Two new compounds from the fruits of Buddleja lindleyana with neuroprotective effect

Article abstract of DOI:10.1080/10286020.2011.653783

Two new triterpenoid glycosides, mimengosides H (1) and I (2), were isolated from the fruits of Buddleja lindleyana Fort. Their structures were determined by extensive spectroscopic methods. Neuroprotective effects of these isolates against 1-methyl-4- phenylpyridinium ion-induced neurotoxicity in PC12 cells were evaluated. Pretreatment with compound 1 had potential protective effect in a concentration range from 0.1 to 1μmol l^-1.

Full text of DOI:10.1080/10286020.2011.653783

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Two new compounds from the
fruits of Buddleja lindleyana with
neuroprotective effect
De-Ling Wu a b , Yang-Kui Wang a c , Jing-Song Liu a b , Xun-Cui
Wang a b & Wei Zhang a b
a Anhui University of Traditional Chinese Medicine , Hefei ,
230031 , China
b Anhui Key Laboratory of Modern Chinese Materia Medica , Hefei ,
230031 , China
c West Anhui College of Health Vocational , Lu'an , 237000 , China
Published online: 01 Mar 2012.
To cite this article: De-Ling Wu , Yang-Kui Wang , Jing-Song Liu , Xun-Cui Wang & Wei Zhang (2012)
Two new compounds from the fruits of Buddleja lindleyana with neuroprotective effect, Journal of
Asian Natural Products Research, 14:4, 342-347, DOI: 10.1080/10286020.2011.653783
To link to this article: http://dx.doi.org/10.1080/10286020.2011.653783
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Journal of Asian Natural Products Research
Vol. 14, No. 4, April 2012, 342–347
Two new compounds from the fruits of Buddleja lindleyana with
neuroprotective effect
De-Ling Wuab*, Yang-Kui Wangac, Jing-Song Liuab, Xun-Cui Wangab and Wei Zhangab
aAnhui University of Traditional Chinese Medicine, Hefei 230031, China; bAnhui Key Laboratory of
Modern Chinese Materia Medica, Hefei 230031, China; cWest Anhui College of Health Vocational,
Lu’an 237000, China
(Received 14 June 2011; final version received 26 December 2011)
Two new triterpenoid glycosides, mimengosides H (1) and I (2), were isolated from the
fruits of Buddleja lindleyana Fort. Their structures were determined by extensive
spectroscopic methods. Neuroprotective effects of these isolates against 1-methyl-4-
phenylpyridinium ion-induced neurotoxicity in PC12 cells were evaluated. Pretreat-
ment with compound 1 had potential protective effect in a concentration range from 0.1
to 1 mmol l21
.
Keywords: Buddleja lindleyana; mimengoside H; mimengoside I; neuroprotective
effect
1. Introduction
reports the isolation and structure elucida-
tion of two new triterpenoid glycosides
mimengosides H (1) and I (2) (Figure 1),
as well as the evaluation of their neuro-
protective abilities against MPPþ-induced
neurotoxicity to PC12 cells by 3-(4,5-
dimethylthiozol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay.
The genus Buddleja (family Loganiaceae)
contained approximately 100 species, in
which 29 are distributed in China [1].
Previous phytochemical studies on the
chemical constituents of Buddleja species
have led to the isolation of various natural
products, including triterpenoids [2],
sesquiterpenoids [3], iridoids [4], pheny-
lethanoids [5], and saponins [6]. Due to the
interesting phytochemical and ethnophar-
macological properties, the genus Bud-
dleja has been investigated extensively.
Buddleja lindleyana Fort. is a herb that has
been traditionally used for its antifungal,
antiprotozoal [7], antispasmodic [8], and
antimicrobial [9] activities. However, little
is known about the chemical constituent of
the fruits of B. lindleyana. Pedicularioside
A from B. lindleyana inhibited cell death
induced by 1-methyl-4-phenylpyridinium
ions (MPPþ) in primary cultures of rat
mesencephalic neurons [10]. This paper
2. Results and discussion
Compound 1 was obtained as a white
amorphous powder. Positive results of both
Liebermann-Burchard and Molish reactions
indicated that 1 could be a triterpenoid
saponin. Its molecular formula was deter-
minedtobeC37H62O8 bythequasimolecular
ion at m/z 669.4125 [M þ Cl]2 in High-
resolution electron ionization mass spec-
trometry (HR-ESI-MS). The IR spectrum
showed the presence of hydroxyl (3425,
1057cm21) and olefinic (1629 cm21) func-
tions. Its 1H and 13C NMR spectra exhibited
six quaternary methyls [dH/dC 0.59(s)/24.8
*Corresponding author. Email: dlwu7375@sina.com.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.653783
http://www.tandfonline.com

