A new lignan glycoside and phenylethanoid glycosides from Strobilanthes cusia BREMEK

Article abstract of DOI:10.1248/cpb.52.1242

The root of Strobilanthes cusia BREMEK. (Acanthaceae), popularly known as Da-Ching-Yeh, has been commonly used in traditional Chinese medicine. It is used to treat influenza, epidemic cerebrospinal meningitis, encephalitis B, viral pneumonia, mumps, and severe acute respiratory syndrome (SARS). In this study, we found a new lignan glycoside (6) and two new phenylethanoid glycosides (7, 8) together with five known compounds as chemical constituents of Strobilanthes cusia root. Some samples were examined for anti-herpes simplex virus type-1 (HSV-1) activity. Among the tested samples, lupeol showed anti-HSV-1 activity (EC₅₀: 11.7 μM) and showed 100% inhibition of virus plaque formation at 58.7 μM.

Full text of DOI:10.1248/cpb.52.1242

1242
Notes
Chem. Pharm. Bull. 52(10) 1242—1245 (2004)
Vol. 52, No. 10
A New Lignan Glycoside and Phenylethanoid Glycosides from
Strobilanthes cusia BREMEK
Tomonori TANAKA, Tsuyoshi IKEDA,* Miho KAKU, Xing-Hua ZHU, Masafumi OKAWA,
Kazumi YOKOMIZO, Masaru UYEDA, and Toshihiro NOHARA
Faculty of Medical and Pharmaceutical Sciences, Kumamoto University; 5–1 Oe-honmachi, Kumamoto 862–0973, Japan.
Received March 12, 2004; accepted June 8, 2004
The root of Strobilanthes cusia BREMEK. (Acanthaceae), popularly known as Da-Ching-Yeh, has been com-
monly used in traditional Chinese medicine. It is used to treat influenza, epidemic cerebrospinal meningitis, en-
cephalitis B, viral pneumonia, mumps, and severe acute respiratory syndrome (SARS). In this study, we found a
new lignan glycoside (6) and two new phenylethanoid glycosides (7, 8) together with five known compounds as
chemical constituents of Strobilanthes cusia root. Some samples were examined for anti-herpes simplex virus
type-1 (HSV-1) activity. Among the tested samples, lupeol showed anti-HSV-1 activity (EC50: 11.7 mM) and
showed 100% inhibition of virus plaque formation at 58.7 mM.
Key words Strobilanthes cusia; Acanthaceae; lignan; phenylethanoid; anti-herpes simplex virus type-1 activity
The root of Strobilanthes cusia BREMEK. (Acanthaceae), scopic data with values reported in the literature.
popularly known as Da-Ching-Yeh, has been commonly used
Compound 6 was obtained as a white amorphous powder,
in traditional Chinese medicine.1) It is used to treat influenza, with an [a]D value of ꢂ2.5° (MeOH). The molecular for-
epidemic cerebrospinal meningitis, encephalitis B, viral mula of 6 was determined to be C33H46O17 based on the
pneumonia, and mumps. Recently, this crude drug has also NMR and high-resolution fast-atom bombardment mass
been used in severe acute respiratory syndrome (SARS). It is spectrum (HR-FAB-MS) data ([MꢀNa]ꢀ, m/z: 737.2632;
1
also used to treat sore throat, aphthae, inflammatory diseases Calcd for C33H46O17Na m/z: 737.2633). The H-NMR spec-
with redness of the skin, etc. However, the chemical con- trum of 6 in CD3OD showed signals similar to those of 3, ex-
stituents of Strobilanthes cusia are not clear,2) and we began cept for an additional anomeric proton signal at d 5.41
a study to isolate the constitutes and elucidate the structure (d, Jꢃ2.0 Hz). The 13C-NMR spectrum of 6 (Table 1) also
of this crude drug. Some compounds obtained from S. cusia showed carbon signals similar to those of 3, except for an ad-
were tested for anti-herpes simplex virus type-1 (HSV-1) ditional pentosyl moiety. Acid hydrolysis of 6 with 1 M HCl
activity.
