Antibacterial Alkaloids from

Zanthoxylum rhoifolium
Wellington de A. Gonzaga, AndrØia D. Weber, Sandro R. Giacomelli,
Ionara I. Dalcol, Solange C. S. Hoelzel, Ademir F. Morel

Abstract

Two new dihydrobenzophenanthridine-type alkaloids, 6-methoxy

[1, 3]dioxolo[4¢,5¢:4,5]benzo[c][1, 3]dioxolo[4,5-j]phenanthridine
(1) and 2,3,13-trimethoxy-5,11a-dihydro[1, 3]dioxolo[4¢,5¢:4,5]-
benzo[c]phenanthridine (2) were isolated from the stem bark of

Letter
Zanthoxylum rhoifolium, together with four other previously
known benzophenanthridine alkaloids, 6-acetonyldihydronitidine
(3) { = 8-acetonyldihydronitidine}, 6-acetonyldihydroavicine (4)
{ = 8-acetonyldihydroavicine}, 6-acetonyldihydrochelerythrine (5)
and xanthoxyline (6). The structures were elucidated mainly by
spectroscopic methods, including 1D and 2D NMR spectroscopy.
For alkaloids 1 and 2 we propose the trivial names rhoifolines A

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and B. The crude plant extracts and the alkaloids 3, 4 and 6 showed
activity against Gram () bacteria, while the new alkaloids 1 and 2
were inactive.

Zanthoxylum rhoifolium (Rutaceae), locally called ªmamica-de-

cadela, mamica-de-porca, jujev†º, is a plant that grows in South
America (Brazil, Uruguay, Paraguay and Argentina). It has been
used in Brazilian folk medicine as teas or infusions against a vari-
ety of disease [1]. Its medicinal properties may be related to its
alkaloid composition [1], [2], [3], [4], [5]. As a continuation of
our chemical studies on Rutaceae plants [6], we now report on 371
the isolation and structural elucidation of two new dihydroben-
zophenanthridine alkaloids, rhoifolines A (1) and B (2) from Z.
rhoifolium.

The molecular formula of rhoifoline A, C20H13NO5, was derived

from HREI-MS data (347.08004, [M]+), in combination with 13C-
NMR spectroscopy, which indicated 20 carbon peaks, including
two methylene, six methine, one methoxy, and eleven quatern-
ary carbon peaks. Analysis of the 1H-NMR spectrum (CDCl3, 400
MHz) of 1 revealed six aromatic hydrogens between d = 7. 0 to
8.0, one methoxy group at d = 3.88, and two methylene at
d = 6.0 and 6.03. The 1H-1H-homonuclear COSY spectrum of 1
revealed the presence of only one spin system in the molecule,
due to the hydrogens H-11 and H-12. The unambiguous position
of the substituents and all non-hydrogenated carbons of 1 were
achieved with the aid of the NOESY and of the HMBC spectra. 1H-
and 13C NMR data are reported in Table 1.

Affiliation: Departamento de Química (NPPN), Universidade Federal de Santa

Maria, Santa Maria RS, Brazil

Correspondence: Prof. Dr. Ademir Farias Morel ´ Universidade Federal de Santa

Maria ´ Departamento de Química ´ Camobi ´ Santa Maria ± RS ´ Brazil - CEP:
97105-900 ´ E-mail: afmorel@base.ufsm.br

Received: August 16, 2002 ´ Accepted: December 15, 2002

Bibliography: Planta Med 2003; 69: 371±374 ´  Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943

Letter ¼ Planta Med 2003; 69: 371 ± 374

Letter

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1
Table 1 H- and 13 C-NMR spectral data of compound 1 and 2 (400/100 MHz, CDCl3, d-values)

1 2
1 13 1 13
H C HMBC (H to C) H C HMBC (H to C)

