Analgesic components of Saposhnikovia root (Saposhnikovia divaricata)

Article abstract of DOI:10.1248/cpb.49.154

By activity-oriented separation using the writhing method in mice, the analgesic components of Saposhnikovia root (Saposhnikovia divaricata Schischkin; Umbelliferae) were identified to be chromones, coumarins, polyacetylenes and 1-acylglycerols. Two new c

Full text of DOI:10.1248/cpb.49.154

154
Chem. Pharm. Bull. 49(2) 154—160 (2001)
Vol. 49, No. 2
Analgesic Components of Saposhnikovia Root (Saposhnikovia divaricata)
Emi OKUYAMA,* Tetsuya HASEGAWA, Takamitsu MATSUSHITA, Haruhiro FUJIMOTO, Masami ISHIBASHI,
and Mikio YAMAZAKI
Faculty of Pharmaceutical Sciences, Chiba University, 1–33 Yayoicho, Inage-ku, Chiba 263–8522, Japan.
Received August 18, 2000; accepted November 7, 2000
By activity-oriented separation using the writhing method in mice, the analgesic components of Saposh-
nikovia root (Saposhnikovia divaricata Schischkin; Umbelliferae) were identified to be chromones, coumarins,
polyacetylenes and 1-acylglycerols.
Two new components, divaricatol and (3؅S)-hydroxydeltoin, were also isolated. The most potent analgesia
was observed in chromones such as divaricatol, ledebouriellol and hamaudol, which inhibited writhing inhibition
at an oral dose of 1 mg/kg in mice. Acylglycerols also showed inhibition significantly at a dose of 5 mg/kg. In some
pharmacological tests using sec-O-glucosylhamaudol, the compound showed analgesia by the tail pressure and
the Randall & Selitto methods, and its writhing inhibition was not reversed by naloxone.
Key words Saposhnikovia divaricata; analgesia; divaricatol; (3ЈS)-hydroxydeltoin; chromone; 1-acylglycerol
In view of their scientific understanding and potential dose of 3 g/kg. The separation of the extract was mainly car-
usage, we have continuously investigated traditional medi- ried out by following the writhing inhibition in mice with
cines to clarify the active components and their pharmaco- oral administration of samples as an isolation guide (Chart
logical properties.1—4) Saposhnikovia root (Saposhnikovia di- 1). Partition using ethyl acetate, n-butanol and water concen-
varicata SCHISCHKIN, syn. Ledebouriella seseloides WOLFF; trated the activity to the ethyl acetate fraction, which was fur-
Umbelliferae) is an important Chinese and Japanese tradi- ther separated by Sephadex LH-20 chromatography. The ob-
tional medicine, called Fang-feng and Bofu, respectively.5,6) tained analgesic fraction, fr. 1-C, also showed the prolonga-
The herb is listed as a high-grade drug in the old Chinese tion effect on pentobarbital-induced sleeping time in mice.
Materia Medica, Shen Nung Pen Tsao Ching, and is applied The following silica gel chromatography, however, gave two
for headaches, vertigo, generalized aching and arthralgia due active fractions, fr. 2-A and fr. 2-B. The latter fraction
to “wind, cold and dampness” in the traditional medical sys- showed only analgesia at a dose of 150 mg/kg and no prolon-
tem.7) It has been used in many prescriptions, including those gation effect at a dose of 200 mg/kg, while the former exhib-
for the treatment of analgesic symptoms.
ited both effects at doses of 300 mg/kg and 400 mg/kg, re-
Pharmacological experiments using the extract reported its spectively. Fraction 2-B was further separated by repeated
suppression of adjuvant arthritis, inhibitory effects on the chromatography using silica gel, ODS and Sephadex LH-20
CNS and peptic ulcers, and fegrifugal analgesic, anti-convul- guided by the analgesic effect. Compounds 1 and 2 were fi-
sant and anti-inflammatory activities, etc.8—11) Chemical nally obtained as the major components, as shown in Chart
study on this herb had been done, and many components 1-1, together with minor components of compounds 3—10,
such as chromones, coumarins and polyacetylenes were iso- and 12 in Chart 1-2. The other active fraction, fr. 2-A, was
lated.12—14) However, it has not been clearly identified which separated by silica gel chromatography to give two types of
components contribute to the pharmacological efficacy, such active fractions, fr. 2-Ab and fr. 2-Ad. Compared with the re-
as analgesia, of this traditional medicine. In Japan, Glehnia sults of Glehnia root, these fractions were found to be poly-
root, named Hama-Bofu (Glehnia littoralis FR. SCHMIDT ex acetylenes- and furanocoumarins-containing fractions, re-
MIQUEL; Umbelliferae), is used for the same purpose as Bofu spectively.2) Fraction 2-Ab showed analgesia at a dose of
(Saposhnikovia root), and in prescriptions the two medicines 150 mg/kg without the sleeping-prolongation effect at
are often confused. With respect to Glehnia root, we recently 200 mg/kg. From this fraction, compound 11 was obtained as
identified the active components which are responsible for a major component. Fraction 2-Ad, showing both analgesic
the analgesia and/or a prolongation effect on pentobarbital- and sleeping-prolongation effects at doses of 80 and 50 mg/
induced hypnosis caused by the inhibition of liver-metaboliz- kg, respectively, was analyzed by ODS-HPLC (acetonitrile–
ing enzymes.2) Some of the active components in Glehnia water) with photodiode array detection (Fig. 1). The follow-
root, such as furanocoumarins and polyacetylenes, are the ing components in fr. 2-Ad were detected by comparison
same as the constituents in Bofu. By applying activity-ori- with each retention time and the UV spectrum of the authen-
ented separation to the extract of Saposhnikovia root, the tic samples: psoralen, xanthotoxin, bergapten, imperatorin,
analgesic components of this herbal medicine can be clari- deltoin and isoimperatorin.
