C-Methylated flavanones from the rhizomes of Matteuccia intermedia and their α-glucosidase inhibitory activity

Article abstract of DOI:10.1016/j.fitote.2019.04.002

One new flavanonol, demethylmatteucinol (1), and nine new flavanone glucoside derivatives, matteflavosides H-J (2–4) and matteuinterates A-F (5–10), were isolated from the rhizomes of Matteuccia intermedia C.Chr., along with 21 known flavanones (11?31). N

Full text of DOI:10.1016/j.fitote.2019.04.002

Fitoterapia136(2019)104147
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
C-Methylated flavanones from the rhizomes of Matteuccia intermedia and their
α-glucosidase inhibitory activity
Xue Lia, Ling-Juan Zhua, Jin-Peng Chena, Chen-Yu Shia, Li-Ting Niua, Xue Zhanga,⁎
,
⁎
Xin-Sheng Yaoa,b,
a School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China
b Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
One new flavanonol, demethylmatteucinol (1), and nine new flavanone glucoside derivatives, matteflavosides H-
J (2–4) and matteuinterates A-F (5–10), were isolated from the rhizomes of Matteuccia intermedia C.Chr., along
with 21 known flavanones (11−31). Notably, all of them contain C-methylation in the A-ring. The structures of
the compounds were elucidated by spectroscopic methods and chemical derivatization. The α-glycosidase in-
hibition assay indicated that compounds 12–17 showed potent inhibitory activity with IC50 values of
12.4–69.7 μM, which suggested their hypoglycemic effect.
Matteuccia intermedia
C-methylated flavanones
α-glycosidase inhibitory activity
1. Introduction
Accumulating evidence suggested that farrerol exerts multiple biolo-
gical activities, including antibechic [10], anti-inflammatory [11], an-
The genus Matteuccia (Onocleaceae) includes about 5 species in-
habitated mostly in north temperate regions. In China, there are 3
species named as Matteuccia struthiopteris, M. orientalis and M. inter-
media [1]. A series of flavonoids, isocoumarins, phthalides, lignans, and
stilbenes have been previously identified from the genus Matteuccia, in
which flavanones with C-methylation at C-6 and/or C-8 are the main
members [2–8]. The methyl groups of the C-methylated flavanone is
derived from a chain extension with methylmalonyl-CoA in the second
and third condensation of the biosynthetic reaction sequence [9]. Up to
now, C-methylated flavanones and their glycosides with 2′‑oxygenated,
4′‑oxygenated, 2′,4′-dioxygenated, 2′,5′-dioxygenated, 3′,4′-dioxyge-
nated, 2′,3′,4′-trioxygenated patterns in B ring have been reported from
the genus Matteuccia [2,3,6,7]. Several C-methylated flavanones gly-
cosides were further derivatized with a characteristic HMG group at C-
3, C-4, or C-6 of the sugar moiety [2,3,6]. These diverse structures have
resulted in wide range of pharmacological activities. In our former
study on M. struthiopteris, a C-methylated flavanone as a potential an-
tiviral agent against influenza A (H1N1) was obtained [7]. And a
number of C-methylated flavanones from M. orientalis were reported to
possess hypoglycemic activity in streptozotocin (STZ)-induced diabetic
rats and anti-influenza virus (H1N1) effect [2,3,6]. Farrerol, a typical C-
methylated flavanone, has been isolated from M. orientalis.
tibacterial [12] and antioxidant activities [13], and a potential to treat
and prevent cardiovascular diseases [14]. Desmethoxymatteucinol,
obtained from M. orientalis, was found to exhibit moderate anti-in-
flammatory and cytotoxic activities [15]. In the course of our continual
research for bioactive constituents from Matteuccia genus, one new
flavanonol, demethylmatteucinol (1), and nine new flavanone gluco-
side derivatives, matteflavosides H-J (2–4) and matteuinterates A-F
(5–10), along with 21 known C-methylated flavanones (11–31) were
isolated from the 60% EtOH extract of the rhizomes of M. intermedia.
The structural elucidation of the new compounds and α-glucosidase
inhibitory activity of 1–9, 11–31 are described herein.
2. Experimental
2.1. General experimental
UV spectra were recorded by a UV-2201 UV-VIS recording spec-
trophotometer (Shimadzu, Kyoto, Japan). IR spectra were acquired on a
Bruker IFS-55 spectrometer (Bruker, Rheinstetten, Germany) with KBr
disks. Optical rotations were measured with an Anton Paar MCP 200
polarimeter (l = 1 cm) (Anton Paar GMBH, Graz, Austria). CD spectra
were given by MOS 450 spectrometer (Bio-Logic Science, Grenoble,
⁎ Corresponding authors at: School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China.
E-mail addresses: syzxalice@163.com (X. Zhang), tyaoxs@jnu.edu.cn (X.-S. Yao).
https://doi.org/10.1016/j.fitote.2019.04.002
Received 9 March 2019; Received in revised form 12 April 2019; Accepted 13 April 2019
Availableonline22April2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

X. Li, et al.
Fitoterapia136(2019)104147
France). NMR spectra were recorded using an AVANCE-600 NMR
spectrometers (Bruker, Rheinstetten, Germany). HRESIMS were mea-
sured on a Waters Synapt G2 QTOF spectrometer (Waters, Milford,
Massachusetts, USA). The analytical High Performance Liquid
Chromatography (HPLC) was collected on an Agilent 1200 (Agilent
Technologies, Santa Clara, California, USA) equipped with a DAD de-
tector and a reversed-phase (RP) C18 column (5 μm, 4.60 × 250 mm;
Phenomenex Luna, CA, USA). Semipreparative HPLC was performed
applying a Shimadzu LC-6 CE with a UV SPD-20A detector, using a RP-
C18 column (5 μm, 10 × 250 mm; Phenomenex Luna, CA, USA).
Macroporous resin (Diaion HP20, Mitsubishi Chemical Corporation,
Tokyo, Japan), silica gel (200–300 mesh, Qingdao Haiyang Chemical,
Qingdao, China), ODS (60–80 μm, YMC, Tokyo, Japan), and Sephadex
LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden) were used for
column chromatography (CC).
gel CC and p-HPLC (MeOH-0.1% formic acid, 45:55, monitored at
280 nm). Fr. D95 (eluted with 50% MeOH) was further separated into 9
subfractions (D951–959) using silica gel CC eluted with CH2Cl2/MeOH/
H2O (93:7:0 to 0:100:0). Subfractions D952 and D955 were subjected to
p-HPLC (MeOH-0.1% formic acid, 50:50, monitored at 280 nm and
monitored with RID, respectively) to yield 19 (1.1 mg, tR 62.5 min) and
21 (1.1 mg, tR 67.5 min), and 4 (2.1 mg, tR 21.5 min) and 24 (5.4 mg, tR
74.5 min), respectively. Purification of subfraction D957 by p-HPLC
(MeOH-0.1% formic acid, 45:55, monitored at 220 nm) gave 25
(30.0 mg, tR 80.0 min), 28 (8.8 mg, tR 85.5 min) and 29 (5.9 mg, tR
90.0 min). Fr. D96 (eluted with 50% MeOH) was purified by silica gel
CC and p-HPLC (MeOH-0.1% formic acid, 55:45, monitored at 280 nm)
to obtain 3 (7.2 mg, tR 35.0 min) and 22 (11.2 mg, tR 28.8 min).
Compound 26 (63.0 mg) was crystallized in MeOH from Fr. D98 (eluted
with 60% MeOH). Fr. D10 was subjected to an ODS column eluted with
a gradient of MeOH/H2O. The 40% MeOH eluate was loaded on a
Sephadex LH-20 column, eluted with CH2Cl2/MeOH (1:1), and purified
by p-HPLC (MeOH-0.1% formic acid, 45:55, monitored at 280 nm) to
give 6 (11.2 mg, tR 25.0 min), 20 (9.2 mg, tR 22.1 min), 5 (8.2 mg, tR
28.8 min), 2 (6.2 mg, tR 40.4 min). Purification of the 50% MeOH eluate
by Sephadex LH-20 CC and p-HPLC (MeOH-0.1% formic acid, 45:55,
monitored at 220 nm) to obtain 30 (22.7 mg, tR 103.8 min) and 31
(23.2 mg, tR 105.0 min).
