A new ionone glycoside and three new rhemaneolignans from the roots of rehmannia glutinosa

Article abstract of DOI:10.3390/molecules200815192

A new ionone glycoside, frehmaglutoside I (1), and three new rhemaneolignans A-C (2-4) were isolated from the 95% EtOH extract of the roots of Rehmannia glutinosa. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and 2D NMR) analyses. In addition, these compounds were evaluated for their protective effects on cardiocytes impaired by doxorubicin in H9c2 cells. Among them, compounds 1-3 exhibited protective effects against DOX-induced cardiotoxicity.

Full text of DOI:10.3390/molecules200815192

Molecules 2015, 20, 15192-15201; doi:10.3390/molecules200815192
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Ionone Glycoside and Three New Rhemaneolignans from
the Roots of Rehmannia glutinosa
Meng Li 1,2, Xiaolan Wang 2,3, Xiaoke Zheng 2,3, Jianchao Wang 2,3, Wei Zhao 2,3, Kai Song 2,3
,
Xuan Zhao 2,3, Yanli Zhang 2,3, Haixue Kuang 1 and Weisheng Feng 2,3,
*
1
School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China;
E-Mails: limeng31716@163.com (M.L.); hxkuang@hotmail.com (H.K.)
2
Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese
Medicine Development of Henan Province, Zhengzhou 450046, China;
E-Mails: wxl_@163.com (X.W.); zhengxk.2006@163.com (X.Z.); 15238397097@163.com (J.W.);
18236159652@163.com (W.Z.); songky5901@163.com (K.S.); lilas616@hotmail.com (X.Z.);
zyl2013hnzy@163.com (Y.Z.)
3
School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450046, China
* Author to whom correspondence should be addressed; E-Mail: fwsh@hactcm.edu.cn;
Tel./Fax: +86-371-6568-0011.
Academic Editor: Derek J. McPhee
Received: 25 June 2015 / Accepted: 14 August 2015 / Published: 20 August 2015
Abstract: A new ionone glycoside, frehmaglutoside I (1), and three new rhemaneolignans
A–C (2–4) were isolated from the 95% EtOH extract of the roots of Rehmannia glutinosa.
Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI-MS, 1D and
2D NMR) analyses. In addition, these compounds were evaluated for their protective effects
on cardiocytes impaired by doxorubicin in H9c2 cells. Among them, compounds 1–3 exhibited
protective effects against DOX-induced cardiotoxicity.
Keywords: Rehmannia glutinosa; ionone glycoside; rhemaneolignans; the protective effects
of cardiomyocytes

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1. Introduction
Rehmannia glutinosa, which belongs to Scrophulariaceae family, has been used as traditional Chinese
herbal medicine for thousands of years. It was recorded in the Chinese medical classic “Shennong’s
Herba” and was thought of in China as a “top grade” herb. Previous phytochemical studies on the dried
and steamed roots of R. glutinosa have led to the isolation and identification of iridoid glycosides, ionone
glycosides, phenethyl alcohol glycosides, and several other components [1–5]. In our previous studies, three
new ursane-type triterpenes glutinosalactone A–C were isolated from the leaves, a new megastigmane,
rehmamegastigmane, from the fresh roots, and two new ionone glycosides frehmaglutoside G and H
from the dried roots [6–8]. Next, the further phytochemical study was undertaken to investigate the
chemical constituents of the 95% EtOH extract of the dried roots of R. glutinosa, which led to the
isolation of four new compounds: frehmaglutoside I (1) and rhemaneolignan A–C (2–4) (Figure 1). Their
structures were elucidated by extensive analysis of HR-ESI-MS, 1D and 2D NMR, CD data, and
chemical methods. In this paper, we describe the isolation, structure elucidation, and biological evaluation
of the new compounds.
Figure 1. Structures of compounds 1–4.