fied in this paper, and the activity-mechanisms of the compo-
nents are also discussed.
The structures of the isolated compounds were estimated
by spectra, including 2D-NMR, and compounds 1, 2, 6 and 7
were identified by comparison with the reported data to be
sec-O-glucosylhamaudol, cimifugin, ledebouriellol and
Results and Discussion
The methanol extract of Saposhnikovia root showed an hamaudol, respectively.12,13,15) Compound 3 was identified
analgesic effect at a dose of 2 g/kg in mice by the acetic acid- with isofraxidin, which has been isolated from Fraxinus
induced writhing method. The extract also caused a prolon- japonica, and compound 5 with fraxidin reported to be pro-
gation effect on pentobarbital-induced hypnosis in mice at a duced by hydrolysis of fraxin methyl ether.16) Compounds 4
To whom correspondence should be addressed. e-mail: emi@p.chiba-u.ac.jp
© 2001 Pharmaceutical Society of Japan

February 2001
155
Chart 1-1. Activity-Oriented Separation Procedure of Saposhnikovia Root [Writhing Inhibition: % vs. Control], (Sleeping Time: % vs. Control)
a) 3 g/kg, b) 2 g/kg, c) 1.5 g/kg, d) 600 mg/kg, e) 500 mg/kg, f) 400 mg/kg, g) 300 mg/kg, h) 200 mg/kg, i) 150 mg/kg, j) 100 mg/kg, k) 80 mg/kg, l) 60 mg/kg, m) 50 mg/kg.
:
pϽ0.05, : pϽ0.01.
MS. The H- and 13C-NMR of 8 were similar to those of 6
(ledebouriellol), except there was no observation of the sig-
nals of an angeloyl group. The signals at d 2.01 (3H, s) in the
1H-NMR and d 21.0 and 170.3 in the 13C-NMR were as-
signed to an acetyl group in 8, instead of an angeloyl group.
The compound, therefore, was elucidated as a 3Ј-O-acetyl
derivative and named divaricatol. The stereochemistry at C-
3Ј position was determined to be S-configuration because of
very similar ORD and CD to those of 3Ј-O-acetylhamaudol
and 6, respectively.12,15)
1
and 11 were directly identified with the authentic scopoletin
and panaxynol, respectively.
The NMR of 9 and 10 suggested the compounds to be
glycerol monoacylates. The Mass spectra gave the molecular
ions of m/z 354 and 356, and characteristic fragment ions of
1
m/z 262 and 264, respectively. The H- and 13C-NMR of
compound 9 were very similar to those of glycerol monoli-
noleate.17) The stereochemistry of the (2R)-glycerol moiety
was considered by comparison of its optical activity, [a]589
Ϫ3°, to the reported data of R(Ϫ)-glycerol monolinoleate,
[a]D Ϫ5.2°.18) Compound 10 was estimated to be glycerol
Compound 12 is a white powder, [a]589 Ϫ45°, the HR-
1
monooleate by comparing the H- and 13C-NMR spectra of
FAB-MS spectrum of which gave the molecular formula,
1
C19H20O6. The H- and 13C-NMR suggested the compound
oleic acid together with those of 9. Compounds 3, 5, 9 and
10 were isolated from this plant for the first time.
Compounds 8 and 12 were new compounds, and the struc-
tures were estimated as follows. Compound 8 is a white crys-
talline powder, mp 168—171 °C, [a]589 Ϫ30°. The molecular
formula, C17H18O7 (MW 334), was obtained by the HR-FAB-
was a coumarin derivative having a hydroxy and an angeloyl
group in the C-ring. The positions at CЈ-3 and CЈ-2, respec-
tively, were determined by the PFG-HMBC cross peaks ob-
served between the methyl groups at d 1.66 and 1.67 and d
98.2 (C-2Ј), and between the hydroxyl group at d 5.05 and d

156
Vol. 49, No. 2
Chart 1-2. Activity-Oriented Separation Procedure of Saposhnikovia Root [Writhing Inhibition: % vs. Control]
k) 80 mg/kg, l) 60 mg/kg, m) 50 mg/kg, n) 40 mg/kg, o) 30 mg /kg, p) 20 mg/kg, q) 5 mg/kg, r) 1 mg/kg. : pϽ0.05, : pϽ0.01.
