Hepatoprotective glycosides from the rhizomes of Imperata cylindrical

Article abstract of DOI:10.1080/10286020.2018.1471065

Three new C-methylated phenylpropanoid glycosides (1, 2), a new 8–4′-oxyneolignan (3), together with two known analogs (4, 5), were isolated from the rhizomes?of Imperata cylindrical Beauv. var. major (Nees) C. E. Hubb. Their structures were determined by spectroscopic and chemical methods. Compounds 1, 2, and 5 (10?μM) exhibited pronounced hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in vitro assays. Furthermore, their antioxidant activities against Fe²⁺-cysteine-induced rat liver microsomal lipid peroxidation and the effects on the secretion of?TNF-α?in murine peritoneal macrophages (RAW264.7) induced by lipopolysaccharides were evaluated.

Full text of DOI:10.1080/10286020.2018.1471065

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Hepatoprotective glycosides from the rhizomes of
Imperata cylindrical
Jie Ma, Hua Sun, Hui Liu, Gao-Na Shi, Ying-Da Zang, Chuang-Jun Li, Jing-Zhi
Yang, Fang-You Chen, Ji-Wu Huang, Dan Zhang & Dong-Ming Zhang
To cite this article: Jie Ma, Hua Sun, Hui Liu, Gao-Na Shi, Ying-Da Zang, Chuang-Jun Li, Jing-
Zhi Yang, Fang-You Chen, Ji-Wu Huang, Dan Zhang & Dong-Ming Zhang (2018) Hepatoprotective
glycosides from the rhizomes of Imperata cylindrical, Journal of Asian Natural Products Research,
20:5, 451-459, DOI: 10.1080/10286020.2018.1471065
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Journal of asian natural Products research, 2018
Vol. 20, no. 5, 451–459
https://doi.org/10.1080/10286020.2018.1471065
Hepatoprotective glycosides from the rhizomes of Imperata
cylindrical
Jie Ma#, Hua Sun#, Hui Liu, Gao-Na Shi, Ying-Da Zang, Chuang-Jun Li, Jing-Zhi Yang,
Fang-You Chen, Ji-Wu Huang, Dan Zhang and Dong-Ming Zhang
state Key laboratory of Bioactive substance and function of natural Medicines, institute of Materia Medica,
chinese academy of Medical sciences and Peking union Medical college, Beijing 100050, china
ABSTRACT
ARTICLE HISTORY
received 19 January 2018
accepted 24 april 2018
Three new C-methylated phenylpropanoid glycosides (1, 2), a
new 8–4′-oxyneolignan (3), together with two known analogs (4,
5), were isolated from the rhizomes of Imperata cylindrical Beauv.
var. major (Nees) C. E. Hubb. Their structures were determined
by spectroscopic and chemical methods. Compounds 1, 2, and 5
(10 μM) exhibited pronounced hepatoprotective activity against N-
acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in vitro
assays. Furthermore, their antioxidant activities against Fe2+-cysteine-
induced rat liver microsomal lipid peroxidation and the effects on
the secretion of TNF-α in murine peritoneal macrophages (RAW264.7)
induced by lipopolysaccharides were evaluated.
KEYWORDS
Graminea; Imperata
cylindrical; c-methylated
phenylpropanoid; lignan
glycoside; hepatoprotective
Imperata cylindrical
1. Introduction
Imperata cylindrical Beauv. var. major (Nees) C.E.Hubb., commonly known as cogongrass
or spear grass, is a perennial rhizomatous plant that can grow on soils with a vast range
of nutrients, moisture and pH [1]. It belongs to the genus Imperata, Graminea family. e
decoction of its rhizomes has the effects of clearing heat and cooling blood, dehumidification
CONTACT dong-Ming Zhang
zhangdm@imm.ac.cn
#these authors contributed equally to this work.
supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2018.1471065
© 2018 informa uK limited, trading as taylor & francis Group

452
J. MA ET AL.
Table 1. 1h and 13c nMr spectroscopic data for compounds 1 and 2 in dMso-d6 (δ in ppm, J in hz).