2. Results and Discussion
Compound 1 was obtained as pale yellow crystalline powder, with a molecular formula of C22H38O9
on the basis of a [M + Na]+ ion peak at m/z 469.2407 (calcd. 469.2414) in the HRESIMS. It showed IR
absorptions for hydroxyl (3361 cm−1), methyl (2923 cm−1), double bond (1698 cm−1) and ether linkages
(1074 and 1025 cm−1). In the 1H- and 13C-NMR spectra of 1 (Table 1), the signal patterns were similar
to those of frehmaglutoside H [8] from R. glutinosa, except for the presence of one methoxy (δH 3.32
(3H, s, OCH3) and δC 58.0 (OCH3)). The NMR chemical shifts at the C-11 position of 1 were shifted
downfield to δH 4.15 (2H, d, J = 7.0 Hz, H-11) and δC 69.1 (C-11) compared with those of frehmaglutoside
H, suggesting that the methoxy was located at the hydroxymethyl group of C-11. This finding was

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supported by the heteronuclear multiple-bond connectivity (HMBC) correlation from H-12 (δ 3.32) to
C-11 (Figure 2).
Figure 2. Key HMBC and 1H-1H COSY correlations of 1–4.
Table 1. 1H- and 13C-NMR data for 1 (500 and 125 MHz, δ ppm).
No.
1
δH
-
δC
No.
12
δH
δC
82.3
75.3
3.32 (3H, s)
1.92 (3H, s)
58.0
21.0
2
-
13
1.96 (1H, m)
1.49 (1H, m)
1.96 (2H, m)
1.88 (2H, m)
3.73 (1H, m)
-
3
4
35.9
26.9
14
15
1.17 (3H, s)
0.95 (3H, s)
18.8
22.6
5
6
85.5
45.0
16
1′
2′
3′
4′
5′
1.03 (3H, s)
27.0
106.7
75.6
78.2
71.6
77.7
4.31 (1H, d, 7.5)
7
6.29 (1H, d, 16.0)
6.68 (1H, d, 16.0)
-
134.1
127.5
137.5
125.4
8
3.16–3.64 (4H, m)
9
10
5.45 (1H, t, 6.5)
3.84 (1H, dd, 2.5, 11.0)
3.67 (1H, dd, 5.0, 11.0)
11
4.15 (2H, d, 7.0)
69.1
6′
62.8
The relative configuration of 1 was confirmed by analysis of the NOESY spectrum, the correlations
between H-5 (δ 3.73) and H-15 (δ 0.95)/H-16 (δ 1.03) revealed that they were β-oriented; on the other
hand, the correlations between H-5 and H-14 (δ 1.17) indicated that these protons were α-oriented
respectively (Figure 3).
The CD spectrum of 1 displayed positive Cotton effect at 204 nm and negative Cotton effect at
232 nm, which were also similar to frehmaglutoside H. Therefore the asymmetric centers of 1 had a 1R,
2R, 5S configuration. Finally, in the acid hydrolysis of 1, D-glucose was obtained as confirmed by TLC
comparison with a reference sample, and the configuration was determined by measurement of the

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optical rotation value. On the basis of the above analysis, 1 was identified as (7E,9E)-7-[(1R,2R,5S)-
trihydroxy-2,6,6-trimethylcyclohexane]-9-methyipenta-7,9-dienoic-11-methoxy-5-O-β-D-glucopyranoside,
and named frehmaglutoside I (Figure 1).
Figure 3. Key NOESY correlations of compounds 1 and 3.
