Four new monoterpenoid glycosides from the flower buds of Magnolia biondii

Article abstract of DOI:10.3390/molecules21060728

Four new monoterpenoid glycosides 1-4, named magnoliaterpenoid A-D, were isolated from a 50% aqueous acetone extract of flower buds of Magnolia biondii, along with one known compound, (1′R,3′S,5′R,8′S,2Z,4E)-dihydrophaseic acid 3-O-β-D-glucopyranoside (5). Their structures and relative configuration were identified by extensive spectroscopic analysis (IR, UV, MS, 1D and 2D NMR). The aglycones of these four new compounds possess seven-membered rings systems, which are very rare. A plausible biosynthetic route for the four new compounds was proposed via the biogenetic isoprene rule. Compounds 1, 2, 3, and 4 showed no antimicrobial activity at the concentration range of 1.95-250 μg/mL.

Full text of DOI:10.3390/molecules21060728

molecules
Article
Four New Monoterpenoid Glycosides from the
Flower Buds of Magnolia biondii
Wei-Sheng Feng 1,2,*, Yu-Huan He 1,2, Xiao-Ke Zheng 1,2, Jian-Chao Wang 1,2, Yan-Gang Cao 1,2
Yan-Li Zhang 1,2 and Kai Song 1,2
,
1
Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine
Development of Henan Province, Henan University of Chinese Medicine, Zhengzhou 450046, China;
13071020601@163.com (Y.-H.H.); zhengxk.2006@163.com (X.-K.Z.); 15238397097@163.com (J.-C.W.);
caoyangang1987@126.com (Y.-G.C.); zyl2013hnzy@163.com (Y.-L.Z.); songky5901@163.com (K.S.)
2
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China
*
Correspondence: fwsh@hactcm.edu.cn; Tel./Fax: +86-371-6568-0011
Academic Editor: Derek J. McPhee
Received: 19 April 2016; Accepted: 30 April 2016; Published: 3 June 2016
Abstract: Four new monoterpenoid glycosides
1–4, named magnoliaterpenoid A–D, were isolated
from a 50% aqueous acetone extract of flower buds of Magnolia biondii, along with one known
1
1
1
1
compound, (1 R,3 S,5 R,8 S,2Z,4E)-dihydrophaseic acid 3-O-β-D-glucopyranoside (5). Their structures
and relative configuration were identified by extensive spectroscopic analysis (IR, UV, MS, 1D and
2D NMR). The aglycones of these four new compounds possess seven-membered rings systems,
which are very rare. A plausible biosynthetic route for the four new compounds was proposed
via the biogenetic isoprene rule. Compounds 1, 2, 3, and 4 showed no antimicrobial activity at the
concentration range of 1.95–250 µg/mL.
Keywords: Magnoliaceae; flower buds of Magnolia biondii; monoterpenoid glycosides; biosynthetic route
1. Introduction
The genus Magnolia (family Magnoliaceae) includes about 90 species worldwide, mainly
distributed in tropical and temperate regions of Asia and North America to South America. In China,
there are about 30 species, mostly distributed in the southern and northern provinces of China [1].
Flos Magnoliae, the flower buds of M. biondii, M. denudata or M. sprengeri, which is unique to China
and mostly distributed in the Henan, Hubei, Zhejiang, Anhui, and Shanxi provinces, has been used
as a traditional Chinese medicine for more than 1500 years to treat nasal congestion, headaches,
sinusitis, and allergic rhinitis [
which are widely used in China [
anti-inflammatory [ ], anti-angiogenic [
antioxidant activities [12 13]. Several types of compounds have been isolated from the plant, including
lignans [ ], neolignans [ ], sesquiterpenoids [14], alkaloids [15 16], and flavonoids [15 16]. In order
2–4
]. Its volatile oil has also been processed into all kinds of cosmetics
]. It has been reported to possess extensive anti-allergy [ ],
], anti-platelet aggregation [ 10], antimicrobial [11] and
4
5,6
7
8
9,
,
9
9
,
,
to further investigate the active components of the herb, a systematic phytochemical study was
carried out on the 50% aqueous acetone extract of flower buds of Magnolia biondii. As a result,
four new monoterpenoid glycosides1–4, named magnoliaterpenoid A–D (Figure 1), were isolated
and structurally identified, along with a known sesquiterpenoid glycoside, (11R,31S,51R,81S,2Z,4E)-
dihydrophaseic acid 3-O-β-D-glucopyranoside (5). Herein, the isolation and structural elucidation of
compounds 1–4 are reported, as well as their antimicrobial activity.