Journal of Asian Natural Products Research
343
29
30
30
29
20
21
22
19
13
14
20
17
19
21
22
18
12
26
28
12
26
O
11
H3CO
25
1
18
28
11
25
13
14
17
16
OH
1
9
2
16
9
6
2
8
7
10
15
8
7
15
10
3
5
27
3
5
4
27
4
6
O
O
23
5′
5′
23
O
OH
O
OH
24
24
CH3
CH3
4′
1′
1′
4′
6′
6′
OH
OH
OH 3′
OH
2′
3′
2′
OH
OH
1
2
Figure 1. Chemical structures of compounds 1 and 2.
(Me-30), dH/dC 0.85(s)/39.3 (Me-29), dH/dC
Compound 2, a white amorphous
powder, has molecular formula
0.86(s)/30.8 (Me-27), dH/dC 0.89(s)/12.9
(Me-24), dH/dC 0.98(s)/18.4 (Me-26), and
dH/dC 1.08(s)/16.9 (Me-25)] and an olefinic
bond [dH/dC 5.25 (br s)/121.7 (C-12), 148.5
(C-13)]. The 13C NMR spectrum displayed
37 signals, aided by distortionless enhance-
ment by polarization transfer spectrum, 31
of which were assigned to a triterpene
skeleton and six to the sugar moieties. The
13C NMR spectrum suggested that com-
pound 1 had a similar structure to that of
3,23,28-trihydroxy-11-methoxy-12-olea-
nene[2],exceptforthepresenceofonesugar
unit in 1. Acid hydrolysis yielded one L-
fucose, which was detected by thin layer
chromatography (TLC) and Gas chromato-
graph-mass spectrum (GC-MS) via compar-
ing with authentic samples. The coupling
constants of the anomeric protons 6.8 Hz
indicated that the glycosidic linkage of
fucose was in b-configuration. The sugar
moiety was placed at C-3 in 1 according to
the observed distinctly downfield-shifted
resonanceofC-3(dC 79.7)ascomparedwith
that of 3,23,28-trihydroxy-11-methoxy-12-
oleanene. The rotating frame overhauser
effect spectroscopy (ROESY) correlations
between H-11 and H-9, H-12, H-18
indicated these protons to be on the same
sideofthemolecule. Therefore,thestructure
of compound 1 was established as 3-O-b-L-
fucopyranosyl-23,28-dihydroxy-11-meth-
oxy-12-oleanene.
a
C36H58O7 determined by the observed
pseudomolecular ion at m/z 637.3854
[M þ Cl]2 in HR-ESI-MS. The 1H and
13C NMR spectra of 2 resembled those of
mimengoside A [6], which indicated that
they had the same aglycone (Table 1). Acid
hydrolysis yielded one L-fucose, which was
detected by TLC and GC-MS with the
authentic samples. The b-linkage configur-
ation for L-fucose was assigned based on
the coupling constants (J ¼ 7.0 Hz) of its
anomeric proton. The 13C NMR chemical
shifts of C-3 (dC 79.6) indicated that 2 was
a saponin with glycosidic linkages at C-3.
The ROESY spectrum of 2 was the
observed correlations (Figure 2) between
H-11 and H-9, and those between H-12 and
H-19 indicated them to be on the same side
of the molecule. Therefore, the structure of
2 was established as 3-O-b-L-fucose-13,28-
epoxy-3b,23-dihydroxy-11-oleanene.
3. Experimental
3.1 General experimental procedures
The optical rotations were measured on a
Horiba SEPA-300 polarimeter (HORIBA
Instruments (Shanghai) Co., Ltd., Shanghai,
China). Melting points were determined on
an XT digital melting point apparatus with
microscope and are uncorrected (Shanghai
Eastern Co., Ltd., Shanghai, China). UV