Compound 2 was isolated as a colorless amorphous powder with [α]2D0 −4.20 (c 0.20, MeOD). Its
molecular formula, C21H24O8, was determined by the observation of a sodiated molecular ion peak at
m/z 427.1367 (calcd. for C21H24O8Na, 427.1369) in the HRESIMS. The IR spectrum showed the
presence of hydroxyl (3355 cm−1), methyl (2956, 2923 and 2853 cm−1), ester carbonyl (1635 cm−1),
and methoxyl groups (1260 and 1022 cm−1). The 1H-NMR data of 2 (Table 2) indicated the presence of
two ABX patterns (δH 6.95 (1H, d, J = 8.5 Hz, H-3), 7.06 (1H, dd, J = 2.0, 8.5 Hz, H-4), 7.15 (1H, d,
J = 2.0 Hz, H-6) and 7.02 (1H, d, J = 2.0 Hz, H-3′), 6.83 (1H, dd, J = 2.0, 8.0 Hz, H-5′), 6.70 (1H, d,
J = 8.0 Hz, H-6′)), trans-olefinic proton signals (δH 7.59 (1H, d, J = 16.0 Hz, H-7), 6.38 (1H, d, J = 16.0 Hz,
13
H-8)), and three methoxy groups (δH 3.81 (3H, s), 3.77 (3H, s), 3.76 (3H, s)) [9]. The C-NMR and
DEPT spectra (Table 2) of 2 showed 21 carbon signals, including three methoxyls (δC 52.0, 56.3, 56.6),
one methylene (δC 62.4), ten methines (δC 74.1, 85.5, 112.0, 112.4, 115.6, 116.5, 117.6, 121.2, 123.5,
129.5), six quaternary carbons (δC 134.0, 146.3, 147.1, 148.7, 151.8, 151.8) and one ester carbonyl (δC
1
13
169.5). These H- and C-NMR data implied that compound 2 should be an oxyneolignane and were
similar to those of 2,2′-dimethoxy-4-(3-hydroxypropenyl)-4′-(1,2,3-trihydroxypropyI) biphenyl ether [10],
except for the fact the hydroxymethyl group (C-9) in it was replaced by an ester carbonyl (δC 169.5),
and one additional methoxy unit was present in compound 2. The HMBC experiment confirmed the
abovementioned suggestion and placed the additional methoxyl unit at C-9 (Figure 2). The relative
configuration of 2 was determined by analysis of the coupling constants. The H-7′ possessed a
β-orientation on the basis of the observed J value of 6.0 Hz between H-7′ and H-8′, indicating that
H-7′ and H-8′ were in a trans-configuration [11]. The NOESY correlations from H-8′ to H-7′ further
supported the conclusion (Figure 3). Thus the relative configuration for 2 was determined. In addition,
the absolute configuration of C-8′ was determined to be S, as the CD spectrum showed a positive
Cotton effect at 253 nm [12,13]. Correspondingly, the absolute configuration of 7′ was elucidated as R.
In summary, the structure of 2 was established as (7′R,8′S)- 3,3′-dimethoxy-1-(3-methl-1-acrylate)-1′-
(1,2,3-trihydroxypropyl)-4-O-4′ neolignan and named rhemaneolignan A (Figure 1).

Molecules 2015, 20
Table 2. 1H- and 13C-NMR data for 2 and 3 (500 and 125 MHz, δ ppm).
15196
2 (CD3OD)
3 (DMSO-d6)
No.
