Neuritogenesis of herbal (+)- and (-)-syringaresinols separated by chiral HPLC in PC12h and neuro2a Cells

Article abstract of DOI:10.1248/bpb.25.791

Syringaresinol isolated from Epimedium koreanum NAKAI and Magnolia officinalis REHB. et WILS. was subjected to optical resolution by chiral HPLC to give (+)- and (-)-enantiomers. The two syringaresinol enantiomers, as well as a mixture of their glucosides, showed dose-dependent neuritogenesis in a concentration range from 0.24 to 24 μM in PC12h cells.

Full text of DOI:10.1248/bpb.25.791

June 2002
Notes
Biol. Pharm. Bull. 25(6) 791—793 (2002)
791
Neuritogenesis of Herbal (؉)- and (؊)-Syringaresinols Separated
by Chiral HPLC in PC12h and Neuro2a Cells
Kenzo CHIBA, ,a Matsumi YAMAZAKI,a Emiko UMEGAKI,a Ming Run LI,b Zhen Wen XU,b
*
a
Sumio TERADA,c Michihiro TAKA,c Noriyuki NAOI,d and Tetsuro MOHRI
a Department of Biodynamics, Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3 Kanagawa-machi,
Kanazawa, Ishikawa 920–1181, Japan: b Tianjin Institute of Medical and Pharmaceutical Science; 96 Gui Zhou Road,
c
Heping District, Tianjin 300070, China: Natural Products Research Laboratory, Research Laboratory, Zenyaku Kogyo
Co., Ltd.; 2–33–7 Ohizumi-machi, Nerima-ku, Tokyo 178–0062, Japan: and d Medical Devices Division, Kaneka
Corporation; 3–2–4 Nakanoshima, Kita-ku, Osaka 530–0005, Japan.
Received November 1, 2001; accepted March 4, 2002
Syringaresinol isolated from Epimedium koreanum NAKAI and Magnolia officinalis REHD. et WILS. was sub-
jected to optical resolution by chiral HPLC to give (؉)- and (؊)-enantiomers. The two syringaresinol enan-
tiomers, as well as a mixture of their glucosides, showed dose-dependent neuritogenesis in a concentration range
from 0.24 to 24 mM in PC12h cells.
Key words syringaresinol; optical isomer; neuritogenesis; PC12h cell; Epimedium koreanum
We have carried out a continuing study of the neuron-stim-
Magnolia officinalis REHD. et WILS., collected in China,
ulative substance in plants and found many lipophilic con- was purchased from Matsuura Yakugyo Co., Ltd. (Nagoya,
stituents that promote the differentiation (neurite outgrowth) Japan). The voucher specimen (No 228110) was deposited in
of paraneurons, such as PC12h cells.1,2) The lipophilic ex- the natural product laboratory of the Research Laboratory,
tracts of the aerial parts of Epimedium koreanum NAKAI or Zenyaku Kogyo Co., Ltd. Syringaresinol-4Ј-O-b-D-glucoside
Epimedium macranthum MORR. et DECNE. have been demon- (100 mg) isolated from the bark of M. officinalis according to
strated to have neuritogenetic activity in PC12h cells and the method of Yahara et al.6) was hydrolyzed by b-glucosi-
stimulate these cells excitedly to respond to carbachol.2) We dase (Sigma, U.S.A.) in 0.1 M acetate buffer (pH 5.0) at 37 °C
have preliminarily found an active component of the Et2O overnight. The reaction mixture was extracted three times
extract of E. koreanum syringaresinol, which was found as it- with EtOAc to afford syringaresinol (31 mg). Each of the two
self or its glucosides in many of traditional Chinese drugs, racemic syringaresinol samples obtained from E. koreanum
3)