in dioxane–H2O (1 : 1) afforded D-glucose and D-apiose in a
The MeOH extract of S. cusia root was chromatographed ratio of 1 : 1 as carbohydrate components upon GLC analysis
on porous-polymer polystyrene resin (Diaion HP-20), and after conversion to the thiazolidine derivatives,11) while the
the MeOH eluate was subjected to column chromatography aglycone (6a) was obtained by enzymatic hydrolysis and
on silica gel and octadecylsilanized (ODS) silica gel and on identified as (2R,3R)-(ꢀ)-lyoniresinol after comparison with
Sepadex LH-20, giving compounds 1—8. Compounds 1—5 an authentic sample.7) The NMR data could be assigned with
were identified as lupeol (1),3) (ꢀ)-5,5ꢁ-dimethoxy-9-O-b-D- the aid of 1H–1H correlation spectroscopy (COSY), heteronu-
glucopyranosyl lariciresinol (2),4—6) (ꢀ)-9-O-b-D-glucopyra- clear multiple-quantum coherence (HMQC), and heteronu-
nosyl lyoniresinol (3),7,8) (ꢀ)-5,5ꢁ-dimethoxy-9-O-b-D-glu- clear multiple-bond correlation spectroscopy (HMBC) exper-
copyranosyl secoisolariciresinol (4),9) and acteoside (5),10) iments. The anomeric centers of the glucose moieties were
respectively. The structural identities of these compounds determined to have the b-configuration from the large 3JH1–H2
were established by comparing the physical and spectro- values. The 4C1-conformation of glucose was shown by com-
* To whom correspondence should be addressed. e-mail: tikeda@gpo.kumamoto-u.ac.jp
© 2004 Pharmaceutical Society of Japan

October 2004
1243
parison of the carbon resonance for monosaccharide. In the of the glucosyl moiety. Two hydroxymethyl groups were ob-
HMBC experiment, the anomeric proton signals at d 4.33 served at d 3.18 and 3.24 (each H, d, Jꢃ10.4 Hz) and 3.47
(H-1ꢄ) and d 5.41 (H-1ꢅ) showed long-range correlation with and 3.59 (each 1H, d, Jꢃ9.2 Hz) in 7 instead of the doublet
the carbon signals at d 70.5 (C-3a) and 77.6 (C-2ꢄ), respec- methyl signals d 0.96 (3H, d, Jꢃ6.1 Hz, rha-H3-6) in 5. Acid
tively. From the evidence presented above, the structure of 6 hydrolysis of 7 with 1 M HCl in dioxane–H2O (1 : 1) afforded
was concluded to be (ꢀ)-lyoniresinol 3a-O-b-D-apiofura- D-glucose and D-apiose in a ratio of 1 : 1 as carbohydrate
nosyl-(1Æ2)-b-D-glucopyranoside, as shown in the formula. components in GLC, as described above. The anomeric cen-
This compound is a new lignan glycoside in our literature ters of the glucosyl moieties were determined to have the
survey.
b-configuration from the large 3JH1–H2 values (7.3 Hz). 1H–1H
Compound 7 was obtained as a white amorphous powder, COSY, HMQC, and HMBC experiments were carried out for
with an [a]D value of ꢂ67.5° (MeOH). The molecular for- 7. In the HMBC experiment, the anomeric proton signals at d
mula of 7 was determined to be C28H34O15 based on the 4.35 (H-1ꢄ) and d 5.25 (H-1ꢅ) showed long-range correla-
NMR and HR-FAB-MS data ([MꢀH]ꢀ, m/z: 611.2041; tions with the carbon signals at d 70.1 (C-8) and 78.6 (C-3ꢄ),
1
Calcd for C28H35O15 m/z: 611.1976). The H- and 13C-NMR respectively. Additionally the H-4ꢄ proton (d 4.68) was cor-
spectra (see Table 2) are similar to those of acteoside (5), ex- related to C-9ꢁ (d 165.6) in the HMBC. From the evidence
cept for the signal due to a pentosyl group attached to 3ꢄ-O presented above, the structure of 7 was concluded to be
[2-(3,4-dihydroxyphenylethyl)]-3-O-a-D-apiofuranosyl-
Table 1. 13C-NMR Data for 3, 6, and 6a (3, 6 in CD3OD, 6a in CDCl3)
(1Æ4)-(4-O-caffeoyl)-b-D-glucopyranoside, and designated
cusianoside A.
