Antiinflammatory actions of cat's claw: the role of nf-kb

Antiinflammatory actions of cat's claw: the role of
NF-kB
M. SANDOVAL-CHACON, J. H. THOMPSON, X.-J. ZHANG, X. LIU, E. E.
MANNICK,
H. SADOWSKA-KROWICKA, R. M. CHARBONNET, D. A. CLARK & M. J. S.
MILLER
LSU Medical Center, Department of Pediatrics and Stanley S. Scott Cancer Center, New Orleans, LA 70112, USA
Accepted for publication 22 July 1998
Background: Uncaria tomentosa is a vine commonly known as cat's claw or 'una de gato'
(UG) and is used in traditional Peruvian medicine for the treatment of a wide range of
health problems, particularly digestive complaints and arthritis.
Purpose: The aim of this study was to determine the proposed anti-inflammatory
properties of cat's claw. Specifically: (i) does a bark extract of cat's claw protect against
oxidant-induced stress in vitro, and (ii) to determine if UG modifies transcriptionally
regulated events.
Methods: Cell death was determined in two cell lines, RAW 264.7 and HT29 in response to
peroxynitrite (PN, 300 ~M). Gene expression of inducible nitric oxide synthase (iNOS) in
HT29 cells, direct effects on nitric oxide and peroxynitrite levels, and activation of NF-
ICBin RAW 264.7 cells as influenced by UG were assessed. Chronic intestinal
inflammation was induced in rats with indomethacin (7.5 mg/kg), with UG administered
orally in the drinking water (5 mg/mL).
Results: The administration of UG (100 ug/mL) attenuated (P < 0.05) peroxynitrite-induced
apoptosis in HT29 (epithelial) and RAW 264.7 cells (macrophage). Cat's claw inhibited
lipopolysaccharide-induced iNOS gene expression, nitrite formation, cell death and
inhibited the activation of NF-KB. Cat's claw markedly attenuated indomethacin-enteritis
as evident by reduced myeloperoxidase activity, morphometric damage and liver
metaflothionein expression.
Conclusions: Cat's claw protects cells against oxidative stress and negated the activation
of NF-KB. These studies provide a mechanistic evidence for the widely held belief that
cat's claw is an effective anti-inflammatory agent.
INTRODUCTION
Among the numerous factors associated with chronic gut inflammation the enhanced
production of oxidants and free radicals have become widely recognized as integral
components of cell and tissue injury. Agents which negate the production and/or effects
of reactive metabolites of oxygen and nitrogen have displayed therapeutic benefits.2-4
Endogenous antioxidants may be depleted during states of chronic inflammation, which
may in part explain the therapeutic efficacy of mesalamine and glucocorticoids.5. 6
Crohn's disease and ulcerative colitis are the two most common forms of inflammatory
bowel disease. Although the aetiology of inflammatory bowel disease remains unclear,
there is mounting evidence to suggest that oxidants, free radicals and bacterial flora may
play a role in the pathogenesis of gut inflammation. Bacterial overgrowth has been
associated with a range of inflammatory disorders of the gut/ Increased intestinal
permeability to luminal contents (bacterial or dietary) may also promote mucosal
inflammation as well as affecting systemic organs, particularly the liver. 8 During states of
inflammation, LPS and cytokines have been reported to induce the synthesis of
metallothionein in the liver. 9 Metallothioneins (MT) are sulfhydryl-rich proteins that bind
heavy metals and oxidants and are considered as acute phase response proteins. 10
Our current therapeutic approaches to gut inflammation remains inadequate. In
developing countries many of the present therapeutic options are beyond the financial
reach of the general population. For this reason we are evaluating traditional herbal
remedies in gut inflammation.
In the present work we have used an aqueous extract from dried bark of cat's claw Uncaria
tomentosa (Willd.) DC. Cat's claw is a plant belonging to the family Rubiaceae, commonly
known as 'una de gato'. It is a vine that grows wild in the Peruvian Amazon. The aqueous
extract and decoctions of cat's claw are widely used in traditional Peruvian medicine for
the treatment of gastritis, arthritis and as an anti-inflammatory.ll11
Similarly, during the last 10 years, cat's claw in different forms (e.g. extracts, tablets and
capsules) has been introduced in Europe to treat patients suffering from cancer and some
viral diseases. In addition to the anti-inflammatory properties of cat's claw, its protective
antimutagenic effects have also been demonstrated in vitro against photomutagenesis.l2
The extract contains a mixture of quinovic acid and glycosides as well as pentacyclic or
tetracyclic of oxindole alkaloids such as pteropodine, isopteropodine, speciophylline,
uncarine F, mitraphylline and isomitraphylline, 13. 14
The purpose of this study was twofold: (i) to investigate whether the bark extract of cat's
claw Uncaria tomentosa (Willd.) DC. is a cytoprotective agent in vitro against oxidant-
induced stress in murine macrophages (RAW 264.7) and human intestinal epithelial cells
(HT29), and (ii) to determine if the anti-inflammatory activity of cat's claw involved an
inhibition of transcriptionally regulated genes.
