Gentisides A and B, two new neuritogenic compounds from the traditional Chinese medicine Gentiana rigescens Franch

Article abstract of DOI:10.1016/j.bmc.2010.02.004

Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These compounds showed a significant neuritogenic activity at 30 μM against PC12 cells that was comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. Gentisides A and B showed parallel activity, indicating that the observed structural difference at the end of their alkyl chain did not affect neuritogenic activity.

Full text of DOI:10.1016/j.bmc.2010.02.004

Bioorganic & Medicinal Chemistry 18 (2010) 2131–2134
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Gentisides A and B, two new neuritogenic compounds from the traditional
Chinese medicine Gentiana rigescens Franch
Lijuan Gao, Jinyou Li, Jianhua Qi *
College of Pharmaceutical Sciences, Zhejiang University, Yu Hang Tang Road 388, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese
medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectro-
scopic methods and chemical derivatization. These compounds showed a significant neuritogenic activity
Received 5 January 2010
Revised 3 February 2010
Accepted 4 February 2010
Available online 10 February 2010
at 30 lM against PC12 cells that was comparable to that seen for the best nerve growth factor (NGF) con-
centration of 40 ng/mL. Gentisides A and B showed parallel activity, indicating that the observed struc-
tural difference at the end of their alkyl chain did not affect neuritogenic activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Neuritogenic compounds
Gentiana rigescens Franch
PC12 cells
Nerve growth factor
1
. Introduction
Neurotrophic factors such as nerve growth factor (NGF), brain de-
tisides A (1) and B (2) (Fig. 1), two new neuritogeniccompounds, were
isolated from a methanol extract of the traditional Chinese medicine
Gentiana rigescens Franch. In this paper, we report the isolation, struc-
rived neurotrophic factor (BDNF), and glial cell line derived neurotro-
phic factor (GDNF) play critical roles in neuronal development,
survival, and thefunctional maintenance ofneurons.1 Neurotrophic
factors have been targeted as potential therapeutic drugs for treat-
ment of neurodegenerative disorders. At this time, NGF has been
the most intensively studied and has been subjected to animal exper-
tures, and biological activity of these substances.
–3
2
2
. Results and discussion
.1. Isolation
4
iments. However, because it is a high molecular weight polypeptide,
A methanol extract of dried roots of G. rigescens was partitioned
between 80% aqueous MeOH and n-hexane. The active n-hexane
5
NGF is unstable and is also unable to cross the blood–brain barrier.
Therefore, its application as a medicine for the treatment of neurode-
generative disorders is assumed to be difficult. To address this issue,
considerable efforts have been made to find small molecules that
have neurotrophic properties similar to those of NGF and/or that
OH
O
1
'
7
HO
R
2
6
O
(CH2)15
can enhance the activity of endogenous neurotrophic factors.
3
1
PC12 cells, a line derived from a rat pheochromocytoma, respond
to NGF by switching from an immature chromaffin-cell-like pheno-
type to a sympathetic-neuron-like one, complete with the outgrowth
of long branching neurites. This cell line is therefore useful as a bioas-
say system for the investigation of potential drugs for neurodegener-
5
R =
21'
2'
7
20'
20'
23'
ative diseases. Our previous search for compounds that mimic and/
1
2'
or enhance the neuritogenic activity of NGF using the PC12 cell line
system resulted in theisolation of novel cerebrosides, thetermitomy-
cesphins, fromtheedibleChinesemushroomTermitomycesalbumino-
2
2
8
,9
sus and a series of steroid glycosides named linckosides from the
HO
(CH2)15
10
Okinawan blue starfish Linckia laevigata. In our present study, gen-
3
*
Corresponding author. Tel./fax: +8657188208627.
E-mail address: qijianhua@zju.edu.cn (J. Qi).
Figure 1. Structures of gentisides A (1) and B (2), and 20S-methyl docosanol (3).
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.004

