Feruloyl sucrose derivatives from the root of Xerophyllum tenax

Article abstract of DOI:10.1016/j.phytochem.2021.112703

A phytochemical investigation of the roots of Xerophyllum tenax led to the isolation of three undescribed feruloyl sucrose derivatives along with two known feruloyl sucrose derivatives, heloniosides A and B. This is the first report of their occurrence in the genus Xerophyllum and the family Melanthiaceae. The structures of these compounds were elucidated on the basis of chemical and spectroscopic analysis including 1D and 2D NMR and analysis of MS-MS fragmentation.

Full text of DOI:10.1016/j.phytochem.2021.112703

Phytochemistry 185 (2021) 112703
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Feruloyl sucrose derivatives from the root of Xerophyllum tenax
,
*
Xueling Liua, Daniel Athab, Benjamin R. Clarka, Robert P. Borrisa
a School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China
b New York Botanical Garden, Bronx, NY, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
A phytochemical investigation of the roots of Xerophyllum tenax led to the isolation of three undescribed feruloyl
sucrose derivatives along with two known feruloyl sucrose derivatives, heloniosides A and B. This is the first
report of their occurrence in the genus Xerophyllum and the family Melanthiaceae. The structures of these
compounds were elucidated on the basis of chemical and spectroscopic analysis including 1D and 2D NMR and
analysis of MS-MS fragmentation.
Xerophyllum tenax
Melanthiaceae
Bear grass
Structure elucidation
Feruloyl sucrose
MS-MS
1. Introduction
mostly produced by the shikimic acid biosynthetic pathway (Seo et al.,
2015). Feruloyl sucrose esters incorporate a central sucrose unit, con-
Xerophyllum tenax (Pursh) Nutt. (Melanthiaceae), commonly known
as bear grass, is a perennial characterized by thickened underground
rhizomes and tufts of long, slender leaves and flowers crowded on ver-
tical spikes. It is not a grass but a member of the Xerophyllae tribe in the
Melanthiaceae family. The family includes the well-known wildflowers
in the genera Trillium and Paris, as well as the genus Veratrum, which is
rich in steroidal alkaloids (Schep et al., 2012). Xerophyllum Michx.
comprises two species endemic to North America: Xerophyllum aspho-
deloides (L.) Nutt in the Appalachian Mountains and coastal pine barrens
from Alabama to New Jersey, and X. tenax in the mountains and foothills
of western North America from British Columbia to central California
(Utech 2012). Indigenous Americans of the western United States use
X. tenax for baskets, food, medicine, cosmetics, and for spiritual and
ceremonial purposes (Hummel et al., 2012).
nected by ester linkages to one or more ferulic acid moieties, and
sometimes other ester groups. In the current report, three undescribed
feruloyl sucrose derivatives were obtained from the roots of X. tenax.
Herein we describe the isolation and structure elucidation of these
undescribed compounds.
2. Results and discussion
The n-BuOH soluble fraction of a methanol extract of the roots of
X. tenax was fractionated by a combination of chromatographic methods
to afford two undescribed feruloyl sucrose esters (1, 3) and one unde-
scribed feruloyl sucrose acetate ester (5), together with two known
compounds (2, 4) (Fig. 1). The known compounds were identified as
helonioside A (2) and helonioside B (4) by comparison of 1D and 2D
NMR, HR-ESI-TOF-MS with literature data (Fig. S29 – S34 of the Sup-
porting Information) (Nakano et al., 1986; Yan et al., 2008). This is the
first time that these compounds have been isolated from the genus
Xerophyllum and the family Melanthiaceae.
Only two phytochemical report on this species is available to date,
one focused on the seeds of X. tenax and reported the isolation, identi-
fication and biological activity of two ecdysteroids (Alison et al., 1997).
This was the first report of the occurrence of ecdysteroids in Xerophyllum
and also in the family Melianthaceae. Another one reported the content
of shikimic acid in several plants, including X. tenax. (Avula et al., 2009).
Given the lack of phytochemical studies on the genus Xerophyllum, it
remains an interesting target for the discovery of new molecules.
