Antibacterial compounds from zanthoxylum rhetsa

Article abstract of DOI:10.1007/s12272-012-0703-9

A new amide, zanthorhetsamide (1), along with nine known compounds (2-10) was isolated from the roots and stem barks of Zanthoxylum rhetsa. The structure was characterized by spectroscopic methods. In addition, the antibacterial activity of the isolates was evaluated. Dihydrochelerythrine (4) exhibited strong activity against methicillin-resistant Staphylococcus aureus SK1 and moderate activity against Escherichia coli TISTR 780 with MIC values of 8 and 16 μg/mL, respectively.

Full text of DOI:10.1007/s12272-012-0703-9

Arch Pharm Res Vol 35, No 7, 1139-1142, 2012
DOI 10.1007/s12272-012-0703-9
Antibacterial Compounds from Zanthoxylum rhetsa
1
1
2
1
1
Cholpisut Tantapakul , Wong Phakhodee , Thunwadee Ritthiwigrom , Kulsiri Yossathera , Suwanna Deachathai ,
1
and Surat Laphookhieo
1
Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai, 57100
Thailand and Department of Chemistry, Faculty of Science, Chiang Mai University, Sutep, Muang, Chiang Mai 50200,
2
Thailand
(Received December 25, 2011/Revised January 17, 2012/Accepted January 19, 2012)
A new amide, zanthorhetsamide (1), along with nine known compounds (2-10) was isolated
from the roots and stem barks of Zanthoxylum rhetsa. The structure was characterized by
spectroscopic methods. In addition, the antibacterial activity of the isolates was evaluated.
Dihydrochelerythrine (4) exhibited strong activity against methicillin-resistant Staphylococ-
cus aureus SK1 and moderate activity against Escherichia coli TISTR 780 with MIC values of
and 16 g/mL, respectively.
8
µ
Key words: Zanthorhetsamide, Zanthoxylum rhetsa, Rutaceae, Antibacterial activity
INTRODUCTION
et al., 1982), 5,7,8-trimethoxycoumarin (
005) and dictamine (10) (Tanaka et al., 1985) from
About 200 species of the plants of the genus the stem barks and roots of Z. rhetsa
9) (Cheng et al.,
2
.
Zanthoxylum are distributed in pantropical countries.
Several species of Zanthoxylum are used in traditional MATERIALS AND METHODS
medicine and consumed as vegetables, especially in
Asian and African countries (Ladino and Suarez, General experimental procedure
2
010). Among them, Z. acanthopodium
,
Z. armatum
,
D
The [α] values were determined with a Bellingham
Z. nitidum, and Z. rhetsa are commonly used as spices & Stanly ADP440 polarimeter. The infrared (IR) and
and condiments in the Northern part of Thailand ultraviolet (UV) spectra were recorded on a Perkin-
(
Smitinand, 2001). Z. rhetsa produces a variety of Elmer FTS FT-IR and a Perkin-Elmer UV-Vis spectro-
1
13
biologically active metabolites including alkaloids, photometers, respectively. H and C nuclear magnetic
lignans, coumarins, and terpenoids (Cheng et al., resonance (NMR) spectra were recorded using a 400
2005). In our ongoing study on chemical constituents MHz Bruker spectrometer. Tetramethylsilane was used
and biological activity of Rutaceae plants, we report as internal reference. A MicroTOF, Bruker Daltonics
herein the isolation and structure elucidation of a new mass spectrometer was used to correct electrospray
amide (
methoxyphenethyl)benzamide (
alatamide ( ) (Maxwell and Ramperad, 1989), dihydro- chromatography (CC) were carried out on silica gel 60
chelerythrine ( ) (Martin et al., 2005), 6-acetonyldi- H (Merck, 5-40 m) and silica gel 100 (Merck, 63-200
hydrochelerythrine ( ) (Chen et al., 2011), 8-acetonyl- m), respectively. Precoated plates of silica gel 60 F254
dihydronitidine ( ) (Nissanka et al., 2001), asarinin ( were used for analytical thin layer chromatography
Gunatilaka et al., 1982), horsfieldine ( ) (Gunatilaka (TLC).
1) along with nine known compounds (N-(4- ionization time-of-flight mass spectra (ESI-TOF-MS).
2) (Vargas et al., 2010), Quick column chromatography (QCC) and column
3
4
µ
5
µ
6
7)
(
8
Plant material
Correspondence to: Wong Phakhodee, Natural Products
Research Laboratory, School of Science, Mae Fah Luang Uni-
versity, Tasud, Muang, Chiang Rai 57100, Thailand
Tel: 66-5391-6238, Fax: 66-5391-6776
The stem barks and roots of Z. rhetsa were collected
in December 2008 from Chiang Rai Province, North-
ern Thailand. The plant was identified by Mr. James
Maxwell, Chiang Mai University Herbarium and the
E-mail: w.phakhodee@sci.mfu.ac.th
1139