1 7,07 s 104.7 C-4a,C-3,C12 7.19 s 104.7 C-3,C4a,C-12

2 ± 147.6 ± ± 147.4 ±
3 ± 147.0 ± ± 147.0 ±
372
4 7.50 s 100.6 C-2,C-4b,C12a 7.65 s 102.6 C-2,C4b,C12a
4a ± 120.8 ± ± 121.0 ±
4b ± 152.4 ± ± 135.8 ±
6 ± 164.0 ± ± 164.3 ±
6a ± 135.9 ± ± 119.0 ±
7 7.81 s 106.6 C-6,C-9,C-10a 7.94 s 108.6 C-6,C-9,C-10a
8 ± 148.2 ± ± 149.6 ±
9 ± 131.1 ± ± 153.5 ±
10 7.53 s 102.6 C-6a,C-8,C-10b 7.60 s 102.7 C-6a,C-8,C10b
10a ± 132.0 ± ± 128.9 ±
10b ± 116.8 ± ± 116.7 ±
11 7.82 d 118.5 C-4b,C-10a 8.00 d 118.3 C-4b,C-10a, C-12a
12 7.44 d 123.2 C-1,C-4a,C10b 7.57 d 123.2 C-1,C4a,C10b
12a ± 120.9 ± ± 131.8 ±
13 3.88 s 41.1 C-6 3.99 s 41.2 C-6
14 6.00 s 101.5 C-2,C-3 6.11 s 101.5 C-2,C-3
15 6.03 s 101.9 C-8,C9 4.06 s 56.2 C-8
16 ± ± ± 4.11 s 56.1 C-9

J (Hz): Compound 1: H-11,12 = 8.78; Compound 2: H-11,12 = 8.93.

The HREI-MS of rhoifoline B gave the molecular ion peak at m/z =
363.11080 corresponding to the molecular formula C21H17NO5. Its
1
H- and 13C-NMR spectral data were in part very close to those of
rhoifoline A (1), but some differences were observed at C-8 and C-
9. The signals of the oxymethylene protons H2 ± 15¢ were replaced
by two methyl singlets at d = 4.06 and 4.11. This were also con-
firmed by the 13C-NMR spectrum which showed similar signals to
those of 1, except for the replacement of the methylene signals of
C-15 by two methyl resonances at d = 56.1 and 56.2. 2D NMR ex-
periments (1H-1H COSY and NOESY, HMQC and HMBC) confirmed
the proposed structure 2 and allowed the complete assignment of
all 1H and 13C resonances (see Table 1).

Letter ¼ Planta Med 2003; 69: 371 ± 374

Table 2 Antibacterial activity: minimum amount required for inhibition on bacteria growth on TLC plates (mg)

Tested samples S. aureus S. epidermidis K. pneumonie S. setubal E. coli M. luteus

1 NA NA NA NA NA NA
2 NA NA NA NA NA NA
3 1.06 1.06 3.12 3.12 1.06 NA
4 1.06 3.12 1.06 3.12 3.12 NA
6 12,5 6,25 6,25 6,25 12,5 3,12
ME* 100 25.0 25.0 50.0 25.0 50.0
CH** 25.0 25.0 12.5 12.5 12.5 12.5
HE*** 12.5 25.0 25.0 12.5 12.5 12.5
Amoxicillin 0.2 0.2 0.2 0.2 0.2 0.2

Letter
* Methanol extract, ** chloroform extract, *** hexane extract; NA: not active.

The other known alkaloids reported, 6-acetonyldihydronitidine Plant material: Zanthoxylum rhoifolium was collected in Novem-
(3), 6-acetonyldihydroavicine (4) and 6-acetonyldihydrocheler- ber 2000, in the town of Jaguari, Rio Grande do Sul, Brazil, and