The present study focused on the isolation of feruloyl sucrose de-
rivatives from the roots of X. tenax. Ferulic acid derivatives are a type of
natural compound composed of a phenylpropanoid nucleus, which are
Compound 1 was isolated as a white amorphous powder. The mo-
lecular formula was determined to be C32H38O17 due to the observation
of an [M + Na]+ quasi-molecular ion at m/z 717.2013 in the HR-ESI-
TOF mass spectrum. All of the individual protons and carbons
(Table 1) were assigned by combined analysis of 1D and 2D NMR. The
1H-NMR spectrum (Table 1) of compound 1 showed two typical ABX
spin systems of aromatic moieties at δ 7.16 (1H, dd, J = 8.3, 2.0 Hz, H-
* Corresponding author.
E-mail address: rborris@tju.edu.cn (R.P. Borris).
https://doi.org/10.1016/j.phytochem.2021.112703
Received 25 November 2020; Received in revised form 6 February 2021; Accepted 9 February 2021
Available online 24 February 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.

X. Liu et al.
Phytochemistry 185 (2021) 112703
= 10.0, 4.0 Hz, H-2′), 3.62 (t, J = 9.5 Hz, H-3′), 3.39 (t, J = 9.5 Hz, H-4′),
and 3.88 (m, H-5′), three oxygenated methylenes at δ 3.60 and 3.65
(each 1H, d, J = 12.0 Hz, H-1), 4.46 (dd, J = 11.9, 3.6 Hz, H-6a), 4.54
(dd, J = 11.9, 7.3 Hz, H-6b), 3.79 (dd, J = 12.0, 4.5 Hz, H-6′a) and 3.87
(dd, J = 12.0, 2.5 Hz, H-6′b) were observed in the 1H-NMR spectrum,
indicating the presence of two sugars. A characteristic anomeric proton
signal at δ 5.43 with a small coupling constant (d, J = 4.0 Hz, H-l’), two
anomeric carbons at δ 105.0 (C-2) and 93.1 (C-1′), together with another
10 oxygenated carbon signals at δ 65.2 (C-1), 79.0 (C-3), 74.8 (C-4), 81.2
(C-5), 66.1 (C-6), 73.2 (C-2′), 74.9 (C-3′), 71.3 (C-4′), 74.4 (C-5′) and
65.2 (C-6′) observed in the 13C-NMR spectrum, established the sugar to
be a sucrose moiety (Cho et al., 2015). The presence of sucrose was
further confirmed by alkaline and acid hydrolysis, and comparison of
the TLC behavior and optical rotations of the resulting sugars with
standard samples. The HMBC correlation from H-3 (δ 5.47) to C-9" (δ
167.3) confirmed that the (Z)-feruloyl group was located at C-3 of the
fructose moiety, while the correlations from H-6a (δ 4.46) and H-6b (δ
4.54) to C-9′′′ (δ 169.1) confirmed that the (E)-feruloyl group was
located at C-6 of the fructose moiety (Fig. 2). Therefore, compound 1
was identified as 3-(Z)-feruloyl-6-(E)-feruloyl sucrose (Fig. 1). This is the
first report of both an E-feruloyl group and a Z-feruloyl group occurring
together in one feruloyl sucrose ester.
Fig. 1. Chemical structures of 1–5.
6′′), δ 6.76 (1H, d, J = 8.3 Hz, H-5′′), δ 7.83 (1H, d, J = 2.0 Hz, H-2′′), and
δ 7.09 (1H, dd, J = 8.3, 2.0 Hz, H-6′′′), δ 6.81 (1H, d, J = 8.3 Hz, H-5′′′), δ
7.19 (1H, d, J = 2.0 Hz, H-2′′′). Together with two pairs of olefinic
protons at δ 6.90 (1H, d, J = 12.0 Hz, H-7′′), 5.89 (1H, d, J = 12.0 Hz, H-
8′′) and 7.65 (1H, d, J = 16.0 Hz, H-7′′′), 6.39 (1H, d, J = 16.0 Hz, H-8′′′),
they indicated the presence of two feruloyl groups in compound 1. As
the coupling constant was 16.0 Hz for protons at δ 7.65/6.39 and 12.0
Hz for protons at δ 6.90/5.89, one of these was an (E)-feruloyl, while the
other was a (Z)-feruloyl group (Kim et al., 2010). The position of the two
methoxy groups in feruloyl units was assigned on the basis of NOESY
correlations, which showed that the correlation between two methoxy
groups and aromatic protons: δ 3.87 with H-2’’; and δ 3.89 with H-2′′′.