Fig. 1. HPLC Profile of Fr. 2-Ad by Photodiode Array Detection
Retention time, min: peak 1 (psoralen), 38.8; peak 2 (xamthotoxin), 41.8; peak 3 (bergapten), 50.3; peak 4 (imperatorin), 84.6; peak 5 (deltoin), 100.6; peak 6 (isoimperatorin),
101.8.
in the structure shown in Chart 2.19) Trans substitution at the
2Ј and 3Ј-position in the C-ring of 12 was estimated by the
NOEs between d 5.51 (3Ј-H) and d 1.66 and 1.67 (4Ј-di-
72.4 (C-3Ј) and d 98.2 (C-2Ј). The 2D-NMRs did not con-
clude a five- or six-membered ring for the C-ring, since no
typical correlation such as a cross peak between d 4.73 and d
164.4 (2Ј-H/C-7 in the case of the former) or d 167.2 (3Ј-
H/carbonyl C in the latter case) was clearly observed in
HMBC. The chemical shifts of the methyl groups, however,
were similar to those of deltoin rather than the six-membered
derivative, L1-2 from Libanotis laticalycina, which resulted
1
methyl) in the H-NMR. The absolute stereochemistry was
obtained by direct comparison of the CD with deltoin, which
showed the similarity in both compounds (see experimental
part). Therefore, compound 12 was determined as (3ЈS)-hy-
droxydeltoin.

February 2001
157
Chart 2. Structures of the Components of Saposhnikovia Root
Figure 2 summarizes the analgesic effects of the compo-
nents by the acetic acid-induced writhing method in mice.
The chromones such as 1 (sec-O-glucosylhamaudol) and 2
(cimifugin) indicated analgesia significantly by oral adminis-
tration of 40 and 80 mg/kg, respectively. Compound 7, the
aglycone part of 1, increased the potency to exhibit a signifi-
cant effect at doses of 1, 5 and 10 mg/kg, although it did not
show clear dose dependency. Both 6 and 8, having a dihy-
dropyran-type C-ring, showed a potent analgesic effect at
doses of 1 and 5 mg/kg, in spite of the modification with
acyl-substituents at the C-3Ј position and a hydroxy group at
C-11. Although the other compounds having a dihydrofuran-
type C-ring, except for cimifugin and deltoin, were not iso-
lated by this activity-oriented isolation, some of them have
been reported as major components in Saposhnikovia
root.12,13) Therefore, the compounds, such as prim-O-gluco-
sylcimifugin, 4Ј-O-glucosyl-5-O-methylvissaminol and 5-O-
methylvissaminol, which were kindly offered by Dr. T.
Deyama, were tested at a dose of 10 mg/kg. None of these
showed significant effects at this dose. For the potency of
analgesic effect of chromones, therefore, dihydropyran-type
C-ring without glycosylation may have an important role.
The writhing inhibition of coumarins, such as scopoletin
(37% inhibition, pϽ0.01, 50 mg/kg), has been reported in
Glehnia root.2) Compound 3 (isofraxidin) also showed the ac-
tivity to a similar extent (40% inhibition, pϽ0.01, 50 mg/kg).
It has been reported in a patent that a mixture of 1-acyl-
glycerols from Aconitum chinense inhibited carrageenan-in-
duced edema by peritoneal injection of 100 mg/kg.20) In our
writhing test, both 1-acylglycerols, 9 and 10, inhibited the
writhing at a lower dose, 5 mg/kg orally.
Our previous paper on Glehnia root has already summa-
rized the writhing inhibitions at doses of 50—100 mg/kg of
11 (panaxynol) and furanocoumarins such as psoralen, xan-
thotoxin, bergapten, imperatorin and isoimperatorin, which
were also detected in Saposhnikovia root by HPLC with a
photodiode array detector. Prolongation effects on pentobar-
bital-induced hypnosis in mice were observed in the case of
furanocoumarins, but not in panaxynol. The effect seems to
be a characteristic property of furanocoumarins that causes
the inhibition of metabolizing enzymes in mouse liver rather
Fig. 2. Analgesic Effects of the Components from Saposhnikovia Root on
Acetic Acid-Induced Writhing in Mice
AP: aminopyrine. : pϽ0.05, : pϽ0.01, nϭ5—6. Each bar represents the meanϮ
S.E.M. The number of squirms in each control was taken as 100% (control squirms
from up to down, 29.5Ϯ3.2, 24.8Ϯ2.0, 22.9Ϯ2.2, 27.6Ϯ1.0, 27.4Ϯ0.6, 28.0Ϯ0.4,
26.8Ϯ1.9 and 29.1Ϯ0.4, respectively).

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Vol. 49, No. 2
than CNS depression.2,25a) We also tested a chromone, 1, on
aminopyrine N-demethylase activity in liver microsomes of
mice, but did not observe the inhibition until 0.5 mM. These
activity differences of the components reflected the observed
activities in the isolation process, in which some fractions
showed both activities of analgesia and sleeping time-prolon-
gation and some others had only the analgesic effect.