δH
δC
δH
δC
1
2
3
4
151.8
151.8
112.4
129.5
-
149.5
150.8
111.2
126.7
7.15 (d, 2.0)
7.32 (d, 2.0)
-
5
7.06 (dd, 2.0, 8.5) 123.5 7.18 (dd, 2.0, 8.0) 122.6
6
7
8
9
6.95 (d, 8.5)
7.59 (d, 16.0)
6.38 (d, 16.0)
117.6
146.3
116.5
169.5
56.6
7.04 (d, 8.0)
7.51 (d, 16.0)
6.52 (d, 16.0)
-
114.4
144.7
115.1
167.0
55.7
OCH3
OCH3′′
3.81 (s)
3.76 (s)
-
3.80 (s)
3.69 (s)
-
52.0
51.2
1′
2′
3′
4′
5′
6′
7′
8′
147.1
148.7
112.0
134.0
145.5
147.0
111.0
132.9
7.02 (d, 2.0)
6.95 (d, 2.0)
-
6.83 (dd, 2.0, 8.0) 121.2 6.74 (dd, 2.0, 8.0) 119.0
6.70 (d, 8.0)
4.81 (d, 6.0)
4.48 (m)
115.6
74.1
85.5
6.66 (d, 8.0)
4.68 (d, 4.0)
4.37 (m)
114.6
76.0
83.8
3.83 (m)
3.76 (m)
3.77 (s)
3.56 (m)
3.23 (m)
3.71 (s)
9′
62.4
56.3
60.1
55.4
OCH3′
Compound 3, isolated as a colorless powder with [α]2D0 +1.79 (c 0.20, MeOD), gave the same molecular
formula C21H24O8 as that of 2 by positive HRESIMS (m/z 427.1366 [M + Na]+, calcd. for 427.1369) and
was found to be an diastereoisomer of compound 2. The two structures differ from each other only by
the stereochemistry at the two chiral centers (C-7′ and C-8′). The 1H-NMR spectrum was almost identical
to that of 2 except for the coupling constant of H-7′ (δH 4.68 (d, J = 4.0)), which suggested that
H-7′ possessed a α-orientation, furthermore indicating that H-7′ and H-8′ were in a cis-configuration.
The CD spectrum of 3 exhibited similar Cotton effects (positive at 258 nm) to that of 2. Therefore the
asymmetric centers of 3 were 7′S, 8′S configuration. Accordingly, the structure of 3 was defined as
(7′S,8′S)-3,3′-dimethoxy-1-(3-methl-1-acrylate)-1′-(1,2,3-trihydroxypropyl)-4-O-4′-neo-lignan and named
rhemaneolignan B (Figure 1).
Compound 4, as pale yellow amorphous powder, was assigned the molecular formula C26H36O12 by
HRESIMS (m/z 563.2101 [M + Na]+, calcd. for 563.2107). The NMR spectroscopic data of 4 (Table 3)
was similar to that of daphneresinol [14], except for additional signals associated with a glucopyranosyl
unit inferred from signals at δ 102.9 (C-1′′), 71.4 (C-2′′), 78.1 (C-3′′), 74.9 (C-4′′), 77.8 (C-5′′), 62.5
(C-6′′). In the 1H-NMR spectrum, the anomeric proton signal at δ 4.80 (1H, d, J = 8.0 Hz) pointed out
the existence of one β-glucopyranose unit. In the HMBC spectrum, a correlation from the anomeric
proton to C-4 (δ 146.1) indicated unequivocally that the β-glycopyranosyl moiety was located at C-4
(Figure 2). Consequently, the structure of 4 was identified as 4-O-β-D-glucopyranoside-2-benzhydryl-3-
hydroxymethyl butane-1,4-diol and named rhemaneolignan C (Figure 1).

Molecules 2015, 20
Table 3. 1H- and 13C-NMR data for 4 (500 and 125 MHz, δ ppm).
15197
No.
δH
δC
No.
