The sample was prepared in four solvents. It
was prepared at a concentration of 1 mg/ml in
0.85% NaCl plus a 10% solution of one of the
three solvents: dimethyl sulfoxide (DMSO),
ethyl acetate or ethanol.
These solutions were assayed against
HSV-1 and HSV-2 using a cell protection
assay. Immortalized African Green Monkey
Kidney (Vero) cells (ATCC) were grown to a
monolayer in minimal essential medimum
(MEM) (Sigma) supplemented with 10% fetal
calf serum (FCS) (Sigma) in 12 well microtiter
plates (Falcon). The cells were infected with
about 100 virus particles of HSV-1 strain E377
or HSV-2 strain MS per well. After one hour,
the cells were treated with 1 ml of the
treatment (for 1 mg/ml compound in 10%
ethanol) or 1 ml of saline (for virus control
and cell control) and given 1 ml 2x MEM. The
cells were incubated for 72 hours and
observed for cytopathic effects from the virus
and cytotoxic effects from the samples. After
72 hours, the cells were stained with 0.05%
crystal violet in 35% methanol.
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115
Journal of Chemistry, Vol. 42 (1), P. 115 - 120, 2004
isomeranzin against Herpes simplex virus in vitro
from clausena heptaphylla (Roxb.) W. & Arn.:
isolation, structure and biological assay
Received 15-1-2003
Luu Duc Huy1, R. Caple2, C. Kamperdick3, Nguyen Thi Diep1, R. Karim2
1Institute of Chemistry, Vietnamese Academy of Science and Technology
2University of Minnesota, Duluth, USA
3Institute of Plant Biochemistry, Halle, Germany
Summary
The isomeranzin, a coumarin was isolated from the leaves of Clausena heptaphylla. The
structure was elucidated by IR, MS, 1H and 13C-NMR. For the first time, the anti-Herpes
simplex virus type 1 & 2 in vitro activity of the compound was reported and discussed.
I - introduction
Clausena heptaphylla (Roxb.) W. & Arn.
(Rutaceae) (Vietnamese name: Giæi 7 l¸) is a
tropical tree [1]. This species is also widely
used in traditional medicine. In Asia (China,
India, Vietnam, etc.), it is used for fever.
Several groups have reported the isolation of
the isomeranzin from Skimmia japonica [2],
Triphasia trifloliata [3], Clausena anisata [4].
In this paper we reporte on the isolation of the
coumarin from the leaves of Clausena
heptaphylla growing in Northern Vietnam, the
structure elucidation and the anti-Herpes
simplex virus type 1 and 2 in vitro biological
assay of the compound.
II - Results and Discussion
Specimens of Clausena heptaphylla were
collected many times, in November 1999, May
2000, October 2001, January 2002, from Ba Vi
forest of Northern Vietnam. The same coumarin,
isomeranzin was isolated simply in high yield
(0.7 ÷0.9%).
The structure of obtained coumarin was
determined by interpretation of its spectral data
(IR, MS, 1H and 13C-NMR) as well as by
comparison with spectral data of an authentic
sample [2, 5].
Thus, there are IR absorptions at 1608 cm-1
(C = O ketone), at 1719 cm
-1 (C = O lactone).
MS data gave a molecular peak at m/z 260
accompanied by ion peak [M+1]+ at m/z 261
and by fragment at m/z 190 corresponding to
the sequential loss of a COCH(CH3)2 of side-
chain, [M+1- COCH(CH3)2]
+.
The NMR spectrum showed signals characte-
ristic of a coumarin; spin-spin decoupling led to
the assignment of doublets at 7.36 and 6.84 ppm
(o-aromatic protons) and at 7.62 and 6.19 ppm
O OMeO
CH2 C
O
CH
CH3
CH3
1 2
3
45
6
7 8
10
9
1' 2' 3' 4'
5'
116
(-pyrone ring protons). The presence of a 3’-
methyl-2’-oxobutyl side - chain was indicated by
the appearance of a two-protons singlet at 4.00 ppm,
a one-proton septet at 2.83 ppm and doublet
signals at 1.21 ppm [-CH(CH3)2]. Signal at 3.85 ppm
(s, 3H) was assigned for 7 -OMe protons.
Figure 1: IR spectrum of isomeranzin (KBr)
Figure 2: MS spectrum of the product (CHCl3)
Wavenumbers (cm-1)
51
71
131
146
175
260
243201 217
89 103
50 100 150 200 250
190
117
Figure 3: 1H-NMR spectrum of the product (500 MHz, CDCl3)
Figure 4: 13C-NMR spectrum of the product (500 MHz, CDCl3)
8,0 m 7,5 7,0 6,5 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0
118
The 13C-NMR spectrum also gave support to
this assignment. Thus, 15 carbon atoms of
obtained compound were indicated by the
appearance of 15 signals. The DEPT 135
spectrum confirmed that the coumarin possesses
01 CH2, 08 CH and CH3; the DEPT 90 spectrum
- 05 CH. So, there are 06 quaternary C in this
compound.