In our preliminary experiment, Saposhnikovia root in-
creased the pain threshold in the tail pressure method by oral
administration of the extract of 3 g/kg. The result suggested
that the analgesia of this herbal drug include the CNS effect.
Compound 1, a major component, was applied to additional
pharmacological tests. The analgesic effect of 1 by the tail
pressure method is shown in Fig. 3. The compound increased
the pain threshold significantly at an oral dose of 80 mg/kg in
mice. The modified Randall & Selitto method in Fig. 4 also
described that 1 raised the pain threshold of both the in-
kg). From this fact, it seems that the analgesic pathway of the
compound is related to an opioid receptor, although the activ-
ity was much less than that of morphine.
In conclusion, the analgesic property of Saposhnikovia
root is a mixture of several types of compounds, such as
chromones, coumarins, furanocoumarins, polyacetylenes,
and monoacylglycerides, although the contents and potencies
of such are variable. The analgesia may involve both anti-in-
flammatory and CNS effects. The inhibitory effect of fura-
nocoumarins on liver metabolizing enzymes also contributes
to prolongation of the activity. The content level of each
component, therefore, may alter the analgesia of this herb
qualitatively and quantitatively, especially in decoctions to-
gether with other herbs.
Experimental
Melting points were determined on a Yanagimoto melting point apparatus
and are uncorrected. UV spectra were recorded on a Hitachi U-3400 spec-
flamed and non-inflamed hind paws in mice when it was ad- trometer, and ORD on a JASCO J-20 polarimeter, CD on a JASCO J-720WI
ministered at a dose of 80 mg/kg.21) Aminopyrine 50 mg/kg
was used as a positive control in both methods. Compound 1,
however, did not show a significant hypothermic effect on
spectropolarimeter. EI-MS and HR-FAB-MS were taken with JEOL JMS-
AM20 and HX-110A spectrometers, respectively. 1H- and 13C-NMR were
measured with a JEOL JNM GSX400A and 500A spectrometers with
tetramethylsilane or a deuterated solvent as an internal standard. Column
lipopolysaccharide-induced pyrexia at doses of 80 and
160 mg/kg, which is not shown here. These experiments sug-
gested that the compound affects the central nervous system.
Figure 5 shows the naloxone counteraction of 1 together
with the effect of morphine as a positive control. The
writhing inhibition of 1 at a dose of 80 mg/kg in mice was re-
versed by subcutaneous (s.c.) injection of naloxone (1 mg/
chromatographies were performed on Sephadex LH-20, Wakogel C-200,
Nacalai Silica Gel 60 and Chromatorex ODS (100—200 mesh). Pre-packed
columns for HPLC, Senshu Pak. ODS-5251-S and Pegasil ODS, were also
used for purification.
Material Saposhnikoviae radix (the chopped and dried root of Saposh-
nikovia divaricata SCHISCHKIN; specimen No. LNP19804-02) was purchased
Fig. 5. Effect of Compound 1 with Naloxone on the Writhing Inhibition in
Mice
Fig. 3. Analgesic Effect of Compound 1 by the Tail Pressure Method in
AP: aminopyrine. : pϽ0.01, nϭ6. Each bar represents the meanϮS.E.M. The
number of control squirms (control–saline, 31.6Ϯ0.9) was taken as 100%, and each
significance was shown versus control–saline.
Mice
AP: aminopyrine. : pϽ0.05, : pϽ0.01, nϭ6.
Fig. 4. Analgesic Effect of Compound 1 by the Randall & Selitto Method in Mice
AP: aminopyrine. : pϽ0.05, : pϽ0.01, nϭ6.

February 2001
159
in April, 1995, from Uchida Wakanyaku, a commercial outlet of traditional
medicines in Japan, which carried out their own identification of the plant
(lot. No. US042323).
2.87 (1H, dd, Jϭ17.1, 5.4 Hz, 4Ј-Hb), 2.92 (1H, ddd, Jϭ8.3, 7.5, 5.3 Hz, 2Љ-
H), 3.02 (1H, td, Jϭ9.2, 5.1 Hz, 4Љ-H), 3.13 (1H, ddd, Jϭ9.3, 6.1, 1.9 Hz, 5Љ-
H), 3.15 (1H, td, Jϭ8.8, 4.9 Hz, 3Љ-H), 3.41 (1H, ddd, Jϭ11.7, 5.9, 5.6 Hz,
6Љ-Ha), 3.67 (1H, ddd, Jϭ11.7, 6.3, 1.9 Hz, 6Љ-Hb), 3.96 (1H, dd, Jϭ6.6,
5.4 Hz, 3Ј-H), 4.32 (1H, d, Jϭ7.5 Hz, 1Љ-H), 4.37 (1H, dd, Jϭ6.3, 5.6 Hz, 6Љ-
OH), 4.84 (1H, d, Jϭ5.3 Hz, 2Љ-H), 4.86 (1H, d, Jϭ5.1 Hz, 4Љ-OH), 4.87
(1H, d, Jϭ4.9 Hz, 3Љ-OH), 6.17 (1H, s, 3-H), 6.36 (1H, s, 8-H), 13.17 (1H, s,
5-OH). 13C-NMR (DMSO-d6) d: 20.0 (C-11), 21.5 (C-4Ј), 21.7 (2Ј-CH3),
25.3 (2Ј-CH3), 61.4 (C-6Љ), 70.3 (C-4Љ), 72.7 (C-3Ј), 73.4 (C-2Љ), 76.90 (C-3Љ
or 5Љ), 76.96 (C-5Љ or 3Љ), 94.3 (C-8), 100.6 (C-1Љ), 103.4 (C-6 or 10), 103.5
(C-10 or 6), 107.7 (C-3), 155.5 (C-9), 158.7 (C-5 or 7), 158.8 (C-7 or 5),
Isolation Saposhnikovia root 2.78 kg was extracted four times with
methanol at room temperature. After evaporation of the solvent, 650 g of the
methanol extract was obtained. Oral administration of the extract induced
analgesia by both the acetic acid-induced writhing and the tail pressure
methods in mice at doses of 2 and 3 g/kg, respectively. The extract at a dose
of 3 g/kg in mice also prolonged the sleeping time in mice, which was in-
duced by pentobarbital sodium. The isolation was carried out by following
the activity-guide of writhing inhibition together with sleeping time-prolon-
gation in mice.