1′
δH
δC
1
2
-
141.3
113.8
150.7
146.1
118.2
-
136.7
113.0
149.1
145.8
116.3
7.02 (d, 2.0)
2′
6.97 (d, 2.0)
3
-
3′
-
4
-
4′
-
5
7.08 (d, 2.0)
5′
6.73 (d, 2.0)
6
6.95 (dd, 2.0, 8.0) 121.4
6′
6.86 (dd, 2.0, 8.0) 121.8
7
4.07 (d, 12.5)
3.69 (2H, m)
1.96 (2H, m)
3.75 (2H, m)
3.57 (1H, m)
3.38 (1H, m)
3.75 (1H, m)
3.39 (1H, m)
52.1 glu′′
8
44.7
43.7
63.6
1′′
2′′
3′′
4.80 (d, 8.0)
3.38 (1H, m)
3.38 (1H, m)
102.9
71.4
78.1
9
10
11
12
60.0
59.7
4′′
5′′
6′′
3.47 (1H, m)
3.48 (1H, m)
74.9
71.8
62.5
3.86 (1H, 2.0, 12.0)
3.70 (1H, 6.0, 11.0)
OCH3
3.87 (3H, s)
3.86 (3H, s)
56.8
56.5
OCH3′
To investigate whether compounds 1–3 could protect cells from DOX-induced cell death, H9c2 cells
were treated with 50 μM DOX in the presence or absence of compounds 1–3 (40 and 80 μM) and the
cell viability was assessed by performing MTT assay. Among them, compound 1–3 exhibited protective
effects against DOX-induced cardiotoxicity with different concentrations (Figure 4).
Figure 4. Protective effects of 1–3 on DOX-induced cytotoxicity in H9c2 cell. n = 3;
* p < 0.05; ** p < 0.01 compared with the DOX group.

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3. Experimental Section
3.1. General Experimental Procedures
1
13
NMR spectra (500 MHz for H-NMR and 125 MHz for C-NMR) were recorded in CD3OD or
DMSO-d6 on an AVANCE III 500 spectrometer (Bruker Daltonics, Bremen, Germany), with TMS as
internal standard. Optical rotations were measured with an AP-IV instrument (Rudolph Research Analytical,
Hackettstown, Madison, WI, USA). IR spectra were determined on a Nicolet iS 10 Microscope Spectrometer
(Thermo Scientific, San Jose, CA, USA). HR-ESI-MS spectra were recorded on a Bruker maxis HD mass
spectrometer. UV spectra were recorded on a UV-2401PC apparatus (Shimadzu Corporation, Kyoto,
Japan). HPLC was performed on a Waters Alliance 2695 separations module (equipped with Empower
software) connected to a Waters 2998 photodiode array (PDA) detector (190–800 nm) equipped with a
Platisil ODS C18 column (250 mm × 4.6 mm I.D. 5 μm) (Waters, Milford, MA, USA). Preparative HPLC
was conducted using a Saipuruisi LC-50 instrument with an UV200 detector (Beijing, China) and a
YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm and 250 mm × 10 mm, 5 μm). Column chromatography
was made use of Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Toyopearl HW-40,
MCI gel CHP-20 (TOSOH Corp., Tokyo, Japan), Sephadex LH-20 (40–70 μm, Amersham Pharmacia
Biotech AB, Uppsala, Sweden), silica gel (160–200 mesh, Marine Chemical Industry, Qingdao, China).
TLC was carried out on self-made silica gel G (Marine Chemical Industry) plates, CH2Cl2/MeOH/H2O
(10:1:0.1, v/v), CH2Cl2/MeOH/H2O (4:1:0.1, v/v) as the eluent, and spots were visualized by spraying
with 10% H2SO4 in ethanol (v/v) followed by heating. The chemical reagents were supplied by Beijing
Chemical Plant (Beijing, China), Tianjin NO. 3 Reagent Plant (Tianjin, China) and Tianjin Shield Fine
Chemicals Company (Tianjin, China).
3.2. Plant Material
The dried roots of R. glutinosa were collected from Jiaozuo, Henan Province in China, in November
2010. The plants were identified by Prof. Chengming Dong of Henan University of TCM. A voucher
specimen (No. 20101101A) has been stored in the Department of Natural Medicinal Chemistry, School
of Pharmacy, Henan University of TCM, Zhengzhou, China.