including Acanthopanax senticosus HARMS and the bark of and M. officinalis was subjected to HPLC using a Chiralcel
Eucommia ulmoides OLIV.4) It was previously shown to have OD column (Daicel Chemical, Japan) and EtOH–n-hexane
a prominent effect on PC12h cells.5) But the syringaresinol (8 : 2) as a mobile phase to give optically pure isomers (col-
samples we isolated from the extract of E. koreanum seemed orless needles).
to be peculiarly racemic mixtures on the data of HPLC
Optical rotations were measured on a Perkin-Elmer 247
analysis with a chiral stationary phase. We report here the polarimeter. High resolution mass spectra were recorded on a
separation of optical isomers of syringaresinol from each of JMS-SX102 MS instrument. NMR spectra were recorded on
two species of plant, and the comparison of the biological ac- a JEOL EX270 NMR spectrometer. HPLC was performed on
tivity of individual enantiomers and a glucoside mixture.
a Chiralcel OD (Daicel Chemical) column (0.46ϫ25 cm)
using a JASCO TRI ROTAR-V PUMP with a UVIDEC-100-
V UV detector (eluent, EtOH–n-hexane (8 : 2) (flow rate,
0.5 ml/min; detection, UV at 254 nm).
MATERIALS AND METHODS
The concentrated Et2O extract (35.2 g) of E. koreanum col-
PC12h cells were generously donated by Dr. Hatanaka,
lected in northern China in July, 1998 was prepared at the Osaka University, Japan, and cultured in Dulbecco’s modified
Tianjin Institute of Medical and Pharmaceutical Science and Eagle medium supplemented with precolostrum calf serum
chromatographed on a silica gel (Kieselgel 60) column by a and heat-inactivated horse serum for growth, as described
successive elution with CHCl3, CHCl3–MeOH (3 : 1) and previously.7) Neuro2a cells were purchased from the Institute
MeOH (each 2 l). The evaporation residue (17.5 g) of the for Fermentation, Osaka, Japan, and cultured in Eagle’s mini-
CHCl3 fraction was chromatographed on a silica gel column mum essential medium supplemented with non-essential
by a succcssive elution with n-hexane, n-hexane–EtOAc amino acids and fetal bovine serum for growth. After 24 h of
(5 : 1), benzene–EtOAc (1 : 1), benzene–EtOAc–MeOH (1 : 1 : culture, the culture medium was replaced by serum-free
0.1) and benzene–EtOAc–MeOH (1 : 1 : 1). The most bioac- medium to treat these cell lines with test compounds as spec-
tive fraction from the benzene–EtOAc–MeOH (1 : 1 : 0.1) ified in the results. Syringaresinol-4Ј-O-b-D-glucoside was
eluate was chromatographed on TSK HW 40 (Tosoh, Japan) dissolved in distilled water and (ϩ)- and (Ϫ)-syringaresinol
with an eluent, MeCN–H2O (1 : 1). The main fraction of the in 75% dimethylsulfoxide before use. The neurite outgrowth
eluate was purified by Sephadex-LH20 chromatography with of these cells was evaluated by measuring the length of the
MeOH–H2O (1 : 1) to give syringaresinol (3 mg), which was longest neurites of individual cells following 48 h of treat-
identified by comparison with UV, MS and NMR spectral ment with the test compounds as described previously.7) One
data of an authentic sample.
hundred cells in ten random fields in two culture dishes were
To whom correspondence should be addressed. e-mail: k-chiba@hokuriku-u.ac.jp
© 2002 Pharmaceutical Society of Japan