The extract was partitioned with ethyl acetate, n-butanol and water, and
the activity was only found in the ethyl acetate fraction. Separation of the ac-
tive fraction (115.5 g) by Sephadex LH-20 with a solvent of acetone–
methanol 1 : 1 concentrated the analgesic effect into fr. 1-C (60.7 g). The
sleeping prolongation was also observed in the fraction at a dose of
500 mg/kg. As the TLC spots in fr. 1-D were overlapped with those of fr. 1-
C, both fractions were combined to be separated by silica gel chromatogra-
phy. Two active fractions by the writhing method, fr. 2-A (43.3 g) and fr. 2-B
(20.2 g), were obtained by eluting with n-hexane/acetone 5 : 1 and 3 : 1—
acetone, respectively. The sleeping prolongation, however, was only indi-
cated in the former fraction and not in the latter.
Fraction 2-B, having analgesia at a dose of 150 mg/kg, was separated
again by silica gel chromatography with n-hexane/ethyl acetate, and one of
two active fractions, fr. 3-B (8.79 g), was applied on ODS flash column chro-
matography. As fr. 4-C (4.73 g) eluted with acetone showed no effect, fr. 4-A
(2.81 g) and fr. 4-B (1.78 g) of the methanol/water 2 : 3 and 3 : 2 eluate, re-
spectively, were further separated. Fraction 4-B was purified by silica gel
with CHCl3/methanol 10 : 1 to give a major compound, 1 (1215 mg). Frac-
tion 4-A was separated by silica gel using CHCl3/methanol eluent, which re-
moved non-active fr. 5-C (716 mg) with 5 : 1 as an eluent. Compound 2
(671 mg) was purified from fr. 5-B (1280 mg) of the eluate 20 : 1 by repeated
chromatography on ODS (methanol/water 1 : 2—2 : 3), Sephadex LH-20
(methanol) and silica gel (n-hexane/water 1 : 1—2 : 3).
Another active fraction, 3-A (9.66 g), was separated by silica gel with the
eluent of n-hexane/ethyl acetate 2 : 1—ethyl acetate and gave active frac-
tions, fr. 8-A (4.87 g) and fr. 8-B (3.60 g). After removal of the non-active
part from the latter (the acetone and methanol eluate on silica gel), fr. 9-B
(2538 mg) which showed fluorescent spots on TLC under UV360 was applied
on ODS column. From fr. 10-B (217 mg) eluted with methanol/water 2 : 3,
3—5 were obtained in yields of 42, 35 and 13 mg, respectively. Fraction 10-
E (201 mg) of the 2 : 1 eluate gave 7 (1 mg), 8 (19 mg) and 12 (2 mg) by sil-
ica gel with CHCl3/methanol 100 : 1 and then ODS-HPLC with acetonitrile/
water 1 : 1.