MATERIALS AND METHODS
Materials
Unless otherwise stated, all chemicals were at least reagent grade and were obtained from
Sigma Chemical Co. (St Louis, MO). All cellular reagents and culture media were from
Gibco BRL(Gaithersburg, MD).
Plant material and aqueous extraction
The bark of cat's claw Uncaria tomentosa (Willd.) DC. was collected in Tingo Maria, Peru
and Identified by
Eng. Raul Araujo of the Universidad Nacional Agraria de la Selva. The extract of Uncaria
tomentosa was prepared from the air-dried bark of cat's claw by boiling it in water (20 g/L)
for 30 min and then leaving it at room temperature overnight. The extract was decanted
and filtered at 10 um. The cat's claw (UG) extract for the cell culture experiments was
filtered at 0.2 Can and diluted to a final concentration of 5 mg/mL, and then refrigerated.
For the in vivo studies the UG extract was filtered at 0.45 ~m. The extract contains
oxindole alkaloids such as pteropodine, isopteropodine, mitraphylline and
isomitraphylline.l5' 16
Cell culture
HT29 and RAW 264.7 cells were obtained from the American Type Culture Collection
(Rockville, MD). Cells were grown in DMEM high glucose, 10% FCS and supplemented with
25 HIM HEPES, pH 7.4; 4 HIM L-glutamine; 40 ~g/mL penicillin; 90 ~g/mL streptomycin;
0.25 ~g/mL fungizone and 1.2 g/L NaHCO3. Cell cultures were maintained in a humidified
5% 002 incubator at 37 °C. Cells were plated at I x 106 cells/mL. Harvested cells were
plated in six-well tissue culture plates and allowed to grow to confluence over 24 h before
use.
Peroxynitrite synthesis
Peroxynitrite (PN) was synthesized by modifying a previously reported methodology."
Briefly, solutions of (a) 0.7 M NaNO2, 0.7 M Hz02 and (b) 0.6 M HCI, were pumped using a
syringe infusion pump (Harvard Apparatus, South Natick, MA) at 25 mL/min, into a Y-
junction and mixed in a 2 mm-diameter by 0.5 cm silica tube. The mixture was collected in
a beaker containing a 1.5-M KOH solution. To destroy the excess H202, the peroxynitrite
solution was filtered in a column containing Mn02 (4 g). The prepared solution contained 3
5-50 HIM peroxynitrite, as determined by absorbance at 302 nm (£302 = 1670/M/cm).18 A
fresh working peroxynitrite solution (5 mM) was prepared in 5 mM KOH for each
experiment and filtered at 0.2 ~m.
Cell viability
Aliquots of treated cells were examined for viability as determined by trypan blue dye
exclusion. Briefly, HT29 cells were detached with trypsin-EDTA and RAW 264.7 cells were
scraped and washed with phosphate buffered saline (154 HIM, NaCI; 2.7 HIM,
Na2HP04'7H20; 1.3 HIM, KH2PC)4), resuspended in medium and 0.4% trypan blue stain
was added. After 5 min of incubation, the number of cells excluding the dye was
expressed as a percentage of total cells counted from three randomly chambers of the
haemocytometer.
Measurement of nitrite/nitrate
For these experiments, HT29 and RAW 264.7 cells were treated with lipopolysaccharide
(LPS, I ~g/mL) and/or UG (100 ~g/mL) and incubated for 18h and 12 h, respectively. The
stable end products of nitric oxide nitrite/nitrate (NO~ and NO 3 ) were assayed in HT29
and RAW 264.7 cells using Griess reagent after the conversion of NO 3 to NOz with a
colourimetric assay kit (Cayman Chemical Co., Ann Arbor, MI).
