Phytomedicine 17 (2010) 11331139

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Phytomedicine
journal homepage: www.elsevier.de/phymed

Anticancer activity of the Uncaria tomentosa (Willd.) DC. preparations with

different oxindole alkaloid composition
Radosaw Pilarski a , Beata Filip b , Joanna Wietrzyk b , Mieczysaw Kuras c , Krzysztof Gulewicz a,
a
b
c

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 str., 61-704 Pozna
n, Poland
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocaw, Poland
Department of Ecotoxicology, Warsaw University, Warsaw, Poland

a r t i c l e
Keywords:
Uncaria tomentosa
Una de gato
Cats claw
Oxindole alkaloids
Anticancer activity

i n f o

a b s t r a c t
The activity of Uncaria tomentosa preparations on cancer cells was studied using in vitro and in vivo models.
IC50 values were calculated for preparations with different quantitative and qualitative oxindole alkaloid
composition: B/W37 bark extracted in water at 37 C, B/Wb bark extracted in boiling water, B/50E37
bark extracted in 50% ethanol at 37 C, B/Eb bark extracted in boiling 96% ethanol, B/96E37 bark
extracted in 96% ethanol at 37 C and B/SRT bark extracted in water and dichloromethane. Generally,
the results obtained showed a high correlation between the total oxindole alkaloid content (from 0.43% to
50.40% d.m.) and the antiproliferative activity of the preparations (IC50 from >1000 g/ml to 23.57 g/ml).
B/96E37 and B/SRT were the most cytotoxic preparations, whereas the lowest toxicity was observed
for B/W37 . B/96E37 were shown to be active against Lewis lung carcinoma (LL/2) [IC50 = 25.06 g/ml],
cervical carcinoma (KB) [IC50 = 35.69 g/ml] and colon adenocarcinoma (SW707) [IC50 = 49.06 g/ml].
B/SRT was especially effective in inhibiting proliferation of cervical carcinoma (KB) [IC50 = 23.57 g/ml],
breast carcinoma (MCF-7) [IC50 = 29.86 g/ml] and lung carcinoma (A-549) [IC50 = 40.03 g/ml]. Further
animal studies on mice bearing Lewis lung carcinoma showed signicant inhibition of tumor growth by
B/W37 administered for 21 days at daily doses of 5 and 0.5 mg (p = 0.0009). There were no signicant
changes in the cell cycles of tumor cells with the exception of cell decrease at the G2 /M phase after the
administration of B/96E37 at a daily dose of 0.5 mg and the G1 /G0 cells cycle arrest demonstrated after
the B/SRT therapy at a daily-dose of 0.05 mg. All tested preparations were non-toxic and well tolerated.
2010 Elsevier GmbH. All rights reserved.

Introduction
Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle is a woody climbing vine belonging to the Rubiaceae family
that grows in the highlands of the Amazon rain forest (Cisneros et
al. 2005). Due to its curved hooks this plant is commonly known
under the Spanish name una de gato that is translated to English
as cats claw (Keplinger et al. 1999).
Since ancient times, the indigenous people of Peru have used
its inner bark and root to prepare medical decoctions (Aguilar et
al. 2002; kesson et al. 2003a; Cisneros et al. 2005; Keplinger
et al. 1999). Recently, cats claw has become more popular and
increasingly distributed all over the world as an immunomodu-

Abbreviations: B/W37 , bark extracted in water at 37 C; B/Wb , bark extracted in

boiling water; B/50E37 , bark extracted in 50% ethanol at 37 C; B/Eb , bark extracted
in boiling 96% ethanol; B/96E37 , bark extracted in 96% ethanol at 37 C; B/SRT, bark
extracted in water and dichloromethane.
Corresponding author. Tel.: +48 61 852 85 03; fax: +48 61 852 05 32.
E-mail address: krysgul@ibch.poznan.pl (K. Gulewicz).
0944-7113/$ see front matter 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2010.04.013

latory, anticancer and anti-inammatory herbal remedy (De Jong

et al. 1999). The list of treated diseases includes gastric ulcers, diarrhoea, gonorrhoea, arthritis and rheumatism, acne, diseases of the
urinary tract and cancers (Heitzman et al. 2005).
A number of chemical studies on U. tomentosa revealed the
presence of diverse compounds such as alkaloids, quinovic acid
glycosides, polyhydroxylated triterpenes, avonoids, catechins and
sterols (Aquino et al. 1989, 1991; De Matta et al. 1976; Heitzman et
al. 2005; Kitajima et al. 2004; Muhammad et al. 2001). Wagner and
co-workers, already in 1985, demonstrated that some of these compounds (i.e. four oxindole alkaloids: isopteropodine, pteropodine,
isomitraphylline, and isorynchophylline) potentiated the phagocytosis by white blood cells (Lemaire et al. 1999; Wagner et al. 1985).
Further in vitro and in vivo investigations undertaken by other
researchers showed a very complex and multilateral activity of the
active constituents of U. tomentosa (Gulewicz et al. 2004; Heitzman
et al. 2005; Kuras et al. 2006; Okuhama et al. 2001; Sandoval et al.
2002).
U. tomentosa preparations have been found to show various
anti-inammatory effects such as inhibiting the production of the
inammatory cytokine TNF and the activation of the nuclear tran-

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R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

scription factor NF-B (kesson et al. 2003a; Miller et al. 1999;