1
167.9 (C-2), 181.9 (C-4). The UV, IR and H-NMR were identical with the
data published, although the sugar moiety was not assigned in the 1H-
NMR.12,15)
Compound 2 (cimifugin), colorless needles, mp 112—113 °C (lit.,22) mp
107—109 °C), [a]2D3 ϩ87° (cϭ0.61, ethanol) (lit.,15) [a]589 ϩ88.5° in
ethanol). 1H-NMR (acetone-d6) d: 1.24 (3H, s, 4Ј-CH3), 1.28 (3H, s, 4Ј-
CH3), 3.27 (1H, dd, Jϭ16.1 Hz, 9.3, 3Ј-Ha), 3.32 (1H, dd, Jϭ16.1, 7.8 Hz,
3Ј-Hb), 3.77 (1H, s, 5-OCH3), 3.88 (3H, s, 5-OCH3), 4.45 (2H, br d, Jϭ
4.8 Hz, 11-H2), 4.74 (1H, br, 11-OH), 4.78 (1H, dd, Jϭ9.3, 7.8 Hz, 2Ј-H),
6.12 (1H, t-like, Jϭ1.0 Hz, 3-H), 6.50 (1H, s, 8-H). The MS, UV, IR and
13C-NMR were identical with the published data.12)
Compound 3 (isofraxidin), white powder, mp 150—152 °C (lit.,23) mp
148.5—150 °C). 1H-NMR (acetone-d6) d: 3.89 (3H, s, 6-OCH3), 3.95 (3H, s,
8-OCH3), 6.20 (1H, d, Jϭ9.5 Hz, 3-H), 6.99 (1H, s, 5-H), 7.85 (1H, d, Jϭ
9.5 Hz, 4-H), 8.60 (1H, br s, 7-OH). 13C-NMR (acetone-d6) d: 56.8 (6-
OCH3), 61.3 (8-OCH3), 105.1 (C-5), 111.7 (C-10), 113.5 (C-3), 135.8 (C-8),
144.4 (C-9), 144.8 (C-7), 145.1 (C-4), 146.4 (C-6), 160.8 (C-2). The UV
and IR were identical with the data published by Takemoto et al.23)
Compound 4 was directly identified with the authentic scopoletin.2)
Compound 5 (fraxidin), white powder, mp 199—201 °C (lit.,16) mp 198—
1
200 °C). H-NMR (acetone-d6) d: 3.89 (6H, s, 6,7-OCH3), 6.29 (1H, d, Jϭ
9.5 Hz, 3-H), 6.80 (1H, s, 5-H), 7.85 (1H d, Jϭ9.5 Hz, 4-H), 8.58 (1H, br s,
8-OH). The UV, IR and 13C-NMR (acetone-d6) were identical with the data
published.16)
Compound 6 (ledebouriellol), white needles, mp 95—99 °C (lit.,12) mp
97—99 °C), HR-FAB-MS (NBA/PEG 200ϩ400): 375.1440 (Mϩ1)ϩ, (err.
Ϫ0.4 mmu for C20H23O7). The 1H- and 13C-NMR and CD were identical
with the published data.12)
Compound 7 (hamaudol), white crystalline powder, mp 202—203 °C
24
(lit.,12) mp 202—202.5 °C), [a]
Ϫ32° (cϭ0.13, ethanol) (lit.,15) [a]589
589
Ϫ19.6° in ethanol). CD (cϭ0.031 mg/ml, methanol) De22 (nm): Ϫ0.15 (332,
trough), Ϫ0.02 (306, max.), Ϫ0.30 (282, trough), 0 (270), ϩ1.74 (257,
max), 0 (241), Ϫ1.13 (230, trough). The 1H-NMR and UV were identical
with the published data.12,24)
Silica gel separation of a part of fr. 8-A, 1489 mg, concentrated the activ-
ity to fr. 12-B (855 mg) by eluting with n-hexane/acetone 5 : 1. Fraction 12-B
was chromatographed on Sephadex LH-20 with methanol to obtain active fr.
13-A (498 mg) and fr. 13-C (101 mg), which showed analgesia at doses of 40
and 20 mg/kg, respectively. Fraction 13-C yielded 6 (16 mg) together with 7
(4 mg) and 8 (4 mg) by ODS flash chromatography using methanol/water
4 : 1 and HPLC with acetonitrile/water 4 : 1 for 6 and 1 : 1 for 7 and 8. Com-
pounds 9 and 10 were obtained from fr. 13-A in yields of 12 and 33 mg, re-
spectively, by separation of silica gel with n-hexane/acetone 3 : 1, ODS with
methanol/water 4 : 1—9 : 1 and then silica gel with n-hexane/ethyl acetate
1 : 1.
Fraction 2-A indicated polyacetylenes and furanocoumarins on TLC,
which were separated as fr. 2-Ab and fr. 2-Ad by silica gel chromatography
with n-hexane/ethyl acetate 30 : 1—10 : 1 and 5 : 1—3 : 1, respectively. Frac-
tion 2-Ab showed analgesia at a dose of 150 mg/kg, but no sleeping prolon-
gation effect, while fr. 2-Ad exhibited both activities. A major component in
fr. 2-Ab, 11, was isolated by silica gel chromatography with n-hexane/ethyl
acetate 80 : 1—50 : 1 and ODS with methanol/water 3 : 1. Fraction 2-Ad was
analyzed by HPLC with a photodiode array detector by comparing the reten-
tion time and UV spectrum of each peak with the authentic samples. Shi-
madzu LC Workstation CLASS-LC10 with SPD-M10AVP Diode Array De-
tector; column, Deverosil ODS-UG-5 (f10ϫ250 mm) with the eluent of
acetonitrile–water in CTO-10A Column Oven at 40 °C; flow rate, 2 ml/min;
samples, ca. 10 ml injection of a 10 mg/ml sample solution or 0.1 mg/ml au-
thentic samples.