1
2
Position
δC
δH
δC
δH
1
156.1
101.6
159.8
116.9
138.5
108.6
88.3
162.0
169.5
23.5
57.2
100.2
73.5
76.9
70.0
156.0
102.0,
159.8
116.7
138.6
108.7
88.3
162.4
169.6
23.6
57.2
100.3
73.5
76.9
70.2
75.9
66.5
100.9
70.8
71.0
72.3
68.8
18.3
2
6.87 (d,2.5)
6.80 (d,2.5)
6.88 (d, 2.5)
6.81 (d, 2.5)
3
4
5
6
7
5.68 s
5.72 s
8
9
10
11
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
2.57 s
3.93 s
2.59 s
3.96 s
4.99 (d,7.5)
4.99 (d, 7.5)
3.25b
3.27b
3.27b
3.28–3.30 m
3.12–3.14 m
3.58b
3.16b
77.5
61.0
3.42b
3.45b, 3.70 (dd,10.5.0)
3.87 (dd,11.0,1.5); 3.39b
4.52 (br.s)
3.60
3.53
3.17
3.42
1.08 (d,6.5)
a1h nMr data (δh) were measured at 500 Mhz, 13c nMr data (δc) were measured at 125 Mhz.
boverlapping signals.
function of eliminating turbid, and was been widely used in traditional Chinese medicine
as a diuretic and hemostatic agent [2]. Previous phytochemical investigation revealed that
the presence of carbohydrates, triterpenes, chromones, flavonoids, lactones, and phenylpro-
panoids in the rhizomes of I. cylindrical, which show diversity in their biological activities
including hemostatic, immunomodulatory, anti-hypertension, anti-hepatitis, neuroprotec-
tive, antioxidant, anti-tumor, anti-inflammatory, and anti-complementary [3–8]. As part of
the investigation on bioactive compounds from Chinese medicinal plants, the phytochem-
icals in the 70% aqueous EtOH extract of I. cylindrical rhizomes were purified, and five
compounds were obtained including two new C-methylated phenylpropanoid glycosides
(1, 2), a new 8–4′-oxyneolignan (3) and two known 8–4′-oxyneolignans (4, 5) (Figure 1). In
addition, Compounds 1, 2, and 5 (10 μM) exhibited pronounced hepatoprotective activity
against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell damage in vitro assays. eir
anti-oxidant and anti-inflammatory potentials were herein discussed.
2. Results and discussion
Compound 1 was isolated as a white powder. It was assigned the molecular formula C17H20O9
based on an ion peak at m/z 369.1174 [M + H]+ in the HRESIMS, indicative of eight degrees
of unsaturation. e IR spectrum of 1 displayed the presence of OH (3383 cm−1) and conju-
gated carbonyl (1695 and 1078 cm−1), and aromatic functionalities (1612 cm−1). Its 1H NMR
spectrum in DMSO-d6 (Table 1) displayed resonances attributable to one singlet olefinic
methine at δH 5.68 (H-7), two meta-coupled aromatic protons at δH 6.87 (d, J = 2.5 Hz, H-2),

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
453
6.80 (d, J = 2.5 Hz, H-4), one methyl at δH 2.57 (s, H3-10), and a methoxy at δH 3.93 (s, H3-
11). A doublet assignable to an anomeric proton at δH 4.99 (d, J = 7.5 Hz, H-1′), partially
overlapped signals attributed to oxymethylene and oxymethine protons between δH 3.16
and 3.70, and exchangeable OH protons between δH 4.50 and 5.35 suggested that there was
a glycosyl moiety in 1. Extensive analysis of HSQC spectrum of 1 provided unambiguous
assignments of proton and carbon signals. In the HMBC spectrum of 1, long-range cor-
relations from H-10 to C-4, C-6, and from H-7 to C-5, C-9 confirmed the methyl (C-10)
was located at C-5. is was further confirmed by the NOE correlation of H3-10 with
H-4. HMBC correlations of C-8 and C-9 with H-7 proved that the ester carbonyl (C-9, δC
169.5) was connected to C-8. e HMBC correlation of C-9 with H3-11 indicated that the
methoxy group (C-11) was attached at C-9. Moreover, in combination with chemical shifs
and unsaturation degrees, C-9 and C-1 was connected through C-9–O–C-1 bond to form
the planar structure.