Detection of Apoptosis by ELISA
HT29 and RAW 264.7 cells were either treated with 300 ~M PN and/or supplemented with
cat's claw extract (UG, 100 ~g/mL) and incubated for 4 h. Apoptosis (DNA fragmentation)
was quantified using a cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN)
as previously described. 19
Electrophoretic mobility shift assay (EMSA)
RAW 264.7 cells, at I x 106/well, were plated in sixwell clusters and treated with LPS (I
~g/mL) and/or UG (50-100 ~g/mL) and incubated at 37 °C. After 2 h, the medium covering
the cells was removed and replaced with ice-cold phosphate-buffered saline. RAW 264.7
cells were harvested by scraping followed by centrifugation (1000 g). Preparation of the
nuclear protein extracts and EMSAs were carried out as previously reported.20' 21 The
protein concentration of the nuclear extracts was determined using a Bio-Rad protein
assay kit (Bio-Rad Laboratories, Hercules, CA). The binding reactions were carried out
with 5 ~g of protein for NFKB per reaction. The consensus sequence of the NF-lcB probe
(the binding site is underlined) was 5 -AGTTGAGGGGACTTTCCCAGGC-3' (Promega,
Madison, Wl).
Evaluation of peroxy nitrite scavenging by cat's claw
Diluted samples from the stock solution of peroxynitrite (40 HIM) were used to prepare
working solutions of
300 11M peroxymtrite containing 5 HIM KOH (pH 12). The final volume was I mL, and the
absorbance at 302 nm was determined or scanned from 200 to 400 nm at I and 10 min,
respectively. A Beckman DU 64 spectrometer (Beckman Instruments Inc., Fullerton, CA)
was used to assess the change in absorbance. In separate experiments, the absorbance of
UG (100 Clg/mL) diluted in 5 HIM KOH or reacted with 300 ~M peroxynitrite was scanned
and the absorbance at 302 nm was determined.
Auto-oxidation of nitric oxide by cat's claw extract
To see if the nitric oxide reacted with the UG extract, the time-dependent depletion of 30
CLM nitric oxide was monitored in two solutions: (i) phosphate solution (pH 7.4)
containing 5 mg/mL UG extract, and (ii) phosphate solution without the UG extract. The
microelectrode experiments were performed at 25 °C. The saturated stock solution
contained 160 ~M nitric oxide, as determined by electrochemistry (BAS 100 B/W,
Bioanalytical Systems, West Lafayette, IN). The conditions for the electrochemical
experiments have been reported previously. 22
Analysis of iNOS gene expression by RT-PCR
HT29 cells 2x10hhh cells/well were seeded in six-well tissue culture plates and incubated
with LPS (I ~g/mL) and/or UG (50-200 ~g/mL). After 12 h, the total RNA was isolated from
cells by the acid guanidine thiocyanate-phenol-chloroform extraction method. zs The
integrity of RNA was assessed on a 1.2% agarose gel and the RNA was visualized by
ethidium bromide. First-strand complementary DNAs were synthesized from I ~C18 of total
RNA using oligo dT and Superscript II Reverse Transcriptase (Gibco BRL, Grand Island,
NY). The firststrand complementary DNA templates were amplified for glyceraldehyde-3-
phosphate dehydrogenase and iNOS by polymerase chain reaction (PCR) using a hot start
(Ampliwax, Perkin Elmer, Foster City, CA). The primers for iNOS were as follows: forward
5'-TCG AAA CAA CAG GAA CCT ACC A-3' (a 22-mer oligonucleotide at position 529) and
reverse 5'-ACR GGG GTG ATG CTC CCA GAC A-3' (a 22-mer oligonucleotide at position
1435), giving rise to a 907 base pair PCR product. These sequence data are available from
GENBANK under accession number D 140 51. The primers for the glyceraldehyde-3-
phosphate dehydrogenase (GADPH) used as an internal standard for rats were as follows:
forward 5'-ATT CTA CCC ACG GCA AGT TCA ATG G-3' and reverse 5'-AGG GGC GGA GAT
GAT GAC CC-3' (GENBANK accession number MI 7701). The PCR cycle was an initial step
of 95 °C for 3 min, followed by 94 °C for 30 s, 60 °C for 45 s, 72 °C for I min, with 30 cycles
and a final cycle of 72 °C for 4 min. The negative control was a cDNA reaction that used
water instead of RNA. PCR products (iNOS 12 ~L, GADPH 6 ~L) equalized to give
equivalent signals from the GADPH mRNA, were electrophoresed through 2% agarose
gels (FMC, Rockland, ME) containing 0.2 ~g/mL of ethidium bromide. Gels were visualized
under UV light and photographed with Polaroid film (Polaroid Corporation, Cambridge,
MA).