Hydrolysis of compound 1 (sec-O-glucosylhamaudol) As the CD spec-
trum of 7 was not identical with the published one,12) the enzymatic hydroly-
sis of 1 was carried out. To a solution of 1 (50 mg) in DMSO (3 ml) and
water (9 ml) was added b-glucosidase (30 mg). After stirring at 37 °C for
4 h, the solution was extracted with ethyl acetate. The extract was chro-
matographed by silica gel with CHCl3 as an eluent to give the aglycone
(19 mg), which was identical with 7.
Compound 8 (divaricatol), white crystalline powder, mp 168—171 °C.
HR-FAB-MS (NBA/PEG 200ϩ400): 335.1116 (Mϩ1)ϩ, (err. Ϫ1.4 mmu
for C17H19O7). EI-MS m/z (%): 334 (Mϩ, 2), 274 (14), 259 (100), 221 (9).
1H-NMR (CDCl3) d: 1.34 (3H, s, 2Ј-CH3), 1.37 (3H, s, 2Ј-CH3), 2.01 (3H, s,
CH3COO), 2.37 (1H, br s, 11-OH), 2.78 (1H, dd, Jϭ17.7, 4.8 Hz, 4Ј-Ha),
2.99 (1H, dd, Jϭ17.7, 5.3 Hz, 4Ј-Hb), 4.55 (2H, br s, 11-H2), 5.11 (1H, t-
like, Jϭ5.0 Hz, 3Ј-H), 6.31 (1H, br s, 3-H), 6.34 (1H, s, 8-H), 12.90 (1H, s,
5-OH). 13C-NMR (CDCl3) d: 21.0 (CH3COO), 22.6 (C-4Ј), 23.3 (2Ј-CH3),
24.7 (2Ј-CH3), 61.4 (C-11), 69.7 (C-3Ј), 76.7 (C-2Ј), 94.9 (C-8), 102.7 (C-
6), 104.9 (C-10), 106.4 (C-3), 155.9 (C-9), 159.0 (C-5 or 7), 159.6 (C-7 or
5), 167.9 (C-2), 170.3 (CH3COO), 182.5 (C-4). UV lmax (ethanol) nm
(log e): 210 (4.39), 230 (4.19), 251 (4.21), 257 (4.20), 295 (3.92), 318 (sh.,
3.65). ORD (cϭ0.13, ethanol) [a]24 (nm): Ϫ30° (589), Ϫ35° (550), Ϫ40°
(500), Ϫ45° (450), Ϫ54° (400). CD (cϭ0.041 mg/ml, methanol) De15 (nm):
ϩ1.02 (294, max.), ϩ0.66 (276, trough), ϩ3.65 (258, max.), 0 (236), Ϫ3.49
(229, trough).
Compound 9 (glycerol monolinoleate), colorless oil. EI-MS m/z (%): 354
(Mϩ, 8), 336 (3), 280 (7), 262 (100), 234 (9); % shown in the case that m/z
262 was taken as a base peak. 1H-NMR (CDCl3)d: 0.89 (3H, t-like, Jϭ
6.9 Hz, 18Ј-H3), ca. 1.25 (m, methylene), 1.61—1.65 (2H, m, 3Ј-H2), 2.05
(4H, m, 8Ј, 14Ј-H2), 2.35 (2H, t-like, Jϭ7.5 Hz, 2Ј-H2), 2.77 (2H, t-like,
Jϭ6.4 Hz, 11Ј-H2), 3.57—3.63 (1H, m, 3-Ha), 3.68—3.71 (1H, m, 3-Hb),
Compound
1 (sec-O-glucosylhamaudol), yellow needles, mp 236—
237 °C (lit.12) mp 229—230 °C). [a]2D6 Ϫ48° (cϭ0.27, methanol) (lit.,12) [a]D
Ϫ48.5° in methanol). 1H-NMR (DMSO-d6) d: 1.27 (3H, s, 2Ј-CH3), 1.32
(3H, s, 2Ј-CH3), 2.34 (3H, s, 2Ј-CH3), 2.59 (1H, dd, Jϭ17.1, 6.6 Hz, 4Ј-Ha),

160
Vol. 49, No. 2
statistical significance was evaluated by Student’s t-test or 1-way ANOVA
followed by the Dunnett test.
3.91—3.94 (1H, m, 2-H), 4.15 (1H, dd, Jϭ11.7, 6.1 Hz, 1-Ha), 4.21 (1H, dd,
11.7, 4.6, 1-Hb), 5.32—5.38 (4H, m, 9Ј, 10Ј, 12Ј, 13Ј-H). 13C-NMR (CDCl3)
d: 14.1 (C-18Ј), 22.6 (C-17Ј), 24.9 (C-3Ј), 25.6 (C-11Ј), 27.17 (C-8Ј or 14Ј),
27.19 (C-14Ј or 8Ј), 29.07 (ϫ2), 29.13, 29.3, 29.6, 31.5 (C-16Ј), 34.1 (C-2Ј),
63.3 (C-3), 65.2 (C-1), 70.3 (C-2), 127.9 (C-10Ј or 12Ј), 128.1 (C-12Ј or
10Ј), 130.0 (C-9Ј or 13Ј), 130.2 (C-13Ј or 9Ј), 174.3 (C-1Ј). ORD (cϭ0.36,
methanol) [a]22 (nm): ϩ3° (589), ca. 0 (535), Ϫ5° (400).