Moreover, eight oxygenated methines at δ 5.47 (d, J = 8.0 Hz, H-3), 4.40
(t, J = 8.0 Hz, H-4), 4.13 (m, H-5), 5.43 (d, J = 4.0 Hz, H-1′), 3.41 (dd, J
It was noted that over time the 1H-NMR spectra of both compounds 1
and 2 were subject to change (Fig. 3). For compound 1, over a period of
several months, characteristic proton signals corresponding to the
presence of the Z-double bond lowered in intensity, and new signals
corresponding to the presence of an E-double bond appeared, suggesting
that compounds 1 was gradually converting into compound 2. Based on
their integrals in the 1H-NMR spectra, the percentage of compounds 1
Table 1
1H (600 MHz) and 13C NMR (150 MHz) data for compounds 1–3 in CD3OD.
1
3
5
Position
H
C
H
C
H
C
β-D-Fru 1a
3.60 (1H, d, 12.0)
3.65 (1H, d, 12.0)
65.2
3.57 (1H, d, 12.0)
3.60 (1H, d, 12.0)
65.1
3.56 (1H, d, 12.0)
3.60 (1H, d, 12.0)
65.4
1b
2
105.0
79.0
74.8
81.2
66.1
105.2
79.4
75.3
81.4
66.1
105.0
79.1
74.8
81.3
65.9
3
5.47 (1H, d, 8.0)
5.46 (1H, d, 7.5)
4.38 (1H, t, 7.5)
5.47 (1H, d, 8.0)
4.43 (1H, t, 8.0)
4
4.40 (1H, t, 8.0)
5
4.13 (1H, m)
4.14 (1H, m)
4.14 (1H, m)
6a
6b
4.46 (1H, dd, 11.9, 3.6)
4.54 (1H, dd, 11.9, 7.3)
5.43 (1H, d, 4.0)
4.45 (1H, dd, 11.9, 3.7)
4.52 (1H, dd, 11.9, 7.5)
5.40 (1H, d, 3.8)
3.42 (1H, dd, 9.8, 3.8)
3.67 (1H, t, 9.5)
4.47 (1H, dd, 11.9, 3.7)
4.51 (1H, dd, 11.9, 7.3)
5.42 (1H, d, 3.8)
3.43 (1H, dd, 9.7, 3.8)
3.63 (1H, t, 9.5)
α
-D-Glu 1′
93.1
73.2
74.9
71.3
74.4
65.2
93.3
73.2
74.9
71.3
74.4
62.7
92.7
73.0
74.9
71.9
72.2
65.8
2′
3.41 (1H, dd, 10.0, 4.0)
3.62 (1H, t, 9.5)
3′
4′
3.39 (1H, t, 9.5)
3.39 (1H, t, 9.5)
3.25 (1H, t, 9.5)
5′
3.88 (1H, m)
3.94 (1H, m)
4.17 (1H, m)
6′a
3.79 (1H, dd, 12.0, 4.5)
3.87 (1H, dd, 12.0, 2.5)
3.77 (1H, dd, 12.0, 4.5)
3.90 (1H, dd, 12.0, 2.6)
6-Z-Feruloyl
4.10 (1H, dd, 11.9, 7.2)
4.57 (1H, dd, 11.9, 1.2)
6-Z-Feruloyl
6′b
3-Z-Feruloyl 1′′
128.0
115.2
148.3
149.7
115.7
127.1
146.7
115.8
167.3
56.4
128.0
115.2
148.3
149.4
115.7
127.0
146.5
116.0
168.0
56.5
128.1
115.1
148.3
149.6
115.7
126.9
145.9
116.1
167.9
56.5
2′′
7.83 (1H, d, 2.0)
7.82 (1H, d, 2.0)
7.84 (1H, d, 2.0)
3′′
4′′
5′′
6.76 (1H, d, 8.3)
6.76 (1H, d, 8.3)
6.76 (1H, d, 8.2)
6′′
7.16 (1H, dd, 8.3, 2.0)
6.90 (1H, d, 12.0)
5.89 (1H, d, 12.0)
7.13 (1H, dt, 8.3, 2.0)
6.88 (1H, d, 12.5)
5.82 (1H, d, 12.5)
7.12 (1H, dt, 8.2, 2.0)
6.86 (1H, d, 12.8)
5.82 (1H, d, 12.8)
7′′
8′′
9′′
OCH3
3.87 (1H, s)
3.87 (1H, s)
3.86 (1H, s)
6-E-Feruloyl 1′′′
127.7
111.7
149.4
150.7
116.5
124.3
147.2
115.2
169.1
56.5
3-E-Feruloyl
7.22 (1H, d, 2.0)
127.7
112.1
149.7
150.8
116.5
124.2
147.8
115.0
168.2
56.4
3-E-Feruloyl
7.24 (1H, d, 2.0)
127.7
112.1
149.4
150.7
116.5
124.3
147.9
114.9
168.2
56.4
2′′′
7.19 (1H, d, 2.0)
3′′′
4′′′
5′′′
6.81 (1H, d, 8.3)
6.80 (1H, d, 8.3)
6.80 (1H, d, 8.2)
6′′′
7.09 (1H, dd, 8.3, 2.0)
7.65 (1H, d, 16.0)
6.39 (1H, d, 16.0)
7.13 (1H, dt, 8.3, 2.0)
7.70 (1H, d, 16.0)
6.42 (1H, d, 16.0)
7.12 (1H, dt, 8.2, 2.0)
7.70 (1H, d, 16.0)
6.43 (1H, d, 16.0)
7′′′
8′′′
9′′′
OCH3
3.89 (1H, s)
3.89 (1H, s)
3.89 (1H, s)