Indomethacin-induced intestinal inflammation
Animal models of nonsteroidal anti-inflammatory drug (NSAID) induced enteropathy are
associated with changes in morphology, microvascular injury and changes in epithelial
penneability. 4' 25 To evaluate the anti-inflammatory activity of cat's claw against
indomethacin-induced intestinal injury, male SpragueDawley rats (225-250 g) were
purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in stainless-steel
cages in an environmentally controlled room (25 °C, 12 h/12 h light/dark cycle). Food and
water were supplied ad libitum for 5 days before the experiment. A chronic model of the
intestinal inflammation was induced by two s.c. injections of indomethacin (7.5 mg/kg)
daily at a 24-h interval as described by Yamada et al. 4 Animals were divided into four
groups: (A) vehicle control group (5% NaHC03), (B) injected s.c. with indomethacin, (C)
injected s.c. with indomethacin and supplemented with cat's claw (UG, 5 mg/mL) in the
drinking water (indomethacin + UG), and (D) injected s.c. twice daily with vehicle and
supplemented with cat's claw (UG, 5 mg/mL) in the drinking water (UG). The rats from
groups A and B were given water, and all animals were fed a standard laboratory rat chow
ad libitum.
Tissue myeloperoxidase activity
Tissue myeloperoxidase (MPO) was quantified as an index of neutrophil infiltration. Tissue
samples were weighed, frozen on liquid nitrogen and then stored at -77 °C until assayed.
Tissue levels of myeloperoxidase
were determined by modifying a previously described technique.2" Briefly, 0.2 g of
midjejunum was homogenized for 45 s at 4 °C with a tissue homogenizer (Virtis, Gardiner,
NY) in 2 mL of ice cold 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM
potassium phosphate buffer, pH 6.0. The homogenate was then sonicated for 10s, freeze-
thawed three times, after which the sonication was repeated; suspensions were then
centrifuged at 40 000 g for 15 min. Myeloperoxidase was measured
spectrophotometrically: 50 fJtL of the supernatant was mixed with 1450 ~L of 50 HIM
phosphate buffer, pH 6.0 containing 0.167 mg/ mL o-dianisidine dihydrochloride and
0.0005% 11202. The change in absorbance at 460 nm was measured with a Beckman DU 64
spectrophotometer (Beckman Instruments, Fullerton, CA).
Determination of intestinal damage
A group of rats was used for a comparison of morphologic studies. At the end of 7 days,
rats were anaesthetized and samples of the midjejunum were taken. The tissue was fixed
in phosphate-buffered formaldehyde, embedded in paraffin, and 5-Gem sections were
prepared. The tissue was routinely stained with haematoxylin and eosin and evaluated by
light microscopy.
Metallothionein protein assay
After 7 days in the study, animals were sacrificed and samples of liver and intestinal
mucosal cells were collected for metallothionein (MT) protein. The metallothionein
concentration of cytosolic fractions of the liver and intestinal cells was determined by
10gCd-Hb affinity assay, as previously described. 27
Statistical analysis
Each experiment was performed at least three times and results are presented as the
mean ± S.E.M. Statistical analyses were performed using one-way ANOVA. Post hoc
comparison of means was carried out by a least significant difference test. A probability of
< 0.05 was considered significant.
Assessment of viability and apoptosis in cell lines
Experiments to examine the cytotoxic effects of peroxynitrite (300 CLM, for 4 h), with and
without cat's claw
(UG, 100 ~g/mL) were conducted to delineate the protective effect of UG extract. Cell
viability was not affected by the experimental conditions (Table 1). However, the results
indicated that HT29 and RAW 264.7 cells receiving 300 ~M peroxynitrite showed a
significant increase (P < 0.05) in cytosolic DNA fragments compared to their
decomposed/peroxynitrite (DC/PN) control group. In both cell lines, the simultaneous
administration of 100 ,clg/mL UG extract and peroxynitrite resulted in a significant (P <
0.05) reduction in apoptosis (Figures I and 2). Table 2 shows the cytoprotective effect of
the UG extract in RAW 264.7 cells treated with LPS (I ~g/mL). Similarly, the concentration
of nuclear protein was greater (P < 0.05) in cells treated simultaneously with LPS and UG
than in cells exposed to LPS alone.