Sandoval-Chacon et al. 1998; Sandoval et al. 2000). In other studies, U. tomentosa extracts induced the production of factors that
regulate lymphocyte-proliferation by human endothelial cells and
the synthesis of IL-1 and IL-6 by rat alveolar macrophages (Lemaire
et al. 1999; Wurm et al. 1998). Proapoptotic and antiproliferative
effects of aqueous extracts on human myeloid leukaemia cells such
as K562 and HL60 have also been described (Sheng et al. 1998). U.
tomentosa constituents exerted cytotoxic activity on lymphoblastic leukaemia and breast cancer cells (Bacher et al. 2006; Riva
et al. 2001). Furthermore, it has been shown that U. tomentosa
extracts enhance DNA repair and protect lymphocytes from apoptosis induced by hydrogen peroxide, diphenyl-2-picrylhydrazyl,
and peroxynitrite (kesson et al. 2005; Sheng et al. 1998). Significant antioxidative properties were also observed in our previous
analyses including trolox equivalent antioxidant capacity (TEAC),
peroxyl radical-trapping capacity (PRTC) and superoxide radical
scavenging activity (SOD) (Pilarski et al. 2006). Aqueous extract
was demonstrated to increase the number of leukocytes in normal rats and splenic lymhocytes subsets CD4+, CD8+ and B cells
in mice (kesson et al. 2005; Eberlin et al. 2005). This increase in
cell viability and DNA stability could be important for the recovery process of cancer patients after myelosuppression caused by
chemotherapy (Allen-Hall et al. 2007; Steinberg 1995).
One of the main problems hindering systematic research on
biological activities of U. tomentosa preparations is the different
composition of extracts, which makes it difcult to draw direct
comparisons between studies. The aim of our study was to produce a series of preparations using different extraction methods
and to investigate quantitative and qualitative differences in their
composition. Next, we studied anticancer activity of these preparations using selected in vitro and in vivo models. Several human and
mouse cancer cell lines were used to investigate antiproliferative
activity of the preparations and the selected ones were then studied for toxicity and antitumor effect in the in vivo mouse model of
Lewis lung carcinoma.
Materials and methods
Plant material
The bark of U. tomentosa originated in Peru was kindly supplied
by Vilcacora omianki Centre (omianki, Poland). The general characteristics of this material were described previously (Pilarski et al.
2006). The voucher material is deposited at the Laboratory of Phytochemistry, Institute of Bioorganic Chemistry, Polish Academy of
Poland.
Sciences, Poznan,
Obtaining B/W37 , B/Wb , B/50E37 , B/Eb and B/96E37 preparations
We prepared several extracts of 1.0 gram of the bark in 10 ml
of water, 50%, or 96% ethanol for 8 h at 37 C (B/W37 , B/50E37 and
B/96E37 , respectively). Water and 96% ethanol extractions were also
performed at boiling temperatures of these solvents for 8 h (preparations B/Wb , and B/Eb ). Then, the extracts were centrifuged for
15 min at 4000 rpm. Supernatants were evaporated on Speed-Vac
and next exsiccated with P2 O5 .
Obtaining alkaloids-rich bark preparation (B/SRT)
10.0 gram of the bark of U. tomentosa was extracted with 50 ml
of water (6 h, 37 C). Next, the sample was centrifuged at 3500 rpm.
50 ml of dichloromethane was added to the supernatant and the
mixture was intensively shaken. The organic layer was evaporated
to dryness under vacuum at 40 C. The residue (150 mg) was dis-

solved in 1.5 ml of 96% of ethanol and ltered through a 0.22 m

lter.
HPLC-quantitative alkaloid analysis
The preparations were analyzed on oxindole alkaloid qualitative
and quantitative contents according to the standardization protocols elaborated for U. tomentosa. To 100 mg of preparation, 15 ml of
2% sulphuric acid solution was added and sonied for 15 min with
an ultrasonic bath (Bandelin Sonorex RK 103H). The mixture was
then centrifuged at 3000 rpm for 10 min and extracted three times
with 10 ml of ethylacetate. The aqueous phase was separated and
adjusted to pH = 10 with 10% ammonium hydroxide and extracted
three times with 10 ml of ethylacetate each. The organic phases
were combined, evaporated to dryness and the residue dissolved
in 1 ml of methanol. The qualitative and quantitative contents of
alkaloids were determined by means of HPLC ngerprint analysis
as desribed previously (Pilarski et al. 2007).
Cancer cell lines
Antiproliferative activity was evaluated using the following
established in vitro cancer cell lines: HT-29 (colon adenocarcinoma), SW707 (human colorectal adenocarcinomas), KB (human
cervical carcinoma), MCF7 (human breast carcinoma), A549
(human non-small cell lung carcinoma), OAW-42 (human ovarian cystoadenocarcinoma), HL60 (human acute promyelocytic
leukemia), LLC (LL/2, mouse Lewis lung carcinoma) and B16 (mouse
melanoma). All cell lines were obtained from the American Type
Culture Collection (Rockville, Maryland, USA) and maintained at
the Cell Culture Collection of the Institute of Immunology and
Experimental Therapy, Wroclaw, Poland. 24 h before adding the
test preparations, the cells were plated in 96-well plates (Sarstedt, USA) at a density of 104 cells per well in 100 l of culture
medium. The SW707, OAW-42, B16, LL/2 cell lines were cultured
in the Dulbeccos Modied Eagles Medium supplemented with
2 mM glutamine (DMEM, Gibco, Warsaw, Poland). The KB, MCF7,
A549 cell lines were cultured in the Minimum Essential Medium
Eagle (MEM, Gibco, Warsaw, Poland). The HL60 and HT-29 cell
lines were cultured in the RPMI-1640 medium (Gibco, Warsaw,
Poland) and McCoys 5 medium supplemented with 2 mM glutamine (Gibco, Warsaw, Poland), respectively. All mediums were
also supplemented with 10% fetal calf serum (Gibco, Grand Island,
USA). The cell cultures were maintained at 37 C humid atmosphere
saturated with 5% CO2 .
Antiproliferative assays in vitro
The in vitro cytotoxic effect of B/W37 , B/Wb , B/50E37 , B/Eb ,
B/96E37 and B/SRT was examined after 72-h of exposure of the cultured cells to varying concentrations of the preparations, using the
SRB assay as described by Skehan et al. (1990). Briey, the cells
were attached to the bottom of plastic wells by xing them with
cold 50% trichloroacetic acid (TCA, SigmaAldrich Chemie GmbH,
Steinheim, Germany) on the top of the culture medium in each well.
The plates were incubated at 4 C for 1 h and then washed ve times
with running water. The background optical density was measured
in the wells lled with culture medium without the cells. The cellular material xed with TCA was stained with 0.4% sulforhodamine B
(SigmaAldrich Chemie Gmbh, Steinheim, Germany) and dissolved
in 1% acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye
was removed by rinsing (4) with 1% acetic acid. The protein-bound
dye was extracted with 10 mM unbuffered TRIS base (POCH, Gliwice, Poland) for determination of optical density (at 540 nm) in
a computer-interfaced, 96-well microtiter plate reader Multiskan
RC photometer (Labsystems, Helsinki, Finland). The in vitro results