2.10 (1H, s)
OAc-6′
173.0
20.9
2

X. Liu et al.
Phytochemistry 185 (2021) 112703
Fig. 2. Selected HMBC (solid arrows) and NOESY (dashed arrows) correlations for compounds 1, 3 and 5.
Fig. 3. Isomerization of double bond in compound 1, as shown by change in 1H-NMR over time (CD3OD).
and 2 was 37% and 63%, respectively. Ferruloyl derivatives have been
shown previously to isomerize from Z to E, in particular when under UV
illumination (Hwang et al., 2016), however in this instance isomeriza-
tion occurred despite the sample being stored in the dark.
4.52) to C-9" (δ 168.0) confirmed that the (Z)-feruloyl is located at C-6 of
the fructose moiety. Therefore, compound 3 was identified as 3-(E)-
feruloyl-6-(Z)-feruloyl sucrose (Fig. 1).
Compound 5 was isolated as a yellowish gum. The molecular formula
was determined to be C34H40O18 from a [M + Na]+ quasi-molecular ion
at m/z 759.2126 in the HR-ESI-TOF mass spectrum. On the basis of the
1H and 13C NMR spectra data (Table 1), compounds 5 and 3 were almost
identical, except that compound 5 had additional signals corresponding
to the presence of an acetate group. Combined with the coupling con-
stants of 16.0 Hz for the protons at δ 7.70/6.43 and of 12.8 Hz for the
protons at δ 6.86/5.82, this indicated that compound 5 possessed a
structure containing one sucrose unit, one (Z)-feruloyl moiety, one (E)-
feruloyl moiety and one additional acetate group. Correlations of the
oxygenated methylene proton signals H-6′a (δ 4.10) and H-6′b (δ 4.57)
with the acetate carbonyl resonance at δ 173.0 indicated that the acetyl
group was located at C-6′ of the glucose moiety. In addition, correlation
Compound 3 was obtained as a white amorphous powder. Based on a
[M+Na]+ quasi-molecular ion at m/z 717.2012 in the HR-ESI-TOF-MS
spectrum, the molecular formula was determined to be C32H38O17
,
indicating that it was an isomer of compound 1. Comparing the 1H and
13C NMR spectra of compounds 1 and 3 (Table 1) suggested that com-
pound 3 was also composed of two feruloyl units and one sucrose. The
coupling constant of 16.0 Hz for the protons at δ 7.70/6.42 and that of
12.5 Hz for the protons at δ 6.88/5.82 again showed the presence of both
(E)-feruloyl and (Z)-feruloyl groups. From the HBMC spectrum, corre-
lations from H-3 (δ 5.46) to C-9′′′ (δ 168.2) confirmed that, in contrast to
compound 1, in compound 3 the (E)-feruloyl group is located at C-3 of
the fructose moiety, while correlations from H-6a (δ 4.45) and H-6b (δ
3

X. Liu et al.
Phytochemistry 185 (2021) 112703
of the proton signal H-3 (δ 5.47) with the carbonyl carbon signal C-9′′′ (δ
168.2) indicated that the (E)-feruloyl group is located at C-3 of the
fructose moiety, and correlation of the proton signal H-6 (δ 4.51/4.47)
with the carbonyl carbon signal C-9" (δ 167.9) indicated that the (Z)-
feruloyl group is located at C-6 of the fructose moiety (Fig. 2). Overall,
the structure of 5 was determined to be 6′-acetyl-3-(E)-feruloyl-6-(Z)-
feruloyl sucrose (Fig. 1).