1140
C. Tantapakul et al.
specimen (MFU-NPR0026) was deposited at Natural Table I. 1H-NMR (400 MHz) and 13C-NMR (100 MHz)
data of zanthorhetsamide (1) in CDCl
3
Products Research Laboratory, School of Science, Mae
Fah Luang University.
Position
δC
δH (mult.,
J
in Hz)
1
2
45.2
74.1
3.82 (m)
5.95 (dd,
-
Extraction and isolation
J = 8.0, 4.4)
The stem barks and roots of Z. rhetsa (6.65 kg) were
extracted with acetone over the period of 3 days at
room temperature. Removal of solvent under reduced
pressure provided an acetone extract (140 g) that was
chromatographed by QCC and eluted with a gradient
of hexanes-acetone (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7,
1
'
129.8
127.9
114.1
159.8
114.1
127.9
167.5
134.3
126.8
128.7
131.7
128.7
126.8
173.6
34.5
2
'
7.32 (d,
J
= 8.4)
= 8.4)
3
'
6.90 (d,
J
4
'
-
5
'
6.90 (d,
7.32 (d,
J
J
= 8.4)
= 8.4)
6
'
2:8, 1:9, 0:10) to yield 14 fractions (A-N). QCC of frac-
1''
2''
3''
4''
5''
6''
7''
-
-
tion D (9.2 g) with 5% ethyl acetate (EtOAc)-hexanes
yielded eight subfractions (DA-DH). Subfraction DC
7.71 (br d,
7.42 (m)
7.49 (br t,
7.42 (m)
7.71 (br d,
-
J = 7.6)
(
1.6 g) was purified by CC with 5% EtOAc-hexanes to
yield compound
(6.2 mg). Fractions G (19.3 g) and H
15.5 g) were combined and subsequently subjected to
QCC with 10% EtOAc-hexanes to yield (400.5 mg)
4
J
= 7.2)
= 7.6)
(
7
J
and 10 (29.4 mg). Fraction J (7.05 g) was further sub-
jected to QCC with a gradient of EtOAc-hexanes (30%
EtOAc-hexanes to 100% EtOAc) to provide 20 subfrac-
tions (JA-JT). Subfraction JM (912.7 mg) was further
purified by CC with 30% CH Cl -hexanes to yield four
1'''
2'''
3'''
4'''
5'''
6'''
2.34 (t,
J
= 7.6)
24.9
29.5
22.8
14.1
55.4
-
1.58 (m)
1.26 (m)
1.27 (m)
2
2
subfractions (JM1-JM4). Subfraction JM3 (274.8 mg)
was subsequently separated by CC with 30% EtOAc-
0.85 (t,
3.80 (s)
J
= 6.4)
4
'-OMe
NH
hexanes to give
2 (39.5 mg) and 3 (3.0 mg). Subfrac-
6.42 (br s)
tion JO (520.2 mg) was further purified by CC using
20% acetone-hexanes to give six subfractions (JO1-
JO6). Compound
9 (2.2 mg) was derived from subfrac- mixture was stirred at room temperature for 10 min
tion JO2 (215.4 mg) by repeated CC using 10% EtOAc- and filtered through pad column chromatography
hexanes. Subfraction JS (363.0 mg) was subjected to (30% EtOAc-hexanes) to give
S-(+)-tembamide (1.3
26
QCC with a gradient of EtOAc-hexanes (30% EtOAc- mg). Yield: 81%; white solid; [
α]
D
+45.2 (
c
= 0.01,
7.75 (2H, dd
= 7.6, 7.2, 1.6,
, H-4'', 6''), 7.32 (2H, br d
(15.1 mg). Fraction N (1.11 g) was subjected = 8.8 Hz, H-2', 6'), 6.90 (2H, br d = 8.8 Hz, H-3', 5'),
to CC with 40% hexanes-CH Cl
tions (NA-NE). Compound (6.2 mg) was obtained from H-2), 3.90 (1H,
1
hexanes to 100% EtOAc) to yield
mg) and 10 subfractions (JS1-JS10). Subfraction JS3
was further purified by CC with 5% CH Cl -hexanes 1.2 Hz, H-5''), 7.41 (2H,
5
(4.2 mg),
8
(52.0 CHCl
3
); H-NMR (400 MHz, in CDCl
3
):
δ
,
J
= 8.8, 1.6 Hz, H-3'', 7''), 7.49 (1H, tt, J
m
, J
2
2
to provide
6
, J
2
2
to afford five subfrac- 6.63 (1H, br s, NH), 4.90 (1H, br dd
,
J
= 5.6, 2.0 Hz,
1
s, HO-2), 3.86 (1H,
m, H-1), 3.79 (3H,
subfraction NA (35.0 mg) by CC with 20% acetone-
hexanes as an eluent.
s
, OMe-4'), 3.50 (1H, , H-1).
m
Antibacterial activity testing
Escherichia coli TISTR 780, Salmonella typhimurium
TISTR 292, and Staphylococcus aureus TISTR 1466
Zanthorhetsamide (1)
White amorphous solid; mp 67-69 C; [α] +55.7 (c =
D
o
30
0
2
.01, CHCl ); UV λmax (MeOH) nm (log
ε
) 228 (4.08), were obtained from the Microbiological Resources,
74 (3.24), 281 (3.16); Fourier transform (FT)-IR (KBr) Centre of the Thailand Institute of Scientific and Tech-
nological Research, and methicillin-resistant S. aureus
Calcd for C H NO , 369.1940); H-NMR (400 MHz, (MRSA) SK1 was obtained from the Department of
3
−1
+
ν
max 1740, 1648 cm ; ESI-TOF-MS m/z 369.3510 [M]
1
(
2
2
27
4
13
in CDCl
Table I.
3
) and C-NMR (100 MHz, in CDCl
3
) data, see Microbiology, Faculty of Science, Prince of Songkla
University, Thailand. Minimum inhibition concentra-
tions (MICs) were determined by a two-fold serial
dilution method using Mueller Hinton broth according
Hydrolysis of zanthorhetsamide
Zanthorhetsamide (2.2 mg), dissolved in methanol to the Clinical and Laboratory Standards Institute re-
1 mL), was added to K CO (20 mg). The resulting commendations (CLSI, 2002). Vancomycin and genta-
(
2
3