Acknowledgements We thank Dr. T. Deyama, Yomeishu Seizo Co.,
Ltd., for test samples, Mr. S. Fujimori and Ms. R. Hara for their preliminary
experiments, and the staff of the Analytical Center of Chiba University for
measurement of HR-FAB-MS.
Compound 10 (glycerol monooleate), white waxy solid. EI-MS m/z (%):
1
356 (Mϩ, 3), 325 (5), 299 (11), 264 (64), 239 (24), 221 (6), 98 (100). H-
References
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NMR (CDCl3) d: 0.87 (3H, t-like, Jϭ7.0 Hz, 18Ј-H3), ca. 1.25 (m, methyl-
ene), 1.58—1.66 (2H, m, 3Ј-H2), 1.98—2.03 (4H, m, 8Ј and 11Ј-H2), 2.34
(2H, t-like, Jϭ7.5 Hz, 2Ј-H2), 3.59 (1H, dd, Jϭ11.5, 5.9 Hz, 3-Ha), 3.69
(1H, dd, Jϭ11.5, 3.9 Hz, 3-Hb), 3.90—3.95 (1H, m, 2-H), 4.14 (1H, dd,
Jϭ11.7, 6.1 Hz, 1-Ha), 4.19 (1H, dd, Jϭ11.7, 4.8 Hz, 1-Hb), 5.30—5.38
(2H, m, 9Ј and 10Ј-H2). 13C-NMR (CDCl3) d: 14.1 (C-18Ј), 22.7 (C-17Ј),
24.9 (C-3Ј), 27.1 (C-8Ј or 11Ј), 27.2 (C-11Ј or 8Ј), 29.07, 29.12, 29.30 (ϫ2),
29.50, 29.66 (ϫ2), 29.74, 31.9 (C-16Ј), 34.1 (C-2Ј), 63.3 (C-3), 65.1 (C-1),
70.3 (C-2), 129.7 (C-9Ј or 10Ј), 130.0 (C-10Ј or 9Ј), 174.3 (C-1Ј). ORD (cϭ
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1
Compound 11 (panaxynol), colorless oil. The H- and 13C-NMR and the
ORD were identical with the authentic sample.2)
Compound 12 ((3ЈS)-hydroxydeltoin), white amorphous solid. HR-FAB-
MS (NBA/PEG 200ϩ400): 345.1338 (Mϩ1)ϩ, (err. 0.0 mmu for C19H21O6).
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H), 6.77 (1H, s, 8-H), 7.66 (1H, s, 5-H), 7.92 (1H, d, Jϭ9.7 Hz, 4-H). 13C-
NMR (acetone-d6) d: 15.6 (C-4Љ), 20.6 (C-5Љ), 21.9 (4Ј-CH3), 22.3 (4Ј-CH3),
72.4 (C-3Ј), 82.3 (C-4Ј), 98.2 (C-2Ј), 98.4 (C-8), 113.1 (C-3), 114.0 (C-10),
126.1 (C-5), 128.7 (C-6), 129.8 (C-2Љ), 137.6 (C-3Љ), 144.9 (C-4), 157.7 (C-
9), 160.9 (C-2), 164.4 (C-7), 167.2 (C-1Љ). UV lmax (ethanol) nm (log e):
221 (4.26), 247 (3.55), 259 (3.47), 301 (3.81), 329 (4.12), 340 (4.00). ORD
(cϭ0.078, CHCl3) [a]26 (nm): Ϫ45° (589), Ϫ50° (550), Ϫ66° (500), Ϫ103°
(450). CD (cϭ0.16 mg/ml, ethanol) De22 (nm): Ϫ3.76 (328, trough), Ϫ1.86
(299, sh.), Ϫ0.12 (266, max.), Ϫ0.52 (259, trough), Ϫ0.33 (254, max.),
Ϫ0.65 (248, trough), 0 (243). Deltoin, CD (cϭ0.092 mg/ml, ethanol) De23
(nm): Ϫ3.72 (332, trough), Ϫ1.39 (299, sh.), Ϫ0.05 (267, max.), Ϫ1.94
(258, trough), Ϫ1.63 (254, max.), Ϫ1.98 (249, trough).
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Pharmacological Assay Male ddy strain mice weighing 24—36 g were
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22—25 °C, water and food being provided at libitum. The following materi-
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cal Industries), pentobarbital sodium (Tanabe), carrageenin (Nacalai
Tesque), naloxone hydrochloride (Sigma), morphine hydrochloride
(Takeda), Tween 80 (Wako Pure Chemical Industries), and saline (Otsuka).
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90 min after the injection of 0.1% carrageenin (0.02 ml) to the left hindpaw
in mice. Then, the pain thresholds of both hindpaws in each mouse were
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before sample administration. The sample administration was followed after
25 min by intraperitoneal injection of 0.7% acetic acid. After 5 min, the
number of squirms was counted in each mouse for the next 15 min. Mor-
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Statistics: After the data were analyzed for outlier by Smirnov-Groubbs,