Levels of nitrite/nitrate
HT29 cells treated with LPS produced higher (P < 0.05) levels of NC)2 /NOs than cells
simultaneously treated with LPS and UG extract (Figure 3). In another set of experiments
with RAW 264.7 cells, the simultaneous administration of cat's claw and LPS caused a
significant (P < 0.05) inhibition of 60% nitrite formation (Figure 4).
Inhibition of NF-KB activation by cat's claw extract
Figure 5 shows the effect of LPS (I ~g/mL) and UG extract on NF-KB activation in RAW
264.7 cells. In the presence of LPS as a source of oxidative stress, the activation of NF-KB
was markedly enhanced, consistent with previous reports 28 On the other hand, RAW
264.7 cells treated with LPS and UG extract (100 ~g/mL) caused an inhibition of NF-KB.
Regulation ofiNOS mRNA in HT29 cells induced by LPS
Figure 6 shows the levels of iNOS mRNA expression from HT29 cells treated with LPS (I
~g/mL) and/or UG extract (50-200 ~g/mL). The expression of iNOS mRNA was increased in
LPS treated cells, as was evident after 12 h incubation. However, the simultaneous
administration of UG extract and LPS significantly decreased the levels of iNOS mRNA.
While the expression of the house-keeping gene, GAPDH, was variable in this gel, the
reduction in iNOS gene expression with UG was evident, and was supported by the
reduced production of nitrite/nitrate (Figures 3 and 4).
Scavenging of oxidants by cat's claw extract
The decomposition of peroxynitrite (pH 12) in the presence and absence of cat's claw (UG
extract) was monitored spectrophotometrically at 302 nm. The addition of UG extract (100
~g/mL) to peroxynitritecontaining 5 HIM KOH resulted in a significant (P < 0.05) decrease
in peroxynitrite concentration (Figure 7). The decomposition of peroxynitrite was
evaluated at pH 12 because peroxynitrite degrades rapidly at pH 7.4. The absorbance of
the UG extract at pH 12 did not vary during the time it was evaluated (10 min). Because of
the time constraints encountered for quantifying PN at pH 7.4 (peroxynitrite has a very
short half-life at physiological pH) we elected to follow the depletion of the UG extract
absorbance determined at 245 nm. As expected, absorbance of the UG extract was
reduced by the presence of peroxynitrite (P < 0.05), suggesting a decomposition or
consumption of the oxindole alkaloids present in the UG extract by peroxynitrite. Figure 8
shows the in vitro reaction of NO, 30 ~M with UG, 5 mg/mL and from these results it was
clear the half-life of nitric oxide was not affected significantly.
Assessment of indomethacin-induced intestinal inflammation
Rats treated with two daily s.c. injections of indomethacin produced mucosal ulcerations
on the mesenteric side of the mid-small intestine, and numerous white nodules located
along the serosal side of the intestine were also observed by day 7 following the injection.
The degree of inflammation, in the midjejunum, was associated with significant increase of
MPO in this section of the gastrointestinal tract (Figure 9). Histological sections of the
midjejunum of rats that received indomethacin (7.5 mg/kg) showed a
pronounced disruption of the mucosal architecture, with loss of villi and a pronounced
inflammatory cell infiltrate. On the other hand, rats receiving UG (5 mg/ mL in the drinking
water, Figure 10) had a normal mucosal architecture. Table 3 shows the hepatic
metallothionein concentration, an index of inflammation. Metallothionein was increased (P
< 0.05) in rats that received indomethacin after 7 days compared to control rats.
Administration of UG (5 mg/mL) in the drinking water to rats treated with indomethacin
resulted in lower (P < 0.05) liver metallothionein. In contrast to the liver metallothionein,
either indomethacin or the UG extract did not affect the content of intestinal
metallothionein. The induction of metallothionein synthesis in the intestine is not
inflammation dependent but rather occurs in response to trace metals such as Zn and
CU.29
DISCUSSION
Inflammatory disorders are characterized by an excessive production of free radicals and
reactive oxygen and nitrogen species. Currently used therapeutics often modify the
actions or production of the reactive species, and in so doing reduce the degree of tissue
injury.'" However, for the developing world, access to these therapeutic agents may be
limited due to financial constraints. These populations tend to use traditional medicines,
often of plant origin, for the therapeutic management of disease. While the Amazon river
basin has proven to be a rich source of valuable pharmacological agents, a great deal of
potential for ethnomedically driven drug discovery still remains. In this study we have
evaluated the potential benefits of a widely used herbal medicine, cat's claw. An aqueous
extract of the cat's claw bark negated the cellular toxicity associated with peroxynitrite, a
powerful oxidant formed from the interaction of nitric oxide and superoxide (the radical
precursors to a family of potent, reactive species). These results demonstrated that the
aqueous extract of cat's claw elicited similar beneficial effects as an antioxidant,
consistent with previous findings using a bark methanoi extract. 31 In addition, cell death
induced by bacterial endotoxin was attenuated by cat's claw.