145.0286 and 117.0339 are also consistent with previous reports
(Fig. 4C) (Asamenew et al., 2019; Qi et al., 2008). Compounds 2–5
showed almost identical MS-MS spectra to 1.
In negative mode, alternative MS2 fragmentation patterns were
observed (Fig. 4B). The parent ion at m/z 693.2052 for compound 1 can
lose a ferulic acid unit to give an ion at m/z 517.1574, which then
eliminates one molecule of glucose to yield a product ion at m/z
337.0939. The ion at m/z 175.0404 is consistent with a ferulic acid
fragment which can fragment further to give the ion at m/z 160.0169
indicating the loss of a further methyl group. (Fig. 4D). Unlike in positive
mode, in negative mode compounds 4 and 5 showed a different frag-
mentation pattern from 1–3, with several of the higher-mass fragments
shifted 42 amu higher, consistent with the presence of an additional
acetyl group. However, in neither positive nor negative mode were there
any significant differences between the MS-MS spectra of the various cis-
trans isomers: MS-MS analysis alone cannot be used to distinguish these
compounds.
The presence of the undescribed feruloyl sucrose derivatives,
together with helonioside A and helonioside B, constitutes the first
report of this compound class in the family Melanthiaceae. Compounds
1, 2 and 3 are isomers, as are compounds 4 and 5. According to HRESI-
LC-MS2 analysis, in both positive and negative mode, compounds 1–5
share a similar fragmentation pathway (Fig. S27 and S28 of the Sup-
porting Information). Based on the results of MS2 analysis, a fragmen-
tation pathway was proposed (Fig. 4). Fig. 4A shows the MS-MS
spectrum of compound 1 under positive mode, which revealed an [M
+ NH4]+ adduct ion at m/z 712.2455 and major fragment ions at m/z
515.1553, 303.0864, 177.0548, 145.0286, and 117.0339. The cleaving
of the glycosidic bond in MS-MS is a common feature of feruloyl sucrose
derivatives, along with the loss of other side chains (Wu et al., 2010).
The ion at m/z 515.1553 was consistent with the elimination of one
glucose unit, which could be followed by loss of a ferulic acid and H2O to
yield a product ion at m/z 303.0864. A product ion at m/z 177.0548 was
consistent with the presence of ferulic acid: smaller fragment ions at m/z
Compounds 1–5 were tested for antimicrobial activity against a
small suite of test microbes, but did not show any activity. Several
studies have already shown that feruloyl sucrose derivatives possess
multiple types of pharmacological activity, including antioxidant (Choi
et al., 2012), cytotoxic (Yan et al., 2008; Kiem et al., 2008),
anti-inflammatory (Zheng et al., 2012), and tyrosinase inhibitory ac-
tivities (Panda et al., 2011; Tanimoto et al., 2006). Thus, the
Fig. 4. Proposed fragmentation pathway of compound 1–5. A. The MS2 spectrum of compounds 1 and 5; positive mode. B. The MS2 spectrum of compound 1 and 5;
negative mode. C. Proposed fragmentation pathway of compound 1; positive mode. D. Proposed fragmentation pathway of compounds 1 and 5; negative mode.
4

X. Liu et al.
Phytochemistry 185 (2021) 112703
undescribed feruloyl sucrose derivatives may possess similar biological
properties, which need to be explored further.
3 × 4 L of methanol at ambient temperature (24 h each time). The
pooled methanol extracts were evaporated to dryness in vacuo to afford a
tarry residue. Then, the methanolic extract (28.0 g) was dispersed in
methanol:water (9:1, 100 ml) and defatted with n-hexane (3 × 100 ml).