344
D.-L. Wu et al.
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR spectral data of 1 and 2 in DMSO-d6.
1
2
No.
dC
dH (J, Hz)
dC
dH (J, Hz)
1
2
3
4
5
6
39.9
33.1
79.7
41.6
46.2
17.9
1.77 (1 H, m) 1.25 (1 H, m)
1.62 (1 H, m) 1.62 (1 H, m)
3.25 (1 H, br)
37.8
24.9
79.6
42.6
45.9
16.9
1.73 (1 H, m) 1.23 (1 H, m)
2.01 (1 H, m) 1.78 (1 H, m)
3.31 (1 H, br)
1.04 (1 H, m)
1.12 (1 H, m)
1.12 (1 H, m)
1.44 (1 H, m)
1.25 (1 H, m)
1.11 (1 H, m)
1.21 (1 H, m)
0.81 (1 H, m)
1.42 (1 H, m)
1.20 (1 H, m)
7
38.9
30.6
8
9
40.2
53.2
39.5
74.9
121.7
148.5
42.6
39.1
22.5
39.7
41.2
42.7
37.2
36.4
33.1
62.7
41.1
52.7
35.4
131.4
130.9
83.9
43.0
30.3
25.0
41.0
50.6
36.7
33.4
31.4
25.1
62.5
1.74 (1 H, m)
1.78 (1 H, m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
3.73 (1 H, dd, 5.0, 3.0)
5.25 (1 H, d, 3.0)
5.27 (1 H, dd, 10.8, 3.0)
5.77 (1 H, d, 10.8)
1.67 (1 H, m) 1.18 (1 H, m)
1.77 (1 H, m) 1.18 (1 H, m)
1.39 (1 H, m) 1.21 (1 H, m)
1.13 (1 H, m) 0.95 (1 H, m)
1.74 (1 H, m)
1.68 (1 H, m) 1.42 (1 H, m)
1.56 (1 H, m)
1.73 (1 H, m) 0.95 (1 H, m)
1.27 (1 H, m) 1.18 (1 H, m)
1.44 (1 H, m) 1.08 (1 H, m)
3.25 (1 H, d, 10)
3.01 (1 H, d, 10)
0.89 (3 H, s)
1.08 (3 H, s)
0.98 (3 H, s)
0.86 (3 H, s)
3.42 (1 H, d, 11.0)
3.09 (1 H, d, 11.0)
0.85 (3 H, s)
0.59 (3 H, s)
3.21 (s)
1.36 (1 H, m) 1.15 (1 H, m)
1.35 (1 H, m) 1.15 (1 H, m)
3.42 (1 H, d, 10.8)
3.06 (1 H, d, 10.8)
0.93 (3 H, s)
0.99 (3 H, s)
1.08 (3 H, s)
0.83 (3 H, s)
24
25
26
27
28
12.9
16.9
18.4
30.8
67.5
12.4
18.1
19.1
19.3
76.1
3.57 (1 H, d, 6.5)
3.13 (1 H, d, 6.5)
0.88 (3 H, s)
29
30
OC
H3
fuc
10
39.3
24.8
51.3
34.3
23.4
0.56 (3 H, s)
104.8
71.2
73.9
70.3
69.7
17.1
4.14 (1 H, d, 6.8)
3.31 (1 H, m)
3.47 (1 H, m)
3.21 (1 H, m)
3.21 (1 H, m)
1.63 (3 H, d, 6.2)
104.8
71.1
73.6
69.7
70.9
16.5
4.14 (1 H, d, 7.0)
3.43 (1 H, m)
3.47 (1 H, m)
3.21 (1 H, m)
3.48 (1 H, m)
1.42 (3 H, d, 6.4)
20
30
40
50
60
spectra were recorded on a Shimadzu UV-
2401 PC spectrophotometer (Shimadzu
Instruments Co., Ltd., Tokyo, Japan). IR
spectra were obtained using a Bruker
Tensor 27 FT-IR spectrometer with KBr
pellets (Bruker Optics GmbH, Ettlingen,
Germany). NMR spectra were measured on
Bruker DRX-500 and AV-400 instruments
(Bruker BioSpin GmbH, Rheinstetten,
Germany). GC experiments were carried
out on a GC-14B Gas Chromatograph
(Shimadzu Instruments Co., Ltd., Suzhou,
China), with an SE30 capillary column
(30 m, 0.32mm i.d., 0.25mm), hydrogen