2.2. Plant materials
The rhizomes of M. intermedia were collected from Hongyuan
County, Aba Tibetan and Qiang Autonomous Prefecture (Sichuan,
China) in November 2013, which were identified by Prof. Hao Zhang
(West China School of Pharmacy, Sichuan University, China). A vou-
cher specimen (YLXMI-2013) was deposited at the School of Traditional
Chinese Materia Medica, Shenyang Pharmaceutical University, China.
2.3.1. Demethylmatteucinol (1)
2.3. Extraction and isolation
20
Light yellow amorphous powder; [α]D −8.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 294 (3.93) nm; CD (c 1.58 × 10−3 M, MeOH) λmax
(△ε) 217 (+19.21), 239 (+9.04), 257 (+4.98), 293 (−26.27), 330
(+3.31) nm; IR (KBr) νmax 3408, 2925, 2853, 1630, 1515, 1462, 1422,
The dried rhizomes of M. intermedia (4.2 kg) were extracted with
60% EtOH (2 × 42 L, 2 h each). The dried extract (619.7 g) was sus-
pended in H2O (3 L) and subjected to a Diaion HP20 macroporous ad-
sorptive resins column by gradient elution with EtOH/H2O (0:100 to
95:5). The 50% EtOH eluate (36.5 g) was then separated over a silica
gel column with a gradient system of CH2Cl2/MeOH (100:0 to 0:100) to
afford 8 fractions (Fr. C1-C8). Fr. C5 was subjected to a Sephadex LH-20
column (CH2Cl2/MeOH, 1:1), followed by an ODS column eluted with
MeOH/H2O. The eluate of 40% MeOH was further purified by pre-
parative HPLC (p-HPLC) (MeOH-0.1% formic acid, 50:50, monitored at
280 nm) to afford 8 (7.6 mg, tR 28.8 min). Fr. C6 was divided into 7
fractions (Fr. C61-C67) by an ODS column with a system of MeOH/H2O
(30:70 to 100:0). Fr. C62 (eluted with 40% MeOH) and Fr. C65 (eluted
with 50% MeOH) were applied to a Sephadex LH-20 column (CH2Cl2/
MeOH, 1:1) followed by p-HPLC (MeOH-0.1% formic acid, 45:55 and
50:50, respectively, monitored with RID) to afford 10 (2.2 mg, tR
30.5 min) and 9 (2.8 mg, tR 40.1 min), respectively.
The 95% EtOH eluate (100.8 g) was loaded on a silica gel column
eluted with CH2Cl2/MeOH (100:0 to 0:100) to afford 11 fractions (Fr.
D1-D11). Compound 11 (120.0 mg) was crystallized in MeOH from Fr.
D1. Fr. D3 was fractionated by an ODS column eluted with MeOH/H2O
(30:70 to 100:0) to afford 8 fractions (D31-D38). Fr. D34 (eluted with
60% MeOH) was further separated into 6 subfractions (D341–346) with
CH2Cl2/MeOH (100:0 to 0:100). Purification of subfraction D341 by p-
HPLC (55% MeOH, monitored at 280 nm) gave 14 (5.0 mg, tR
63.7 min). Subfractions D342 and D343 were subjected to p-HPLC (62%
MeOH, monitored at 220 nm) to yield 1 (5.0 mg, tR 22.0 min), 12
(25.0 mg, tR 25.0 min), 15 (284.3 mg, tR 31.8 min), and 13 (58.8 mg, tR
40.3 min), 16 (12.8 mg, tR 26.3 min), 18 (10.0 mg, tR 45.0 min), re-
spectively. Fr. D6 and Fr. D8 were passed over a Sephadex LH-20
column eluted with CH2Cl2/MeOH (1:1), and a successive ODS column
eluted with MeOH/H2O, respectively. The 60% MeOH eluate of Fr. D6
and 40% MeOH eluate of Fr. D8 were purified by p-HPLC to yield 17
(11.3 mg, tR 16.5 min) (55% MeOH, monitored at 220 nm) and 23
(4.5 mg, tR 13.7 min) (MeCN-0.1% formic acid, 60:40, monitored at
280 nm), respectively. Fr. D9 was run on an ODS column with a gra-
dient of MeOH/H2O (20:80 to 100:0) to afford 10 fractions (D91-D910).
Compounds 7 (6.4 mg, tR 44.3 min) and 27 (105.8 mg, tR 40.5 min)
were afforded from the eluent of 40% MeOH (Fr. D94) purified by silica
1384, 1250, 1179, 1114, 1030, 833 cm−1
;
1H and 13C NMR data, see
+
Table 1; HRESIMS m/z 317.1023 [M + H]
317.1025).
(calcd. for C17H17O6,
2.3.2. Matteflavoside H (2)
20
Yellow amorphous powder; [α]D −38.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 282 (3.99) nm; CD (c 1.02 × 10−3 M, MeOH) λmax
(△ε) 213 (+12.35), 233 (−3.02), 250 (+0.36), 287 (−9.03), 356
(+1.91) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z
+
493.1705 [M + H] (calcd. for C24H29O11, 493.1705).
2.3.3. Matteflavoside I (3)
20
Yellow amorphous powder; [α]D −28.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 285 (4.02) nm; CD (c 1.02 × 10−3 M, MeOH) λmax
(△ε) 211 (+26.72), 219 (−25.01), 251 (+0.66), 291 (+13.36), 319
(+1.60) nm; IR (KBr) νmax 3386, 2920, 2851, 1630, 1522, 1431, 1384,
1280, 1166, 1125, 1068, 1038, 896, 832 cm−1 1H and 13C NMR data,
;
-
see Table 1; HRESIMS m/z 491.1549 [M - H] (calcd. for C24H27O11
491.1553).
,
2.3.4. Matteflavoside J (4)
20
Yellow amorphous powder; [α]D −54.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 283 (4.02) nm; CD (c 1.08 × 10−3 M, MeOH) λmax
(△ε) 219 (+30.97), 235 (−1.93), 249 (+3.35), 277 (−18.75), 350
(+4.64) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z
+
463.1617 [M + H] (calcd. for C23H27O10, 463.1604).
2.3.5. Matteuinterate A (5)
20
Light yellow amorphous powder; [α]D -32.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 282 (4.35) nm; CD (c 0.77 × 10−3 M, MeOH) λmax
(△ε) 218 (+12.12), 235 (−3.17), 250 (+1.01), 289 (−15.97), 354
(+4.26) nm; IR (KBr) νmax 3424, 2924, 1721, 1631, 1514, 1443, 1402,
1384, 1275, 1176, 1126, 1071, 1024, 889, 833 cm−1
;
1H and 13C NMR
+
data, see Table 2; HRESIMS m/z 637.2126 [M + H]
30H37O15, 637.2132).
(calcd. for
C
2

X. Li, et al.
Fitoterapia136(2019)104147
Table 1
1H and 13C NMR data of compounds 1–4 (1H 600 MHz, 13C 150 MHz).
No.