Molecules 2016, 21, 728; doi:10.3390/molecules21060728
www.mdpi.com/journal/molecules

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Figure 1. Structures of compounds 1–4.
2. Results and Discussion
2.1. Structure Elucidation of Compounds 1–4
Compound , obtained as a colorless gum, was determined to have the molecular formula
1
´
C16H26O8Cl by HRESIMS (m/z 381.1301 [M + Cl] , calcd. 381.1310),with four degrees of unsaturation.
Its UV spectrum showed an absorption maximum at 217 nm, indicating the presence of an
α,β
-unsaturated carbonyl moiety. The IR spectrum supported the presence of hydroxyl (3367 cm´1),
carbonyl (1682 cm´1) and double bond (1652 cm´1) groups. The 1H-NMR spectroscopic data of
1
(Table 1) displayed the existence of two methyl singlets [δH 1.25 (3H, s, H-8), 1.23 (3H, br. s, H-9)]
and one olefinic proton [δH 6.98 (1H, t, J = 2.2, 4.9Hz, H-2)] (See the Supplementary Materials).
1
1
Figures S1–S36 showing the H-NMR, 13C-NMR , DEPT, HSQC, HMBC, H-1H COSY, NOESY,
HR-ESI-MS, UV and IR spectra of compounds 1–4 can be found in the Supplementary Materials.
1
Table 1. H- and 13C-NMR data for compounds 1–4 (500/125 MHz, in CD3OD).
Position
1
2
3
4
δH
δC
δH
δC
δH
δC
δH
δC
1
2
3
131.5 (s)
141.3 (d)
28.6 (t)
138.2 (s)
124.6 (d)
28.3 (t)
135.8 (s)
148.3 (d)
28.6 (t)
1.40 (1H, m)
1.40 (2H, m)
1.01 (1H, m)
1.76 (1H, m)
1.93 (1H, m)
37.9 (d)
29.6 (t)
28.3 (t)
6.98 (1H, t, 2.2, 4.9)
2.02 (1H, m)
2.41 (1H, m)
5.44 (1H, d, 4.2)
1.84 (1H, m)
2.05 (1H, m)
1.76 (1H, m)
6.87 (1H, d, 5.4)
2.28 (1H, m)
2.48 (1H, m)
2.16 (1H, m)
4
5
6
1.72 (1H, m)
44.5 (d)
80.5 (s)
24.5 (t)
44.6 (d)
80.3 (s)
35.1 (t)
46.7 (d)
79.3 (s)
40.4 (t)
41.3 (d)
81.1 (s)
35.1 (t)
2.15 (2H, m)
1.21 (1H, m)
2.27 (1H, dd, 5.7, 11.6)
4.08 (1H, br. s)
1.21 (3H, s)
1.21 (3H, s)
1.70 (3H, s)
4.45 (1H, d, 7.8)
3.12 (1H, m)
3.20 (1H, m)
3.23 (1H, m)
3.34 (1H, m)
3.62 (1H, dd, 5.6, 11.9)
3.78 (1H, dd, 2.1, 11.9)
2.28 (1H, m)
2.66 (1H, m)
1.23 (1H, m)
2.17 (1H, m)
3.84 (1H, m)
1.23 (3H, s)
7
8
9
10
11
21
31
41
51
61
2.45 (2H, m)
1.25 (3H, s)
1.23 (3H, s)
26.3 (t)
24.8 (q)
23.3 (q)
170.9 (s)
98.5 (d)
75.3 (d)
77.5 (d)
71.8 (d)
78.3 (d)
62.9 (t)
71.9 (d)
24.1 (q)
23.6 (q)
19.2 (q)
98.4 (d)
75.2 (d)
77.5 (d)
71.7 (d)
78.3 (d)
62.8 (t)
203.5 (s)
24.9 (q)
23.5 (q)
15.6 (q)
98.5 (d)
75.2 (d)
77.5 (d)
71.6 (d)
78.2 (d)
62.8 (t)
71.9 (d)
24.1 (q)
23.6 (q)
18.8 (q)
98.6 (d)
75.4 (d)
77.8 (d)
72.2 (d)
78.2 (d)
63.2 (t)
1.28 (3H, s)
1.24 (3H, s)
1.72 (3H, s)
4.45 (1H, d, 7.8)
3.12 (1H, m)
3.20 (1H, m)
3.26 (1H, m)
3.33 (1H, m)
3.61 (1H, m)
3.78 (1H, m)
1.14 (3H, s)
0.91 (3H, d, 6.2)
4.45 (1H, d, 7.7)
3.10 (1H, m)
3.23 (1H, m)
3.16 (1H, m)
3.33 (1H, m)
3.56 (1H, m)
3.81 (1H, m)
4.46 (1H, d, 7.8)
3.11 (1H, m)
3.21 (1H, m)
3.26 (1H, m)
3.35 (1H, m)
3.63 (1H, m)
3.80 (1H, m)
Closer examination of the 1H/13C/1H-1H COSY/HSQC/HMBC NMR data (Table 1) obtained
for
1
1revealed the presence1of a glucose moiety [
δ
4.46 (H-11),198.5 (C-11);13.11 (H-21), 75.3 (C-21); 3.21
1
1
1
1
1
(H-3 ), 77.5 (C-3 ); 3.26 (H-4 ), 71.8 (C-4 ); 3.35 (H-5 ), 78.3 (C-5 ); 3.80 (H-6 a), 3.63 (H-6 b), 62.9 (C-6 )],
along with 10 carbon resonances, including two methyl [δC 24.8 (C-8), 23.3 (C-9)], three methylene
[