3.3. Extraction and Isolation
The dried roots (20.0 kg) of R. glutinosa were cut into small pieces and extracted twice with 95%
EtOH (200 L) under reflux for 2 h. After concentrating under reduced pressure on a vacuum evaporator, the
concentrated solution (1500 mL) was centrifuged and subjected to Diaion HP-20 column chromatography,
eluting successively with H2O and MeOH, to afford water and methanol extracts (101.6 g). Next the
methanol extracts were dissolved in 10% MeOH/H2O (1000 mL), and subjected to Diaion HP-20 column
chromatography once again, eluting successively with 10%, 30%, 50%, 70%, 100% (v/v) MeOH/H2O
(10 column volumes each). After removing the solvents, extracts of 65.0 g, 16.6 g, 11.5 g, 4.2 g, 2.0 g,
were obtained, respectively.
The 10% MeOH/H2O (65.0 g) fraction was subjected to silica gel column chromatography eluting
with CH2Cl2/MeOH (30:1, 20:1, 10:1, 5:1) to afford Fr. 1.1–1.4. Fr. 1.3 (8.6 g) was purified over

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Sephadex LH-20 using MeOH/H2O (4:6) to afford Fr. 2.1–2.4. Fr. 2.2 was separated by preparative
HPLC with MeOH/H2O (33:67) (YMC-Pack ODS-A, Kyoto, Japan, 10 mm × 250 mm) to afford
compound 1 (11 mg).
The 30% MeOH/H2O (16.6 g) fraction was subjected to Toyopearl HW-40 column chromatography
eluting with MeOH/H2O from 0% to 100% to give Frs. 1.1–1.4. Fr. 1.4 was fractionated repeatedly by
column chromatography on MCI gel CHP-20 using MeOH/H2O (2:8) to yield Frs. 2.1–2.5. Fr. 2.4 was
repeatedly chromatographed on a silica gel column with CH2Cl2/MeOH (10:1) to yield 4 (3.5 mg).
The 50% MeOH/H2O (11.5 g) fraction was fractionated on a silica gel column and eluted with mixtures
of CH2Cl2 and MeOH of increasing polarity to afford fractions 1.1–1.6. Fraction 3 was chromatographed
on a Sephadex LH-20 column eluting with MeOH/H2O (7:3), followed by purification by semi-preparative
HPLC with MeOH/H2O (52:48) at a flow rate of 3 mL/min to give compounds 2 (12.0 mg) and 3 (8.6 mg).
3.4. Spectral Data
Frehmaglutoside I (1): Pale yellow crystalline powder; [α]2D0 −13.7 (c 0.20, CH3OH); UVmax 237 nm;
CD (MeOH): 204 (Δε +3.09), 232 (Δε −5.23) nm; IR (MeOH)νmax: 3361, 2923, 2854, 1698, 1613, 1453,
1074, 1025 cm−1; HRESIMS: m/z 469.2407 [M + Na]+, C22H38O9; 1H-NMR (500 MHz, CD3OD) spectral
data and 13C-NMR (125 MHz, CD3OD) spectral data, see Table 1.
Rhemaneolignan A (2): Colorless amorphous powder, [α]2D0 −4.20 (c 0.20, MeOD); UVmax 325 nm; CD
(MeOH): 253 (Δε +2.12) nm; IR (MeOH)νmax: 3356, 2956, 2923, 2853, 1635, 1508, 1456, 1259, 1022 cm−1;
1
HRESIMS: m/z 427.1367 [M + Na]+, C21H24O8; H-NMR (500 MHz, CD3OD) spectral data and
13C-NMR (125 MHz, CD3OD) spectral data, see Table 2.
Rhemaneolignan B (3): Colorless amorphous powder, [α]2D0 +1.79 (c 0.20, MeOD); UVmax 322 nm; CD
(MeOH): 258 (Δε +2.73) nm; IR (MeOH)νmax: 3363, 2956, 2923, 2853, 1635, 1600, 1508, 1457, 1259,
1032 cm−1; HR-ESI-MS: m/z 427.1366 [M + Na]+, C21H24O8; 1H-NMR (500 MHz, DMSO-d6) spectral
data and 13C-NMR (125 MHz, DMSO-d6) spectral data, see Table 2.