To date there have been few studies evaluating the mechanisms for the proposed
beneficial effects of cat's claw. Here we have defined the potential loci. Firstly, cat's claw
directly degrades peroxynitrite and attenuates peroxynitrite-induced cell death, similar to
that which we have recently described for mesalamine.22 Mesalamine does not modify
nitric oxide oxidative degradation, implying that its anti-inflammatory properties are not
mediated by direct effects on nitric oxide, a relatively weak free radical.22 Rather,
peroxynitrite, which is highly reactive, is a site of action. Similar results were noted with
cat's claw. However, cat's claw slowed the rate of oxidative degradation of nitric oxide,
while directly degrading peroxynitrite. This is a pattern of effects that we have seen also
with ascorbic acid. 32 We and others have demonstrated the contributions of reactive
nitrogen oxides to inflammatory bowel disease and gastritis. 33, 34 Indeed these species
may be critical components in the development of gastritis and gastric cancer in response
to Helicobacter pylori infection which is endemic in South America. 35, 36
The second mechanism by which cat's claw may afford benefit appears to be unique
amongst natural products. Cat's claw, by preventing the activation of the transcriptional
factor NF-lcB, inhibits the expression of inducible genes associated with inflammation,
specifically, cat's claw negated the expression of inducible nitric oxide synthase, thereby
attenuating nitric oxide production. Together with the direct degradation of peroxynitrite
once it is formed, the cytotoxic consequences of this pathway will be greatly diminished.
Other pro-inflammatory pathways regulated by NF-lcB, but not investigated directly here
(cytokines, adhesion molecules) should also be modified.
Suppression in the liver by cat's claw of the indomethacin-induced acute phase response
protein, metallothionein, demonstrates that these transcriptiondependent responses are
registered in vivo as well as in vitro. Metallothionein levels in the intestine were not altered
by indomethacin-induced inflammation, nor by cat's claw. This is not an unexpected
finding, as intestinal metallothionein is regulated almost exclusively by heavy metals,
whereas the hepatic expression of metallothionein is also influenced by the cytokines
released in states of inflammation.3 77 Glucocorticoids can negate the induction of iNOS
expression through transcriptional mechanisms, e.g. by the inhibition of NF-TcB,38 as
seen in this study with cat's claw. As NF-lcB controls the expression of a wide range of
pro-inflammatory signals, including the adhesion molecules and cytokines which were not
evaluated here, 39 it is reasonable to assume that the anti-inflammatory effects of cat's
claw involves a generalized reduction in proinflammatory mediators and effectors.
The anti-inflammatory actions of cat's claw were registered at doses that are consistent
with the practice of traditional medicine. Indeed, rats evaluated in the indomethacin
enteritis model were treated with a 'tea' made from cat's claw prepared in a manner that
was identical to the ethno-medical use of cat's claw in Peru and neighbouring regions.
This oral administration of cat's claw 'tea' had an impressive protective effect on
indomethacin-induced enteritis in rats; normalizing mucosal architecture and attenuating
granulocyte infiltration. This tea has a palatable taste and is widely consumed in South
America, and is becoming quite accessible in North America. Anecdotal reports have
indicated that it is useful in the treatment of refractory gut inflammation. It is also
important to note that the beneficial effects observed in the present study were at doses
that did not compromise cellular function or viability. Thus there was no suggestion of
toxicity.
There have been reports that the active ingredients of cat's claw may be subject to
regional and seasonal variability." In addition, there are differences between the use of
bark and roots 40. However, it is also appreciated that the proposed active ingredients
cannot account for the known efficacy of cat's claw.41 Thus, it is not clear if the present
report of antioxidant properties and transcriptional inhibition with this Peruvian extract of
cat's claw are due to the proposed active ingredients or to novel chemical entities. The
possibility that novel chemical structures participate in these antiinflammatory effects
warrants a continued evaluation of this herbal medicine. However, beyond the search for
new chemical leads, this study offers definitive evidence that the anecdotal reports of anti-
inflammatory properties of cat's claw has a basis in fact and are sufficiently diverse to be
considered an important therapeutic entity. Cat's claw is available in most Western
countries and further research in other models of inflammation (gastrointestinal and
systemic) including clinical studies, should be evaluated. For developing countries, where
health care funds are stretched, herbal medicines like cat's claw deserve serious
consideration.