δC 28.6 (C-3), 24.5 (C-6), 26.3 (C-7)], two methine [δC 141.3 (C-2), 44.5 (C-4)], one carbonyl [δC 170.9
(C-10)], one oxygenated quaternary carbon [δC 80.5 (C-5)] and one olefinic quaternary carbon [δC 131.5
(C-1)]. Apart from one double bond, one carbonyl and one glucose moiety, the remaining unsaturation
1
of
1
required it must contain a ring. Inspection of the H- and 13C-NMR spectra of
1
indicated a
monoterpenoid glycoside with a structure similar to that of paeoveitol B [17]. The 7.8 Hz coupling
constant for the anomeric proton H-11 of the glucose at δH 4.46 confirmed its axial orientation. Acid
hydrolysis and GC-MS analysis of the corresponding thiazolidine derivative substantiated the sugar
unit as being β
-D-glucose. In the 1H-1H COSY spectrum (Figure 2), the correlations of H-2/H-3/H-4

Molecules 2016, 21, 728
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and H-6/H-7 displayed the key spin systems. In the HMBC spectrum (Figure 2), the correlation from
H-8 to C-5 indicated that the 8-CH3 was located to C-5; the correlation from H-9 to C-4 indicated that
the 9-CH3 was connected to C-4; the correlation from H-11 to C-5 indicated that the
β-D-glucose was
linked to C-5; the correlations from H-2 and H-7 to C-10 indicated that the carbonyl (C-10) was linked
to C-1; the oxygenated quaternary carbon (C-5) was located between C-4 and C-6 from the HMBC
correlations from H-4 and H-6 to C-5. The relative configuration of compound
the NOESY experiment (Figure 3) in which the correlation of H-4 and H-8 was observed. Thus, the
structure of compound was determined to be as shown in Figure 1, and it was assigned the trivial
name magnoliaterpenoid A.
1 was established by
1
Figure 2. Key COSY and HMBC correlations of compounds 1–4.
Figure 3. Key NOESY correlations of compounds 1–4.
Compound
2
was isolated as a colorless gum and had a molecular formula C16H28O7Cl by
HRESIMS (m/z 367.1519 [M + Cl]´, calcd. 367.1518), indicating three degrees of unsaturation. The IR
spectrum showed the presence of hydroxyl (3345 cm´1) and double bond (1385 cm´1) groups.
The 1H-NMR spectroscopic data of
2 (Table 1) revealed the existence of three methyl singlets [δH 1.21
(3H, s, H-8), 1.21 (3H, br. s, H-9), 1.70 (3H, s, H-10)] and one olefinic proton [δH 5.44 (1H, d, J = 4.2 Hz,
H-2)] (See the Supplementary Materials). Comparison of its 13C-NMR and DEPT data with those of
compound
1
indicated that the two compounds possessed a similar skeleton. One of the differences
was replaced
between these two compounds was that the methylene signal at C-7 (δC 26.3) in
1
by an oxygenated methine at C-7 (δC 71.9) in
2
, which was established by the HMBC correlations
1
(Figure 2) from H-2, H-6 and H-10 to C-7, along with the H-1H COSY correlations (Figure 2) of
H-6/H-7. The other main difference was that the carbonyl at C-10 (δC 170.9) in
methyl at C-10 (δC 19.2) in , which was determined on the basis of the HMBC correlations (Figure 2)
from H-10 to C-1, C-2 and C-7. The relative configuration of compound was deduced from the
NOESY experiment (Figure 3) in which the correlationof H-4/H-8 and H-7/H-8 was detected. Similarly,
1 was replaced by a
2
2
the sugar unit of compound
Therefore, the structure of
magnoliaterpenoid B.