C no.
3
6
6a
Sugar
3
6
Compound 8 was obtained as a white amorphous powder,
with an [a]D value of ꢂ41.9° (MeOH). The molecular for-
mula of 8 was determined to be C28H34O15, which was the
same as that of 7, based on the HR-FAB-MS data ([MꢀH]ꢀ,
1
2
2a
3
3a
4
33.8
40.7
66.4
46.7
72.2
33.9
40.1
65.7
46.6
70.5
33.5
40.4
64.1
49.4
66.8
glc-1
104.8
75.2
78.0
71.7
78.3
62.9
103.7
77.6
78.0
71.5
78.4
62.7
2
3
4
5
6
1
m/z: 611.2041; Calcd for C28H35O15 m/z: 611.1976). The H-
and 13C-NMR spectra of 8 (see Table 2) are essentially the
same as those of 7, except for the signal due to a terminal
pentosyl group attached to 3ꢄ-O of the glucosyl moiety. An
anomeric proton was seen at d 4.30 (1H, d, Jꢃ7.3 Hz) in-
stead of d 5.25 (1H, br s, apiose H-1) as in 7. Acid hydrolysis
of 8 with 1 M HCl in dioxane–H2O (1 : 1) afforded D-glucose
and D-xylose in a ratio of 1 : 1 as carbohydrate components in
GLC, as described above. The anomeric centers of the gluco-
syl and xylosyl moieties were determined to have the b-con-
42.7
43.0
43.0
5
6
7
8
147.6
139.0
148.7
108.0
130.2
126.4
139.3
107.1
149.0
134.7
149.0
107.1
148.2
138.5
147.2
107.5
129.9
126.2
139.2
106.7
148.6
134.1
148.6
106.7
145.6
137.1
146.3
106.0
128.7
125.3
138.2
105.6
146.9
133.1
146.9
105.6
api-1
111.1
79.6
80.3
75.0
65.9
2
3
4
5
9
10
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
6ꢁ
3
figuration from the large JH1–H2 values (7.9 and 7.3 Hz, re-
spectively). In the HMBC experiment, the anomeric proton
signals at d 4.39 (H-1ꢄ) and d 4.30 (H-1ꢅ) showed long-
Table 2. 13C-NMR Data for 5, 7, and 8 (in DMSO-d6)
C no.
5
7
8
Sugar
5
7
8
1
2
3
4
5
6
7
129.0
116.2
144.9
143.4
115.7
119.4
34.9
129.1
116.2
144.9
143.4
115.7
119.4
35.0
129.3
116.3
144.9
143.5
115.8
119.5
35.0
glc-1
102.2
74.4
79.0
69.0
74.4
60.6
102.4
74.0
78.6
69.2
74.5
60.7
102.2
73.2
84.2
69.4
74.5
60.8
2
3
4
5
6
8
70.2
70.1
70.1
rha-1
101.1
70.4
70.3
71.6
68.6
18.0
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
6ꢁ
7ꢁ
8ꢁ
9ꢁ
125.4
114.6
145.5
148.4
115.4
121.3
145.5
113.5
165.6
125.5
114.7
145.5
148.4
115.4
121.2
145.2
113.9
165.6
125.7
114.6
144.8
143.5
115.5
121.2
145.6
114.2
165.6
2
3
4
5
6
api-1
109.5
76.1
78.9
73.6
63.8
2
3
4
5
xyl-1
105.7
74.2
76.9
69.4
65.9
2
3
4
5

1244
Vol. 52, No. 10
Table 3. Anti-herpes Activities
Concentration (mg/ml)
EC50
(mM)
IC50
(mM)
Selective index
(IC50/EC50)
Compound
100
50
25
12.5
6.25
53
5
14
8
3.13
30
14
10
24
1
Toxic
Toxic
100
23
13
83
11
14
10
11.7
ꢇ172
49.3
4.20
—
—
2
3
6
1
0
0
0
15
0
N.T.a)
N.T.a)
N.T.a)
ꢇ172
ꢇ141
12
—
Acyclovir
1.72b)
15860b)
9220b)
a) Not tested. b) Ref. 13.