Rhemaneolignan C (4): Pale yellow amorphous powder, [α]2D0 −35.6 (c 0.14, MeOD); UV (MeOH) λmax
:
278.3 nm; IR (MeOH)νmax: 3397, 2924, 2851, 1601, 1513, 1384, 1268, 1073, 1029; HR-ESI-MS m/z
1
563.2021 [M + Na]+, C26H36O12; H-NMR (500 MHz, CD3OD) spectral data and 13C-NMR (125 MHz,
CD3OD) spectral data, see Table 3.
3.5. Activity Assay
3.5.1. Cell Culture
The rat cardiac H9c2 myocardial cells, were spontaneously immortalized ventricular rat embryo
myoblasts, were purchased from Biowit Technologies (Shenzhen, China). The cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in
a water-saturated 5.0% CO2 incubator.

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3.5.2. Measurement of Cell Viability
Cells were split when a confluence of ~80% was achieved using trypsin-EDTA and seeded onto
96-well plates at a density of 1.0 × 104 cells·L−1 (200 μL/well), incubated for 24 h before treatment.
Thereafter, the cells were exposed to DOX (50 μM) for 24 h and then incubated in a fresh medium with
compound 1–3 (80 and 40 μM) for an additional 24 h. The effects of compounds 1–3 on DOX induced
cytotoxicity were assessed using the MTT assay, as previously described [15,16]. The optical density of
each well was then measured on a microplate spectrophotometer at a wavelength of 490 nm. Cell viability
was determined as the percentage of surviving cells compared with that of the DOX-treated control.
3.5.3. Statistical Analysis
Experiments were performed in triplicate, and the values are the averages of three (n = 4) independent
experiments. Individual data were expressed as mean ± standard deviation (SD). A post-hoc Dunnett’s
test was used to obtain corrected p-values in group comparisons. Statistical analyses were performed
with one-way ANOVA (SPSS version 13.0). A p value of 0.05 or less was considered significant.
4. Conclusions
In conclusion, a new ionone glycoside. frehmaglutoside I (1). and three new rhemaneolignans A–C
(2–4), were isolated from the dried roots of Rehmannia glutinosa. Among them, different concentrations
of compounds 1–3 exhibited protective effects against DOX-induced cardiotoxicity.
Supplementary Materials
The IR, HR-ESI-MS, NMR (1D and 2D) and CD spectra of compounds 1–3 are available as supporting
information. Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/08/15192/s1.
Acknowledgments
Our work were supported by the Key Projects in the National Science & Technology Pillar Program
during the Twelfth Five-Year Plan Period (2011BAI06B02) and the Collaborative Innovation Center of
Diagnosis, Treatment and Drug Research for Respiratory Disease, Henan province, China.
Author Contributions
All listed authors contributed to this work. M.L. was responsible for the extraction, isolation and
identification of the plant, and preparation of the manuscript; X.W. contributed to perform the activity
assay; J.W., W.Z. and K.S. were also responsible for the extraction and isolation for the plant; X.Z. and
Y.Z. were responsible for NMR spectrum; W.F., X.Z. and H.K. designed and obtained financial support.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
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References
1. Liu, Y.F.; Liang, D, Luo, H.; Hao, Z.Y.; Wang, Y.; Zhang, C.L.; Chen, R.Y.; Yu, D.Q. Chemical
constituents from root tubers of Rehmannia glutinosa. Chin. Tradit. Herb. Drugs 2014, 45, 16–22.
2. Liu, Y.F.; Liang, D; Luo H.; Hao, Z.Y.; Wang Y.; Zhang, C.L.; Zhang, Q.J.; Chen, R.Y.; Yu, D.Q.
Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J. Nat. Prod. 2012, 75,
1625–1631.