Enzymatic hydrolysis of 1 produced a sugar. e sugar gave a positive optical rotation
([α]+47.2) and the same Rf value as the authentic glucose by TLC analysis, indicating that
it was d-glucose [9]. e glucosyl unit in 1 was attached to the aglycone via a β-linkage
based on the coupling constant (7.5 Hz) of the anomeric proton (H-1′). e location of the
glucose moiety to the aglycone was deduced as C-3 based on the HMBC correlation of C-3
with the anomeric proton (H-1′) and the chemical shif of C-3. us, the structure of 1 was
determined to be a C-methyled benzo[b]furan derivative, named as 5-methyl coumarilic
acid methyl ester 3-O-β-d-glucopyranoside.
Compound 2 was obtained as an amorphous powder, and the molecular formula
C23H30O13 was indicated by HRESIMS at m/z 515.1755 [M + H]+. Comparison of the 1H
and 13C NMR data (Table 1) with those of 1, revealed that they share the same skeleton except
for an additional set of rhamnopyranosyl resonances with an anomeric proton signal at δH
4.52 (br.s), a typical methyl signal (C-6″: δC/H 18.3/1.08 (d, J = 6.5 Hz)) and downfield shif
of C-6′ (+ 5.5 ppm) of Glc′. e sugar units were confirmed to be d-glucose and l-rhamnose
in a ratio of 1:1 via GC analysis of their derivatives according to the procedure as previ-
3
ously reported [10]. Furthermore, their coupling constants (3JH-1′, H-2′ = 7.5 Hz and JH-1″,
H-2″ ≈ 0 Hz) of the anomeric protons of the sugar units demonstrated that the glucopyranosyl
unit has a β-configurated anomeric carbon and the rhamnopyranosyl unit was α-anomeric
configuration. e linkages of the sugars and the sugars with the aglycone were established
from the HMBC correlations from Rha-H-1″ (δH 4.52) to Glc-C-6′ (δC 66.5), and from Glc-
H-1′(δH 4.99) to C-3 (δC 159.8) (Figure 1). us, compound 2 was elucidated as 5-methyl
coumarilic acid methyl ester 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside.
Compound 3 was isolated as a gum. It had a molecular formula of C27H38O13, determined
by HRESIMS at m/z 569.2244 [M − H]−, inferring nine degrees of unsaturation. e IR
spectrum of 3 indicated the existence of OH (3346 cm−1) and aromatic (1601 and 1512 cm−1)
groups. e 1H NMR spectrum of 1 in DMSO-d6 showed signals attributed to a symmetric
1,3,4,5-tetrasubstituted and 1′,3′,4′-trisubstituted aromatic rings at δH 6.76 (H-2, 6), and 6.77
(H-2′), 6.96 (H-6′), and 6.64 (H-5′), as well as three aromatic methoxy groups resonated at
δH 3.70 (CH3 × 2), and 3.72 (Table 2). A propanoxy unit was assigned based on the chemical
shifs (δH/C: 2.50–2.52 m/31.7 (C-7′); δH/C: 1.66–1.68 m/34.9 (C-8′); δH/C: 3.65–3.67 m/60.6
(C-9′)), which was further confirmed by the HMBC correlations of H-9′/C-7′, H-7′/C-2′
and H-7′/C-6′. Similarly, a glyceryl moiety was indicated by signals of a vicinal spin system
attributed to two oxymethines at δH/C 4.96/76.3 (C-7) and δH/C 4.38/82.9 (C-8) and one

454
J. MA ET AL.
Figure 1. structures of compounds 1–5 from Imperata cylindrical.