The effectiveness and cytotoxicity of the
compound in different solvents is summarized in
Table 1. As can be observed from the table, this
compound is cytotoxic to Vero cells. Although
some of the solvents used were cytotoxic at the
concentrations used, synergistic cytotoxic effects
were observed when the compound was
combined with the solvent, especially in the case
of ethanol. Within 24 hours the monolayer of
cells was completely destroyed in the 10%
ethanol plus 1 mg/ml compound, while in the
10% ethanol, the cells were about 40%
destroyed. At this point in time viral growth had
only been observed in cells treated with DMSO
and infected with HSV-1. However, in other
solvents with 1mg/ml compound, the monolayer
was 40% or less destroyed after 24 hours. Long
term exposure of the cells to the solvent/
compound mixture at the maximum concentra-
tion of each destroyed the cells.
Table 1: Anti-HSV activity of the product
Sample Anti-HSV-1 Anti-HSV-2
10% ethanol +/+ ++/+
10% ethanol + 1 mg/ml cmpd ?/+++ ?/+++
5% ethanol +++/- +++/+
5% ethanol + 0.5 mg/ml cmpd ++/++ ?/+++
2.5% ethanol + 0.25 mg/ml cmpd +++/++ +/+
10% DMSO ?/+++ ?/+++
10% DMSO + 1 mg/ml cmpd ?/+++ ?/+++
5% DMSO ++/++ ++/+
5% DMSO + 0.5 mg/ml cmpd ?/+++ ?/+++
2.5% DMSO + 0.25 mg/ml cmpd +/++ ++/++
10% ethyl acetate +++/- ++/-
10% ethyl acetate + 1 mg/ml cmpd NT/+++ ?/+++
5% ethyl acetate +++/- +++/-
5% ethyl acetate + 0.5 mg/ml cmpd ++/+ ++/+
2.5% ethyl acetate + 0.25 mg/ml cmpd +++/- ++/-
0.85% NaCl + 1 mg/ml* cmpd ?/+++ ?/+++
*did not fully dissolve, so less than 1 mg/ml
- = no viral growth/cytotoxicity ( 0%)
+/- = very little viral growth/cytotoxicity (< 5%)
+ = definite viral growth/cytotoxicity (< 25%)
++ = major viral growth/cytotoxicity (< 60%)
+++ =extreme viral growth/cytotoxicity (up to 100%)
? = cannot determine viral growth due to extreme cytotoxic effects
NT = not tested
119
Although the compound does not demons-
trate spectacular effects against either virus in
Vero cells, 0.5 mg/ml compound in 5% ethyl
acetate or 0.25 mg/ml compound in 2.5% ethyl
acetate generally provided about 40% protection
from both HSV-1 and HSV-2 with little to no
cytotoxic effects. The other combination that
proved effective was 0.25 mg/ml compound in
2.5% DMSO. This combination provided about
40% protection against HSV-2, though it was
also more cytotoxic than either ethyl acetate
mixture. Against HSV-1, it provided about
70% protection, though it was fairly ( 40%)
cytotoxic. Finally, 0.25 mg/ml in 2.5% ethanol
provided about 70% protection against HSV-2
with minor cytotoxic effects.
First symbol refers to viral toxicity, second
symbol to cytotoxicity. Thus +++/- would show
extreme viral growth, but no cytotoxic effects
and +/+++ shows some viral growth but
extreme cytotoxic effects
Based upon this study, this compound
showed some promise as an anti-HSV agent,
though it was not as effective as other plant-
originated compounds have been. The
combinations most effective against HSV-1
(0.25 mg/ml in 2.5% DMSO) or HSV-2 (0.25
mg/ml in 2.5% ethanol) were also cytotoxic.
Whether this cytotoxicity is due to trace
chemicals remaining from the extraction
process, or from the compound itself is
uncertain. When the compound was dissolved
in ethyl acetate, it was effective against both
viruses, albeit to a lesser extent, and not as
cytotoxic. This compound was cytotoxic to
cells even when it was at its maximum limit of
solubility in water (with 0.85% NaCl). Further
studies should focus on examining the
compound at lower concentrations (0.5 mg/ml
down to 0.05 mg/ml or lower), examining the
role of solvent at lower concentrations (2.5%
ethyl acetate plus virus), examining the
effectiveness of a water extract of the plant, or
elucidating the mechanism of synergistic
cytotoxicity of ethanol and the compound. It is
possible that the compound reacts with the
ethanol, changing the compound to a more
cytotoxic form.