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ythrine (5), isolated here as racemic compounds, were identified
mainly by their 1H- and 13C-NMR spectral data, and by compari-
son with literature data [7], [8], [9]. Alkaloid 6 was identified by
direct comparison with an authentic sample of zanthoxyline [6].
To determine whether compounds 3, 4 and 5 were artifacts from
the extraction and isolation procedure, the same plant material
was extracted with MeOH at room temperature followed by ex-
traction under neutral conditions (absence of acetone). From this
procedure, alkaloids 3, 4 and 5 were also present.
By comparison of the spectral proprieties it can be deduced that
the previously reported 8-acetonyldihydronitidine and 8-aceto-
nyldihydroavicine [8] are identical to 6-acetonyldihydronitidine
[7] and 6-acetonyldihydroavicine, respectively. Therefore, we
suggest that the latter nomenclature should be adopted, to avoid
misunderstanding and duplication of reports.
The antimicrobial activities were evaluated by means of direct
bioautography in a TLC bioassay [10]. Table 2 shows the results
obtained with three Gram-positive, S. aureus, S. epidermidis, M.
luteus, and with three Gram-negative bacteria, K. pneumoniae, S.
setubal and E. coli. The extracts (methanol, chloroform, hexane)
and alkaloid 6 were active on all tested bacteria, while alkaloids
3 and 4 were inactive against M. luteus at the highest sample
amount tested (50 mg). In contrast to the significant activity of al-
kaloids 3, 4 and 6, alkaloids 1 and 2 were ineffective when eval-
uated against the tested bacteria.
Materials and Methods
General: Melting points were determined with an MQAPF-301 ap-
paratus and are uncorrected. Optical rotations were measured on a
Perkin Elmer-341 digital polarimeter. 1H- and 13C-NMR spectra
were recorded on a Bruker DPX 400 (400 MHz/100 MHz) NMR
spectrometer, in CDCl3 with TMS as internal standard; chemical
shifts are give in ppm and coupling constants in Hz. Low resolu-
tion MS were obtained with a GC/MS HP Mass Selective Detector,
coupled to a HP 6890 GC. HREIMS were recorded on a VG Autospec
mass spectrometer operating in the mode at 70 eV.
was identified by Prof. Renato Zµchia. The voucher specimen
(HDFI 194) is deposited in the Herbarium of the Federal Univer-
sity of Santa Maria.
Extraction and isolation: Dried and powdered bark (650 g) was
exhaustively extracted with MeOH at room temperature. The
solvent was evaporated under vacuum to afford 98 g of the
crude extract. To this extract, H2O/Et2O (1 : 1) (1000 mL) was
added and acidified with 2N HCl to pH 2.0. The organic phase
was separated and the H2O layer extracted with Et2O (3 ” 100
mL). The Et2O phases were not analyzed. The aqueous acidic so-
lution was basified with NH4OH to pH 9.0 and successively par-
titioned with n-hexane (yield 0.9 g) and CHCl3 (yield 2.4 g). The
hexane fraction (0.5 g) was chromatographed on silica gel col- 373
umn (230 ± 400 mesh, 30 g) and eluted with CHCl3 (1500 mL) to
give 15 fractions (ca. 100 mL each). Fractions 2 ± 4, 5 + 6, 7 + 8,
and 9 + 10 were recombined and concentrated under vacuum
to give 1 (60 mg), 2 (20 mg), 3 (25 mg), 4 (15 mg), respectively.
The CHCl3 fraction (2.4 g) was submitted to CC on silica gel
(230 ± 400 mesh, 100 g) and eluted with CHCl3 (2000 mL) con-
taining increasing amounts of MeOH (84 mL, up to 10 %) to give
15 fractions (ca. 130 mL each). Fractions 2 ± 4 (CHCl3 : MeOH,
98 : 2), 5 ± 7 (CHCl3 : MeOH, 95 : 5), 8 ± 12 (CHCl3 : MeOH, 95 : 5),
13 + 14 (CHCl3 : MeOH, 92 : 8), were recombined and concentrated
under vacuum to afford 3 (90 mg), 4 (500 mg), 5 (15 mg), and 6
(300 mg), respectively.
Rhoifoline A (1) was obtained as a white solid; m. p. 168 ± 169 8C.
For 1H- and 13C-NMR spectral data see Table 1. HREI-MS: m/z =
347.08004 (calcd. for C20H13NO5 : 347.07937).
Rhoifoline B (2) a colorless powder; m. p. 258 ± 259 8C. For 1H- and
13
C-NMR spectral data see Table 1. HREI-MS: m/z = 363.11080
(calcd. for C21H17NO5 : 363.110673).
6-Acetonyldihydronitidine (3): a pale yellow powder; m. p. 216 8C;
[a]D25: ±0.58 (c 0.23, MeOH {Lit.[8] m. p. 165 ± 167 8C, [a]D25: 08
(CHCl3)}. EI-MS: m/z = 405 [M]+, 348 (100 %). The 1H- and 13C-
NMR spectral data are in agreement with those reported in the
literature [7], [8].