Figure 2. Key 2d hMBc correlations of compounds 1 and 3.
oxymethylene at δH/C 3.16–3.18 m, 3.38–3.40 m/60.5 (C-9), which was also indicated by the
HMBC correlations from H-7 to C-9, and from H-2 to C-7. A long-range HMBC correlation of
H-8 with C-4′, in combination with the chemical shifs suggested that 3 was a 8–4′-oxyneolignan
derivative. e HMBC correlations between OMe-3′ and C-3′, and between OMe-3/5 to C-3/5
led to the determination of connecting positions of the methoxy groups. Furthermore, a doublet
sugar anomeric proton signal at δH 4.04 (d, J = 7.2 Hz) and partially overlapped signals attributed
to oxymethylene and oxymethine protons between δH 3.01–3.69 suggested that there was a β-glu-
cosyl moiety in structure of 3. In the 1H NMR spectrum, the coupling constant (J7,8 = 4.2 Hz) in
DMSO-d6 was not unambiguous to elucidate the threo- or erythro- configuration of the arylg-
lycerol with substituents at C-7 [11]. To determinate the absolute configurations of 3, hydrolysis
of 3 with snailase liberated 3a and d-glucose. e NMR spectra of 3a in MeOD showed con-
sistence with (7R,8R)-threo-4,7,9,9′-tetrahydroxy-3,5,2′-trimethoxy-8-O-4′-neolignan, isolated
from the roots of Alangium chinense (Lour.) Harms [12]. e CD spectra of 3 and 3a displayed
a negative Cotton effect at 242.5 and 247 nm, indicating that 3 possessed a 8R configuration
[13,14]. e HMBC correlation (Figure 2) from H-1″ to C-7 revealed that the glucopyranosyl
moiety was connected at C-7 of the aglycone. us, the structure of 3 was established to be
(–)-(7R,8R)-4,7,9,9′-tetrahydroxy-3,5,3′-trimethoxy-8-O-44,7,9,9′-tet7-O-β-d-glucopyranoside.
e known compounds were elucidated by detailed inspection of one- and two-dimen-
sional NMR spectral data and comparison of spectroscopic data with those reported in

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
455
Table 2. 1h and 13c nMr spectroscopic data for compound 3 (δ in ppm, J in hz).
a
a
b
b
Position
δC
δH
δH
δC
1
128.2
105.0
147.8
134.8
147.8
105.0
76.3
129.9
106.3
149.2
136.1
149.2
106.3
77.8
2
6.76 sc
6.81 s
3
4
5
6
6.76 sc
4.96 (d, 4.2)
4.38 (dt, 4.2,3.6)
3.16–3.18 m, 3.38–3.40 m
6.81 s
5.19 (d, 4.2)
7
8
82.9
60.5
4.35 (dt, 4.2,5.4)
85.9
61.9
9
3.56c, 3.70 (dd, 11.4,5.4)
1′
135.5
113.1
149.7
146.1
120.6?
116.0?
31.7
34.9
60.6
101.6
73.9
77.0
70.6
77.1
61.6
56.1 × 2
55.9
137.9
113.7
151.4
147.6
121.8
118.8
32.7
35.5
62.2
101.2
75.2
77.9
71.9
78.0
62.8
56.7 × 2
56.4
2′
6.77c
6.81c
3′
4′
5′
6.64 (dd, 8.4,1.8)
6.96 (d, 8.4)
2.50–2.52 m
1.66–1.68 m
3.65–3.67 m
4.04 (d, 7.2)
3.09c
6.66 (dd, 8.4,1.8)
6.79 (d, 8.4)
2.61 (t, 7.8)
1.79–1.81 m
3.55 (t, 6.0)c
4.20 (d, 7.8)
3.14–3.16 m
3.34c
6′
7′
8′
9′
1″
2″
3″
4″
5″
6″
–OCH3
–OCH3
3.07c
3.08c
3.27c
3.01c
3.34c
3.46c, 3.69c
3.70 s
3.82c, 3.84c
3.817 s
3.72 s
3.821 s
a1h nMr data (δh) were measured at 600 Mhz in dMso-d6, 13c nMr data (δc) were measured at 150 Mhz in dMso-d6.
b1h nMr data (δh) were measured at 600 Mhz in Meoh-d4, 13c nMr data (δc) were measured at 150 Mhz in Meoh-d4.
coverlapped signals.
the literature as (7R,8R)-4,7,9,9′-tetrahydroxy-3,3′-dimethoxy-8–4′-oxyneolignan-7-O-β-
d-glucopyranoside (4), (7R,8S)-4,7,9,9′-tetrahydroxy-3,3′-dimethoxy-8–4′-oxyneolignan-
7-O-β-d-glucopyranoside (5) [15].