The hydroalcoholic phase was freed of solvent, dispersed in water and
successively extracted with dichloromethane (DCM) and n-BuOH (each
3 × 100 ml) to afford n-hexane (1 g), DCM (2.5 g), n-BuOH (6.0 g) and
aqueous (12.5 g) fractions.
The botanical classification of X. tenax has been changed in the past,
being assigned to both the Melanthiaceae (Brummitt, 1992) and Lil-
iaceae (Cronquist, 1981). In 2000, it was assigned to the family Mel-
anthiaceae according to molecular phylogenetic analysis (Rudall et al.,
2000; Vance et al., 2004). While our study has established that feruloyl
sucrose derivatives are a part of the chemical composition of X. tenax,
previous studies have shown that feruloyl sucrose derivatives also
appear in Liliaceae (Nakano et al., 1986; Satou et al., 1996; Chen et al.,
2000; Shu et al., 2002; Ono et al., 2007; Yan et al., 2008; Sun et al.,
2012; Munafo and Gianfagna, 2015; Luo et al., 2018). This is consistent
with the close relationship between the Liliaceae and Melanthiaceae
families.
The n-BuOH fraction was subjected to column chromatography on
Diaion HP-20 (150 g) eluting with a H2O–MeOH step gradient (each 3 ×
200 ml) to give sub-fractions H2O (1.2 g), 20% MeOH–H2O (0.4 g), 40%
MeOH–H2O (0.15 g), 60% MeOH–H2O (0.2 g), 80% MeOH–H2O (2.5 g),
100% MeOH–H2O (0.9 g) successively.
The 80% MeOH–H2O fraction (2.5 g) was separated using flash
chromatography (Spherical C18, 20–45
μm, 80 g) with a H2O-ACN
3. Experimental
gradient solvent system (90:10 → 0:100, v/v, 81.9 min, 25 ml/min) to
give fractions 1, 2, 3 and 4. Fractions 1–3 were separated on preparative
3.1. General
RP-18 column (5
μ
m, 21.2 × 250 mm, 40 ◦C, 10 ml/min) using H2O-ACN
(80:20, v/v) and afforded compound 1 (7.5 mg), compound 2 (20.5 mg),
and compound 3 (6.0 mg). Fraction 4 was separated following the same
procedure and afforded compound 4 (16.3 mg) and compound 5 (6.4
mg). HPLC retention times were 4.27 min, 4.70 min, 5.10 min, 7.59 min
and 8.39 min, respectively (Hypersil Gold C18, 25% MeCN:H2O con-
taining 0.1% formic acid under 0.2 ml/min, 40 ◦C).
The melting points were measured on a Micro-melting point appa-
ratus with corrections. Optical rotations were recorded on an AUTOPOL
II Polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA).
UV spectra were measured using a Hitachi U3900 spectrophotometer
(Hitachi, Kyoto, Japan). The high-resolution electrospray ionization
mass spectra (HR-ESI-MS) were measured using a MicroTOF-QII spec-
trometer (Bruker Daltonics, Bremen, Germany) or a Q Exactive HF
Orbitrap LC-MS (Thermo Fisher Scientific, Waltham MA, USA). NMR
spectra were recorded on an Avance III 600 MHz spectrometer (Bruker
BioSpin, Billerica MA, USA) using TMS or the residual solvents as in-
ternal standard. For column chromatography, Diaion HP-20 (Mitsubishi
Chemical Industries, Tokyo, Japan) was used. Medium pressure liquid
chromatography (MPLC) was performed using an ISCO CombiFlash Rf
+ instrument (Teledyne ISCO, Lincoln NE, USA) equipped with pre-
3.3.1. Compound 1
White, amorphous powder; mp 136–138 ◦C; [α D25
-10.3 (c 0.30,
]
MeOH); UV (MeOH) λmax (log ε) 218 (3.88), 239 (3.85), 299 (3.94), 328
(4.12); 1H-NMR (CD3OD, 600 MHz), see Table 1; 13C-NMR (CD3OD, 150
MHz), see Table 1; HRESIMS m/z 717.2013 [M + Na]+ (calcd. for
C32H38O17Na, 717.2001).