2
132
L. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2131–2134
fraction was chromatographed on silica gel, then on ODS to yield
an active fraction. This was further purified by repeated reversed
phase HPLC to yield gentisides A (1, 0.00075% of dry wt.) and B
Table 1
1
H and 13C NMR data for gentisides A (1) and B (2) in CDCl
3
Carbon No.
1
2
(2, 0.0012%).
1Ha
13Cb
1Ha
13Cb
1
2
3
4
5
6
7
—
—
—
112.7
148.9
145.1
—
—
—
112.7
149.0
145.1
2
.2. Structure elucidation
Gentiside A (1) was obtained as a colorless powder with the
7.10 dd (8.0, 1.5) 119.7
6.80 t (8.0) 119.1
7.38 dd (8.0, 1.5) 120.6
7.10 dd (8.0, 1.5) 119.7
6.80 t (8.0) 119.1
7.37 dd (8.0, 1.5) 120.6
molecular formula C29
surement. The IR absorption band at 3469 cm
presence of a hydroxyl group. The proton nuclear magnetic reso-
nance ( H NMR) spectrum indicated the presence of two methyl
50 4
H O as determined by HR FT-ICR MS mea-
ꢀ
1
suggested the
—
170.5
65.8
28.6
26.0
—
170.5
65.8
28.6
0
0
0
0
1
2
3
4
4.34 t (6.5)
1.77 m
1.43 m
4.34 t (6.5)
1.78 m
1.44 m
1
(
CH
3
) groups (d
H
0.86), many aliphatic methylene (CH
1.14–1.77), a benzene ring (d
.38), one oxygen functionality (CH –O, d 4.34), and two singlet
5.65 and 11.00). The sharp signal of a hydroxyl
2
) and/or
26.0
0
-18
1.22–1.38 m
27.4–
1.10–1.38 m
27.1–
methine (CH) groups (d
7
hydroxyl groups (d
group at d
carboxyl group. The signals at d
ylenes d 1.14–1.77, d 26.0–39.1 suggested is the presence of a
long alkyl in this molecule. Two fully overlapped methyl doubleted
signals (d 0.86) revealed the presence of an isopropyl group at the
H
H
6.80–
c
d
30.0
30.1
n
H
0
1
9
1.14 m
39.1
1.10–1.38 m
27.1–
d
H
30.1
34.5
36.7
11.4
19.3
—
0
0
0
0
H
11.00 is due to the formation of hydrogen-bond with a
20
1.51 m
0.86 d (7.0)
0.86 d (7.0)
—
11.00 s
5.65 s
28.0
22.7
22.7
—
—
—
1.33 m
2
2
2
2
1
2
3
1.09, 1.30 m
0.85 t (7.0)
0.84 d (6.0)
11.00 s
H
0.86 (d) and many aliphatic meth-
Y
C
-OH
H
3-OH
5.68 s
—
C
end of the long alkyl chain. The signal at d 170.5 supported the
a
b
c
5
1
00 MHz, coupling constants (J in Hz) are in parentheses.
25 MHz.
presence of one carboxyl group. The 13C and DEPT NMR data com-
bined with the 2D NMR as well as HR-MS data, confirmed the pres-
d: 27.4, 29.2, 29.5, 29.6, 29.7, 30.0.
d: 27.1, 29.2, 29.5, 29.6, 29.7, 30.1.
d
3 2 2
ence of two CH , eighteen CH , one CH, one CH –O, one benzene
ring with three substituted positions, and one carboxyl group. Tak-
ing the molecular formula into consideration, two of the three
substituted positions of benzene ring turned out to be two hydro-
xyl groups. The analysis of COSY and HOHAHA spectra led to the
the minimum concentration with detectable activity and the max-
imum concentration without cytotoxicity observed 48 h after
treatment. The gentisides showed a dose-dependent increasing
activity ranging across the test range. At the best concentration
without obvious cytotoxicity, gentisides exhibited neurite out-
growth equivalent to that seen with the positive NGF control at
its best concentration (40 ng/mL). The solvent control also induced
few neurite outgrowths. Gentisides A (1) and B (2), differing only in
the isopropyl group and isobutyl group at the end of side chain,
respectively, showed significant neuritogenic activities of 82%
(Fig. 3a), and 87% (Fig. 3b) at a concentration of 30 lM on day 2
after the treatment. Moreover, gentisides A (1) and B (2) showed
quite similar activity at all concentrations tested. At the concentra-
tion of 100 lM, although cytotoxicity was clearly observed, almost
0
0
determination of the partial structures (C-4 to C-6, C-1 to C-3 ,
0
0
0
C-19 to C-21 and 22 , and a long alkyl chain) depicted with the
bold bonds in Figure 2. These partial structures were connected
by the long-range H–C correlations obtained by an HMBC experi-
ment to give a gross structure of 1. The HMBC correlations estab-
lishing the gross structure of 1 were as follows: a proton of
hydroxyl group at C-2 to C-1, C-2, and C-3; a proton of hydroxyl
0
group at C-3 to C-2, C-3, and C-4; H-6 to C-7; H-1 to C-7. Important
HMBC correlations are summarized in Figure 2 with arrows. Thus,
the structure of 1 was elucidated as shown in Figure 1.
Gentiside B (2) was obtained as a colorless powder with the
all of the living cells possessed long neurite outgrowths in the pres-
ence of either compound. Thus, the small structural difference at
the end of the alkyl chain did not affect the biological activity of
the gentisides on PC12 cells.
52 4
molecular formula C30H O as determined by an HR FT-ICR MS
1
13
measurement. The H and C NMR of 2 were superimposable on
those of 1 (Table 1) except for the signals at the end of the long al-
0
kyl chain. The 2 possesses an isobutyl group at C-19 , as deter-
Subsequently, possible synergistic effects of gentisides were
examined on the neurite outgrowth of PC12 cells induced by low
concentration of NGF (1 ng/mL). No significant synergistic activity
was observed, which is probably due to the high activity of genti-
sides alone (data not shown).
Interestingly, 2,3-dihydroxybenzoic acid obtained from alkaline
hydrolysis of gentiside B did not induce obvious neurite outgrowth
in PC12 cells (data not shown).
mined by analysis of 2D NMR. Subsequently, 2 was hydrolyzed
under weakly basic conditions to afford a 20S-methyl docosanol
1
(
3) molecule, which was confirmed by the comparison of
H
1
1,12
NMR, MS as well as specific rotation, with those reported.
The structure of 2 was thus determined as shown in Figure 1.
2
.3. Biological activity
Figure 4 shows the morphological changes of PC12 cells after
treatment with gentisides A (1, Fig. 4c) and B (2, Fig. 4d) in compar-
ison with the solvent control (1% DMSO, Fig. 4a) and the positive
NGF control (40 ng/mL, Fig. 4b). When treated with gentisides at
The neuritogenic activity of gentisides A (1) and B (2) was eval-
uated using PC12 cells and compared to the activity of 40 ng/mL
NGF. Figure 3 shows the dose–dependent increase in the percent-
age of PC12 cells with long neurite outgrowths. The gentiside con-
30 lM, the cells extended long multipolar and bipolar neurite out-
centrations were varied from 1 to 30 lM, which corresponded to
Figure 2. Gross structure of gentiside A (1) with selected HMBC correlations.

L. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2131–2134
2133
1
00
100
9
0
0
0
0
0
0
0
0
0
a
90
80
70
60
50
40
30
20
10
0
b
8
7
6
5
4
3
2
1
0
C
1(1)
3(1) 10(1) 30(1) NGF
40ng/ml)
C
1(2) 3(2) 10(2) 30(2) NGF
(40ng/ml)
(
Concentration (µM)
Concentration (µM)
Figure 3. Dose-dependent response of the NGF-mimicking activity of gentisides A (1, a) and B (2, b) 48 h after treatment. Activity is represented by percentages of PC12 cells
with neurite outgrowths longer than the diameter of the cell body. C: control (1% DMSO); NGF (40 ng/mL): positive control.
jiang province, China. The sample was powdered and soaked in
MeOH (10 L) for five days at room temperature with occasional
stirring. The supernatant was separated by filtration and concen-
trated to give 353.6 g of a crude extract, which was divided into
two portions and subjected to solvent partition. In a typical case,
a portion (176.8 g) of extract was dissolved in 80% aqueous MeOH
(
1.5 L) and then washed three times with n-hexane (500 mL for
each). The n-hexane fraction from the two portions were combined
and concentrated to give a light green viscous oil (14.6 g), which
was chromatographed on silica gel (200–300 mesh, Yantai Chemi-
cal Industry Research Institute) and eluted with a n-hexane/EtOAc
step gradient (100:0, 90:10, 85:15, 80:20, 70:30, 50:50) to afford
2
(
4 fractions. The active sample, eluted with n-hexane/EtOAc
90:10, 85:15), was separated through ODS (Cosmosil 75 C18-
OPN, Nacalai Tesque) and eluted with a MeOH/H O step gradient
80:20, 85:15, 90:10, 95:5, 100:0), and MeOH/CHCl (1:1), succes-
sively, to afford 42 fractions. The active sample (203.4 mg) eluted
with MeOH/H O (90:10, 95:5) was subjected to HPLC [Develosil
ODS-HG-5 (u 10/250 mm), Nomura chemical, flow rate: 3 mL/
min, MeOH/H O (98:2)] to give two active fractions. The active
2
(
3
2
2
fraction with shorter retention time was purified by HPLC [Develo-
sil ODS-HG-5 (u 10/250 mm), Nomura chemical, flow rate: 3 mL/
min, 85–100% aqueous EtOH in 90 min linear gradient] to give pure
Figure 4. Photomicrographs of PC12 cells under a phase-contrast microscope 48 h
after treatment. (a) Solvent control (1% DMSO), (b) NGF (40 ng/mL), (c) gentiside A
(1, 30 lM), and (d) gentiside B (2, 30 lM).
R
gentiside A (1) (14.8 mg, t = 43.0 min). Another active fraction was
also purified by HPLC [Develosil ODS-HG-5 (u 10/250 mm), Nom-
ura chemical, flow rate: 3 mL/min, 90–100% aqueous EtOH in
growths 48 h after treatment, which were similar to those pro-
duced following treatment with NGF at 40 ng/mL. Control cells
cultured without added compounds induced few short neurite
outgrowths.
90 min linear gradient] to yield gentiside
B (2) (24.2 mg,
tR = 31.9 min) as the active component.
3
.2.1. Gentiside A (1)
Colorless powder, IR (KBr): 3469, 2920, 1671, 1469, 1311, 1155,
3
3
. Experimental
ꢀ1
1
071 cm ; UV (MeCN) kmax (log e): 314 nm (5.82); HR FT-ICR MS
+
1
.1. General procedures
50 4
m/z 485.3610 (M+Na) , calcd for C29H O Na 485.3601; for H and
1
3
C NMR see Table 1.
Preparative HPLC was performed using ELITE P-230 pumps, and
the optical rotations were measured on a JASCO P-1030 digital
polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 and
the UV spectra were recorded on a PGENERAL TU-1901. High-res-
olution (HR) MS measurements were performed on a Apex III (7.0
Tesla) FT-ICR mass spectrometer (Bruker, Billerica, MA, USA) using
3.2.2. Gentiside B (2)
2
D
2
Colorless powder, ½
aꢁ
+2.4 (c 2.04, CHCl
3
); IR (KBr): 3476,
):
ꢀ1
2920, 1674, 1468, 1309, 1153, 1073 cm ; UV (MeCN) kmax (log
e
+
321 nm (6.01); HR FT-ICR MS m/z 499.3775 (M+Na) , calcd for
C H O Na 499.3758; for H and C NMR see Table 1.
30 52 4
1
13
3
CF COONa as an external calibration standard. The NMR spectra
were recorded on a Bruker AV III-500 spectrometer, and the NMR
3.3. Degradation of 2
chemical shifts in d (ppm) were referenced to the solvent peaks
of d
C
77.0 and d
H
7.26 for CDCl
3
.
Gentiside B (2) (8 mg) in methanol (4 mL) was treated with
potassium carbonate (20 mg) at room temperature for 27 h. The
3
.2. Extraction and isolation
2
crude product was dissolved in H O (10 mL) and extracted three
times with n-hexane (10 mL). The n-hexane layers were combined,
A commercial preparation of dried roots of G. rigescens (1980 g,
dried wt.) was purchased from HuQingYuTang in Hanzhou, Zhe-
concentrated, and purified by silica gel open column eluted with
2
2
CHCl
3
to afford the 3 (4.4 mg): colorless powder, ½
a
ꢁ
+3.7 (c
D