Compounds 1–3, 3a, and 5 were bioassayed for their hepatoprotective activity
against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 (human hepatocellular
liver carcinoma cell line) cells in vitro, using the hepatoprotective activity drug bicyclol as the
positive control [16]. As shown in Table 3, compounds 1, 2, and 5 exhibited significant hepato-
protective activities. In addition, compounds 1–3, 3a, and 5 were assessed for the effects on
the secretion of TNF-α in murine peritoneal macrophages (RAW264.7) induced by lipopol-
ysaccharides [17] and their antioxidant activity in Fe2+-cystine-induced rat liver microsomal
lipid peroxidation [18], but they were inactive (IC50 > 10 μM is defined as “inactive”).
3. Experimental
3.1. General experimental procedures
Optical rotations were recorded on a JASCO P2000 automatic digital polarimeter (Tokyo,
Japan). e UV spectra were measured on a JASCO V-650 spectrophotometer (Tokyo,
Japan). CD spectra were recorded on a JASCO J-810 spectropolarimeter (Tokyo, Japan). e
IR data were implemented on a Nicolet 5700 spectrometer (ermo Electron, Madison, WI,
USA) using an FT-IR microscope transmission method. e NMR spectra were acquired
on Bruker AV500-III, Bruker AV III HD 600 (Bruker, Billerica, Massachusetts, USA), and

456
J. MA ET AL.
Varian INOVA VNS 600 spectrometers (Palo Alto, USA) in DMSO-d6, CD3OD with solvent
peaks used as references. HRESIMS data were obtained on a ermo Qexactive Focus ion
trap mass spectrometer (ermo Electron, Madison, WI, USA). Preparative HPLC was
performed on a Shimadzu LC-6AD instrument with an SPD-20A detector (Japan) and a
YMC Pack ODS-A column (2 × 25 mm, 5 μm). Silica gel (200–300 mesh, Qingdao Marine
Chemical Inc., Qingdao, China), macroporous resin HPD100 (Tianjin Haiguang Resin Inc.,
Beijing, China), macroporous resin PRP512A (Beijing Jufu Resin Inc., Beijing, China), MCI
(Mitsubishi Chemical Cleansui Co., Ltd., Japan), Sephadex LH-20 (Amersham Pharmacia
Biotech AB, Sweden), and ODS (50 μm, YMC, Japan) were used for column chromatography
(CC). TLC was performed using glass precoated silica gel GF254 plates (Qingdao Marine
Chemical Inc., China). Spots were visualized under UV light or by spraying with 10% H2SO4
in 95% EtOH followed by heating.
3.2. Plant material
e rhizomes of Imperata cylindrica were purchased from the Pharmaceutical Co., Ltd.,
Beijing City, China. is medicinal herb was identified by associate Prof. Lin Ma (the
Institute of Materia Medica, Beijing City, China). A voucher specimen (No. ID-S-2868)
has been deposited with the Institute of Materia Medica, Chinese Academy of Medical
Sciences, Beijing.
3.3. Extraction and isolation
e plant material (25 kg) consisting of the rhizomes of I. cylindrica was extracted with
70% aqueous EtOH (100 L) under reflux for 2 h and repeated three times. e extracted
solution was filtered and evaporated under reduced pressure to give a residue (5 kg). is
extract was suspended in H2O (20 L) and fractionated in a separating funnel with EtOAc
(20 L) twice. e EtOAc-soluble portion was concentrated to dryness in vacuo to give an
EtOAc fraction (300 g). Next, the remaining H2O layer was further passed over a HPD100
macroporous resin column (18 L) eluted with H2O (36 L), 15% EtOH (36 L), 35% EtOH
(54 L) and 95% EtOH (36 L) successively. e 35% EtOH-eluted fraction (B, 200 g) was
chromatographed on a PRP-512A resin column (3 L) using 10, 20, 30, and 40% EtOH
gradient elution to give relative fractions (BA, BB, BC, BD). BB (63 g) fraction was sub-
jected to a MCI column chromatography (eluted in MeOH/H2O with gradient) to give six
fractions (BBA–BBF). BBD fraction (10 g) was chromatographed by a Sephedax LH-20
column (eluted in 50% aqueous MeOH) to give eight subfractions (fr. BBD1-fr. BBD8). Fr.