3.3.2. Compound 3
White, amorphous powder; mp 136–138 ◦C; [α D25
]
-7.3 (c 0.30,
packed spherical ODS silica gel (20–45 μm) column (SepaFlash, Santai
Technologies, Changzhou, China). Analytical and semipreparative HPLC
separations were performed on an Agilent 1260VL quad gradient system
(G1311C pump, G1329B autosampler, G1316A thermostatted column
compartment and G1315D photodiode array detector, Agilent Tech-
MeOH); UV (MeOH) λmax (log ε) 218 (3.88), 239 (3.85), 299 (3.94), 328
(4.12); 1H-NMR (CD3OD, 600 MHz), see Table 1; 13C-NMR (CD3OD, 150
MHz), see Table 1; HRESIMS m/z 717.2012 [M + Na]+ (calcd. for
C32H38O17Na, 717.2001).
nologies, Santa Clara CA, USA), using Hypersil Gold C18 column (3 μm,
2.1 × 150 mm, Thermo Scientific, Waltham MA, USA). Preparative
HPLC separations were performed on an Agilent 1260VL quad gradient
system (G1311C pump, and G1315D photodiode array detector)
equipped with a Rheodyne 7725i manual injection valve (IDEX Health &
Science, Middleboro MA, USA) and Shimadzu CTO-20A column oven
(Shimadzu Scientific Instruments, Kyoto, Japan). Preparative separa-
3.3.3. Compound 5
Yellowish gum; mp 179–181 ◦C; [α D25
-10.3 (c 0.30, MeOH); UV
]
(MeOH) λmax (log
ε
) 218 (3.90), 239 (3.88), 299 (3.97), 328 (4.15); 1H-
NMR (CD3OD, 600 MHz), see Table 1; 13C-NMR (CD3OD, 150 MHz), see
Table 1; HRESIMS m/z 759.2126 [M + Na]+ (calcd. for C34H40O18Na,
759.2107).
tions were conducted using a Hypersil Gold C18 column (5 μm, 21.2 ×
250 mm). All solvents used were of HPLC grade (Concord Technologies,
Tianjin, China). Thin-layer chromatography (TLC) was carried out on
silica gel GF254 plates (Haiyang Chemicals Corp., Qingdao, China). TLC
spots were visualized by heating silica gel plates sprayed with 5% H2SO4
in EtOH (v/v).
3.3.4. Alkaline hydrolysis of 1, 3 and 5
Compounds (2 mg, each, individually) were dissolved in 3%
KOH–MeOH (2 mL), after 6 h, the reaction solutions were neutralized
with 1N HCl and then extracted with CHCl3. From the H2O layer, sucrose
was identified by comparing its TLC behavior with standard samples [Si
gel, developed with CHCl3–MeOH–H2O (6:4:1)] (Shu et al., 2002).
3.2. Plant material
3.3.5. Acid hydrolysis of 1, 3 and 5
The roots of Xerophyllum tenax (Pursh) Nutt. (Melanthiaceae) were
collected in Plumas County, CA, USA (lat 39.8283, lon ꢀ 121.304, 1625
m elev) on 20 Aug 2003. Specimens were authenticated by Daniel Atha
of the New York Botanical Garden. Voucher specimens (Lowell Ahart
10520) have been deposited in the herbarium of the New York Botanical
Garden (CHSC, NY-199257). Plant material was air dried in the shade,
freed of extraneous material and ground to a coarse powder prior to
extraction.
A solution of 1, 3 and 5 (2 mg each, individually) in 3% HCl (2 mL)
was heated for 4 h. Then, the solution was evaporated in vacuo and the
residues were subjected to TLC analysis. The neutralization solutions
were centrifuged, filtrated and furtherly isolated on a LC-2030 C
Prominence-I system (Shimadzu, Kyoto, Japan) equipped with a RID-20
A refractive index detector, using a Waters XBridge BEH Amide column
(10 × 250 mm, 5 μm). The flow rate was 3 mL/min, and the mobile
phase was acetonitrile-water (75:25). D-glucose and D-fructose were
confirmed by compared optical rotation with authentic samples: D-
3.3. Extraction and isolation
glucose, [
α
]25 D +52.5 (c 0.05, H2O); D-fructose, [α D
]
25 -67 (c 0.05, H2O)
(Zhao et al., 2019), and by comparing their TLC behavior with standard
A one kg dried sample of the root part of X. tenax was extracted with
5

X. Liu et al.
Phytochemistry 185 (2021) 112703
inhibition activity. Chem. Nat. Compd. 51, 1094–1098. https://doi.org/10.1007/
s10600-015-1500-8.
samples [Si gel, developed with CHCl3–MeOH–H2O (6:4:1)] (Shu et al.,
2002).