ACKNOWLEDGEMENTS
We gratefully acknowledge the assistance of Eng. Alberto Silva Del Aguila, Rector of
Universidad Nacional Agraria de la Selva, Tingo Maria, Peru for advice and helpful
discussions. Special thanks are due to Eng. Raul Araujo, Natural Resources Faculty,
Universidad Nacional Agraria de la Selva, Tingo Maria, Peru, for providing samples of cat's
claw Uncaria tomentosa.
This study was supported by grants ROI HD 31885 and POI CA 28842 from the National
Institutes of Health, Bethesda, MD (to M.J.S.M.).
REFERENCES
1. Conner EM, Brand SJ, Davis JM, Kang DY, Grisham MB. Role of reactive
metabolites of oxygen and nitrogen in inflammatory bowel disease: Toxins,
mediators, and modulators of gene expression. Inflammatory Bowel Dis 1996; 2:
133~7.
2. Daflegri F, Ottonello L, Ballestero A, Bogliolo F, Ferrando F, Patrone F.
Cytoprotection against neutrophil derived hypochlorous acid: a potential
mechanism for the therapeutic action of 5-aminosalicylic acid in ulcerative colitis.
Gut 1990; 31:184-6.
3. Sastre J, Millan A, de Garcfa la Asuncion J, et al. A ginkgo biloba extract (Egb 761)
prevents mitochondrial aging by protecting against oxidative stress. Free Rad Biol
Med 1998; 24:298-304.
4. Burres GC, Musch MW, Jurivich DA, Welk J, Chang EB. Effects of mesalamine on
the hsp72 stress response in rat IEC-18 intestinal epithelial cells. Gastroenterology
1997; 113:1474-9.
5. Heck S, Bender K, Kullman M, Gottlicher M, Herrlich P, Cato AC. I kappaB alpha-
independent downregulation of NF-kappaB activity by glucocorticoid receptor.
EMBO J 1997; 16:4698-707.
6. McKenzie SJ. Baker MS, Buffinton GD, Doe WF. Evidence of oxidant-induced injury
to epithelial cells during inflammatory bowel disease. J Clin Invest 1996; 98: 136-
41. colitis. J Gastroenterol Hepatol 1993; 8:116-18.
7. Deitch EA, Kemper AC, Specian RD. Berg RD. A study of the relationships among
survival, gut-origin sepsis, and bacterial translocation in a model of systemic
inflammation. J Trauma 1992; 32:141-7.
8. De SK, McMaster MT, Andrews GK. Endotoxin induction of murine metaflothionein
gene expression. J Biol Chem 1990; 265:15267-74.
9. Bremner 1. Interactions between metallothionein and trace elements. Progr Food
Nutr Sci 1987; II: 1-37.
10. Aquino R, De Feo V, De Simone F, Pizza C, Cirino G. Plant metabolites. New
compounds and anti-inflammatory activity of Uncaria tomentosa. J Nat Prod 1991;
11. Rizzi R, Re F, Bianchi A, et (d. Mutagenic and antimutagenic activities of Uncaria
tomentosa and its extracts. J Ethnopharmacol 1993; 38:63-77.
12. Yepez AM, Lock de Ugaz 0, Alvarez A, et (d. Quinovic acid glycosides from Uncaria
guianensis. Phytochemistry 1991; 30:1635-7.
13. Stuppner H, Sturm S. Capillary electrophoretic analysis of oxindole alkaloids from
Uncaria tomentosa. ] Chromat 1992; 609:375-80.
14. Laus G, Keplinger D. Separation of stereoisomeric oxindole alkaloids from Uncaria
tomentosa by high performance liquid chromatography. J Chromat A 1994; 662:
243-9.
15. Stuppner H, Sturm S, Konwalinka G. HPLC analysis of the main oxindole alkaloids
from Uncaria tomentosa. Chromatography 1992; 34:597-600.
16. Beckman JS. Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent
hydroxylradical production by peroxynitrite: Implications for endothelial injury
from nitric oxide and superoxide. Proc Nati Acad Sci USA 1990; 87: 1620-4. Hughes
MN, Nicklin HG. The chemistry of pernitrites. Part I, Kinetics of Decomposition of
pernitrous acid. J Chem Soc 1968; A: 450-52.