2
2
was confirmed as
β-D-glucose using the same method as for compound 1.
was assigned as shown in Figure 1, and it was given the trivial name
Compound
HRESIMS (m/z 365.1357 [M + Cl] , calcd. 365.1361), with four degrees of unsaturation. Its UV spectrum
showed an absorption maximumat 240 nm, indicating the presence of an -unsaturated carbonyl
3 was isolated as a colorless gum and had a molecular formula C16H26O7Cl by
´
α,β

Molecules 2016, 21, 728
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moiety. The IR spectrum supported the presence of hydroxyl (3364 cm´1), carbonyl (1655 cm´1
and double bond (1368 cm´1) groups. The 1H-NMR spectroscopic data of
(Table 1) revealed the
)
3
existence of three methyl singlets [δH 1.28 (3H, s, H-8), 1.24 (3H, br. s, H-9), 1.72 (3H, s, H-10)] and one
olefinic proton [δH 6.87 (1H, d, J = 5.4 Hz, H-2)] (See the Supplementary Materials). The 13C-NMR and
DEPT spectroscopic data of
compound and was that the oxygenated methine at C-7 (δC 71.9) in
C-7 (δC 203.5) in
to C-7. The relative configuration of compound
which the correlation of H-4 and H-8 was observed. Similarly, the sugar unit of compound
confirmed as -D-glucose using the same method as for compound . Therefore, the structure of
established as shown in Figure 1, and the compound was trivially named magnoliaterpenoid C.
3
(Table 1) were highly similar to those of
2
. The only difference between
2
3
2
was replaced by a carbonyl at
3
, which was established by the HMBC correlations (Figure 2) from H-2, H-6 and H-10
was assigned by the NOESY experiment (Figure 3) in
was also
was
3
3
β
1
3
Compound 4 was isolated as a colorless gum. Its molecular formula was established as C16H30O7Na
by HRESIMS (m/z 357.1878 [M + Na]+, calcd. 357.1883), with two degrees of unsaturation. The IR
spectrum displayed the existence of hydroxyl (3362cm´1) groups. The 1H-NMR spectroscopic data
of
0.91 (3H, d, J = 6.2 Hz, H-10)] (See the Supplementary Materials). Critical analysis of 13C-NMR and
DEPT spectroscopic data of (Table 1) demonstrated that its structure was closely related to that of
compound , except that the double bond at C-1 (δC 138.2) and C-2 (δC 124.6) in was reduced to a
4 (Table 1) supported the existence of three methyl singlets [δH 1.23 (3H, s, H-8), 1.14 (3H, br. s, H-9),
4
2
2
methane at C-1 (δC 37.9) and a methylene at C-2 (δC 29.6) respectively in 4, which was established by
the HMBC correlations (Figure 2) from H-10 to C-1 and C-2, along with the 1H-1H COSY correlations
(Figure 2) of H-1 and H-2. The relative configuration of compound
was deduced from the NOESY experiment (Figure 3) in which the correlation of H-4/H-8, H-7/H-8
and H-1/H-7 was detected. Similarly, the sugar unit of compound was also confirmed as -D-glucose
using the same method as for compound . Thus, the structure of was established as shown in
Figure 1, and the compound was given the trivial name magnoliaterpenoid D.
The identity of compound was determined by spectroscopic analysis and comparison with
literature data [18].
4was similar to compound 2, which
4
β
1
4
5
2.2. Plausible Biogenetic Pathway
Since the aglycones of these four new compounds contained 10 carbon atoms and belonged to
the monoterpenoid class of compounds, we propose a plausible biosynthetic route via the biogenetic
isoprene rule, as shown in Scheme 1.
Scheme 1. Plausible biogenetic route of 1–4.

Molecules 2016, 21, 728
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2.3. Antimicrobial Activity
The four new compounds were tested for their antimicrobial activity against Gram-positive and
negative bacteria and fungi at the concentration range of 1.95–250
antimicrobial activity at the tested concentrations.