R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

were presented as inhibitory concentration 50% (IC50 ) values. Each

compound at a given concentration was tested in triplicates in each
experiment, which was repeated two times.
Animal experiments
[C57Bl/6 DBA/2]F1 (BDF1 ) male, 1216 weeks old mice, weighing 2025 g, supplied by the Department of Genetics and Laboratory
Animal Breeding, M. Sklodowska-Curie Memorial Cancer Center
and Institute of Oncology, Warsaw, Poland, were maintained in
standard laboratory conditions. All experiments were performed
according to Interdisciplinary Principles and Guidelines for the Use
of Animals in Research, Marketing and Education issued by the New
York Academy of Sciences Ad Hoc Committee on Animal Research
and were approved by the 1st Local Committee for Experiments
with the Use of Laboratory Animals, Wroclaw, Poland.
Therapeutic efcacy of the B/W37 preparation in different
administration schedules to mice bearing LLC tumors
The B/W37 preparation was dissolved in physiological salt solution with the addition of 10% DMSO to nal concentration of
25 mg/ml. Lower doses were prepared using serial dilutions. LLC
cells derived from in vitro culture were inoculated subcutaneously
(s.c.) into the right ank region with 3 105 cells per mouse (day
0). On days 121 animals were treated intraperitoneally (i.p.)
with B/W37 preparation at doses of 5 mg/day (n = 5, one mouse
died due to the trauma during the experiment), 0.5 mg/day (n = 6),
0.05 mg/day (n = 6) in the 200 l volume. One group (n = 6) received
therapy by the preparation at the dose of 0.05 mg/day from days
7 to 21. Two groups were treated with equivalent volume of
physiological salt solution (control, n = 6) and DMSO 10% (n = 6).
Animals were monitored for food and water consumption, weight,
and general behavioral status.
Therapeutic responses of mice bearing LLC tumors on the B/96E37
and B/SRT intraperitoneal injections
5 mg amounts of B/96E37 and B/SRT were dissolved in 200 l
DMSO and lled up to 2 ml with the physiological salt solution. LLC
cells derived from in vitro culture were inoculated subcutaneously
(s.c.) into the right ank region with 2 105 cells per mouse (day
0). At the days 221 animals were treated i.p. B/96E37 (n = 8) and
B/SRT (n = 8) with a doses 0.05 mg/day and 0.5 mg/day (the control
mice (n = 8) were injected with 200 l of the physiological salt).
Animals were monitored for food and water consumption, weight,
and general behavioral status.
Tumor cell cycle analysis
LLC lung carcinoma cells from primary tumors of mice treated by
on the B/96E37 and B/SRT were obtained after nishing the experiment by mincing fresh tumor tissue with a scalpel, passing them
through a plasma lter, and suspending in phosphate-buffered
saline (PBS). The cell suspension was washed once with PBS supplemented with 2% of fetal bovine serum. Then the cells were washed
in PBS and counted in a hemacytometer. 1 106 cells were xed for
24 h in 70% ethanol at 20 C. Next, the cells were washed twice in
PBS and incubated with RNAse (50 g/ml, Fermentas, Germany)
at 37 C for 1 h. The cells were stained for 30 min with propidium iodide (50 g/ml, SigmaAldrich Chemie GmbH, Steinheim,
Germany) at 4 C and cellular DNA content of the samples was measured using the FACS Calibur ow cytometer (Becton Dickinson,
San Jose, CA, USA). The results were analyzed using WinMDI 2.8
software.

1135

Data processing and statistical analysis

The tumor volume was calculated using the formula TV = (a2
b)/2, where a means shorter diameter and b means longer diameter in millimeters. Tumors were collected when the animals were
sacriced 22 days after the inoculation of the LLC cells. Dynamics
of tumor growth in animal experiments were analyzed using the
multiple analysis of variance (MANOVA) for the B/W37 preparation
and one-way analysis of variance (ANOVA) for B/96E37 and B/SRT
preparations. Blood morphology and cell cycle were assessed using
ANOVA. Data transformations were applied to meet assumptions
of parametric tests whenever applicable. Values of p < 0.05 were
considered to be signicant.