1a
2b
3b
4b
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
2
3
4
85.4 CH
74.6 CH
199.5C
5.03 d (11.3)
4.54 d (11.3)
78.8 CH
43.2 CH2
199.4C
5.52 dd (12.3, 3.0)
3.30 m, 2.86 dd (17.0, 3.0)
74.2 CH
42.0 CH2
199.4C
5.65 dd (12.9, 2.9)
3.30 m, 2.75 dd (17.1, 2.9)
74.8 CH
42.3 CH2
199.5C
5.76 dd (12.8, 3.0)
3.28 m, 2.88 dd (17.0, 3.0)
5
163.7C
158.7C
158.3C
158.8C
6
7
97.5 CH
167.7C
6.00 s
112.1C
162.3C
111.6C
161.9C
112.0C
162.3C
8
105.7C
111.0C
110.5C
111.1C
9
162.1C
158.1C
158.2C
158.5C
10
102.6C
105.7C
105.2C
105.6C
1′
131.7C
132.2C
117.9C
125.9C
2′
3′
4′
5′
131.0 CH
115.7 CH
162.5C
115.7 CH
131.0 CH
7.51 d (8.6)
7.01 d (8.6)
114.8 CH
147.4C
148.7C
113.0 CH
118.3 CH
9.6 CH3
10.2 CH3
56.6 CH3
6.99 d (2.3)
156.3C
105.2 CH
160.8C
101.8 CH
128.4 CH
9.2 CH3
9.7 CH3
55.5 CH3
155.2C
6.48 m
116.4 CH
130.2 CH
120.1 CH
127.6 CH
9.6 CH3
10.2 CH3
6.93 d (7.9)
7.24 td (7.9, 1.5)
6.92 t (7.9)
7.51 dd (7.9, 1.5)
2.14 s
7.01 d (8.6)
7.51 d (8.6)
6.98 d (8.3)
6.92 dd (8.3, 2.3)
2.12 s
2.11 s
3.81 s
6.46 m
7.36 d (8.3)
2.10 s
2.06 s
3.73 s
6′
6-CH3
8-CH3
4′-OCH3
5-OH
3′-OH
2′-OH
1′′
2′′
3′′
4′′
5′′
8.3 CH3
56.6 CH3
1.93 s
3.86 s
2.12 s
12.15 s
9.15 s
12.13 s
12.14 s
9.88 s
10.03 s
4.61 d (7.8)
3.29 m
3.23 m
3.14 m
105.1 CH
75.0 CH
77.3 CH
70.8 CH
77.9 CH
62.0 CH2
4.62 d (7.7)
3.33 m
3.26 m
3.17 m
3.09 m
104.7 CH
74.6 CH
76.8 CH
70.3 CH
77.5 CH
61.5 CH2
105.1 CH
75.0 CH
77.3 CH
70.8 CH
77.9 CH
62.0 CH2
4.64 d (7.4)
3.33 m
3.26 m
3.17 m
3.10 m
3.06 m
3.61 t (10.0), 3.42 m
6′′
3.66 m, 3.45 m
3.66 m, 3.46 m
a
Data measured in CD3OD.
Data measured in DMSO‑d6.
b
2.3.6. Matteuinterate B (6)
2.3.10. Matteuinterate F (10)
20
Yellow amorphous powder; [α]D −30.0 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 284 (4.26) nm; CD (c 0.83 × 10−3 M, MeOH) λmax
(△ε) 219 (+23.11), 233 (−3.04), 251 (+1.74), 288 (−12.25), 353
(+3.14) nm; IR (KBr) νmax 3361, 2923, 1728, 1633, 1519, 1444, 1381,
Brown amorphous powder; [α] −108.0 (c 0.1, MeOH); UV (MeOH)
λmax (log ε) 282 (4.09), 359 (3.56) nm; CD (c 0.63 × 10−3 M, MeOH)
λmax (△ε) 217 (+23.18), 288 (−19.77), 359 (+2.90) nm; IR (KBr)
νmax 3411, 2949, 2835, 1631, 1428, 1272, 1172, 1125, 1031, 1008,
1347, 1278, 1193, 1170, 1124, 1088, 1055, 1005, 824 cm−1
;
1H and
881, 832 cm−1
799.2668 [M + H] (calcd. for C36H47O20, 799.2661).
;
1H and 13C NMR data, see Table 3; HRESIMS m/z
13C NMR data, see Table 2; HRESIMS m/z 607.2035 [M + H] + (calcd.
+
for C29H35O14, 607.2027).
2.4. Biological assays
2.3.7. Matteuinterate C (7)
20
Yellow amorphous powder; [α]D −38.0 (c 0.05, MeOH); UV
2.4.1. α-Glycosidase inhibition assay
(MeOH) λmax (log ε) 287 (4.20) nm; CD (c 0.77 × 10−3 M, MeOH) λmax
(△ε) 218 (+17.06), 232 (−11.41), 247 (−0.69), 281 (−19.82), 305
(+0.77), 319 (−1.85), 352 (+3.31) nm; IR (KBr) νmax 3423, 2923,
1631, 1510, 1402, 1384, 1273, 1206, 1174, 1127, 1073, 1040,
The assay was conducted in 96-well microtiter plates referring to
the procedures as previously described [16]. Briefly, test compounds (in
DMSO) of different concentrations, the enzyme (0.5 U/mL, from Sac-
charomyces cerevisiae, Sigma, Germany), and p-nitrophenyl-α-D-gluco-
pyranoside (p-NPG) (5.0 mM, Sigma, Switzerland) were dissolved by
67.0 mM sodium phosphate buffer (pH 6.8), respectively. Four kinds of
wells were made. Test wells contained 112 μL of buffer, 20 μL of en-
zyme, and 8 μL of sample. Test blank wells contained 132 μL of buffer
and 8 μL of sample. Negative control wells contained 112 μL of buffer,
20 μL of enzyme, and 8 μL of DMSO. Negative blank wells contained
132 μL of buffer and 8 μL of DMSO. The plates were preincubated for
5 min at 37 °C. Then the reaction was initiated by the addition of 20 μL
of p-NPG and incubated for 30 min at 37 °C. The absorbance of released
product (p-nitro phenol) by measured at 405 nm. The α-glucosidase
832 cm−1
;
1H and 13C NMR data, see Table 2; HRESIMS m/z 635.1977
-
[M - H] (calcd. for C30H35O15, 635.1976).
2.3.8. Matteuinterate D (8)
20
Light yellow amorphous powder; [α]D −75.0 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 281 (3.86), 360.0 (3.43) nm; CD (c
0.81 × 10−3 M, MeOH) λmax (△ε) 214 (+7.72), 228 (−0.65), 279
(+2.99), 321 (−1.17), 372 (+1.84) nm; 1H and 13C NMR data, see
+
Table 3; HRESIMS m/z 621.2183 [M + H]
621.2183).
(calcd. for C30H37O14,
inhibition rate (%) = [1- (ODtest
-
ODtest blank)/(ODnegative
-
blank
ODblank)] × 100%. All tests were performed in triplicate, with acarbose
used as a positive control.
2.3.9. Matteuinterate E (9)
20
Yellow amorphous powder; [α]D −98.0 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 285 (4.03), 360.0 (3.48) nm; CD (c
0.77 × 10−3 M, MeOH) λmax (△ε) 218 (+13.26), 234 (−6.33), 250
(+2.08), 284 (−8.03), 303 (0.31), 320 (−1.88), 363 (+3.88) nm; IR
(KBr) νmax 3397, 2947, 1631, 1432, 1276, 1206, 1175, 1126, 1029,
2.4.2. Molecular docking
Molecular docking was performed to predict the binding mode be-
tween the α-glucosidase protein and flavanones in the Discovery Studio
3.5 software. The crystal structure of Gal1p (PDB Code: 2AJ4) was
obtained from the Protein Data Bank [17] and then minimized using the
832 cm−1 1H and 13C NMR data, see Table 3; HRESIMS m/z 651.2283
;
+
[M + H] (calcd. for C31H39O15, 651.2289).
3

X. Li, et al.
Fitoterapia136(2019)104147
Table 2
1H and 13C NMR data of compounds 5–7 (1H 600 MHz, 13C 150 MHz, DMSO‑d6).
No.
