by sodium dodecyl sulfate-gel electrophoresis showed that it consists of subunits of 63 kDa. After gel electrophoresis under non-denaturing conditions, enzymatically active molecules were shown to consist of at least two of these subunits. Molecules aggregated up to about 106 Da also had enzyme activity. Enzyme assays using avenacosides as substrate showed a pH optimum at pH 6.0. The calculatedKm value for this substrate was 1.2·10-5 M. The high affinity to the avenacosides and the high specificity for the C-26 bound glucose indicate that avenacosides are the natural substrates for thisß-glucosidase. Assuming that the avenacosides in oat leaves play a role as preformed chemical inhibitory substances against phytopathogenic microorganisms, a model is presented showing the stromacentre with a central role in activating the fungitoxicity of avenacosides.
Article
Avenacosides A (AA) and B (AB) as well as 26-desglucoavenacoside A (26dAA) were quantified in oats using rapid and sensitive method utilising UPLC-TQ-MS. In the grain, AA and AB were revealed as the primary saponins, whereas in the husks, dAA was predominant. Inconsistent with the published data, observed concentrations of AA and AB in the grain were very similar. Presumably, this is due to higher sensitivity and better selectivity of the mass spectrometry-based quantification method, thus allowing for more precise measurements. Elevated level of an active fungicidal form of saponin, dAA in the husks possibly indicates they are more prone to fungal attacks.
Article
The methanol extract of the fresh bulbs of Lilium brownii has yielded a 27-acyloxyspirostanol saponin, named brownioside and a furospirostanol saponin, which appear to be new constituents. The respective structures have been shown by the spectroscopic evidence, and alkaline- and acid-catalysed degradations to be (25R)-27-O-(3-hydroxy-3-methylglutaroyl)-spirost-5-en-3β,27-diol 3-O-[α-l-rhamnopyranosyl(1→2)]-β-d-glucopyranoside and (22S,25S)-26-O-β-d-glucopyranosyl-22,25-epoxyfurost-5-en-3β, 26-diol 3-O-[α-l-rhamnopyranosyl(1→2)]-β-d-glucopyranoside. Several known phenolic compounds have also been isolated and identified.
Article
Cereal Chem. 80(5):542-543 Antioxidant products such as simple phenolic compounds and avenan- thramides in oat (Avena sativa L.) may have health-promoting effects on humans. Therefore, it is very important to determine simple phenolics and avenanthramide concentrations of different genotypes of oats from different regions of the world. The aim of this research was to determine the concentrations of simple phenolics and avenanthramides of Turkish oat genotypes. According to the results, Turkish oat genotypes were significantly different for three major avenanthramides (Bc, Bp, and Bf) and the simple phenolic, ferulic acid (FA), while not significantly differ- ent for p-coumaric acid (PCA). Ferulic acid concentrations of Turkish oat genotypes were higher than a standard U.S. cultivar, Belle. However, the major avenanthramide concentrations of Turkish oat genotypes were significantly lower than Belle.
Article
Summary New results obtained in course of studies on steroidal saponins of oat indicate that in contrast to our former conclusions the prolamellar body (PLB) is not built up by steroidal saponins. Saponin content of whole leaves is similar in green as well as in etiolated leaves and does not change significantly during development of leaves. In contrast to chloroplast, isolated purified etioplasts contain high amounts of the desgluco-avenacosides, which were thought to be responsible for the tubular structure of PLBs. The amount of these “PLB-saponins”, however, is 10–60 times higher in isolated etioplasts than in etiolated leaves. Evidence is presented that the “PLB-saponins” originate from leaf-saponins during disruption of cells and attach to etioplasts or PLBs. This attachment to PLB-components explains our former results that the PLB-destruction during greening is accompanied by a decline in the amount of the desgluco-avenacosides in isolated PLB-fractions of greening leaves.
Article
The revised structures of avenacosides A and B and a new sulfated steroidal saponin isolated from grains of Avena sativa L. were elucidated. Their structures and complete NMR assignments are based on 1D and 2D NMR studies and identified as nuatigenin 3-O-{α-l-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-d-glucopyranoside}-26-O-β-d-glucopyranoside (1), nuatigenin 3-O-{α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→3)-β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside}-26-O-β-d-glucopyranoside (2), and nuatigenin 3-O-{α-l-rhamnopyranosyl-(1→2)-[β-d-6-O-sulfoglucopyranosyl-(1→4)]-β-d-glucopyranoside}-26-O-β-d-glucopyranoside (3). Copyright © 2012 John Wiley & Sons, Ltd.
Article
Aus den Avenacosiden-A und -B des Hafers (Avena sativa) wird durch Inkubation mit einem wasserunlöslichen Enzym der Blätter die in der Seitenkette am 26-ständigen OH gebundene Glucose abgespalten, und es werden ohne Umlagerung zur Isoform die entsprechenden Nuatigeninglycoside erhalten. Die Desgluco-avenacoside-A und -B sind sowohl hämolytisch wie antibiotisch wirksam. Steroid Saponins with More than One Sugar Chain, XI. Desgluco-avenacosid-A and -B, Biologically Active Nuatigenin Glycosides The avenacosides A and B from the oat (Avena sativa) split off glucose with a water insoluble enzyme of the leaves from OH at C-26 and give without rearrangement to the iso form the corresponding nuatigenin glycosides. The desgluco-avenacosides A and B show as well hemolytic as antibiotic activity.
Article
Die Struktur von Avenacosid B wurde durch stufenweisen enzymatischen Abbau, durch Hydrolyse des permethylierten Glykosids und mit Hilfe der Massenspektrometrie bestimmt. Danach hat es den Aufbau eines 3-O-{[α-L-Rhamnopyranosyl-(1→4)]-[〈β-D-glucopyranosyl-(1→3)〉-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl}-3β.26-dihydroxy-22.25-epoxy-(22S: 25S)-Δ5-furostene-26-β-D-glucopyranosid (5). Steroid Saponines Containing more than one Shugar Chain, V. Avenacoside B, a Second Bisdesmosidical Steroid Saponine from Avena Sativa The structure of avenacoside B was determinated by stepwise enzymatic degradation, by hydrolysis of the permethylated glycoside and with the help of mass spectrometry. It has the constitution 3-O-{[α-L-rhamnopyranosyl-(1→4)]-[〈β-D-glucopyranosyl-(1→3)〉-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl}-3β.26-dihydroxy-22.25-epoxy-(22S: 25S)-Δ5-furostene-26-β-D-glucopyranosid (5).