792
Vol. 25, No. 6
averaged for each treatment.
to the elution profile on chiral chromatography.
The Neuritogenic Activity of Optical Isomers of Sy-
ringaresinol in PC12h and Neuro2a Cells The neurito-
genicity of (ϩ)-syringaresinol obtained from S. baicalensis
RESULTS AND DISCUSSION
Optical Resolution by HPLC of Syringaresinol Sy- and (Ϫ)-syringaresinol from Scutellaria scandens BUCH.-
ringaresinol samples isolated from E. koreanum and M. offic- HAM. ex D. DON was already reported with PC12h cells.5)
inalis were identified by comparison with UV, MS and NMR Here, we elaborate upon the effect of isolated enantiomers of
spectral data of an authentic sample. Their optical rotation syringaresinol as well as their glucoside mixture from the
was estimated at almost naught, but varied from sample to bark of M. officinalis on PC12h and Neuro2a cells. Consis-
sample. So they were then subjected to chiral chromatograhy tent with our previous results, all of the glucosides and the
(Fig. 1). On the basis of the results, the syringaresinol sam- two enantiomers were individually effective on the neurite
ples were supposed to be racemic mixtures.
outgrowth in both PC12h and Neuro2a cells (Fig. 2). At
Actually, identificatin of (ϩ)- and (Ϫ)-syringaresinol on about 0.2 mM of these compounds the effect was comparable
the chromatograms was done by comparison with those of between them in either cell line. But at higher doses (about 2
syringaresinol samples isolated from M. officinalis, because and 20 mM) of each, two enantiomers showed a generally
the amount was not enough to determine the optical rotation stronger effect than the glucoside, especially, the (Ϫ)-isomer
of separate enatiomers of samples from E. koreanum. The was discernibly more effective than any of the other com-
physical data of the enantiomers (in preceding and subse- pounds at a concentration of 24 mM in either cell line. The
quent eluates, respectively) from M. officinalis are (ϩ)-sy- neurite outgrowth activity of the mixture containing two
ringaresinol ([a]D20 ϩ53.3°, cϭ0.36, CHCl3; mp 187.5—189.5 enantiomers of syringaresinol was about similar in extention
°C) and (Ϫ)-syringaresinol ([a]D20 Ϫ50.6°, cϭ0.32, CHCl3; mp to the additive activity of each isomer (data not shown).
188.5—190.5 °C), occurring in the ratio of ca. 2 : 1 according
All of these results indicate that both (ϩ)- and (Ϫ)-sy-
ringaresinol, as well as a mixture of optical isomers of sy-
ringaresinol-4Ј-O-b-D-glucoside, are effective on neurito-
genicity in cultured paraneurons, and that a racemic mixture
of (ϩ)- and (Ϫ)- syringaresinol would be responsible, at least
partly, for the neurite outgrowth, i.e. cell differentiation or
activation, of the paraneurons with the Et2O extract of E. ko-
reanum. It may be interesting to investigate the effect of
these isomers on such activities of the central nervous system
of animals as memory and behaviors, because the Chinese
raw drug “Japanese name; Inyokaku” made of the dried herb
of Epimedium species is an important element in many ton-
ics, and is used in the treatment of aging-related amnesia and
impotence.8)
Syringaresinol and its related compounds have several
pharmacological activities including an inhibitory effect on
cyclic AMP phosphodiesterase (PDE),9) a prolonging effect
on the exercise time to exhaustion in chronic swiming
stressed rat,10) and an inhibitory effect on gastric ulcer.11) The
inhibitory activity on PDE of (ϩ)-syringaresinol-di-b-gluco-
side and the enzymatic hydrolysate (syringaresinol) was con-
sidarable (IC50: 12.5ϫ10Ϫ5 M and 17.5ϫ10Ϫ5 M, respectively),
and the activity of (Ϫ)-syringaresinol-di-b-glucoside was
Fig. 1. Optical Resolution of Syringaresinol Samples by HPLC
The syringaresinol samples were applied to a Chiralcel OD column (0.46ϫ25 cm)
and eluted isocratically with EtOH–n-hexane (8 : 2) at a flow rate of 0.5 ml/min, with
absorption monitored at 254 nm. The elution profiles (A and B) represent sy-
ringaresinol samples isolated from E. koreanum and M. officinalis, respectively. I and II
indicate (ϩ)- and (Ϫ)-syringaresinol, respectively.
Fig. 2. The Neuritogenesis of Syringaresinol-4Ј-O-b-D-glucoside and (ϩ)- and (Ϫ)-Syringaresinol in PC12h and Neuro2a Cells
Cells were cultured with or without (control) concentrations of test compounds as indicated for 2 d. The neurite outgrowth was measured as described in Materials and Methods
for 100 cells in each group and compared with the control, or between treated groups. The values are shown by the meansϮS.E.M. All of the means of treated groups are signifi-
cantly (pϽ0.01) large compared with the control. Statistical analysis was performed by ANOVA and Bonferroni’s test.

June 2002
793
less (IC50: Ͼ50ϫ10Ϫ5 M), with specificity in the chiral iso-
mers. Neuritogenesis was induced by cAMP in both PC12
and Neuro 2a cells used in the present study.12,13) Therefore
syringaresinol may be able to induce cAMP-dependent neuri-
togenesis through PDE inhibition. However, in contrast to the
inhibitory activity on PDE mentioned above, the neuriogenic
activity of the (Ϫ) isomer was more effective than the (ϩ)
isomer. Additional studies will be required to show the
mechanism of syringaresinol.
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Acknowledgment This work was supported in part by
grants of the special research fund of Hokuriku University.