5
6
7
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
2
3
4
79.0 CH
43.5 CH2
199.4C
5.49 dd (12.4, 2.6)
3.25 m, 2.83 dd (16.7, 2.6)
79.0 CH
43.2 CH2
199.5C
5.52 dd (12.7, 2.7)
3.32 m, 2.84 dd (17.3, 2.7)
74.7 CH
42.2 CH2
199.5C
5.74 dd (13.0, 2.9)
3.30 m, 2.86 dd (17.0, 2.9)
5
158.8C
158.8C
158.8C
6
111.9C
112.0C
112.2C
7
162.0C
162.0C
162.0C
8
111.1C
111.0C
111.0C
9
158.2C
158.3C
158.4C
10
105.7C
105.8C
105.7C
1′
132.3C
130.0C
126.6C
2′
3′
4′
5′
114.8 CH
147.8C
148.8C
113.0 CH
118.1 CH
9.5 CH3
10.1 CH3
56.6 CH3
7.05 brs
129.0 CH
116.2 CH
158.6C
116.2 CH
129.0 CH
9.5 CH3
10.1 CH3
7.37 d (8.7)
6.85 d (8.7)
148.8C
117.1 CH
115.1 CH
153.1C
113.2 CH
9.6 CH3
10.1 CH3
6.85 m
6.84 m
6.96 d (8.3)
6.89 dd (8.3, 1.3)
2.09 s
2.06 s
3.81 s
6.85 d (8.7)
7.37 d (8.7)
2.09 s
6′
7.08 d (2.5)
2.14 s
2.13 s
6-CH3
8-CH3
4′-OCH3
4′-OH
5-OH
2′-OH
5′-OCH3
1′′
2′′
3′′
4′′
5′′
2.05 s
9.64 s
12.15 s
12.13 s
12.14 s
9.40 s
3.74 s
4.79 d (7.7)
3.52 m
4.90 t (9.9)
3.38 m
3.25 m
56.3 CH3
104.6 CH
72.9 CH
78.2 CH
68.6 CH
77.5 CH
61.6 CH2
171.0C
104.9 CH
74.8 CH
76.9 CH
70.8 CH
74.5 CH
64.0 CH2
171.2C
4.67 d (7.4)
3.35 m
3.30 m
3.22 m
3.34 m
104.9 CH
74.8 CH
76.9 CH
70.7 CH
74.5 CH
63.9 CH2
171.2C
4.66 d (7.6)
3.34 m
3.28 m
3.24 m
3.35 m
6′′
1′′′
4.23 d (9.9), 4.07 m
4.25 d (10.3), 4.07 m
3.66 m, 3.48 m
2′′′
46.8 CH2
2.56 overlapped, 2.47 d (13.6)
46.5 CH2
2.63 d (14.4),
2.49 d (14.4)
47.3 CH2
2.72 d (13.6),
2.58 d (13.6)
3′′′
4′′′
5′′′
6′′′
69.8C
46.8 CH2
173.8C
69.8C
46.4 CH2
173.5C
70.2C
46.3 CH2
174.0C
2.40 s
1.19 s
2.44 m
1.21 s
2.47 m
1.33 s
28.1 CH3
28.1 CH3
28.5 CH3
CharMM27 force field and the MMFF94 charge with a distance-de-
pendent dielectric and conjugate gradient method. The optimized
structures were used for all subsequent calculations. The Libdock
module embedded in Discovery Studio was used to dock the compounds
to the binding site of α-glucosidase, which was defined by the location
of AMPPNP. The default parameters were used in the docking process.
peaks of the standard D-glucose and L-glucose derivatives were recorded
at tR 20.9 (L-Glc) and 23.6 (D-Glc) min. The glucosidic derivatives of
5–10 were identified as D-glucose based on the tR = 22.8 min [19].
2.6. Determination of the absolute configuration of HMG in compounds
5–8, and 10
2.5. Acid hydrolysis and determination of the absolute configuration of the
monosaccharides of compounds 2–10
To establish the absolute configuration of the HMG group, com-
pounds 5–8 (5 mg each) and 10 (1.5 mg) were dissolved by 0.3 mL DMF
containing (S)-1-phenylethylamine (Aladdin, USA) (2 equiv), Et3N
(Aladdin, USA) (3 equiv), PyBOP (Aladdin, USA) (1.5 equiv), and HOBt
(Aladdin, USA) (2 equiv) under ice-cooling, respectively. The reaction
was quenched with diluted HCl after stirring for 9 h at room tempera-
ture, and the residue was dried in vacuo. Amide A was obtained by
separation of the residue using a Waters Sep-Pak®Vac normal-phase
column (CH2Cl2/MeOH, 30:1, 20:1, 15:1 and 10:1) and was identified
by LC-MS analysis. The THF (0.3 mL) solution containing amide A was
added to LiBH4 (15 equiv. of A) under ice cooling which was stirred for
24 h at 25 °C and quenched with dilute HCl. The resulting mixture was
extracted with EtOAc and concentrated. Purification of the extract was
achieved by a Waters Sep-Pak®Vac normal-phase column (CHCl3/
MeOH, 40:1, 30:1, 20:1 and 15:1) to furnish B. Then B was acetylated
by Ac2O (5 equiv. of A) in pyridine (30 μL) and stirred for 24 h at 25 °C.
The reaction mixture was diluted with H2O and extracted with EtOAc.
Further purification of the product was conducted by analytical HPLC
with 75% MeOH (monitored at 220 nm) under isocratic condition at
0.8 mL/min to obtain C. The 1H NMR spectrum (Fig. S98, Supporting
Information) of C was found to be consistent with that of (3R)-5-O-
acetyl-1-[(S)-phenylethyl]mevalonamide upon comparison, rather than
Glucosyl moiety identification in the structures was carried out
according to the methods of literatures [18,19]. Compound (2−10)
(1.0 mg each) was hydrolyzed with 2 M HCl for 2 h at 90 °C. After
evaporating under vacuum, the residue was dissolved in H2O and ex-
tracted with CHCl3, then the aqueous layer was dried in vacuo. The
residue of 2–4 was dissolved in distilled water (1.0 mL), and analyzed
by LC-NetII/ADC HPLC with OR-4090 optical rotation detector
equipped with a Shodex Asahipak NH2P-50 4E column (1.0 mL/min,
CH3CN-H2O, 3:1, 20 μL), respectively. The sugar moiety was identified
to be D-glucose by comparison of the retention time and orientation
with those of authentic samples at (tR 9.0 min, positive peak) [18]. The
residue of 5–10 was dissolved in 0.5 mL pyridine containing L-cysteine
methyl ester (3.0 mg, Sigma, USA) and heated at 60 °C for 1 h. Then, o-
tolyl isothiocyanate (5 μL) (Alfa Aesar, U.K.) was added and directly
analyzed by HPLC after heating for 1 h at 60 °C. Analytical HPLC was
acquired on an RP-C18 column (5 μm, 4.60 × 250 mm; Phenomenex
Luna) at 35 °C with isocratic elution (CH3CN-0.1% formic acid, 25:75,
0.8 mL/min, monitored at 250 nm). The authentic glucoses, D-glucose
and L-glucose (Sigma, USA), were subjected to the same process. The
4

X. Li, et al.
Fitoterapia136(2019)104147
Table 3
1H and 13C NMR data of compounds 8–10 (1H 600 MHz, 13C 150 MHz, DMSO‑d6).
No.