Letter ¼ Planta Med 2003; 69: 371 ± 374

 6-Acetonyldihydroavicine (4): a pale yellow powder; m. p. 193 ±
194 8C; [a]D25: ±18 (c 0.34, MeOH) {Lit. [8] m. p. 184 ± 185 8C, [a]D25:
±6.68 (CHCl3)}. EI-MS: m/z = 389 [M+], 332 (100 %), 316, 274, 166.
Anal. Found: C 70.84 %, H 4.90 %, N 3.59 %; calcd: C 70.94 %, H
4.92 %, N 3.60 %. The 1H- and 13C-NMR spectral data are in agree-
ment with those reported in the literature [8].
6-Acetonyldihydrochelerythrine (5): a yellow solid; m. p. 199.5 ±
200.5 8C {Lit. [7] m. p. 185 ± 191 8C); [a]D25: ±0.58 (c 0.01, MeOH).
The 1H- and 13C-NMR spectral data are in agreement with those
reported in the literature [7].
Alkaloid 6 was identified by direct comparison with authentic
sample of zanthoxyline [6].
Letter
5
Cushman M, Mohan P, Smith ECR. Synthesis and biological activity of
structural analogues of the anticancer benzophenanthridine alkaloid
nitidine chloride. Journal of Medicinal Chemistry 1984; 27: 544 ± 7
6
Morel AF, Moura NF, Ribeiro HB, Machado ECSM, Ethur EM, Zanatta N.
Benzophenanthridine alkaloids from Zanthoxylum rhoifolium. Phyto-
chemistry 1997; 46: 1443 ± 6
7
Waterman GP, Khalid SA. The biochemical systematics of Fagaropsis
angolensis and its significance in the Rutales. Biochemical Systematics
and Ecology 1981; 9: 45 ± 51
8
Ajith PKN, Veranja K, Bandara BMR, Viajaya K, Tsutomu N, Masatoshi
N, Akira I, Tillekeratne LMV, Wijesundara DSA, Gunatilaka AAL. Anti-
microbial alkaloids from Zanthoxylum tetraspermum and caudatum.
Phytochemistry 2001; 56: 857 ± 61
9
Decaudain N, Kunesch K, Poison J. Alcaloïdes de Zanthoxylum
tsihanimposa. Phytochemistry 1974; 13: 505 ± 7
10
Hamburger OM, Cordell AG. Direct bioautography TLC assay for com-
pounds possessing antibacterial activity. Journal of Natural Products

11
Antibacterial activity: The antibacterial activity of the crude plant
extracts, and of alkaloids 1, 2, 3, 4 and 6 against three Gram-po-
sitive bacteria Staphylococcus aureus (ATCC 6538p),
Staphylococcus epidermidis (ATCC 12 228), Micrococcus luteus
1987; 50: 19 ± 22
Rahalison L, Hamburger M, Hostettmann K, Monod M, Frenk E. A
bioautographic agar overlay method for the detection of antifungal
compounds from higher plants. Phytochemical Analysis 1991; 2:
199 ± 203

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(ATCC 9341), the three Gram-negative bacteria Klebsiella
pneumoniae (ATCC 10 031), Salmonella setubal (ATCC 19196)
and Escherichia coli (ATCC 11103), were determined using the
bioautographic technique [10]. The microorganisms used in the
antibacterial assay have been maintained at the Chemistry De-
partment of the University of Santa Maria, RS, Brazil. For the an-
timicrobial assay, 50.0, 25.0, 12.5, 6.25, 3.12, 1.06 mg of alkaloids 1,
2, 3, 4, 6, and 200, 100, 50.0 25.0 and 12.5 mg of the extracts were
applied to pre-coated TLC plates, without elution of the samples.
The inoculum was prepared by culturing each organism in tryp-
tone soya agar (TSA, Oxoid) at 37 8C to a turbidity equivalent to
McFarland 0.5 standard (1.5 ” 108 CFU/mL). One microliter of
each diluted inoculum (104 ± 106 CFU) was applied onto Mueller-
374 Hinton Agar medium (MHA-DIFCO), and distributed over TLC
plates (5 ” 5 cm). After solidification of the media, the TLC plates
were incubated overnight at 37 8C [11]. Subsequently, bioauto-
grams were stained with an aqueous solution of 2,3,5-triphenyl-
tetrazolium chloride (TCC, 1 mg/mL) and incubated at 37 8C for
1 h. Amoxicillin was used as positive control.

Acknowledgements

This work was supported by FAPERGS (Fundacˈo de Amparo a

Pesquisa do Rio Grande do Sul), and CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico).

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Letter ¼ Planta Med 2003; 69: 371 ± 374