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Antimicrobial Activity of Isopteropodine

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Abstract

Bioassay-directed fractionation for the determination of antimicrobial activity of Uncaria tomentosa, has led to the isolation of isopteropodine (0.3%), a known Uncaria pentacyclic oxindol alkaloid that exhibited antibacterial activity against Gram positive bacteria.
Antimicrobial Activity of Isopteropodine
Rube
´n Garcı
´a
a,
*, Cesia Cayunao
a
, Ronny Bocic
a
, Nadine Backhouse
a
,
Carla Delporte
a
, Mercedes Zaldivar
b
, and Silvia Erazo
a
a
Department of Pharmacological and Toxicological Chemistry, School of Chemical and
Pharmaceutical Sciences, University of Chile, P. O. Box 233, Santiago 1, Chile.
Fax: 56-2-222 7900. E-mail: rgarciam@uchile.cl
b
Department of Biochemistry and Molecular Biology, School of Chemical and
Pharmaceutical Sciences, University of Chile, Santiago, Chile
* Author for correspondence and reprint requests
Z. Naturforsch. 60 c, 385Ð388 (2005); received October 18/November 29, 2004
Bioassay-directed fractionation for the determination of antimicrobial activity of Uncaria
tomentosa, has led to the isolation of isopteropodine (0.3%), a known Uncaria pentacyclic
oxindol alkaloid that exhibited antibacterial activity against Gram positive bacteria.
Key words: Uncaria tomentosa, Isopteropodine, Antibacterial Activity
Introduction
Uncaria tomentosa (Willd. ex Roemer and
Schultes DC.), commonly known as “cat’s claw”, is
a large climbing shrub, belonging to the Rubiaceae
family. Cat’s claw is indigenous to the Amazon
rainforest and other tropical areas of South and
Central America, including Peru, Colombia,
Ecuador, Guyana, Trinidad, Venezuela, Suriname,
Costa Rica, Guatemala, and Panama (Keplinger
et al., 1999; Lock de Ugaz, 1995).
The bark of cat’s claw is used in either folk
medicine or in procuring phytotherapeutic drugs.
This species contain several active compounds
which were tested widely for possible medicinal
value (rheumatism, tumors, anti-inflammatory ef-
fect and breast cancer) (Williams, 2001; Riva et al.,
2001; Aguilar et al., 2002; Gattuso et al., 2004).
Chemical studies on Uncaria tomentosa revealed
the presence of diverse compounds as quinovic gly-
cosides, polyoxygenated triterpenoids, catechins,
sterols and alkaloids, with diverse pharmacological
actions (Aquino et al., 1989; Wagner et al., 1985;
Keplinger et al., 1999; Kang et al., 2002; Sandoval et
al., 2002; Mur et al., 2002; Montoro et al., 2004).
However, of all the extensive biological activi-
ties investigated in this plant, specially for penta-
cyclic oxindol alkaloids (Kang et al., 2002; Lee
et al., 1999; Falkiewicz and Lukasiak, 2001) Ðonly
the quinovic glycosides were reported to have an-
timicrobial (antiviral) (Aquino et al., 1989 ; Wil-
liams, 2001) activity Ðreputed in folk medicine for
Uncaria tomentosa. These instances reinforced our
0939Ð5075/2005/0500Ð0385 $ 06.00 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com ·
D
interest for evaluating the antimicrobial activity of
“cat’s claw” bark against bacteria and fungi.
Materials and Methods
General experimental procedures
The solvent used for NMR studies was CDCl
3
.
The measurements of the NMR spectra were
carried out on a Bruker AMX-300 [
1
H NMR
(300 MHz),
13
C NMR (75 MHz)] spectrometer.
Column chromatography (CC) was carried out
using silica gel 60 G (Merck, 7734). TLC was per-
formed on silica gel GF 254 plates (Merck, 5554);
the spots were detected by UV light (254, 366 nm),
Liebermann Burchard test and/or p-anisaldehyde
and Dragendorf reagents.
GC-MS analyses were made on a Fisons MB 800
mass spectrometer (MS) coupled with a Hewlett-
Packard mod. 5890 series II gas chromatograph
(GC), equipped with a 25 m ¥0.2 mm i.d. HP U-2
column, with 0.25 µm film thickness. The initial
oven temperature was held at 40 C for 6 min, it
was then increased at 7 /min up to 200 C, at
110 KPa with helium as carrier gas.
HPLC was performed on a Shimadzu LC-10
AD, with a Shimadzu SPD-10 AV UV-VIS detec-
tor. Column: C
18
(Phenomenex); solvent system:
acetonitrile/buffer K
2
HPO
4
(pH 7) 50:50 with a
flow of 0.75 ml/min to 27 C.
Plant material
U. tomentosa bark was kindly provided by
Hochstetter Laboratory (collected from Peruvian
386 R. Garcı
´aet al. · Antimicrobial Activity of Isopteropodine
rainforest), and a voucher is kept at the botany
faculty laboratory.
Extraction and isolation
Ground dried bark (720 g) was sequentially ex-
tracted at room temperature with n-hexane, di-
chloromethane and methanol, yielding after re-
moval of the solvents in vacuo, 2.10 g (0.3%),
2.86 g (0.4%) and 82.5 g (11.5%), respectively.
Dichloromethane and methanol extracts were
active in the preliminary bio-assay, showing the
presence of a single bioactive alkaloid substance.