8
9
10
δC, type
δH, (J in Hz)
δC, type
δ
H, (J in Hz)
δC, type
δH, (J in Hz)
2
3
4
79.1 CH
43.2 CH2
199.6C
5.52 dd (12.6, 3.2)
3.34 m, 2.84 dd (17.0, 3.2)
74.8 CH
42.3 CH2
199.4C
5.72 dd (13.1, 2.7)
3.25 m, 2.87 dd (17.0, 2.7)
74.4 CH
43.4 CH2
199.6C
5.93 dd (13.3, 2.6)
3.11 dd (16.9, 13.3), 2.77 dd (16.9, 2.6)
5
158.9C
158.8C
158.8C
6
112.0C
112.1C
112.1C
7
162.0C
162.0C
162.0C
8
111.0C
110.9C
111.0C
9
158.3C
158.4C
158.6C
10
105.8C
105.7C
105.8C
1′
130.0C
126.6C
121.5C
2′
3′
4′
5′
129.0 CH
116.1 CH
158.6C
116.1 CH
129.0 CH
9.5 CH3
10.1 CH3
7.37 d (8.5)
6.85 d (8.5)
148.8C
155.7C
102.8 CH
161.1C
108.6 CH
127.7 CH
9.6 CH3
10.1 CH3
56.1 CH3
117.0 CH
115.1 CH
153.1C
113.1 CH
9.6 CH3
10.0 CH3
56.3 CH3
6.85 d (8.8)
6.83 dd (8.8, 2.5)
6.84 d (2.4)
6.85 d (8.5)
7.37 d (8.5)
2.08 s
6.75 dd (8.7, 2.4)
7.54 d (8.7)
2.10 s
2.09 s
3.80 s
12.18 s
4.66 d (7.8)
3.35 m
6′
7.08 d 2.5
2.094 s
2.091 s
3.73 s
6-CH3
8-CH3
4′-OCH3
5-OH
1′′
2′′
3′′
4′′
5′′
2.05 s
12.15 s
4.65 d (7.5)
3.35 m
3.29 m
3.22 m
3.33 m
104.9 CH
74.8 CH
76.9 CH
70.7 CH
74.5 CH
64.0 CH2
104.9 CH
74.7 CH
76.9 CH
70.7 CH
74.5 CH
64.0 CH2
4.68 d 7.7
3.34 m
3.30 m
3.23 m
3.37 m
104.9 CH
74.8 CH
76.9 CH
70.8 CH
74.5 CH
64.1 CH2
3.30 m
3.22 m
3.38 m
6′′
4.26 dd (11.8, 2.0), 4.07 dd (11.8, 6.1)
4.28 brd (10.5),
4.07 m
4.26 brd (10.4), 4.06 dd (11.8, 6.4)
1′′′
171.1C
171.1C
171.3C
2′′′
3′′′
4′′′
5′′′
46.5 CH2
69.9C
46.0 CH2
171.8C
28.2 CH3
52.0 CH3
2.64 d (14.0), 2.48 d (14.0)
2.56 d (2.1)
46.6 CH2
69.8C
46.0 CH2
171.8C
28.1 CH3
52.0 CH3
2.65 d (14.0), 2.49 d (14.0)
2.55 overlapped
46.6 CH2
69.8C
46.8 CH2
174.4C
2.59 d (14.0), 2.49 d (14.0)
2.41 brs
1.21 s
6′′′
1.22 s
3.58 s
1.21 s
3.58 s
28.2 CH3
5′′′-CH3
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
102.6 CH
74.1 CH
77.2 CH
70.9 CH
78.2 CH
61.8 CH2
4.80 d (7.2)
3.26 m
3.28 m
3.16 m
3.40 m
3.78 m, 3.48 m
the (3S) isomer that was previously reported [2,20].
1H NMR spectrum clearly showed an AA'XX′ coupling system at δH 7.01
(2H, d, J = 8.6 Hz, H-3′, 5′) and 7.51 (2H, d, J = 8.6 Hz, H-2′, 6′)] in B-
ring, a singlet proton at δH 6.00 (1H, s, H-6) in A-ring, one methyl at δH
1.93 (3H, s, 8-CH3) and one methoxy group at δH 3.86 (3H, s, 4′-OCH3).
The HMBC correlations for –CH3 (δH 1.93)/C-7, C-8, C-9, and –OCH3
(δH 3.86)/C-4′ revealed a methyl located at C-8 and a methoxy group
located at C-4′ (Fig. 2). And the molecular formula of 1, in combination
with the 1H and 13C NMR data, indicated that three hydroxy groups
should be located at C-3, C-5 and C-7, respectively. The circular di-
chroism (CD) spectrum of 1 showed negative Cotton effect at 293 nm
and positive Cotton effect at 330 nm, suggesting the absolute config-
urations at C-2 and C-3 were R, R [21]. Based on the foregoing evi-
dence, the structure of 1 was elucidated as (2R,3R)-3,5,7-trihydroxy-8-
methyl-4′-methoxydihydroflavanol, named as demethylmatteucinol.
Compound 2 was isolated as a yellow amorphous powder with the
molecular formula of C24H28O11 on the basis of HRESIMS at m/z
(3R)-5-O-Acetyl-1-[(S)-phenylethyl]-mevalonamide (5C): Colorless
oil; 1H NMR (CDCl3, 600 MHz) δH 7.28–7.37 (5H, m, 1′-C6H5), 6.04
(1H, d, J = 6.0 Hz, NH), 5.15 (1H, quintet, J = 7.1 Hz, H-1′), 4.24 (2H,
m, H-5), 2.41 and 2.29 (each 1H, d, J = 14.5 Hz, H-2), 2.05 (3H, s, 5-
OCOCH3), 1.86 (2H, m, H-4), 1.51 (3H, d, J = 7.1 Hz, H-2′), 1.24 (3H,
-
s, 3-CH3); ESIMS m/z 292.0 [M - H] .
3. Results and discussion
The 60% EtOH extract of the rhizomes of M. intermedia was parti-
tioned by macroporous adsorptive resins eluted with EtOH/H2O (0:100
to 95:5). The 95% EtOH eluate was subjected to chromatographic se-
paration using silica gel, ODS, Sephadex LH-20, and semipreparative
HPLC to yield 31C-methylated flavonoids. The presence of D-glucosyl
in compounds 2–10 was determined by acid hydrolysis and HPLC
analyses [18,19]. The configuration of the anomeric carbon was in-
dicated to be β based on the large coupling constant of the anomeric
proton (J = 6.0–8.0 Hz).
-
491.1549 [M - H] . The UV spectrum was suggestive of a flavanone
derivative with absorption band at 282 nm. The 1H and 13C NMR data
of 2 were similar to those of 16, except for an additional β-D-gluco-
pyranosyl moiety. The HMBC correlation between the anomeric proton
at δH 4.61 (H-1′′) and C-7 indicated that the glycosidation of 7-OH in 2
(Fig. 2). The CD spectrum of 2 showed a negative Cotton effect at
287 nm, suggesting that the absolute configuration at C-2 was S [21].
Thus, compound 2 was elucidated as (2S)-3′-hydroxymatteucinol-7-O-
β-D-glucopyranoside, named as matteflavoside H.
Compound 1 was obtained as a light yellow amorphous powder. Its
molecular formula was deduced as C17H16O6 by 13C NMR data and
+
HRESIMS, giving
a
quasimolecular ion peak [M + H]
at m/z
317.1023 (calcd 317.1025). The UV absorption band was at 294 nm,
characteristic of a flavanonol. 1H NMR spectrum displayed a pair of
protons at δH 4.54 and 5.03 with J = 11.3 Hz, which suggested that 1
was a flavanonol derivative. Apart from these two proton signals, the
Compound 3 was obtained as a yellow amorphous powder. The
5

X. Li, et al.
Fitoterapia136(2019)104147
-
HRESIMS of 3 showed a quasimolecular ion peak [M - H] at m/z
1,3,4-trisubstituted B-ring, which deduced the position of the hydroxy
group was at C-4′ in 6. The absolute configuration of the HMG group
was also determined to be S using the method mentioned above. The CD
spectrum of 6 showed a negative Cotton effect at 288 nm, suggesting
that the absolute configuration at C-2 was S [21]. Thus, compound 6
was established as (2S)-farrerol-7-O-[6′′-O-((S)-3-hydroxy-3-methyl-
glutaryl)]-β-D-glucopyranoside, named as matteuinterate B.
491.1549 (calcd 491.1553), supporting the same molecular formula of
C
24H28O11 as 2. Its 1H and 13C NMR data were similar to those of 2,
with the differences in data attributed to B-ring. The HMBC spectrum of
3 showed correlations of H-2/C-1′, C-2′, C-6′, H-5′/C-3′, C-4′, H-6/C-2′,
C-4′, C-5′, and δH 3.73 (4′-OCH3)/C-4′, revealing the locations of the
methoxy group at C-4′, and the hydroxy group at C-2′ in 3 instead of at
C-3′ in 2. The CD spectrum of 3 showed a positive Cotton effect at
288 nm, suggesting that the absolute configuration at C-2 was R [21].