Results and discussion

HPLC-ngerprint analysis of alkaloids
The oxindole alkaloids proles of all preparations used in cytotoxicity evaluations were presented in Table 1.
All of these preparations (B/W37 , B/Wb , B/50E37 , B/Eb , B/96E37 ,
and B/SRT) were produced from the same bark material. However, it might be easily expected that using different concentration
of ethanol or applying dichloromethane in case of B/SRT would
result in their chemical composition being signicantly different.
In fact, the alkaloid participation increases with the ethanol concentrations reaching 3.48% for the B/96E37 preparation obtained by
extraction with nearly pure ethanol. The highest alkaloid content
was shown for B/SRT where over 50% of dry mass was pure oxindole
alkaloids. On the other hand, extraction with water results in the
lowest alkaloid content of 0.43% observed for B/W37 . The temperature of extraction was generally not signicant for total alkaloid
contents, however, this parameter was pivotal to quantitative representations of particular alkaloids. In general, the preparations
obtained by extraction in boiling solvents have a lower speciophylline content and a higher contribution of isopteropodine. For
instance, taking into account speciophylline, using boiling ethanol
caused a decrease in its participation in total alkaloid content by
over 6 times in comparison with the preparation obtained at 37 C.
This observation is in agreement with literature in which nonstability of speciophylline is reported (Keplinger et al. 1999; Laus
1998; Laus et al. 1996). The above-presented changes in alkaloid
proles are probably a result of isomerisation by retro-Mannich
ring opening, rotation and Mannich ring closure (Keplinger et al.
1999).
It must be emphasized that all presented ngerprints did not
show the presence of any tetracyclic oxindole alkaloids conrming the high pharmacological quality of the obtained preparations
(ukasiak et al. 2001). A number of studies have shown that U.
tomentosa occurs in nature in two different chemotypes characterized by pentacyclic (POA) or tetracyclic (TOA) pattern of indole
and oxindole alkaloids (Hemingway and Phillipson 1974; Keplinger
et al. 1999; Laus and Keplinger 1997). As shown, POA are more biologically active than TOA, particularly if their anti-inammatory
and antiproliferative potency is considered (Wurm et al. 1998).
Both groups of alkaloids may act antagonistically and the selection
of proper cats claw material seems to be pivotal for a successful
therapeutic approach (Keplinger et al. 1999).
Comparison of our HPLC determinations with chemical analyses
of other cats claw preparations available on the phytopharmaceutical market is not possible due to the lack of literature data.
According to the standards informally adopted in Western Europe
the concentration of oxindole alkaloids determined in relation
to pteropodine should be below 1.75% in medicinal preparations
(ukasiak et al. 2001). This criterion is met only by the B/W37 ,

1136

R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

Table 1
The oxindole-alkaloid proles of the studied preparations determined by HPLC [expressed as percentages and in mg/100 g of the preparations].
Alkaloid

B/W37
mg

Uncarine F
Speciophylline
Mitraphylline
Pteropodine
and isomitraphylline
Isopteropodine
Total

B/Wb
%

28 1.4
68 2.3
33 4.2
230 12.5

70 0.9
430

mg

6.59
15.89
7.74
53.44

39 3.2
32 2.1
13 0.2
249 7.9

16.34

157 5.6

100

B/50E37

509

mg

7.89
6.60
2.58
50.88

131 5.3
217 8.2
151 1.9
987 11.2

32.05

354 23.2

100

B/Eb

1896.8

B/25E37 preparations and possibly B/50E37 . Preparations with a

higher content of this alkaloid such as B/Eb and B/96E37 , and especially B/SRT containing over 50% of alkaloids, should not be used
according to these recommendations, due to the risk of overdose or
side effects. On the other hand, this recommendation is not necessarily well-grounded, as the proposed method of standardization is
not fully justied by the existing toxicological data which indicate
that even pure oxindole alkaloids are safe to use. The LD50 value
determined in mice which is higher than 16 g per kg body mass
(Keplinger et al. 1999) suggests that lethal poisoning of an adult
person weighing 70 kg is hardly possible as it could occur after consuming 2 kg of the B/SRT preparation. It must be taken into account,
however, that these animal data cannot be simply transferred to
human beings.
Antiproliferative activity of preparations on various cancer cell
lines
The obtained IC50 values for all studied cell lines and preparations are presented in Table 2.
Various cytotoxicities of the preparations related to their total
alkaloid content were observed. The presented results demonstrated the highest antiproliferative activity for B/96E37 and B/SRT
whereas the lowest one for B/W37 (total alkaloid content of these
preparations in 100 g d.m. are 3480 mg, 50,401 mg and 430 mg,
respectively). Our expectation that the highest over 50% oxindole alkaloid content of this preparation should correlate with
the lowest IC50 , was not fully conrmed. In fact, B/SRT was shown
to have an inconsistent activity that can be explained by increased
selectivity of this preparation for some cell lines or its low solubility
in water and problems with reliable IC50 determinations.
Differentiated activity of the preparations according is not
surprising and was conrmed in our previous paper (Pilarski
et al. 2007), whereas nding a correlation between IC50 values
and cancer cell types is difcult and requires more studies. In case of B/96E37 the highest growth inhibition was
observed for Lewis lung carcinoma (LL/2) [IC50 = 25.06 g/ml],
cervical carcinoma (KB) [IC50 = 35.69 g/ml] and colon adenocarcinoma (SW707) [IC50 = 49.06 g/ml]. On the other hand, colon
adenocarcinoma (HT-29), ovary cystoadenocarcinoma (OAW42) and breast carcinoma (MCF-7) were the most resistant
to this preparation [IC50 = 499.05 g/ml, IC50 = 457.87 g/ml,
IC50 = 375.60 g/ml, respectively]. Other results were obtained for
B/SRT that mainly inhibited proliferation of cervical carcinoma (KB)
[IC50 = 23.57 g/ml], breast carcinoma (MCF-7) [IC50 = 29.86 g/ml]
and lung carcinoma (A-549) [IC50 = 40.03 g/ml]. However, this
preparation was less efcient than B/96E37 for Lewis lung carcinoma (LL/2) [IC50 = 246.79 g/ml] and colon adenocarcinoma
(SW707) [IC50 = 133.67 g/ml].
Comparison of the data obtained with other investigations is
not easy due to the limited availability of IC50 determinations of
U. tomentosa preparations and constituents. In one available study