BBD2 (1 g) was performed by silica gel (50 g, 3 cm × 30 cm) column chromatography using
CHCl3–MeOH–H2O (8:2:0.2, lower layer) to yield subfrations BBD1a-BBD1d. Fraction
BBD1b (0.4 g) was repeatedly subjected to reversed-phase preparative HPLC (YMC-ODS-A
5 μm, 2 × 25 cm, detection at 210 nm), using a mobile phase of MeOH:H2O (25:75, 7 ml/
min) to afford 1 (210 mg, tR = 60 min), 4 (10 mg, tR = 73 min), and 5 (7 mg, tR = 75 min).
Similarly, subfraction BBD1c (0.3 g) was chromatographed on semi-preparative HPLC
column with the solvent CH3CN:H2O (14:86, 3 ml/min, detection at 210 nm) to obtain
compounds 2 (25 mg, tR = 14.5 min) and 3 (7 mg, tR = 23 min).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
457
Table 3. hepatoprotective effects of compounds 1, 2, and 5 (10 μM) against aPaP-induced toxicity in
hepG2 cells.a
Compounds
Value of OD
Cell survival rate (% of normal)
control
normal(aPaP 8 mM)
Bicyclol
1
2
5
1.012 0.200
100.00
29.23
57.87
52.90
41.40
51.71
0.296 0.045***
0.586 0.013##
0.535 0.031##
0.419 0.031##
0.523 0.024##
aresults are expressed as means sd (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM)
***P < 0.001;
##P < 0.01.
3.3.1. 5-Methyl coumarilic acid methyl ester 3-O-β-d-glucopyranoside (1)
White amorphous powder; [α]20D −65.2 (c 0.12, MeOH:H2O = 1:1); UV (MeOH:H2O = 1:1)
λ
max (log ε): 207 (4.65), 220 (4.44), 289 (4.15), 304 (4.20), 315 (4.11) nm; IR (microscope)
ν
max 3383, 2916, 1695, 1612, 1384, 1266, 1078, 1046 cm−1. 1H NMR (DMSO-d6, 500 MHz)
and 13C NMR (DMSO-d6, 125 MHz) spectral data, see Table 1. HRESIMS: m/z 369.1174
[M + H]+ (calcd for C17H21O9, 369.1180).
3.3.2. 5-Methylcoumarilic acid methyl ester 3-O-α-l-rhamnopyranosyl (1→6)-β-d-
glucopyranoside (2)
White amorphous powder; [α]20D −36 (c 0.10, MeOH:H2O = 1:1); UV (MeOH:H2O = 1:1)
λ
max (log ε): 206 (4.30), 230 (4.08), 290 (3.76), 304 (3.80), 316 (3.70) nm; IR (microscope)
ν
max 3387, 2921, 1684, 1608, 1381, 1261, 1071 cm−1. 1H NMR (DMSO-d6, 500 MHz) and 13C
NMR (DMSO-d6, 125 MHz) spectral data, see Table 1. HRESIMS: m/z 515.1755 [M + H]+
(calcd for C23H31O13, 515.1759).