(4H, overlapped, glc H-5, glc Ha-6, api Hb-4, Hb-2a), 3.63 (1H, d,
Jꢃ9.8 Hz, api Ha-5), 3.75 (6H, s, 3ꢁ,5ꢁ-OCH3), 3.79—3.81 (2H, overlapped,
Hb-3a, glc Hb-6), 3.85 (3H, s, 7-OCH3), 3.99 (1H, d, Jꢃ2.0 Hz, api H-2),
4.02 (1H, d, Jꢃ9.8 Hz, api Hb-5), 4.33 (1H, d, Jꢃ7.9 Hz, glc H-1), 4.42 (1H,
d, Jꢃ6.3 Hz, H-4), 5.41 (1H, d, Jꢃ2.0 Hz, api H-1), 6.42 (2H, s, H-2ꢁ,6ꢁ),
range correlations with the carbon signals at d 70.1 (C-8)
and 84.2 (C-3ꢄ), respectively. In addition, the H-4ꢄ proton
(d 4.71) was correlated to C-9ꢁ (d 165.6) in the HMBC.
From the evidence presented above, the structure of 8 was
concluded to be [2-(3,4-dihydroxyphenylethyl)]-3-O-b-D-xy- 6.57 (1H, s, H-8). 13C-NMR (in CD3OD) d: Table 1.
Acid Hydrolysis of 6, 7, and 8 Each sample (1 mg) was hydrolyzed
lopyranosyl-(1Æ3)-(4-O-caffeoyl)-b-D-glucopyranoside, and
designated cusianoside B.
with 2 mol/l of HCl in H2O for 4 h at 80 °C. The reaction mixture was neu-
tralized with 2 mol/l of NaOH in H2O and extracted with CHCl3. The aque-
ous layer was concentrated to dryness in vacuo to give a residue that was
dissolved in dry pyridine, to which was added L-cysteine methyl ester hy-
drochloride.11) The reaction mixture was heated for 2 h at 60 °C and concen-
trated to dryness by blowing with N2 gas. To the residue was added
trimethylsilylimidazole, followed by heating for 1 h at 60 °C. The residue
was extracted with hexane and H2O, and the organic layer was analyzed
using GLC: column, OV-17 (0.32 mmꢆ30 m); detector, FID; column tem-
perature, 230 °C; detector temp., 270 °C; injector temp., 270 °C; carrier gas,
He (2.0 kg/cm2). Peaks were observed at tR (min) for 6 at 12.4 min (D-apiose)
and 15.5 min (D-glucose), for 7 at 12.5 min (D-apiose) and 15.4 min (D-glu-
cose), and for 8 at 7.3 min (D-xylose) and 15.6 min (D-glucose). The standard
monosaccharides were subjected to the same reaction and GLC analysis
under the same conditions.
It was noteworthy that the 13C-NMR data of 6, 7, and 8
were different depending on the terminal saccharide.
Compounds 1, 2, 3, and 6 were examined for anti-HSV-1
activity.12) Among the tested samples, lupeol (1) was exhib-
ited anti-HSV-1 activity (EC50: 11.7 mM) and showed 100%
inhibition of virus plaque formation at 58.7 mM (Table 3). Al-
though lupeol was less active than acyclovir and had a low
selective index (IC50/EC50),13) lupeol was the major consti-
tuent (yield: 0.048%) and might be the active principle in this
crude drug. We plan to synthesize lupeol derivatives and to
test their anti-viral and/or antiinflammatory activity in the
near future.