B/96E37

mg

7.12
11.79
8.22
53.62

303 2.4
78 5.6
223 8.3
1855 89.0

19.25

830 14.4

100

3408.7

mg

9.21
2.38
6.79
56.38

322 10.3
553 9.12
322 23.2
1782 14.3

25.24

501 37.2

100

B/SRT

3581.12

mg

9.26
15.89
9.24
51.21

2712 123.3
10937 342.2
8014 271.2
19303 54.9

14.40

9435 541.0

100

51733.6

5.38
21.7
15.9
38.3

18.72
100

(Riva et al. 2001), IC50 of U. tomentosa extracts fractionated on

Sephadex and determined on breast carcinoma (MCF-7) ranged
from 10 to 270 g/ml, and these values are similar to the results
obtained in this study for B/SRT and B/96E37 (29.86375.60 g/ml,
respectively). The authors of the cited paper do not indicate
any constituents responsible for the observed cytotoxic properties except for the conclusion that they must be more soluble in
methanol than in water, which may suggest oxindole alkaloids.
Anyway, their results are in agreement with ours and suggest that
ethanol preparations have increased activity as compared to aqueous ones (Riva et al. 2001).
We also compared our results with IC50 obtained by other
authors for pure oxindole alkaloids. These IC50 values ranged from
105 to 104 mol for HL-60 and human leukemic monocytes (U937) (Stuppner et al. 1993) and from 30 to 40 g/ml for human skin
melanoma (SK-MEL), cervical carcinoma (KB), human breast carcinoma (BT-549), human ovarian adenocarcinoma (SK-OV-3) and
monkey kidney epithelial (VERO) cells (Muhammad et al. 2001).
Similar results were obtained for alkaloids isolated from U. guianensis that were tested on non-transformed mouse broblasts (BALB/c
3T3), human lung carcinoma (H460), cervical cancer (ME180),
mice reticular lymphosarcoma (LSR), human prostate cancer
(DU145) and mouse stomach carcinoma (C678). Importantly,
in all these evaluations, isopteropodine was more active than
pteropodine (IC50 = 1742 g/ml vs. IC50 = 3851 g/ml, respectively) (Lee et al. 1999). Another cytotoxicity assay was conducted
on wild-type (RS188N) and engineered yeasts that lack the
RAD52 DNA repair pathway, one of the three major DNA
repair pathways that have been dened in yeast (RS321, RS322)
[pteropodine: IC12 = 140 g/ml (RS321), IC12 = 680 g/ml (RS322),
IC12 8000 g/ml (RS188N); isopteropodine: IC12 = 120 g/ml
(RS321), IC12 = 364 g/ml (RS322), IC12 8000 g/ml (RS188N)]
(Lee et al. 1999). The demonstrated selectivity of the alkaloids
against the yeast mutants should be taken carefully due to the limitations of these evaluations in reference to IC50 of mammalian cell
lines.
Therapeutic effect of the B/W37 preparation in mouse Lewis lung
carcinoma model
The therapeutic activity of the B/W37 preparation has been studied in mouse Lewis lung carcinoma model. Mice bearing LLC tumors
were treated using different doses of the preparation during entire
duration of the experiment. We found that mice which received
B/W37 preparation at the doses of 5 and 0.5 mg/day starting from
day 1 of the experiment had statistically signicant inhibition of
tumor growth when compared to the rest of the groups (MANOVA,
p = 0.0009, Fig. 1).
Morbidity and body weight of experimental animals during the
run of the experiment were monitored. No animals died due to the
therapy and we only observed a slight decrease in body weight for
approximately 10% in the group treated with the highest 5 mg/day

R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

1137

Table 2
IC50 values for B/W37 , B/Wb , B/50E37 , B/Eb , B/96E37 and B/SRT [g/ml].
Cell line

HT-29 (DSMZ ACC 136)

KB (ATCC CCL 17)
LL/2 (LLC1) (ECACC 90020104)
B16 (4A5) (ECACC 94042254)
MCF 7
A-549 (ATCC CCL 185)
OAW-42 (ECACC 85073102)
SW707

IC50 [g/ml]
B/W37

B/Wb

B/50E37

B/Eb

B/96E37

B/SRT

803.47
417.19
461.26
732.41
>1000
566.71
734.60
419.10

552.24
81.43
234.15
479.27
759.19
550.42
670.00
378.22

597.64
59.22
75.72
427.08
631.60
404.06
602.02
320.00

514.78
41.64
39.16
273.29
416.32
168.65
594.28
67.41

499.05
35.69
25.06
205.65
375.60
93.17
457.87
49.06

423.13
23.57
246.79
313.82
29.86
40.03
313.72
133.67

Fig. 1. Growth of tumors in experimental groups of mice bearing Lewis lung carcinoma and treated with B/W37 extract at different doses and schedules. The tumor
volumes and standard deviations shown in mm3 (in the log10 and log20 scales,
respectively). Mice which received B/W37 extract at the doses of 5.0 and 0.5 mg/day
starting from day 1 of the experiment had statistically signicant inhibition of
tumor growth when compared to 0.5 mg of *B/W37 started on day 7 and rest
of the groups (MANOVA, p = 0.0009).