µg/mL. None of them exhibited
3. Experimental Section
3.1. General Procedures
Optical rotations were measured on an AP-IV polarimeter (Rudolph, NJ, USA). UV spectra
were measured with a Thermo EVO 300 spectrometer (Thermo Fisher Scientific, Madison, WI, USA).
IR spectra were recorded on a Thermo Nicolet IS 10 spectrometer (Thermo Fisher Scientific). NMR
spectra were scanned on an Avance III spectrometer (500 MHz for 1H-NMR and 125 MHz for 13C-NMR,
Bruker, Zug, Switzerland). HR-ESI-MS was recorded on a Bruker MicroTOF-Q II spectrometer
(Bruker Daltonics, Bremen, Germany). GC analysis was carried out on a Shimadzu GC-MS-QP2010E
instrument (Shimadzu Corporation, Tokyo, Japan) using a RXI-5 ms capillary column (30 m
0.25 m, Restek, Bellefonte, PA, USA). Preparative HPLC was performed on a LC-3000 instrument
(Chuangxintongheng, Beijing, China) equipped with a UV-3000 detector using YMC HPLC columns
(5 m, 10.0 250 mm and 5 m, 20.0 250 mm), with a flow rate of 2.0 mL/min and 8.0 mL/min,
respectively. Column chromatography was performed using DiaionHP-20 (Mitsubishi Chemical
Corporation, Tokyo, Japan), ODS-B (50 m, Daiso Co., Ltd., Tokyo, Japan), MCI GEL CHP20p
75–150 µm, Mitsubishi Chemical Corporation), and Toyopearl HW-40 (TOSOH Corporation, Tokyo,
ˆ
0.25 mm
ˆ
µ
µ
ˆ
µ
ˆ
µ
(
Japan). Thin-layer chromatography was carried out on self-made silica gel G (Qingdao Marine
Chemical Industry, Qingdao, China) plates. The chemical reagents were supplied by Beijing Chemical
Plant (Beijing, China) and Tianjin No. 3 Reagent Plant (Tianjin, China).
3.2. Plant Material
The flower buds of Magnolia biondii were collected from Nanzhao, Henan Province, China, and
identified by Prof. Cheng-Ming Dong of the Henan University of Traditional Chinese Medicine, China.
A voucher specimen (No. 20140609) has been deposited in Department of Natural Medicinal Chemistry,
School of Pharmacy, Henan University of Chinese Medicine.
3.3. Extraction and Isolation
The air-dried flower buds of Magnolia biondii (5.0 kg) were extracted with aqueous acetone
(50% v/v, 3
to give a crude extract (463 g). The crude extract was suspended in H2O (2 L) and then successively
extracted with petroleum ether, EtOAc and n-BuOH (2 L 5), respectively. The n-BuOH fraction
(60.0 g) was subjected to Diaion HP-20 column chromatography (10.0 60.0 cm) and eluted with
EtOH–H2O (0:100, 20:80, 40:60, 80:20, 95:5, v/v, each 20 L) to afford five fractions (Fr. I–Fr. V). Fr. II
(14.0 g) was then subjected to ODS column chromatography (4.0 30.0 cm) and eluted successively
ˆ
20 L) at room temperature. The combined solutions were evaporated under vacuum
ˆ
ˆ
ˆ
with a MeOH–H2O (0:100, 5:95, 10:90, 15:85, 20:80, 25:75, 35:65,45:55, 100:0, v/v, each 1.5 L) gradient
to give nine fractions (Fr. 1–Fr. 9). Fr. 6 (1.3 g) was further separated by Toyopearl HW-40 column
chromatography (2.0
(Fr. 6A–Fr. 6H). Fr. 6B (50.0 mg) was further purified by preparative HPLC on a YMC HPLC column
(5 m, 20.0 250 mm, flow rate 8 mL/min) with MeOH–H2O (25:75) to afford compound (4.6 mg,
tR = 77.5 min). Fr. 6C (110.0 mg) was further isolated by preparative HPLC on a YMC HPLC column
(5 m, 20.0 250 mm, flow rate 8 mL/min) with MeOH–H2O (30:70) to afford compound (36.0 mg,
tR = 59.5 min). Fr. 6E (120.0 mg) was also further purified by preparative HPLC on a YMC HPLC
column (5 m, 20.0 250 mm, flow rate 8 mL/min) with MeOH–H2O (35:65) to afford compound
(50.0 mg, tR = 26.0 min) and compound (2.0 mg, tR = 15.0 min). Compound (12.0 mg, tR = 60.0 min)
ˆ
50.0 cm) with MeOH–H2O (25:75) as the eluent to obtain eight subfractions
µ
ˆ
1
µ
ˆ
2
µ
ˆ
3
4
5

Molecules 2016, 21, 728
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was obtained from Fr. 6G by preparative HPLC on a YMC HPLC column (5
rate 8 mL/min) with MeOH–H2O (15:85).