Antibacterial Compounds from Zanthoxylum rhetsa
1141
mycin were used as standard antibacterial agents.
6''') and C=O at δC 173.6 (C-1''') instead of the acetyl
H C 3
group of tembamide acetate [δ 2.12/δ 21.5 (CH ) and
RESULTS AND DISCUSSION
C
δ 172.0 (C=O)] (Maxwell and Ramperad, 1989). These
data were suggested by COSY and HMBC correlations
3
0
Compound
1
, [
α
]
D
+55.7 (CDCl
3
), was isolated as a as shown in Fig. 2. The mass fragment ion at m/z
+
white amorphous solid. ESI-TOF-MS gave an molecular 254.1 [M-C
H O
6 11 2
] confirmed the presence of hexanoyl
+
ion peak [M] at m/z 369.3510, consistent with a moiety, which is placed on C-2 due to the correlations
molecular formula of C22 . The UV absorptions of H-2 ( 5.95), H-2''' ( 2.34) and H-3''' ( 1.58) with C-1'''
at max 228, 274, and 281 nm suggested the presence 173.6) in HMBC spectrum. Therefore, the structure 1
H
27NO
4
δ
δ
δ
λ
(δ
of a conjugated system in the molecule. The IR absorp- was identified to be zanthorhetsamide (2-benzamido-1-
tions showed stretching frequency of carbonyl func- (4-methoxyphenyl)ethylhexanoate). Zanthorhetsamide
−
1
tionalities at 1740 (ester) and 1648 (amide) cm . The
1
was partially hydrolyzed with K
2
CO
3
to yield tem-
1
1
H-NMR spectrum of
6.42, br s), a monosubstituted benzene group [
2H, br d, = 7.6 Hz, H-3'' and H-7''), 7.49 (1H, br t,
7.2 Hz, H-5'') and 7.42 (2H, m, H-4'' and H-6'')], a
,4-disubstituted benzene group [ 7.32 (2H, d, = 8.4
Hz, H-2' and H-6') and 6.90 (2H, d, = 8.4 Hz, H-3'
and H-5')], a methoxyl group ( 3.80, s, 4'-OMe), an
oxymethine ( 5.95, dd, = 8.0, 4.4 Hz, H-2) and
methylene protons ( 3.82, 2H, m, H-1). These signals
1
showed the presence of a N
H
bamide, which had a similar H-NMR spectrum and
2
6
(
(
δ
δ
7.71 specific rotation ([
α
]
]
+45.2, CHCl ) with synthetic S-
+56.9, CHCl ) (Kamal et al.,
3
D 3
25
J
J
(+)-tembamide ([
α
D
=
1
δ
J
J
δ
δ
J
δ
were similar to the signals described previously for
tembamide acetate isolated from Piper guayranum
1
13
(
Maxwell and Ramperad, 1989). The H- and C-NMR
spectra of displayed the signals for a hexanoyl
moiety (Kanokmedhakul et al., 2007) at δH 2.34 (t, 7.6
Hz, H-2''')/ 34.5 (C-2'''), 1.26 (m, H-3''')/ 29.5 (C-
'''), δH 1.58 (m, H-3''')/ C 24.9 (C-3''') δH 1.26 (m, H-
''')/δC 29.5 (C-4'''), δH 0.85 (t, 6.4 Hz, H-6''')
δC 14.1 (C- Fig. 2. Key HMBC correlations for zanthorhetsamide (
1
δ
C
δ
H
δ
C
3
4
δ
,
/
1).
Fig. 1. Structures of compounds 1-10.