3. Li, Y.S.; Chen, Z.J.; Zhu, D.Y. A novel bis-furan derivative, two new natural furan derivatives from
Rehmannia glutinosa and their bioactivity. Nat. Prod. Res. 2005, 19, 165–170.
4. Sasaki, H; Nishimura, H; Morota, T; Katsuhara, T; Chin, M; Mitsuhashi, H. Norcarotenoids of
Rehmannia glutinosa Ver. Hueichingensis. Phytochemistry 1991, 30, 1997–2001.
5. Liu, Y.F.; Liang, D.; Luo, H.; Hao, Z.Y.; Wang, Y.; Zhang, C.L.; Ni, G.; Chen, R.Y.; Yu, D.Q.
Ionone glycosides from the roots of Rehmannia glutinosa. J. Asian Nat. Prod. Res. 2014, 16, 11–19.
6. Zhang, Y.L.; Feng, W.S.; Zheng, X.K.; Cao, Y.G.; Lv, Y.Y.; Chen, H.; Kuang, H.X. Three new
ursane-type triterpenes from the leaves of Rehmannia glutinosa. Fitoterapia 2013, 89, 15–19.
7. Feng, W.S.; Lv, Y.Y.; Zheng, X.K.; Zhang, Y.L.; Cao, Y.G.; Pei, Y.Y. A new megastigmane from
fresh roots of Rehmannia glutinosa. Acta Pharm. Sin. B 2013, 3, 333–336.
8. Feng, W.S.; Li, M.; Zheng, X.K.; Zhang, N.; Song, K.; Wang, J.C.; Kuang, H.X. Two new ionone
glycosides from the roots of Rehmannia glutinosa Libosch. Nat. Prod. Res. 2015, 29, 59–63.
9. Fang, J.J.; Reichelt, M.; Kai, M.; Schneider, B. Metabolic Profiling of Lignans and Other Secondary
Metabolites from Rapeseed (Brassica napus L.). Food Chem. 2012, 60, 10523–10529.
10. Morita, H.; Kishi, E.; Takeya, K.; Itokawa, H. Biphenylneolignans from wood of Eurycoma longifolia.
Phytochetistry 1992, 31, 3993–3995.
11. Sun, X.G.; Ma, G.X.; Zhang, D.W.; Huang, W.H.; Ding, G.; Hu, H.G.; Tu, G.Z.; Guo, B.L. New
lignans and iridoid glycosides from Dipsacus asper Wall. Molecules 2015, 20, 2165–2175.
12. Yang, Y.D.; Hu, Z.X.; Luo, Z.W.; Xue, Y.B.; Yao, G.M.; Wang, Y.Y. A New Sesquilignan
Glucoside from Uraria sinensis. Molecules 2014, 19, 1178–1188.
13. Matsuda, N.; Kikuchi, M. Studies on the constituents of Lonicera. species. X. neolignan glycosides from
the leaves of Lonicera gracilipes var. glandulosa Maxim. Chem. Pharm Bull. 1996, 44, 1676–1679.
14. Liang, S.; Shen, Y.H.; Tian, J.M.; Wu, Z.J.; Jin, H.Z.; Zhang, W.D.; Yan, S.K. Phenylpropanoids from
Daphne feddei and their inhibitory activities against NO production. J. Nat. Prod. 2008, 71, 1902–1905.
15. Han, X.; Ren, D.; Fan, P.; Shen, T.; Lou, H. Protective effects of naringenin-7-O-glucoside on
doxorubicininduced apoptosis in H9C2 cells. Eur. J. Pharmacol. 2008, 581, 47–53.
16. Wang, W.C.; Uen, Y.H.; Chang, M.L.; Cheah, K.P.; Li, J.S.; Yu, W.Y.; Lee, K.C.; Choy, K.C.;
Hu, C.M. Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin
in vitro. BMC Complement. Altern. Med. 2012, 12, 138, doi:10.1186/1472-6882-12-138.
Sample Availability: Samples of the compounds 1–4 are available from the authors.
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