The obtained results of blood parameters indicate that B/W37

is well tolerated and non-toxic for the used doses. It is worth noting that the decrease in leukocyte count observed in the present
study only partially conrms the results obtained in our previous
experiments on chicken embryos, in which no signicant differences in blood parameters were found, with the exception of MCV,
MCH and MCHC (Pilarski et al. 2009). Furthermore, a number of
data that are not in the accordance with the presented results can
be found in the literature. The results of research on immunomodulatory properties of U. tomentosa that encompassed the analyses of
selected blood parameters in calves after inducing local pneumonia can serve as an example here. In this experiment, a signicant
decrease in the number of thrombocytes was found in the group
receiving cats claw (Bednarek et al. 2002, 2004). Many investigations reported increased leukocyte numbers as well (Wagner et al.
1985; Wurm 1997; Wurm et al. 1998). For example the increase in
leukocyte population was found in mice which were administered
the C-Med-100 preparation following the induction of leukopenia
by means of doxorubicin (Sheng et al. 2000). Stimulation of immune
system following the administration of U. tomentosa preparation
was observed in a mouse model of listeriosis infection (Eberlin et
al. 2005). Finally, an increase in the leukocyte content was found in
the spleen in one study of effect of C-Med-100 on healthy C57/BL/6
mice (kesson et al. 2003b).

dose of the B/W37 preparation (Fig. 2).

In the last day of the experiment blood samples were collected
for morphology tests. The two groups treated with DMSO and
B/W37 at 0.05 mg/day, had signicantly lower leukocytes numbers
than the rest of the groups (ANOVA, p = 0.00002, Table 3). On the
contrary, we have not found any signicant differences in erythrocytes, platelets or hemoglobin between the experimental groups
(data not shown).
Table 3
Leukocytes in the blood samples from mice bearing Lewis lung carcinoma and
treated with B/W37 extract at different doses and schedules.
Group

Leukocytes [103 /l]

Control
DMSO
B/W37 , 5 mg/day, started at day 1
B/W37 , 0.5 mg/day, started at day 1
B/W37 , 0.05 mg/day, started at day 1
*B/W37 , 0.5 mg/day, started at day 7

16.44
8.87
17.80
17.28
9.47
14.37

9.36
1.56**
2.80
11.26
2.14**
3.85

*The preparation name with asterisk (*B/W37 ) indicates start of treatment on day
7.
**
These groups had signicantly lower leukocytes numbers than the rest of the
animals (p = 0.00002).

Fig. 2. Changes in average body weight of mice bearing Lewis lung carcinoma and
treated with the B/W37 preparation at different doses and schedules. The preparation
name with asterisk (*B/W37 ) indicates start of treatment on day 7.

1138

R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

Fig. 3. Phases of mitotic cycle of LLC cells taken on day 21 from mice treated with
B/96E37 and B/SRT. The asterisks (*) indicate signicant statistical differences at
p < 0.05.

This preparation was almost two times less active than B/96E37
(IC50 = 25.06 g/ml) and that is why the results of the in vivo tests
are rather surprising. A possible explanation of the discrepancies
shown can be related to the availability of the chemical compounds
introduced into the body. The B/96E37 preparation, containing
non-polar compounds are less assimilable under physiological conditions than B/W37 obtained by means of water extraction.
On the other hand the explanation that anticancer activity of
the U. tomentosa preparations is only associated with their total
alkaloid content may be erroneous. Low and ambiguous activity
of B/SRT containing mainly pure alkaloids suggest that other phytochemicals are also responsible for cats claw pharmacological
potency. Finding these synergistic compounds by fractionation of
U. tomentosa preparations is the most important task in near future.
Acknowledgements

B/96E37 and B/SRT preparations lack antitumor activity in mouse

Lewis lung carcinoma model
We also checked the activity of B/96E37 and B/SRT preparations
in a mouse Lewis lung carcinoma model. Both preparations were
tested at the doses of 0.05 and 0.5 mg/day. As opposed to the B/W37
preparation, B/96E37 and B/SRT did not reveal any signicant antitumor activity. No animals died due to the therapy and we did not
observe a decrease in body weight of the animals treated with these
preparations (data not shown).
Tumor cell cycle analysis
The results of the cell cycle analysis in tumor cells are unclear
and need to be veried by a larger study. There were no signicant changes in the cell cycle distributions with the exception of
cell decrease at the G2 /M phase after the administration of B/96E37
at a daily-dose of 0.5 mg and the G1 /G0 cells cycle arrest demonstrated after the B/SRT therapy at a daily-dose of 0.05 mg (Fig. 3).
The inhibition of the cells extracted from the tumor at these phases
can constitute a response to stressogenic factors and be a result
of the extended time of DNA repair (phase G1 ) or a considerable
inhibition of anabolic processes and cell adaptation to the unfavorable environmental conditions (phase G0 ). Such a cell-cycle
arrest is very often a response of tumor cells on cytostatic drugs
and one of the most frequent reasons of failure in chemotherapies
because cancer cells may continue their proliferative activity after
withdrawal of the drug (most chemotherapeutic agents induce
apoptosis selectively in replicating cells). Therefore, the observed
G0 /G1 cell accumulation may be a good explanation of lower
effectiveness of B/SRT than expected from IC50 determinations.
This explanation remains unsatisfactory taking into consideration
normal distribution of cell-cycles in case of tumors exposed to
higher (0.5 mg/day) dose of B/SRT. Furthermore, it has been shown
recently that oxindole alkaloids exerted cytotoxic effect on acute
leukaemic lymphoblastic cells by induction of apoptosis in both,
proliferating and G0 /G1 arrested stages (Bacher et al. 2006).
Conclusions
The results obtained for therapeutic effectiveness of B/W37 ,
B/96E37 and B/SRT showed different anticancer properties of the
tested preparations. Our studies showed that B/W37 was the most
active preparation under in vivo conditions as it inhibited the development of tumors at p = 0.0009.
Comparing the in vivo results with the in vitro IC50 values
for LLC cells presented in Table 2, some signicant differences
need to be indicated. First of all, in the case of in vitro tests
B/W37 (IC50 = 461.26 g/ml) was the least active preparation.