17. Sandoval M, Zhang X-J, Liu X, Mannick EE, dark DA, Miller MJS. Peroxynitrite-
induced apoptosis in T84 and RAW 264.7 cells: attenuation by L-Ascorbic acid.
Free Rad Biol Med 1997; 22:489-95.
18. Schreiber E, Matthias P, Muller MM. Schaffner W. Rapid detection of octamer
binding proteins with 'mini extracts', prepared from a small number of cells. Nucl
Acids Res 1989; 17: 6419.
19. Jobin C, Haskill S, Mayer LI Panja A, Sartor RB. Evidence for altered regulation of
lkB« degradation in human coionic epithelial cells. ] Immunol 1997; 158: 226-34.
20. Sandoval M, Liu X, Mannick EE, Clark DA, Miller MJS. Peroxynitrite-induced
apoptosis in human intestinal epithelial cells is attenuated by mesalamine.
Gastroenterology 1997; 113:1480-8.
21. Chomczynki P, Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Analytical Biochem 1987:162:156-9.
Yamada T, Deitch E, Specian RD, Perry MA, Sartor RB, Grisham MB. Mechanisms
of acute and chronic intestinal inflammation induced by indomethacin.
Inflammation 1993; 17:641-62.
22. Renter BK, Davies NM, Wallace JL. Nonsteroidal anti-inflammatory drug
enteropathy in rats: Role of permeability, bacteria and enterohepatic circulation.
Gastroenterology 1997; 112:109-17.
23. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous
inflammation: Estimation of Neutrophil content with an enzyme marker. J Invest
Dennatol 1982; 78:206-9.
24. Eaton DL. Toal BF. Evaluation of the Cd/Hemoglobin affinity assay for the rapid
determination of metallothionein in biological tissues. Toxicol Appi Pharmacol
1982; 66: 134—42. Beliezo JM, Britton RS, Bacon BR, Fox ES. LPs-mediated NF-
feB. activation in rat Kupffer cells can be induced independently of CD14. Am ]
Physiol 1996; 33: G956-G961. Richards MP: Role of metallothionein in copper and
zinc metabolism. J Nutr 1989; 119: 1062-70.Pearson DC, Jourd'heuil D, Meddings
JB. The antioxidantproperties of 5- minosalicylic acid. Free Rad Biol Med 1996;71-
?h7-71
25. Desmarchelier C. Mongelli E. Coussio J. Ciccia G. Evaluatio of the in vitro
antioxidant activity in extracts of Uncaria tomentosa (Wilid.) DC. Phytotherapy Res
1997; II: 254-6.
26. Liu X, Lancaster JR, Jr Miller MJS, et aL Effects of the antioxidants, L-ascorbate
and 5-aminosalicylic acid on nitric oxide oxidation in water. The 214th Am Chem
Soc Nat Meeting 1997; ANYL: 92. Miller MJS, Thompson JH, Zhang X-J, et al. Role
of inducible nitric oxide synthase expression and peroxynitrite formation in guinea
pig ileitis. Gastroenterology 1995; 109: 1475-83. Boughton-Smith NK, Evans SM.
Hawkey CJ, et aL Nitric oxide synthase activity in ulcerative colitis and Crohn's
disease. Lancet 1993; 341:338-40.
27. Mannick EE, Bravo LE, Zarama G, et al. Inducible nitric oxide synthase,
nitrotyrosine and apoptosis in Helicobacter pylori gastritis: effects of antibiotics
and antioxidants. Cancer Res 1996; 56:3238-44.
28. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and
the risk of gastric carcinoma. N Engi J Med 1991; 325:1127-31.
29. DiSilvestro RA, Cousins RJ. Mediation of endotoxin-induced changes in zinc
metabolism in rats. Am J Physiol 1984: 247:E436-E441.
30. Unlap MT, Jope RS. Dexamethasone attenuates NF-kappa B DNA binding activity
without inducing I kappa B levels in rat brain in vivo. Mol Brain Res 1997: 45:83-9.
Baeuerle PA. Henkel T. Function and activation of NF-kB in the immune system.
Annu Rev Immunol 1994; 12: 14179. Laus G, Brossner D, Keplinger K. Alkaloids of
Peruvian Uncaria tomentosa. Phytochemistry 1997:45:855-60. Wagner H,
Krentzkamp B, Jurcic K. Die alkaloide von Uncaria tomentosa und ihre
phagozytose-steigernde wirkung. Planta Med 1985; 44:419-23.

Source: http://inmedis.gr/articles/pdf/Antiinflacatsclaw.pdf