Choi, H., Moon, J.K., Park, B.S., Park, H.W., Park, S.Y., Kim, T.S., Kim, D.H., Ryu, T.H.,
Kweon, S.J., Kim, J.H., 2012. Comparative nutritional analysis for genetically
modified rice, Iksan483 and Milyang204, and nontransgenic counterparts. J. Korean
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of the UV light conversion of feruloyl amides from Portulaca oleracea and their
inhibitory effect on IL-6-induced STAT3 activation. Molecules 21. https://doi.org/
10.3390/molecules21070865.
3.3.6. LC-MS/MS analysis
A Q Exactive HF orbitrap mass spectrometer coupled with an Ulti-
mate3000 RSLC nano (Thermo Fisher Scientific, USA) was used for LC-
MS/MS analysis in both positive and negative ion modes. An ODS col-
umn (Pursuit XRs C18, 4.6 mm × 150 mm, Agilent, USA) was used for LC
separation. The mobile phase A was 100% acetonitrile with 0.1% formic
acid and the mobile phase B was water with 0.1% formic acid. The
separation was performed as follows, 70% (B) in 25 min and then up to
100 (B) from 25 min to 35 min. Mass resolutions of 120,000 and 30,000
were applied for precursors and fragments. Data dependent MS/MS
acquisition with fragmentation of the top five most intense precursors
was performed in the analysis. MS/MS spectra were acquired with
stepped normalized collision energy (NCE) of 20, 40 and 60. The other
detailed ion source parameters are as follows: spray voltage, 3.8 kV for
positive (and 3.0 kV for negative); capillary temperature, 250 ◦C; sheath
gas flow rate (arb), 45; aux gas flow rate (arb), 12; probe heater tem-
perature, 300 ◦C; mass range (m/z), 100–1500.
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Beargrass (Xerophyllum Tenax), p. 80. USFS PNW-GTR-864, October.
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3.3.7. Antimicrobial testing
Antimicrobial assays were carried out according to literature pro-
tocols (Li and Clark, 2020) against the following test microbes: Staphy-
lococcus epidermidis (ATCC 14990), Pseudomonas aeruginosa (ATCC
15692), Pseudomonas fluorescens (ATCC 13525) Bacillus subtilis (ATCC
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Rudall, P.J., Stobart, K.L., Hong, W.P., Conran, J.G., Furness, C.A., Kite, G.C., Chase, M.
W., 2000. Consider the lilies: systematics of Liliales. In: Wilson, K.L., Morrison, D.A.
(Eds.), Monocots – Systematics and Evolution. CSIRO Publishing, Melbourne,
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Acknowledgement
Satou, T., Mimaki, Y., Kuroda, M., Sashida, Y., Hatakeyama, Y., 1996. A pyrroline
glucoside ester and steroidal saponins from Lilium martagon. Phytochemistry 41,
1225–1230. https://doi.org/10.1016/0031-9422(95)00723-7.
This work was supported in part by the National Basic Research
Program of China (2015CD856500). The authors acknowledge the
contribution of Mr. Lowell Ahart for collecting the plant material used in
this study. The authors also acknowledge Yan Gao for assistance in
acquisition and analysis of the MS-MS data.
Shu, Y.L., Fuchino, H., Kawahara, N., Sekita, S., Satake, M., 2002. New phenolic
constituents from Smilax bracteata. J. Nat. Prod. 65, 262–266. https://doi.org/
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new phenylpropanoid glycosides from Smilax riparia. J. Asian Nat. Prod. Res. 14,
165–170. https://doi.org/10.1080/10286020.2011.641536.
Appendix A. Supplementary data
Seo, K.H., Lee, D.Y., In, S.J., Lee, D.G., Kang, H.C., Song, M.C., Baek, N.I., 2015.
Phenylethanoid glycosides from the fruits of Magnolia obovata. Chem. Nat. Compd.
51, 660–665. https://doi.org/10.1007/s10600-015-1379-4.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2021.112703.
Tanimoto, S., Tominaga, H., Okada, Y., Nomura, M., 2006. Synthesis and cosmetic
whitening effect of glycosides derived from several phenylpropanoids. Yakugaku
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