3.3.3. (–)-(7R,8R)-Threo-4,7,9,9′-tetrahydroxy-3,5,2′-trimethoxy-8-O-4′-neolignan
7-O-β-d-glucopyranoside (3)
Amorphous gum; [α]20D −63 (c 0.3, MeOH); UV (MeOH) λmax (log ε): 206 (4.32), 230 (3.76),
278 (3.26) nm; CD (MeOH) Δε207.5 nm −0.24, Δε222 nm + 0.47, Δε242.5 nm −0.28, Δε273 nm + 0.28;
IR (microscope) νmax 3346, 2918, 1601, 1512, 1024 cm−1. 1H NMR (DMSO-d6, 600 MHz)
and 13C NMR (DMSO-d6, 150 MHz) spectral data; 1H NMR (methanol-d4, 600 MHz) and
13C NMR (methanol-d4, 150 MHz) spectral data, see Table 2. HRESIMS: m/z 569.2244
[M − H]− (calcd for C27H37O13, 569.2240).
3.4. Enzymatic hydrolysis of compounds 1–3 and determination of absolute
configurations of monosaccharides
A solution of each compound in H2O (2 ml) was individually hydrolyzed with snailase
(6 mg, biodee code S0100, Beijing biodee biotechnology co., Ltd.) at 37 °C for 48 h. Each
reaction mixture was extracted with EtOAc (3 × 3 ml) to yield the individual EtOAc extract
and the H2O phase afer removing the solvents.
e residues of H2O phase were separately dissolved in MeOH, then filtered to obtain
the MeOH-soluble fractions which were respectively subjected to CC over silica gel eluted
with MeCN–H2O (9:1) to obtain the glucose from the hydrolysates of 1 and 3, and a sugar

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J. MA ET AL.
mixture from 2. e solvent system MeCN–H2O (6:1) was used for TLC identification of the
glucose from 1 and 3 (Rf, 0.33). e [α]20D values ranged from + 43.1 to + 47.2 (c 0.05–0.1,
H2O). e sugar mixture from 2 was prepared as trimethylsilyl l-cysteine derivatives and
the absolute configuration of the sugar moiety was conducted as described previously by
our group [10].
e EtOAc extract from the hydrolysate of 3 (2 mg) was chromatographed over silica
gel eluting with CH3CN–H2O (8:1) and then separated by semi-preparative HPLC column
(YMC C18 column (1 × 25 cm, 5 μm, Φ × L)) with the solvent MeOH–H2O (45׃55) to yield
its aglycone 3a (1 mg; 3.0 ml/min, 210 nm, tR = 35 min).
3a: White amorphous powder; 1H NMR (600 MHz, CD3OD) δ 6.74 (2H, s, H-2, 6), 4.88
(1H, d, J = 5.4 Hz, H-7), 4.25 (1H, dt, J = 10.0, 5.4 Hz, H-8), 3.48 (1H, dd, J = 5.4, 11.4 Hz,
H-9a), 3.74 (1H, overlap, J = 5.4, 11.4 Hz, H-9b), 6.86 (1H, d, J = 1.8 Hz, H-3′), 6.97 (1H,
d, J = 8.4 Hz, H-5′), 6.71 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 2.63 (2H, t, J = 7.8 Hz, H-7′), 1.82
(2H, m, H-8′), 3.57 (2H, m, H-9′), 3.86 (6H, s, 3,5-OMe), 3.86 (3H, s, 2′-OMe); 13C NMR
(150 MHz, CD3OD) δ 129.6 (C-1), 104.9 (C-2,6), 148.8 (C-3,5), 135.7 (C-4), 73.5 (C-7),
87.0 (C-8), 61.5 (C-9), 147.2 (C-1′), 151.2 (C-2′), 113.5 (C-3′), 137.7 (C-4′), 121.6 (C-5′),
118.9 (C-6′), 32.3 (C-7′), 35.2 (C-8′), 61.8 (C-9′), 56.3 (C-3,5-OMe), 56.1 (C-2′-OMe);
CD (MeOH) Δε215 nm + 0.46, Δε237 nm −0.57; HRESIMS: m/z 407.1711 [M − H]− (calcd for
C21H27O8, 407.1713).
Acknowledgements
We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, for measuring the IR, UV, NMR,
MS, and CD spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is project was financially supported by the National Natural Science Foundation of China [grant
number NNSFC 81630094] and CAMS Initiative for Innovative Medicine Project [grant number
CAMS-I2M-1-010].
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