Enzymatic Hydrolysis of Compound 6 A mixture of 6 (10 mg) and
b-glucosidase (10 mg; Sigma Chemical, EC 3.2.1.21 from almonds) in ac-
etate buffer (1.0 ml, 100 mM, pH 4.0) was incubated at 37 °C for 3 d. The
mixture was concentrated in vacuo to dryness, and the residue was chro-
matographed over Chromatorex ODS (30% MeOH) to afford a 6a (3.2 mg).
Compound 6a [(2R,3R)-(ꢀ)-lyoniresinol]: Amorphous powder, [a]D25
Experimental
The optical rotations were measured on a JASCO DIP-360 automatic digi-
tal polarimeter. The NMR spectra were recorded at 500 MHz for 1H- and
125 MHz for 13C-NMR spectra on a JEOL a-500 spectrometer, and chemi-
cal shifts were given on a d (ppm) scale with tetramethylsilane as an internal
standard. Standard pulse sequences were employed for the DEPT, HMQC,
and HMBC experiments. NOESY spectra were measured with mixing times
of 600 ms. The FAB-MS were measured with a JEOL DX-300 and/or
SX102A spectrometer. The HR-FAB-MS were measured with a JEOL DX-
303 HF spectrometer in a glycerol, triethylene glycol, and m-nitrobenzyl al-
cohol matrix. GLC was performed on an HP5890A gas chromatograph with
a flame ionization detector. TLC was performed on precoated Kieselgel 60
F254 plates (Merck). Column chromatography was carried out on Kieselgel
60 (70—230 mesh and 230—400 mesh), Diaion HP-20 (Mitsubishi Chemi-
cal Industries), Sephadex LH-20 (Pharmacia), and Chromatorex ODS-DU
3050MT (Fuji Silysia). b-Glucosidase from almonds was purchased from
Sigma Chemical. Fetal calf serum (FCS) was purchased from Gibco BRL.
Sulfonated g-globulin (Venilon) was supplied by the Chemo-Sero Therapeu-
tic Institute.
1
ꢀ67.2° (cꢃ0.05, MeOH), EI-MS (m/z): 420 [M]ꢀ, H-NMR (in CDCl3) d:
1.76 (1H, m, H-2), 1.92 (1H, m, H-3), 2.60 (1H, dd, Jꢃ4.9, 11.0 Hz, Ha-1),
2.66 (1H, dd, Jꢃ11.0, 15.3 Hz, Hb-1), 3.29 (3H, s, 5-OCH3), 3.57 (1H, dd,
Jꢃ6.1, 11.0 Hz, Ha-2a), 3.63 (1H, dd, Jꢃ6.7, 11.0 Hz, Ha-3a), 3.75 (1H,
dd, Jꢃ4.3, 11.0 Hz, Hb-3a), 3.79 (6H, s, 3ꢁ,5ꢁ-OCH3), 3.81 (1H, dd, Jꢃ4.9,
11.0 Hz, Hb-2a), 3.88 (3H, s, 7-OCH3), 4.01 (1H, d, Jꢃ7.3 Hz, H-4), 5.40
(2H, br s, 2ꢆOH), 6.34 (2H, s, H-2ꢁ,6ꢁ), 6.44 (1H, s, H-8). 13C-NMR (in
CDCl3) d: Table 1.