2
134
L. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2131–2134
) [lit.11
½
aꢁ
23
D
+4.0], H NMR (CDCl
1
, 500 MHz) d: 3.64 (2H,
2
.04, CHCl
3
3
Acknowledgments
t, J = 6.5 Hz), 1.57 (2H, m), 1.20–1.38 (35H, m), 1.07–1.14 (2H, m),
0
.85 (3H, t, J = 7.0 Hz), 0.84 (3H, d, J = 6.0 Hz).12
This work was financially supported by NSFC (30873152) and
Science Foundation of Chinese University (2009QNA7023).
3
.4. Bioassay methods
References and notes
Biological activity was evaluated according to the methods de-
scribed in our previous paper.8 Briefly, 20,000 of PC12 cells in
,9
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2
3
.
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Berninger, B.; Poo, M. M. Nature 1999, 401, 862.
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2
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by 1 mL of serum-free DMEM medium containing a test sample
and DMSO (1%). In the case for evaluation of the NGF-enhancement
effects, the medium was replaced by 1 mL of serum-free DMEM
medium containing 1.0 ng of NGF (Recombinant Human b-NGF,
SIGMA) in addition to a test sample. The morphological changes
of the cells were monitored under a phase-contrast microscope
at every 24 h. About one hundred cells were counted from a ran-
domly chosen sight and this was repeated three times.
6. Brinton, R. D.; Yamazaki, R. S. Pharm. Res. 1998, 15, 386.
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Greene, L. A.; Tischler, A. S. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2424.
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