III - Experimental
1. General experimental procedures
Solvents were purified and distilled prior to
use.
Optical rotation was measured on ATAGO
POLAR- D.
Melting point was determined on Boetius
apparatus (D. Germany).
IR spectra were recorded on FT-IR-
IMPACT-410 as KBr pellets .
1H-NMR and 13C-NMR spectra were
recorded on Bruker AM 500 spectrometer in
CDCl3 with TMS as internal reference.
MS spectra were recorded as solutions in
CHCl3 on MS-5989B spectrometer.
Analytical thin-layer and column chromato-
graphy was performed using silicagel Merck
GF254.
Biological assay was performed in the
University of Minnesota, Duluth, USA.
2. Plant material
The dried leaves of Clausena heptaphylla
used in this investigation was collected from
the Ba Vi Forerst, Northern Vietnam many
times. The plant was determined by Botanist,
Dr Tran Ngoc Ninh, Vietnamese Academy of
Science and Technology.
3. Extraction and isolation
Leaves of Clausena heptaphylla were dried
at 40 ÷50
0C and powdered. The powder (500 g)
was extracted with 3 litres MeOH 900 at 800C.
The extract was filtered and concentrated in
vacuo to yield 84 g extract. The concentrated
extract was fractionated into Hexan, EtOAc,
BuOH, and H2O-soluble fractions, sequentially.
The EtOAc-soluble fractions (28.7 g) was sub-
jected to silicagel column chromatography.
Elution with Hexan / EtOAc 10 : 1 ÷10 : 2 gave
isomeranzin (4 g, 0.8%). Recrystallization from
Hexan / EtOAc 10 : 1 gave needles melting at 58 ÷
600C (60oC ÷620C (from pentane) [2]), []D 00
(CHCl3) (0
0 [2]). IR (, cm-1, KBr) : 838, 1028,
1115, 1250, 1291, 1394, 1502, 1608, 1719,
2968. MS (m/z, CHCl3) : 261 [M+1]
+, 260 [M+],
120
190 [M+1-COCH(CH3)2]
+. 1H-NMR (, ppm,
CDCl3): 1.21 [d, 6H, j = 6.93 Hz, 3’ - CH(CH3)2];
2.83 (septet, 1H, 3’-H); 3.85 (s, 3H, 7-OCH3);
4.00 (s, 2H, 1’ - CH2); 6.19 (d, 1H, 4-H, j = 9.46
Hz); 6.84 (d, 1H, 6-H, j = 8.60 Hz); 7.36 (d, 1H,
5-H, j = 8.60 Hz); 7.62 (d, 1H, 3-H, j = 9.46 Hz).
13C-NMR (, ppm, CDCl3): 18.42 (4’-C, 5’-C);
34.69 (1’-C); 40.88 (3’-C); 56.12 (7 - OCH3);
107.26 (6-C); 111.95 (10-C); 112.91 (8-C);
112.97 (3-C); 127.55 (5-C); 143.80 (4-C); 153.20
(9-C); 160.44 (7-C); 160.97 (2-C); 210.76 (2’-C).
4. Methods of biological activity assay [6 - 9]
The sample was prepared in four solvents. It
was prepared at a concentration of 1 mg/ml in
0.85% NaCl plus a 10% solution of one of the
three solvents: dimethyl sulfoxide (DMSO),
ethyl acetate or ethanol.
These solutions were assayed against
HSV-1 and HSV-2 using a cell protection
assay. Immortalized African Green Monkey
Kidney (Vero) cells (ATCC) were grown to a
monolayer in minimal essential medimum
(MEM) (Sigma) supplemented with 10% fetal
calf serum (FCS) (Sigma) in 12 well microtiter
plates (Falcon). The cells were infected with
about 100 virus particles of HSV-1 strain E377
or HSV-2 strain MS per well. After one hour,
the cells were treated with 1 ml of the
treatment (for 1 mg/ml compound in 10%
ethanol) or 1 ml of saline (for virus control
and cell control) and given 1 ml 2x MEM. The
cells were incubated for 72 hours and
observed for cytopathic effects from the virus
and cytotoxic effects from the samples. After
72 hours, the cells were stained with 0.05%
crystal violet in 35% methanol.
IV - conclusion
1. The isomeranzin was isolated many
times and simply from the leaves of the
Clausena
heptaphylla in high yield (0.7 ÷0.8%). The
structure of the compound was determined by
interpretation of its spectral data (IR, MS, 1H
and 13C-NMR) and by comparison with spectral
data of an authentic sample.
2. The isomeranzin showed some promise
as an anti-Herpes simplex virus agent.
This work was supported by the National
Basic Research Program in Natural Science,
Vietnam.
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5. F. A. Macias, G. M. Massanet, and F.
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