We would like to thank Dr. Dmitry Nevozhay for his technical assistance, statistical evaluations and thorough review of this
manuscript. We are grateful for the generous supply of Uncaria
tomentosa from Vilcacora omianki Centre (omianki, Poland).
References
Aguilar, J.L., Rojas, P., Marcelo, A., Plaza, A., Bauer, R., Reininger, E., Klaas, C.A., Merfort, I., 2002. Anti-inammatory activity of two different extracts of Uncaria
tomentosa (Rubiaceae). J. Ethnopharmacol. 81, 271276.
kesson, C., Lindgren, H., Pero, R.W., Leanderson, T., Ivars, F., 2003a. An extract
of Uncaria tomentosa inhibiting cell division and NF-[kappa]B activity without
inducing cell death. Int. Immunopharm. 3, 18891900.
kesson, C., Lindgren, H., Pero, R.W., Leanderson, T., Ivars, F., 2005. Quinic acid is a
biologically active component of the Uncaria tomentosa extract C-Med 100 . Int.
Immunopharm. 5, 219229.
kesson, C., Pero, R.W., Ivars, F., 2003b. C-Med 100, a hot water extract of Uncaria
tomentosa, prolongs lymphocyte survival in vivo. Phytomedicine 10, 2333.
Allen-Hall, L., Cano, P., Arnason, J.T., Rojas, R., Lock, O., Lafrenie, R.M., 2007.
Treatment of THP-1 cells with Uncaria tomentosa extracts differentially regulates the expression if IL-1[beta] and TNF-[alpha]. J. Ethnopharmacol. 109,
312317.
Aquino, R., De Feo, V., De Simone, F., Pizza, C., Cirino, G., 1991. Plant metabolites.
New compounds and anti-inammatory activity of Uncaria tomentosa. J. Nat.
Prod. 54, 453459.
Aquino, R., De Simone, F., Pizza, C., Conti, C., Stein, M.L., 1989. Plant metabolites.
Structure and in vitro antiviral activity of quinovic acid glycosides from Uncaria
tomentosa and Guettarda platypoda. J. Nat. Prod. 52, 679685.
Bacher, N., Tiefenthaler, M., Sturm, S., Stuppner, H., Ausserlechner, M.J., Koer, R.,
Konwalinka, G., 2006. Oxindole alkaloids from Uncaria tomentosa induce apoptosis in proliferating, G0/G1-arrested and bcl-2-expressing acute lymphoblastic
leukaemia cells. Br. J. Haematol. 132, 615622.

Bednarek, D., ukasiak, J., Kondracki, M., Zurowska,

K., Falkiewicz, B., Niemczuk, K.,
2002. Modulating effects of Uncaria tomentosa in experimentally-induced local
pneumonia in calves. Bull. Vet. Inst. Pu. 46, 6577.

Bednarek, D., ukasiak, J., Kondracki, M., Zurowska,

K., Falkiewicz, B., Niemczuk, K.,
2004. Analysis of phenotype and functions of peripheral blood leukocytes in
cellular immunity of calves treated with Uncaria tomentosa. Bull. Vet. Inst. Pu.
48, 289296.
Cisneros, F.J., Jayo, M., Niedziela, L., 2005. An Uncaria tomentosa (cats claw) extract
protects mice against ozone-induced lung inammation. J. Ethnopharmacol. 96,
355364.
De Jong, W., Melnyk, M., Lozano, L.A., Rosales, M., Garcia, M., 1999. Una de gato:
fate and future of a Peruvian forest resource. CIFOR Occasional Paper, Indonesia,
Center for International Forestry Research, pp. 114.
De Matta, S.M., Monache, F.D., Ferrari, F., Marini-Bettolo, G.B., 1976. Alkaloids and
procyanidins of an Uncaria sp. from Peru. Farmaco [Sci] 31, 527535.
Eberlin, S., dos Santos, L.M., Queiroz, M.L., 2005. Uncaria tomentosa extract increases
the number of myeloid progenitor cells in the bone marrow of mice infected
with Listeria monocytogenes. Int. Immunopharm. 5, 12351246.

Gulewicz, K., Tykarska, T., Wysocki, W., Augustynowicz, J., Zurowska,

K., Kuras, M.,
2004. Developmental and ultrastructural effect of Uncaria tomentosa (Willd.) DC
extract on the paprika Capsicum annuum L. Ind. Crop Prod. 19, 5967.
Heitzman, M.E., Neto, C.C., Winiarz, E., Vaisberg, A.J., Hammond, G.B., 2005.
Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae). Phytochemistry 66, 529.
Hemingway, S.R., Phillipson, J.D., 1974. Proceedings: alkaloids from S. American
species of Uncaria (Rubiaceaes). J. Pharm. Pharmacol. 26 (Suppl), 113P.
Keplinger, K., Laus, G., Wurm, M., Dierich, M.P., Teppner, H., 1999. Uncaria tomentosa
(Willd.) DC. Ethnomedicinal use and new pharmacological, toxicological and
botanical results. J. Ethnopharmacol. 64, 2334.

R. Pilarski et al. / Phytomedicine 17 (2010) 11331139

Kitajima, M., Hashimoto, K., Sandoval, M., Aimi, N., Takayama, H., 2004. New oleanantype triterpene and cincholic acid glycosides from Peruvian Una de Gato
(Uncaria tomentosa). Chem. Pharm. Bull. (Tokyo) 52, 12581261.