Compound 7, a yellow amorphous powder, had a molecular formula
of C30H36O15 based on its HRESIMS analysis (m/z 635.1977, [M - H] −
,
Therefore, the structure of
3
was elucidated as (2R)-2′-hydro-
calcd for 635.1976). The UV, 1H NMR, and 13C NMR data closely re-
sembled those of the known flavanone glucoside matteuorienate J (30),
which revealed that they possessed identical skeleton, but differed in
the linkage position between the glucosyl moiety and the HMG frag-
ment. The HMBC correlation of H-3′′/C-1′′′ indicated that the HMG
fragment was attached to the C-3′′ of the glucosyl moiety. The down-
field shift of C-3′′ from 76.7 in 30 to 78.2 in 7 further confirmed the
above conclusion. The 3S-configuration of the HMG moiety was de-
termined as described above. The CD spectrum of 7 showed a negative
Cotton effect at 281 nm, suggesting that the absolute configuration at C-
2 was S [21]. Therefore, the structure of 7 was defined as (2S)-meth-
oxymatteucin-7-O-[3′′-O-((S)-3-hydroxy-3-methylglutaryl)]-β-D-gluco-
pyranoside, named as matteuinterate C.
xymatteucinol-7-O-β-D-glucopyranoside, named as matteflavoside I.
Compound 4 was isolated as a yellow amorphous powder. The 1H
and 13C NMR spectra of 4 exhibited spectroscopic features similar to
those of the known flavanone glucoside matteucin 7-O-β-D-glucoside
[6]. Analyses of the 2D NMR suggested an identical planar structure to
that of 4, which was also supported by the molecular formula of
C
23H26O10 determined by HRESIMS (m/z 463.1617 [M + H] +, calcd
for C23H27O10, 463.1604). The CD spectrum showed a negative Cotton
effect at 277 nm, being opposite to matteucin 7-O-β-D-glucoside, which
indicated as S configuration at C-2 in 4 [21]. Therefore, compound 4
was identified as (2S)-matteucin 7-O-β-D-glucopyranoside, named as
matteflavoside J.
Compound 5, a light yellow amorphous powder, had a molecular
Compound 8, obtained as a light yellow amorphous powder, was
assigned the molecular formula C30H36O14 by HRESIMS data (m/z
621.2183, [M + H] +, calcd for 621.2183). A detailed comparison of
1H and 13C NMR spectral data between 8 and 6 indicated that com-
pound 8 was a methylated derivative of 6 based on an additional
methoxy group at δH 3.58 (3H, s) and δC 52.0. The HMBC correlation
between –OCH3 (δH 3.58) and the carbonyl carbon at δC 171.8 con-
firmed the methylation of C-5′′′ in the HMG unit. Experiment for de-
termining the absolute configuration of the HMG moiety in 8 was at-
tempted, but appropriate derivatives could not be obtained due to the
methylation of C-5′′′ in the HMG moiety. Due to the isolates with the
HMG group from the genus Matteuccia both in our study and in the
literature possessing S-configuration in the HMG unit [2], the asym-
metric C-3′′′ in compound 8 was proposed to be an S-configuration
because of a shared biogenesis. In turn, the positive Cotton effect of 8 at
279 nm in the CD spectrum revealed the structure of 8 as 2R config-
uration [21]. Thus, compound 8 was assigned as (2R)-farrerol-7-O-[6′′-
O-((S)-3-hydroxy-3-methyl-methylglutaryl)]-β-D-glucopyranoside,
named as matteuinterate D.
formula of C30H36O15 based on a quasi-molecular ion peak at m/z
+
637.2126 [M + H]
in HRESIMS. The UV spectrum revealed the
presence of a flavanone skeleton with absorption band at 282 nm.
Detailed analyses of the 1D NMR (Table 2) revealed that 5 was similar
to 2, and the key differences were the presence of two carbonyls, two
methylenes, one oxygenated quaternary carbon, and one methyl. The
HMBC correlations for H-2′′′/C-1′′′, H-4′′′/C-5′′′, and 3′′′-CH3/ C-2′′′, C-
3′′′, C-4′′′, in combination with the molecular formula, revealed the
presence of a 3-hydroxy-3-methylglutaryl (HMG) fragment. The HMG
substituent was placed at C-6′′ of the glucosyl unit by HMBC correla-
tions between H-6′′ (δH 4.23 and 4.07) and C-1′′′ (δC 171.2) (Fig. 2). The
absolute configuration of the chiral carbon in the HMG group was de-
termined with the literature method including amidation, reduction
and acetylation to yield 5-O-acetyl-1-[(S)-phenylethyl]mevalonamide
(Scheme 1). Its 1H NMR data were identical to those of (3R)-5-O-acetyl-
1-[(S)-phenylethyl]-mevalonamide rather than the (3S) isomer [20].
The CD spectrum of 5 showed a negative cotton effect at 289 nm,
suggesting that the absolute configuration at C-2 was S [21]. Therefore,
compound 5 was named as (2S)-3′-hydroxymatteucinol-7-O-[6′′-O-((S)-
3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside, named as mat-
teuinterate A.
Compound 9 was obtained as a yellow amorphous powder. The
molecular formula of
C31H38O15 was confirmed on the basis of
HRESIMS data (m/z 651.2283, [M + H] +, calcd for 651.2289). The 1H
and 13C NMR data of 9 were similar to those of 7, except for the pre-
sence of an extra methoxy group at δH 3.58 (3H, s) and δC 52.0.
Compound 9 was a methylated derivative at C-5′′′ of 7 confirmed by the
HMBC correlation between –OCH3 (δH 3.58) and C-5′′′ in the HMG unit.
The absolute configuration of C-3′′′ in the HMG group was also
Compound 6 was obtained as a yellow amorphous powder. The
+
HRESIMS ion at m/z 607.2035 [M + H]
established its molecular
formula as C29H34O14. On careful analyses of the 1D NMR data and
molecular formula between 6 and 5, the differences in 6 involved the
absence of a methoxy group, and a 1,4-disubstituted B-ring replacing a
Scheme 1. Determination of the absolute configuration of HMG group in compound 5 (representive). Reagents and conditions: (a) (S)-1-phenylethylamine, PyBOP,
HOBt, Et3N, DMF; (b) LiBH4, THF; (c) Ac2O, pyridine [2,20].
6

X. Li, et al.
Fitoterapia136(2019)104147
Fig. 1. Chemical structures of compounds 1–31 isolated from Matteuccia intermedia.
proposed to be S because of a shared biogenesis. The negative Cotton
effect at 283 nm implied a 2S absolute configuration [21]. Thus, 9 was
determined as (2S)-methoxymatteucin-7-O-[6′′-O-((S)-3-hydroxy-3-me-
thyl-methylglutaryl)]-β-D-glucopyranoside, named as matteuinterate E.
Compound 10 was obtained as a brown amorphous powder, with a
molecular formula of C36H46O20 by HRESIMS (m/z 799.2668 [M + H]
+, calcd for 799.2661). The 1H and 13C NMR data were similar to those
of 28, except for an additional β-D-glucosyl unit. The extra D-glucose
moiety was linked to the aglycone at C-2′, which was indicated by the
HMBC correlation of H-1′′′/C-2′ (Fig. 2). The CD spectrum of 10 showed
a negative Cotton effect at 288 nm, suggesting that the absolute con-
figuration at C-2 was S [21]. Further determination of the absolute
configuration of the HMG group as described above confirmed the
structure of 10 to be (2S)-2′-hydroxymatteucinol-7-O-[3′′-O-((S)-3-hy-
droxy-3-methylglutaryl)]-β-D-glucopyranoside-2′-O-β-D-
glucopyranoside, named as matteuinterate F.