1142
C. Tantapakul et al.
2
004). This result may imply that zanthorhetsamide
sesquiterpenoids and anti-HIV principles from the root
should have the same configuration as synthetic -(+)-
S
bark of Zanthoxylum ailanthoides. Bioorg. Med. Chem.,
13, 5915-5920 (2005).
tembamide. The absolute configuration of zanthorhets-
amide was, therefore, proposed to be 2S-(+)-zanthorhets-
amide.
CLSI, Method for dilution antimicrobial susceptibility tests
for bacteria that grow aerobically; Approved Standard
M7-A4. http://isoforlab.com/phocadownload/csli/M7-A7.pdf
All isolated compounds, except compound
6, were
(2006).
evaluated for their antibacterial activity against both
Gram-positive bacteria (Staph. and MRSA SK1) and
Gram-negative bacteria (S. typhimurium and E. coli).
The standard drugs were vancomycin (MIC = 1 and
Gunatilaka, A. A. L., Jasmin De Silva, A. M. Y., Sotheeswaran,
S., and Tellederatne, L. M. V., Horsfieldin, a lignin and other
constituents from Horfieldia iryaghedhi
1, 2719-2723 (1982).
Kamal, A., Shaik, A. A., Sandbhor, M., and Malik, M. S.,
Chemoenzymatic synthesis of ( )- and ( )-tembamide,
. Phytochemistry,
2
0.25
µ
g/mL against MRSA SK1 and Staph., respectively)
and gentamicin (MIC = 0.25 and 0.125
E. coli and S. typhimurium, respectively). All isolates
displayed weak activity against S. typhimurium with
µg/mL against
R
S
aegeline and denopamine by a one-pot lipase resolution
protocol. Tetrahedron Asymmetry, 15, 3939-3944 (2004).
Kanokmedhakul, S., Kanokmedhakul, K., and Buayairaksa,
M., Cytotoxic clerodane diterpenoids from fruits of Casearia
the same MIC value of 128
against Staph. Only compound
against MRSA (MIC of 8 g/mL), while the remaining
compounds were inactive. Compound also displayed Ladino, O. J. P. and Suarez, L. E. C., Isoquinoline alkaloids
moderate activity against E. coli (MIC = 16
whereas the rest of compounds were weakly active with
the same MIC value of 128
that the phenanthridine alkaloid
µ
g/mL, but were inactive
4
showed strong activity
µ
grewiifolia. J. Nat. Prod., 70, 1122-1126 (2007).
4
µg/mL),
of Zanthoxylum quinduense (Rutaceae). Biochem. Syst.
Ecol., 38, 853-856 (2010).
µ
g/mL. It should be noted Martin, M. T., Rasoanaivo, L. H., and Raharisololalao, A.,
without substitu-
Phenanthridine alkaloids from Zanthoxylum madagas-
cariense Fitoterapia, 76, 590-593 (2005).
Maxwell, A. and Ramperad, D., -Phenylethylamine-derived
amides from Piper guaranum J. Nat. Prod., 52, 411-414
1989).
4
.
ent located at C-6 plays an important role in both Gram-
positive and Gram-negative antibacterial activity.
β
.
(
ACKNOWLEDGEMENTS
Nissanka, A. P. K., Karunaratne, V., Bandara, B. M. R., Kumar,
V., Nakanishi, T., Nishi, M., Inada, A., Tillekeratne, L. M.
V., Wijesundara, D. S. A., and Gunatilaka, A. A. L., Anti-
microbial alkaloids from Zanthoxylum tetraspermum and
caudatum. Phytochemistry, 56, 857-861 (2001).
Smitinand, T., Thai Plant Names, Rivised ed. Prachachon
Co, Bangkok, (2001).
Tanaka, H., Ahn, J. W., Katayama, M., Wada, K., Marumo,
S., and Osaka, Y., Isolation of two ovicidal substances
against two-spotted spider mite, Tetranychus urticae
Koch, from Skimmia repens Nakai. Agric. Biol. Chem., 49,
We would like to thank National Research Council
of Thailand (NRCT) for financial support. Mae Fah
Luang University is also acknowledged for the gradu-
ate student research grant and laboratory facilities.
We appreciate to Mr. Nitirat Chimnoi, Chulabhorn
Research Institute, Bangkok, for recording mass spectra.
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