Kuras, M., Nowakowska, J., Sliwi

nska,
E., Pilarski, R., Ilasz, R., Tykarska, T., Zobel,
A., Gulewicz, K., 2006. Changes in chromosome structure, mitotic activity and
nuclear DNA content from cells of Allium Test induced by bark water extract of
Uncaria tomentosa (Willd.) DC. J. Ethnopharmacol. 107, 211221.
Laus, G., 1998. Kinetics of isomerization of tetracyclic spiro oxindole alkaloids. J.
Chem. Soc., Perkin Trans. 2, 315317.
Laus, G., Brossner, D., Senn, G., Wurst, K., 1996. Analysis of the kinetics of isomerization of spiro oxindole alkaloids. J. Chem. Soc., Perkin Trans. 2, 19311936.
Laus, G., Keplinger, K., 1997. Radix Uncariae tomentosae (Willd.) DC. a monographic
description. Zeit. Phyt. 18 (2), 122126.
Lee, K.K., Zhou, B.N., Kingston, D.G., Vaisberg, A.J., Hammond, G.B., 1999. Bioactive indole alkaloids from the bark of Uncaria guianensis. Planta Med. 65,
759760.
Lemaire, I., Assinewe, V., Cano, P., Awang, D.V.C., Arnason, J.T., 1999. Stimulation
of interleukin-1 and -6 production in alveolar macrophages by the neotropical
liana, Uncaria tomentosa (Una de Gato). J. Ethnopharmacol. 64, 109115.
ukasiak, J., Falkiewicz, B., Prusakowski, M., 2001. Vilcacora w s wietle badan

naukowych. Tower Press, Gdansk.

Miller, M.J.S., Zhang, X.J., Charbonnet, R.M., Clark, D.A., Sandoval, M., 1999. The antiinammatory actions of the herbal medicine, cats claw, are due to a suppression
of NF-kappa B activation and inhibition of gene expression. Pediatr. Res. 45,
114a1114a.
Muhammad, I., Dunbar, D.C., Khan, R.A., Ganzera, M., Khan, I.A., 2001. Investigation of Una De Gato I. 7-Deoxyloganic acid and 15N NMR spectroscopic studies
on pentacyclic oxindole alkaloids from Uncaria tomentosa. Phytochemistry 57,
781785.
Okuhama, N., Angeles, F., Lao, J., Miller, M.J.S., Sandoval, M., 2001. An extract of
Uncaria tomentosa protects against oxidative stress-induced cell damage. Free
Radic. Biol. Med. 31, S35.
Pilarski, R., Bednarczyk, M., Gulewicz, K., 2009. Evaluation of biological activity
of Uncaria tomentosa (Willd.) DC. using the chicken embryo model. Folia Biol.
(Krakw) 57, 16.
Pilarski, R., Poczekaj-Kostrzewska, M., Ciesioka, D., Szyfter, K., Gulewicz, K., 2007.
Antiproliferative activity of various Uncaria tomentosa preparations on HL-60
promelocytic leukemia cells. Pharmacol. Rep. 59, 565572.

1139

Pilarski, R., Zielinski, H., Ciesioka, D., Gulewicz, K., 2006. Antioxidant activity of
ethanolic and aqueous extracts of Uncaria tomentosa (Willd.) DC. J. Ethnopharmacol. 104, 1823.
Riva, L., Coradini, D., Di Fronzo, G., De Feo, V., De Tommasi, N., De Simone, F., Pizza,
C., 2001. The antiproliferative effects of Uncaria tomentosa extracts and fractions
on the growth of breast cancer cell line. Anticancer Res. 21, 24572461.
Sandoval-Chacon, M., Thompson, J.H., Zhang, X.J., Liu, X., Mannick, E.E.,
Sadowska-Krowicka, H., Charbonnet, R.M., Clark, D.A., Miller, M.J.S., 1998. Antiinammatory actions of cats claw: the role of NF-kappa B. Aliment. Pharmacol.
Ther. 12, 12791289.
Sandoval, M., Charbonnet, R.M., Okuhama, N.N., Roberts, J., Krenova, Z., Trentacosti, A.M., Miller, M.J.S., 2000. Cats claw inhibits TNF[alpha] production and
scavenges free radicals: role in cytoprotection. Free Radic. Biol. Med. 29, 7178.
Sandoval, M., Okuhama, N.N., Zhang, X.J., Condezo, L.A., Lao, J., Angeles, F.M., Musah,
R.A., Bobrowski, P., Miller, M.J., 2002. Anti-inammatory and antioxidant activities of cats claw (Uncaria tomentosa and Uncaria guianensis) are independent
of their alkaloid content. Phytomedicine 9, 325337.
Sheng, Y., Pero, R.W., Amiri, A., Bryngelsson, C., 1998. Induction of apoptosis and
inhibition of proliferation in human tumor cells treated with extracts of Uncaria
tomentosa. Anticancer Res. 18, 33633368.
Sheng, Y., Pero, R.W., Wagner, H., 2000. Treatment of chemotherapy-induced
leukopenia in a rat model with aqueous extract from Uncaria tomentosa. Phytomedicine 7, 137143.
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T.,
Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay
for anticancer-drug screening. J. Natl. Cancer Inst. 82, 11071112.
Steinberg, P.N., 1995. Cats claw: an herb from the Peruvian Amazon. Sidahora,
3536.
Stuppner, H., Sturm, S., Geisen, G., Zmian, U., Konwalinka, G., 1993. A differential
sensitivity of oxindole alkaloids to normal and leukemic cell lines. Planta Med.,
59.
Wagner, H., Kreutzkamp, B., Jurcic, K., 1985. The alkaloids of Uncaria tomentosa and
their phagocytosis-stimulating action. Planta Med., 419423.
Wurm, M., 1997. Immunologisch aktive pentazyklishe Oxindolalkaloide aus Uncaria
tomentosa. Faculty of Medicine at the Leopold-Franzens-University, Insbruck.
Wurm, M., Kacani, L., Laus, G., Keplinger, K., Dierich, M.P., 1998. Pentacyclic oxindole
alkaloids from Uncaria tomentosa induce human endothelial cells to release a
lymphocyte-proliferation-regulating factor. Planta Med. 64, 701704.