The extracts were separately worked up dissolving
the dried extracts in acetic acid (2 n), filtering, ba-
sifying with concentrated ammonia solution, and
extracting with chloroform (5 ¥25 ml). The chlo-
roform extracts were joined and subjected to CC
over silica gel, eluting an unpurified alkaloid with
dichloromethane/ethyl acetate 9.5:0.5 to dichloro-
methane/ethyl acetate 8:2, in fractions 49Ð72,
respectively. Preparative TLC on silica gel led to
the isolation of the purified alkaloid compound.
The purity of this substance was evaluated by gas
chromatography-mass spectroscopy, showing a sin-
gle peak at the retention time (Rt) of 21.5 min.
Crystallization from MeOH gave colorless needles.
Isopteropodine: M.p. 207Ð209 C. Ð[α]
24
D
Ð85
(c= 0.764, CHCl
3
). ÐUV(MeOH): λ
max
= 208, 243,
283 nm (sh). ÐMS: m/z (% rel. int.) = 368 (4.5)
[M
+*
], 223 (13), 208 (11), 180 (13), 146 (55), 130
(61), 117 (58), 103 (25), 77 (72), 69 (100), 55 (74);
C
21
H
2
N
2
O
4
.Ð
13
C NMR (ppm): δ= 181.43,
167.64, 154.98, 140.23, 133.76, 127.67, 124.53,
122.52, 109.82, 109.70, 72.14, 71.25, 56.93, 54.14,
53.51, 51.02, 37.83, 34.84, 30.17, 30.42, 18.65. Ð
1
H
NMR (ppm): δ= 8.56 (1H, brs), 7.42 (1H, s), 7.28
(1H, d, J= 7.5 Hz), 7.19 (1H, t, J= 7.6 Hz), 7.02
(1H, t, J= 7.5 Hz), 6.93 (1H, d, J= 7.6 Hz), 4.35
(1H, m), 3.60 (3H, s), 3.29 (2H, m), 2.46 (5H, m),
2.01(1H, m), 1.59 (2H, m), 1.41 (3H, d, J= 6.2 Hz),
0.87 (1H, appq, J= 12.8 Hz) (* brs: broad singlet;
appq: apparent quartet).
Antimicrobial assays
The antimicrobial activity of the extracts was de-
termined against Escherichia coli (ATCC 8739),
Klebsiella pneumoniae (isolated from a patient),
Salmonella aviatum (ATCC 2228), Pseudomonas
aeruginosa (ATCC 14207), Staphylococcus aureus
(ATCC 6538P), Micrococcus flavus (ATCC 10290)
and Bacillus subtilis (ATCC 6633), Candida albi-
cans and Saccharomyces cerevisiae.
Dilutions of 100 and 200 µg/ml of DMSO ex-
tract’s solution were added to a fixed volume of
Plate Count Agar (PCA) and Tryptic Soy Broth
(TSB). They were then superficially inoculated
with a single line of an overnight culture of the
different microorganisms and incubated at 37 C
for 24 h for bacteria and 28 C for fungi. Results
were recorded as growth or growth inhibition at
each extract concentration.
The active extracts were submitted to a bio-
autography agar overlay bioassay in order to deter-
mine the active compounds (Rahalison et al., 1995).
The turbidimetric method (Balows et al., 1991)
was used for determining MIC values of the iso-
lated substance against S. aureus and B. subtilis. It
consisted in preparing serial dilutions of the com-
pound (Table I) in TSB culture medium. Each
tube (carrying the same volume) was inoculated
with a fixed volume of the culture medium to ob-
tain 2 ml of final volume. Each dilution of the sam-
ple in study was assayed in triplicate.
Results and Discussion
U. tomentosa active extracts (dichloromethane
and methanol extracts) showed antimicrobial ac-
tivity only against M. flavus and B. subtilis and was
inactive against C. albicans and S. cerevisiae.
The methanol extract presented the major anti-
bacterial activity at concentrations of 100 µg/ml and
200 µg/ml. A bioautographic agar overlay in TLC of
the extracts and the isolated alkaloid was carried
out on a silica gel 60 F
254
plate developed with di-
chloromethane/ethyl acetate 1:1. The bioautograms
were sprayed with an aqueous solution of thiazolyl
blue (MTT). A positive antimicrobial reaction was
observed as a clear inhibition zone against a purple
background (Rahalison et al., 1995).
The isolated alkaloid was identified unambigu-
ously as isopteropodine (Uncarine E), by its mass
fragmentation,
1
H NMR,
13
C NMR and literature
data (Chan et al., 1966; Shamma and Foley, 1967;
Wagner et al., 1985; Seki et al., 1993).
The isopteropodine bark quantification (0.3%)
was assayed by HPLC using the isolated alkaloid as
internal reference (Rt: 12.1; Ganzera et al., 2001).
Isopteropodine was inactive, in the same way as
Uncaria extracts, against C. albicans and S. cerevi-
siae, and was active only against the Gram positive
bacteria S. aureus and B. subtilis, with a MIC value
R. Garcı
´aet al. · Antimicrobial Activity of Isopteropodine 387
Table I. Isopteropodine MIC value determinations.
Bacteria Isopteropodine concentration [µm]
353 380 408 435 462 489 516 543 571 598 625 652 679
S. aureus ++ÐÐÐÐÐÐÐÐÐÐÐ
B. subtilis ++++++++++++Ð
+, Bacterial growth.
Ð, No bacterial growth.
of 150 µg/ml (408 µm) and 250 µg/ml (679 µm),
respectively (Table I) [ampicillin: MIC 5 µg/ml
(14 µm) for S. aureus and 10 µg/ml (29 µm) for B.
subtilis].
In conclusion, these results support scientifically
the use of this species in popular medicine as anti-
microbial, despite its weaker activity compared
with the standard antibiotic.
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