µm, 20.0
ˆ
250 mm, flow
3.4. Compound characterization
20
Magnoliaterpenoid A (
1
): colorless gum; rαsD –28.8 (MeOH, 0.10); UV
λ
max
nm (log ε): 217 (0.8); IR
(iTR): 3367, 2943, 2879, ´1682, 1652, 1424, 1203, 1140 cm´1; 1H- and 13C-NMR, see Table 1; HRESIMS:
m/z = 381.1301 [M + Cl] , (C16H26O8Cl, calcd.381.1310).
20
Magnoliaterpenoid B (
2
): colorless gum; rαsD –13.4 (MeOH,0.72); UV
λ
max
nm (log ε): 203 (0.5); IR
(iTR): 3345, 2969, 2917, 2884, 1385,1367, 1073, 918, 860cm´1; 1H- and 13C-NMR, see Table 1; HRESIMS:
m/z = 367.1519 [M + Cl] , (C16H28O7Cl, calcd.367.1518).
´
20
Magnoliaterpenoid C (
3
): colorless gum; rαsD –15.5 (MeOH, 1.0); UV
λ
max
nm (log ε): 240 (0.8); IR
(iTR): 3364, 2973, 2923,´2886, 1655, 1368, 1073, 904 cm´1; 1H- and 13C-NMR, see Table 1; HRESIMS:
m/z = 365.1357 [M + Cl] , (C16H26O7Cl, calcd. 365.1361).
20
Magnoliaterpenoid D (
4
): colorless gum; rαsD –6.6 (MeOH, 0.03); UV
λ
max
nm (log ε): 202 (0.6); IR (iTR):
3362, 2926, 2871, 1100, 995 cm´1; 1H- and 13C-NMR, see Table 1; HRESIMS: m/z = 357.1878 [M + Na]+,
(C16H30O7Na, calcd. 357.1883).
3.5. Acid Hydrolysis and Sugar Analysis
Compounds
reaction mixture was extracted with EtOAc (2 mL
Then the residue was dissolved in pyridine (0.3 mL) containing L-cysteine methyl ester hydrochloride
(1.5 mg) and heated at 60 ˝C for 1 h. A 0.3 mL solution of phenyl isothiocyanate (1.5 mg) in pyridine
was added to the mixture, which was heated at 60 C for 1 h. The reaction mixture was directly
1
–
4
(1.0 mg, respectively) were refluxed with 8% HCl (2 mL) for 3 h. After the
3), the aqueous layer was dried under vacuum.
ˆ
˝
analyzed by gas chromatography (GC). The D-configuration of glucose was confirmed by comparing
the retention time with a standard sample [tR (min): D-glucose (8.5)].
3.6. Antimicrobial Assay
The antimicrobial activity of compounds 1–4 was evaluated against Gram-positive bacteria
(Staphylococcus aureus ATCC25923), Gram-negative bacteria (Escherichia coli ATCC35150 and Proteus
vulgaris ATCC33420) and fungi (Aspergillusniger ATCC6257 and Candida albicans ATCC90029) by a
microdilution titre technique [19]. Kanamycin and fluconazole was used as positive controls. All tests
were performed in triplicate.
4. Conclusions
In conclusion, although these four new compounds did not show antimicrobial activity at the
tested concentration range of 1.95–250
µg/mL, the aglycones of these four new compounds possess
seven-membered rings system which are very rare and a plausible biogenetic pathway of
1–4
was
proposed via the biogenetic isoprene rule. This kind of skeleton was also isolated from the Magnoliaceae
for the first time.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
6/728/s1.
Acknowledgments: This work was supported by the Key Project of Chinese Ministry of Education of
China (2003078).
Author Contributions: W.-S.F. and Y.-H.H. conceived and designed the experiments; Y.-H.H. performed the
experiments; X.-K.Z., J.-C.W., Y.-G.C. and Y.-L.Z. analyzed the data; K.S. contributed reagents/materials/analysis
tools; W.-S.F. wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare that there are no conflicts of interest.

Molecules 2016, 21, 728
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Sample Availability: Samples of the compounds 1–4 are available from the authors.
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