Journal of Asian Natural Products Research
345
H3CO
O
OH
H
H
O
H
H
O
O
OH
O
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
2
1
Figure 2. Key HMBC correlations of compounds 1 and 2.
flame ionization detector (2708C), column
temperature of 2308C, and carrier gas of N2
(30 ml/min). HR-ESI-MS was detected on a
API QSTAR Pulsar spectrometer (MDS
Sciex, Concord, Ontario, Canada). Silica
gel (200–300 mesh, Qingdao Marine
Chemical Inc., Qingdao, China) and Sepha-
dex LH-20 (Amersham Biosciences Co.,
Ltd., Uppsala, Sweden) were used for
column chromatography. Fractions were
monitored by silica gel TLC, visualized
with vanillin–H2SO4 in ethanol under
heating. Cell viability was quantitatively
measured by the MTT assay.
extracted successively with petroleum
ether (55 g), EtOAc (198 g), and n-BuOH
(300 g). The EtOAc fraction (198 g) was
repeatedly isolated on silica gel column
using a gradient of CHCl3–Me2CO (from
100:0 to 50:50) to give six fractions (I–
VI). Fraction IV (6.5 g) was isolated on
silica gel column using a gradient of
CHCl3–MeOH (50:1 to 10:1) to give four
fractions (IV-1, IV-2, IV-3, and IV-4).
Fraction IV-2 (1 g) was purified using
preparative HPLC (MeOH–H2O, 70:30),
flow rate 35 ml/min, wavelength 210 nm)
to afford compounds 1 (40 mg, 25 min)
and 2 (40 mg, 30 min).
3.2 Plant material
3.3.1 Mimengoside H
The fruits of B. lindleyana were collected
in December 2008 from Wanfou Mountain
of Shucheng County, Anhui Province of
China, and identified by Professor Shou-
Jin Liu, Department of Pharmacy, Anhui
University of Traditional Chinese Medi-
cine. The voucher specimen (AHTCM
200812) has been deposited in the
Herbarium of Anhui University of Tra-
ditional Chinese Medicine.
White amorphous powder; mp 205.6–
206.68C; a1D2:9 þ 3.3 (c 0.23, MeOH); UV
(MeOH) lmax 205.4 nm, IR (KBr) ymax
(cm21): 3425, 2972, 2943, 2922, 2901,
2876, 1629, 1465, 1386, 1347, 1327, and
1082. 1H NMR (DMSO-d6, 500 MHz) and
13C NMR (DMSO-d6, 125 MHz) spectral
data, see Table 1. HR-ESI-MS m/z:
669.4125
C37H62O8Cl, 669.4133).
[M þ Cl]2
(calcd
for
3.3 Extraction and isolation
3.3.2 Mimengoside I
Dried fruits of B. lindleyana (7 kg) were
extracted with 95% and 50% ethanol by
percolation. Concentrated under vacuum
to remove the part of ethanol, the
resulting aqueous suspension was
White amorphous powder; mp 231–
2328C; a1D2:9 þ 71.9 (c 0.23, MeOH),
UV (MeOH) lmax 202.0 nm; IR (KBr)
y
max(cm21): 3469, 3522, 2984, 2943,