Compound 7: Amorphous powder, [a]D25 ꢂ67.5° (cꢃ0.11, MeOH), posi-
tive FAB-MS (m/z): 611 [MꢀH]ꢀ. HR-FAB-MS (m/z): 611.2041 [MꢀH]ꢀ
1
(Calcd for C28H35O15, 611.1976). H-NMR (in DMSO-d6) d: 2.71 (2H, m,
H2-7), 3.18 (1H, d, Jꢃ10.4 Hz, api Ha-4), 3.22 (1H, dd, Jꢃ7.3, 9.8 Hz, glc
H-2), 3.24 (1H, d, Jꢃ10.4 Hz, api Hb-4), 3.43—3.47 (3H, overlapped, glc
Ha-6, glc Hb-6, glc H-5), 3.47 (1H, d, Jꢃ9.2 Hz, api Ha-5), 3.59 (1H, d,
Jꢃ9.2 Hz, api Hb-5), 3.63 (1H, m, Ha-8), 3.67 (1H, dd, Jꢃ9.2, 9.8 Hz, glc
H-3), 3.71 (1H, br s, api H-2), 3.88 (1H, m, Hb-8), 4.35 (1H, d, Jꢃ7.3 Hz,
glc H-1), 4.68 (1H, dd, Jꢃ9.8, 9.8 Hz, glc H-4), 5.25 (1H, br s, api H-1),
6.21 (1H, d, Jꢃ15.9 Hz, H-7ꢁ), 6.50 (1H, d, Jꢃ7.9 Hz, H-6), 6.62 (1H, br s,
H-2), 6.63 (1H, d, Jꢃ7.9 Hz, H-5ꢁ), 6.77 (1H, d, Jꢃ7.9 Hz, H-5), 6.99 (1H,
d, Jꢃ7.9 Hz, H-6ꢁ), 7.05 (1H, br s, H-2ꢁ), 7.47 (1H, d, Jꢃ15.9 Hz, H-8ꢁ).
13C-NMR (in DMSO-d6) d: Table 2.
Extraction and Isolation The root of S. cusia (2.0 kg) was extracted
with MeOH, and the methanol extract (92.7 g) was subjected to Diaion HP-
20 column chromatography (eluted with H2O, MeOH, and acetone) to give
three fractions. The acetone eluate (2.23 g) was purified by silica gel chro-
matography to give compound 1 (960 mg). The MeOH eluate (20.4 g) was
further purified by Sephadex LH-20 (MeOH), chromatorex ODS (20%
MeOHÆ50% MeOH), silica gel [CHCl3–MeOH–H2Oꢃ9 : 1 : 0.1Æ8 : 2 :
0.1] column chromatography to give compounds 2 (37 mg), 3 (559 mg), 4
(30 mg), 5 (42 mg), 6 (13 mg), 7 (66 mg), and 8 (27 mg), respectively.
Compound 6: Amorphous powder, [a]D25 ꢂ2.5° (cꢃ0.12, MeOH), positive
FAB-MS (m/z): 715 [MꢀH]ꢀ, HR-FAB-MS (m/z): 737.2632 [MꢀNa]ꢀ
Compound 8: Amorphous powder, [a]D25 ꢂ41.9° (cꢃ0.09, MeOH), posi-
tive FAB-MS (m/z): 633 [MꢀNa]ꢀ. HR-FAB-MS (m/z): 611.2041 [MꢀH]ꢀ
1
(Calcd for C28H35O15, 611.1976). H-NMR (in DMSO-d6) d: 2.70 (2H, m,
H2-7), 2.92—2.96 (2H, overlapped, xyl H-2, xyl Ha-5), 3.08 (1H, dd, Jꢃ8.6,
9.2 Hz, xyl H-3), 3.16 (1H, m, xyl H-4), 3.34 (1H, dd, Jꢃ7.9, 9.2 Hz, glc H-
2), 3.42—3.50 (4H, overlapped, xyl Hb-5, glc Ha-6, glc Hb-6, glc H-5), 3.62
(1H, m, Ha-8), 3.72 (1H, dd, Jꢃ9.2, 9.2 Hz, glc H-3), 3.89 (1H, m, Hb-8),
4.30 (1H, d, Jꢃ7.3 Hz, xyl H-1), 4.40 (1H, d, Jꢃ7.9 Hz, glc H-1), 4.71 (1H,
dd, Jꢃ9.2, 9.8 Hz glc H-4), 6.22 (1H, d, Jꢃ15.9 Hz, H-7ꢁ), 6.50 (1H, d,
Jꢃ7.9 Hz, H-6), 6.63 (1H, br s, H-2), 6.64 (1H, d, Jꢃ7.9 Hz, H-5ꢁ), 6.77
1
(Calcd for C33H46O17Na, 737.2633). H-NMR (in CD3OD) d: 1.72 (1H, m,
H-2), 2.04 (1H, m, H-3), 2.62 (1H, dd, Jꢃ11.7, 14.6 Hz, Ha-1), 2.72 (1H,
dd, Jꢃ4.4, 14.6 Hz, Hb-1), 3.23 (1H, dd, Jꢃ7.9, 9.2 Hz, glc H-2), 3.30 (3H,
s, 5-OCH3), 3.31 (1H, overlapped, glc H-4), 3.38 (2H, overlapped, glc H-3,
Ha-3a), 3.47 (1H, d, Jꢃ9.8 Hz, api Ha-4), 3.56 (1H, m, Ha-2a), 3.59—3.62

October 2004
1245
surements.