Twenty-one known structures were identified as demethox-
ymateucinol (11) [22], farrerol (12) [23], matteucin (13) [24], mat-
teucinol (14) [25], methoxymatteucin (15) [25], 3′-hydroxy-matteu-
cinol (16) [26], cyrtominetin (17) [27], 5,7-dihydroxy-4′-methoxy-6-
methyl-flavanone (18) [28], demethoxymateucinol 7-O-glucoside (19)
[2], farrerol 7-O-glucoside (20) [29], matteucinol 7-O-glucoside (21)
[2], myrclacitrin II (22) [2], matteflavoside G (23) [7], matteuorienate
A (24) [3], matteuorienate B (25) [3], matteuorienate D (26) [2],
matteuorienate F (27) [2], matteuorienate H (28) [2], matteuorienate I
(29) [2], matteuorienate J (30) [2] and matteuorienate K (31) [2]
(Fig. 1) by comparing their chemical and spectroscopic characteristics
with the reported data. Among the isolated C-methylated flavanones
with substituted B ring, the 2′-hydroxyl, 4′-hydroxyl, 3′,4′-dihydroxyl
and 3′,5′-dihydroxyl substituted derivatives were found. Compounds 12
7

X. Li, et al.
Fitoterapia136(2019)104147
OCH3
OH
4'
3'
OCH3
4'
8
O
O
O
HO
O
HO
HO
1'
2
2
OH
10
OH
OH
O
OH
OH
O
1
2
OCH3
O
O
O
1''
7
HO
2'
2
OH
OH
HO
OH
O
OH
3
OCH3
OH
O
5'''
O
3'''
O
7
O
O
O
1''
HO
6''
O
OH
OH
HO
OH
OH
5
OCH3
OH
OH
O
OH
O
O
3'
HO
HO
OH O
O
1'''
7
OH
O
O
OH
O
O
O
O
O
O
5'''
H
3CO
O
OH
OH
HO
OH
OH
8
OCH3
O
O
O
O
O
H
3CO 5'''
O
OH
OH
OH
HO
OH
O
OH
9
OCH3
O
O
O
O
7
O
O
1''
1'''
HO
O
6''
2'
O
OH
OH
OH
HO
1''''
OH
O
OH
HO
OH
OH
10
Fig. 2. Selected key HMBC (→) correlations of 1–3, 5, 7–10.
8

X. Li, et al.
Fitoterapia136(2019)104147
Fig. 3. The binding modes of compounds 12–17 with the key residues on α-glucosidase. (A) compound 12. (B) compound 13. (C) compound 14. (D) compound 15.
(E) compound 16. (F) compound 17.
and 14 were converted into compounds 17 and 16 by flavonoid 3′-
hydroxylase (F3′H), respectively [30]. And previous reports suggested
that the B ring appeared to undergo stepwise oxidation from 2′-hy-
droxyl to 2′,4′-dioxygenated derivatives, as represented by compound
13 to 15 [31].
Table 4
α-Glucosidase inhibitory activity of compounds 1–9, 11–31.
Compound
IC50 (μM)a
Compound
IC50 (μM)a
1
2
3
4
5
6
7
8
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Acarbose
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
α-Glucosidase is the most important enzyme in carbohydrate di-
gestion in vivo. Inhibition of α-glucosidase can lead to prevent excess
glucose absorption at the small intestine, which controls the blood
glucose level and is considered an effective way to prevent diabetes and
obesity exacerbation [32]. The hypoglycemic activity in vitro of all
isolates except for 10 (due to limited amount of sample available) was
evaluated via an α-glucosidase inhibitory assay. Acarbose, a common α-
glycosidase inhibitor, was used as a positive control and showed IC50
value of 172.3 μM. Compounds 12–17 showed potent inhibitory ac-
tivity (IC50 values of 12.4–69.7 μM) (Table 4), revealing their hy-
poglycemic effect. In our study, all the active flavanones in the α-glu-
cosidase inhibitory assay possessed 7-OH, and all the 7-O-glycoside
derivatives were inactive. These results suggested that the hydroxy
group at C-7 of flavanones should be crucial for the potent inhibitory
activity on α-glucosidase, which was consistent with previous report
[33]. The decreasing inhibitory effect after glycosylation may be caused
by increasing molecular size and polarity, and resulting the increase of
the steric hindrance, which weakens the binding interaction between
flavanones and α-glucosidase. Compounds 12–17, possessing 6- and 8-
methyl in A-ring and hydroxy or methoxy group in B-ring, showed
stronger activity than other aglycones (1, 11, 18). Therefore, the hy-
droxylation or methoxylation in the B-ring of flavanones and the si-
multaneous methylation at C-6 and C-8 may contribute to the inhibitory
activity. Compound 17, with two hydroxy groups in Bring, displayed
9
11
12
13
14
15
16
17
44.1
37.6
28.0
69.7
43.6
12.4
0.42
2.67
3.56
7.86
3.96
2.66
172.3
14.7
a
IC50 values represent the means standard deviation (SD) of three parallel
measurements.
the strongest α-glucosidase inhibitory activity. Compounds 15 and 16
with both hydroxy and methoxy groups in the B-ring were less active
than 17. For compounds 12–14 possessing mono oxygenated B ring,
compound 14 with a methoxy group showed higher inhibitory activity
than compounds 12 and 13 with a hydroxy group. Based on the above
results, it is reasonable to hypothesize that the methylation of solitary
hydroxy group in B-ring is favourable to increase the binding stability
of compounds and α-glycosidase, and further enhance the inhibitory
9

X. Li, et al.
Fitoterapia136(2019)104147
capacity. On the contrary, the methylation of hydroxy group in the
disubstituted B ring significantly increased the steric hindrance, espe-
cially at C-5′ position in the 2,5-disubstituted pattern, which was in
accordance with the result of 15.
(1995) 1558–1564.
[7] B. Li, Y. Ni, L.J. Zhu, F.B. Wu, F. Yan, X. Zhang, X.S. Yao, Flavonoids from
Matteuccia struthiopteris and their anti-influenza virus (H1N1) activity, J. Nat. Prod.
78 (2015) 987–995.
[8] L.J. Zhu, F. Yan, J.P. Chen, N. Zhang, X. Zhang, X.S. Yao, 8-O-4′ Neolignan gly-
cosides from the aerial parts of Matteuccia struthiopteris, Chin. Chem. Lett. 27 (2016)
63–65.
[9] J. Schroder, S. Raiber, T. Berger, A. Schmidt, J. Schmidt, A.M. Soares-Sello,
E. Bardshiri, D. Strack, T.J. Simpson, M. Veit, G. Schroder, Plant polyketide syn-
thases: a chalcone synthase-type enzyme which performs a condensation reaction
with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones,
Biochemistry 37 (1998) 8417–8425.
[10] J. Zhu, D. Li, J. Jin, L. Wu, Binding analysis of farrerol to lysozyme by spectroscopic
methods, Spectrochim. Acta A Mol. Biomol. Spectrosc. 68 (2007) 354–359.
[11] C.P. Chung, J.B. Park, K.H. Bae, Pharmacological effects of methanolic extract from
the root of Scutellaria baicalensis and its flavonoids on human gingival fibroblast,
Planta Med. 61 (1995) 150–153.
[12] Y. Cao, Q. Chu, J. Ye, Chromatographic and electrophoretic methods for pharma-
ceutically active compounds in Rhododendron dauricum, J. Chromatogr. B. Analyt.
Technol. Biomed. Life Sci. 812 (2004) 231–240.
[13] Y. Hanasaki, S. Ogawa, S. Fukui, The correlation between active oxygens scaven-
ging and antioxidative effects of flavonoids, Free Radic. Biol. Med. 16 (1994)
845–850.
[14] X. Qin, X. Hou, M. Zhang, T. Liang, J. Zhi, L. Han, Q. Li, Relaxation of rat aorta by
farrerol correlates with potency to reduce intracellular calcium of VSMCs, Int. J.
Mol. Sci. 15 (2014) 6641–6656.
[15] Z. Liu, X. Zheng, Y. Wang, M. Tang, S. Chen, F. Zhang, L. Li, C. Zhang, Y. Sun,
Lignans and isoflavonoids from the stems of Pisonia umbellifera, RSC Adv. 8 (2018)
16383–16391.
[16] L.J. Zhu, S. Yi, X. Li, H.F. Chen, M. Ming, X. Zhang, X.S. Yao, C-glycosides from the
stems of Calophyllum membranaceum, J. Asian Nat. Prod. Res. 20 (2017) 1–6.