346
D.-L. Wu et al.
Table 2. Effects of compounds 1 and 2 on MPPþ-induced cytotoxicity in PC12 cells.
Compound
Concentration (mM)
Absorbance (x ^ s)
ꢀ
Cell viability (% of control)
Control
Model
Compound 1
0
0
1.068 ^ 0.068
0.638 ^ 0.047
100
59.7##
0.1
1
10
100
0.658 ^ 0.083
0.782 ^ 0.061
0.533 ^ 0.061
0.602 ^ 0.080
64.2
65.5
56.4
58.0
**
**
*
*
Compound 2
0.1
1
10
100
0.620 ^ 0.080
0.632 ^ 0.098
0.645 ^ 0.072
0.630 ^ 0.093
58.1
59.1
60.4
58.9
*
*
**
*
Notes: values were þpresented as the mean ^ standard error of the mean (n ¼ 6). #P , 0.05, ##P , 0.01,
compared with MPP group; P , 0.05, P , 0.01, compared with control group.
* **
2892, 2858, 1639, 1467,1389, 1365, 1306,
and 1064. 1H NMR (DMSO-d6, 500 MHz)
and 13C NMR (DMSO-d6, 125 MHz)
spectral data, see Table 1. HR-ESI-MS
m/z: 637.3854 [M þ Cl]2 (calcd for
C36H58O7Cl, 637.3871).
3.5 MTT assay for cell viability
PC12 cells, obtained from the Chinese
Academia Sinica (Shanghai, China), were
cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% (v/v)
fetal bovine serum with humidified atmos-
phere of 5% CO2 at 378C. The differ-
entiated PC12 cells were pretreated with
different concentrations of compounds 1
and 2 (dissolved in DMSO to a final
concentration of DMSO ,0.3%) for 1 h
prior to exposure to MPPþ(1 mM). After
treatment with compound for 48 h, the
subsequent experiments were conducted in
triplicate. PC12 cells were seeded in a 96-
well culture plate at 1 £ 105 cells/well, and
the cell viability was measured by MTT
assay [11]. The viability of PC12 cells
decreased in a concentration-dependent
manner 48 h after MPPþ treatment. Com-
pound 1 showed potential protective
effects on MPPþ-induced cell injury in
the concentrations ranging from 0.1 to
1 mmol l21 (Table 2).
3.4 Acid hydrolysis of compounds 1
and 2
Each compound (2 mg) was hydrolyzed in
10 ml of 2 M HCl at 808C for 4 h. The
reaction mixture was extracted with
CHCl3 (10 ml £ 3). The aqueous layer
was concentrated to dryness to give a
residue, which was dissolved in pyridine
(1 ml), and then L-cysterine methyl ester
hydrochloride (2 mg) was added to the
solution. The mixture was heated at 608C
for 1 h, and trimethylchlorosilane (0.2 ml)
was added, followed by heating at 608C for
1 h. Then the solution was concentrated to
dryness and dissolved in water (1 ml £ 3),
followed by extraction with n-hexane
(1 ml £ 3). The hexane extract was sub-
jected to GC/MS analysis. The absolute
configuration of the monosaccharide was
confirmed to be L by comparison of the
retention times of monosaccharide
(14.39 min) derivatives with the standard
sample L-fucose (14.42 min).
Acknowledgments
This research was financially supported by the
Natural Science Foundation of Education
Department of Anhui Province, China (No.
KJ2011A185), and by the Natural Science
Foundation of Anhui University of Traditional
Chinese Medicine (No. 2010zr009B).

Journal of Asian Natural Products Research
347
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