(1H, d, Jꢃ7.9 Hz, H-5), 6.99 (1H, d, Jꢃ7.9 Hz, H-6ꢁ), 7.05 (1H, br s, H-2ꢁ),
7.45 (1H, d, Jꢃ15.9 Hz, H-8ꢁ). 13C-NMR (in DMSO-d6) d: Table 2.
Cell and Virus HSV-1 strain KOS and Vero cells were provided by the
Chemo-Sero Therapeutic Institute.
References
1) Ho Y.-L., Kao K.-C., Tsai H.-Y., Chueh F.-Y., Chang Y.-S., Am. J.
Chin. Med., 31, 61—69 (2003).
2) Xia Z. Q., Zenk M. H., Phytochemistry, 31, 2695—2697 (1992).
3) Sholichin M., Yamasaki K., Kasai R., Tanaka O., Chem. Pharm. Bull.,
28, 1006—1008 (1980).
4) Yuasa K., Ide T., Otsuka H., Ogimi C., Hirata E., Takusi A., Takeda Y.,
Phytochemistry, 45, 611—615 (1997).
5) Abe F., Yamauchi T., Phytochemistry, 28, 1737—1741 (1989).
6) Achenbach H., Benirschke M., Torrenegra R., Phytochemistry, 45,
325—335 (1997).
7) Kato Y., Chem. Pharm. Bull., 11, 823—827 (1963).
8) Miyamura M., Nohara T., Nishioka I., Phytochemistry, 22, 215—218
(1983).
9) Shibuya H., Takeda Y., Zhang R.-S., Tanitame A., Tsai Y.-L., Kita-
gawa I., Chem. Pharm. Bull., 40, 2639—2646 (1992).
10) Sticher O., Lahloub M. F., Planta Med., 46, 145—148 (1982).
11) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501—507
(1987).
Antiviral Assays The antiviral activity of test samples against HSV-1
(KOS) was determined using the plaque reduction assay.12) Confluent mono-
layers of Vero cells in 6-well plates were infected with HSV-1 at 100 plaque-
forming units per cell. After a 1 h adsorption period, the cultures were over-
laid with Dulbecco’s modified Eagle minimum essential medium (DMEM)
containing 2% heat-inactivated FCS and 2% sulfonated g-globulin including
various concentrations of the test samples. The plates were incubated in the
CO2 incubator for 3 d, then fixed with formalin, and stained with crystal vio-
let in methanol. Infectious HSV-1 production was quantified by observing
the virus-induced cytopathic effect.
Cytotoxic Assays The anticellular activity was examined as described
below. Confluent monolayers of Vero cells were seeded in 96-well plates at
5ꢆ104 cells per well. After 1 d, the cells were refed with DMEM containing
5% FCS and various concentrations of the test samples. After 3 d of incuba-
tion, cells were fixed with formalin and stained with crystal violet in
methanol. Excess dye was washed off, and the dye incorporated by the
viable cells was eluted with dimethyl sulfoxide. The optical densities were
read at 560 nm.
12) Schinazi R. F., Peres J., Williams C. C., Chance D., Nahmias A. J., An-
timicrob. Agents Chemother., 22, 499—507 (1982).
13) Ikeda T., Ando J., Miyazono A., Zhu X.-H., Tsumagari H., Nohara T.,
Yo k omizo K., Ueda M., Biol. Pharm. Bull., 23, 363—364 (2000).
Acknowledgments We are grateful to Mr. K. Takeda and Mr. T. Iriguchi
of the Analytical Center of Kumamoto University for NMR and MS mea-