[17] J.B. Thoden, C.A. Sellick, D.J. Timson, R.J. Reece, H.M. Holden, Molecular structure
of Saccharomyces cerevisiae Gal1p, a bifunctional galactokinase and transcriptional
inducer, J. Biol. Chem. 280 (2005) 36905–36911.
[18] M. Bai, G.D. Yao, S.F. Liu, D. Wang, Q.B. Liu, X.X. Huang, S.J. Song, Lignans from a
wild vegetable (Patrinina villosa) able to combat Alzheimer's disease, J. Funct. Foods
28 (2017) 106–113.
[19] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, I. Kouno, Facile discrimination of
aldose enantiomers by reversed-phase HPLC, Chem. Pharm. Bull. 55 (2007)
899–901.
[20] Y. Hattori, K.H. Horikawa, H. Makabe, N. Hirai, M. Hirota, T. Kamo, A refined
method for determining the absolute configuration of the 3-hydroxy-3-methylglu-
taryl group, Tetrahedron-Asymmetr. 18 (2007) 1183–1186.
To further reveal the potential binding mode of α-glucosidase and
the candidate compounds, molecular docking was performed using
Discovery Studio 3.5 software package. The 3′,4′-phenolic hydroxy
groups of B-ring in compound 17 formed two vital hydrogen bonds with
the side chains of Asn-95 and Ser-170 located in the bottom of binding
pocket. In addition, a Pi-Pi stacking interaction was observed between
the aromatic A-ring of 17 and the side chain of Phe-100. The 5,7-phe-
nolic hydroxy groups of A-ring oriented towards solvent polarizable
surface, which further stabilized the complex (Fig. 3). The above in-
teractions suggested that compound 17 could bind with α-glucosidase
with a reasonable conformation. Then the aromatic A and/or B-ring of
compounds 12–17 shared common Pi-Pi stacking interactions with Phe-
100, and compound 14 was discovered an extra Pi-Pi stacking inter-
action between the B-ring and Phe-174. There are two H-bond inter-
actions between the 7-hydroxyl and Ser-121, and the carbonyl and Gly-
165 in 12; two H-bond interactions between 5-hydroxyl and Ser-121,
and 7-hydroxyl and Glu-268 in 14. There is one H-bond interaction
between the 2′-hydroxyl of 13 and Ser-170, the carbonyl of 15 and Gly-
165, the methoxy group of 16 and Asn-95, respectively. These results
show compounds 12–17 could form stable interactive conformation
with the binding site of α-glucosidase, and Pi-Pi stacking interaction of
A- and B-ring as well as the H-bond interaction of the substituted hy-
droxy groups are vital to stability of the complex. The results further
support the inhibitory activity obtained by the α-glucosidase assay and
structure-activity relationship rules.
Conflict of interest
The authors declared that they have no conflicts of interest to this
[21] W. Gaffeld, Circular dichroism, optical rotatory dispersion and absolute config-
uration of flavanones, 3-hydroxyflavanones and their glycosides: Determination of
aglycone chirality in flavanone glycosides, Tetrahedron 26 (1970) 4093–4108.
[22] P. Basnet, S. Kadota, M. Shimizu, H.X. Xu, T. Namba, 2′-Hydroxymatteucinol, a new
C-methyl flavanone derivative from Matteccia orientalis; potent hypoglycemic ac-
tivity in streptozotocin (STZ)-induced diabetic rat, Chem. Pharm. Bull. 41 (1993)
1790–1795.
[23] Y.Z. Tang, J.H. Ling, P. Zhang, X.R. Zhang, N. Zhang, W.L. Wang, J.Y. Li, N. Li,
Potential therapeutic agents for circulatory diseases from Bauhinia glauca
Benth.subsp. pernervosa, Bioorg. Med. Chem. Lett. 25 (2015) 3217–3220.
[24] W.S. Feng, X.W. Cao, X.K. Zheng, H.X. Kuang, A new flavanone from Dryopteris
sublaeta, Acta Pharm. Sin. 40 (2005) 443–446.
work.
Acknowledgements
The authors thank Prof. Hao Zhang, at Sichuan University, China for
the botanical identification. We also thank Institute of Traditional
Chinese Medicine & Natural Products, College of Pharmacy, Jinan
University for the HRESIMS measurement.
[25] H. Wada, N. Tanaka, T. Murakami, T. Uchida, K. Kozawa, Y. Saiki, C.M. Chen,
Chemical and chemotaxonomical studies of filices. LXXVI.: An unusual flavanone
derivative from Wagneriopteris japonica LOEVE et LOEVE, Yakugaku Zasshi 108
(1988) 740–744.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
[26] W.S. Feng, X.W. Cao, H.X. Kuang, X.K. Zheng, Isolation and structure identification
of a new flavanone from Dryopteris sublaeta, Chin. J. Nat. Med. 3 (2005) 336–339.
[27] T. Iwashina, J. Kitajima, S. Matsumoto, Flavonoids in the species of Cyrtomium
(Dryopteridaceae) and related genera, Biochem. Syst. Ecol. 34 (2006) 14–24.
[28] S. Matsuura, Ring closure of chalcones to flavanones in the presence of concentrated
orthophosphoric acid. II. Ring closure of chalcones derived from phloroglucinol
monomethyl ethers, Yakugaku Zasshi 77 (1957) 296–298.
[29] X.W. Lou, Q.H. Lin, G.Y. Zhang, W.Y. Liu, F. Feng, W. Qu, Identification and
characterization of three new flavonoids from Rhododendron dauricum, Chin. J. Nat.
Med. 13 (2015) 628–633.
[30] K. Stich, G. Forkmann, Studies on columnidin biosynthesis with flower extracts
from Columnea hybrida, Zeitschrift fuer Naturforschung C, J. Biosci. 43 (1988)
311–314.
[31] M. Linuma, M. Mizuno, Natural occurrence and synthesis of 2′-oxygenated flavones,
flavonols, flavanones and chalcones, Phytochemistry 28 (1989) 681–694.
[32] W. Hakamata, M. Kurihara, H. Okuda, T. Nishio, T. Oku, Design and screening
strategies for α-glucosidase inhibitors based on enzymological information, Curr.
Top. Med. Chem. 9 (2009) 3–12.
doi.org/10.1016/j.fitote.2019.04.002.
References
[1] Flora of China Editorial Committee, Chinese Academy of Sciences, vol. 4, Science
Press, 1999, p. 164.
[2] J. Huh, T. Ha, K.B. Kang, K.H. Kim, W.K. Oh, J. Kim, S.H. Sung, C-Methylated
flavonoid glycosides from pentarhizidium orientale rhizomes and their inhibitory
effects on the H1N1 influenza virus, J. Nat. Prod. 80 (2017) 2818–2824.
[3] S. Kadota, P. Basnet, K. Hase, T. Namba, Matteuorienate A and B, two new and
potent aldose reductase inhibitors from Matteuccia orientalis (Hook.) Trev, Chem.
Pharm. Bull. 42 (1994) 1712–1714.
[4] P. Shao, X. Zhang, B. Li, W.H. Jiao, L.J. Wu, X.S. Yao, New isocourmarin and
phthalide derivatives from the rhizomes of Matteuccia orientalis, Chem. Pharm. Bull.
58 (2010) 1650–1654.
[5] L.J. Zhu, Y. Song, P. Shao, X. Zhang, X.S. Yao, Matteucens I-J, phenolics from the
rhizomes of Matteuccia orientalis, J. Asian Nat. Prod. Res. 20 (2017) 62–66.
[6] P. Basnet, S. Kadota, K. Hase, T. Namba, Five new C-methyl flavonoids, the potent
aldose reductase inhibitors from Matteuccia orientalis Trev, Chem. Pharm. Bull. 43
[33] H. Cao, X. Chen, Structures required of flavonoids for inhibiting digestive enzymes,
Anti-Cancer Agent Me. 12 (2012) 929–939.
10