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Manuel Sandoval-Chacón,
Jane H. Thompson, Xiao-Jing Zhang, Xiaoping Liu, Elizabeth E.
Running Title: Cat's claw and oxidative stress Corresponding author: Manuel Sandoval-Chacón,
Ph.D.
Background:
Methods:
Results:
INTRODUCTION Among the numerous factors associated with chronic gut inflammation the enhanced production of oxidants and free radicals has become widely recognized as integral components of cell and tissue injury. 1 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 (IBD). Although the etiology of IBD 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. 7 Increased intestinal permeability
to luminal contents (bacterial or dietary) may also promote
8 During states of inflammation, LPS and cytokines have been reported to induce the synthesis of metallothionein in liver. 9 Metallothionein (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 addition, in developing countries many of these 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 ?uña 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. 11 Similarly, during the last 10 years, cats 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. 12 The extract contains a mixture
of quinovic acid and glycosides as well as pentacyclic or
13, 14 The purpose of this study was two fold 1) 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 2) 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 medium 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 María, Peru and identified by Eng. Raúl Araujo of the Universidad Nacional Agraria de la Selva. The extract of Uncaria tomentosa was prepared from air-dried bark of cat?s claw by boiling in water (20 g/L) for 30 min and left at room temperature overnight. The extract was decanted and filtered at 10 mm. The cat's claw (UG) extract for the cell culture experiments was filtered at 0.2 mm and diluted to a final concentration of 5 mg/ml, and refrigerated. For the in vivo studies the UG extract was filtered at 0.45 mm. The extract contains oxindole alkaloids such as pteropodine, isopteropodine, mitraphylline and isomitraphylline. 15, 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 mM HEPES, pH 7.4; 4 mM L-glutamine; 40 mg/ml penicillin; 90 mg/ml streptomycin; 0.25 mg/ml fungizone and 1.2 g/L NaHCO3. Cell cultures were maintained in a humidified 5% CO2 incubator at 37oC. Cells were plated at 1x106 cells/ml. Harvested cells were plated in 6-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. 17 Briefly, solutions of (a) 0.7 M NaNO2, 0.7 M H2O2 and (b) 0.6 M HCl, 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 received in a beaker containing a 1.5 M KOH solution. To destroy the excess of H2O2 the peroxynitrite solution was filtered in a column containing MnO2 (4 g). The prepared solution contained 35-50 mM peroxynitrite as determined by absorbance at 302 nm (E302 = 1,670 M-1.cm-1). 18 A fresh working peroxynitrite solution (5 mM) was prepared in 5 mM KOH for each experiment and filtered at 0.2 mm. Cell viability. Aliquots of treated cells were examined for viability as determined by trypan blue dye exclusion. Briefly, HT29 were detached with trypsin-EDTA and RAW 264.7 cells were scraped and washed with phosphate buffered saline (154 mM, NaCl; 2.7 mM, Na2HPO4?7H2O; 1.3 mM, KH2PO4), resuspended in medium and added 0.4% trypan blue stain. Within 5-min incubation, the number of cells excluding dye was expressed as a percentage of total cells counted from three randomly chambers of the hemocytometer. Measurement of Nitrite/Nitrate. For these experiments, HT29 and RAW 264.7 cells were treated with lipopolysaccharide (LPS, 1 mg/ml) and/or UG (100 mg/ml) and incubated for 18 h and 12 h, respectively. The stable end products of nitric oxide nitrite/nitrate (NO2- and NO3-) were assayed in HT29 and RAW 264.7 cells using the Griess reagent after conversion of NO3- to NO2- with a colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI). Detection of Apoptosis by ELISA. HT29 and RAW 264.7 cells were either treated with 300 mM PN and/or supplemented with cat's claw extract (UG, 100 mg/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 1 x 106/well were plated in six-well clusters and treated with LPS (1 mg/ml) and/or UG (50-100 mg/ml) and incubated at 37oC. After 2 h, the medium covering the cells was removed and replaced with ice-cold phosphate-buffered saline. RAW 264.7 cells were harvested byscraping followed by centrifugation (1000 g). Preparation of nuclear protein extracts and EMSAs were carried out as previously reported. 20, 21 The protein concentration
of the nuclear extracts was determined using the Bio-Rad
Evaluation of peroxynitrite scavenging by cat's claw. Diluted samples from the stock solution of peroxynitrite(40 mM) were used to prepare working solutions of 300 mM peroxynitrite containing 5 mM KOH (pH 12). Finalvolume was 1 ml, and absorbance at 302 nm was determined or scanned from 200 to 400 nm at 1 and 10 min,respectively. A Beckman DU 64 spectrometer (Beckman Instrument Inc., Fullerton, CA) was used to assess the change in absorbance. In separate experiments, the absorbance of UG (100 mg/ml) diluted in 5 mM KOH or reacted with 300 mM PN was scanned and absorbance at 302 nm was determined. Auto-oxidation of NO by cat's claw extract. To evaluate if NO reacts with UG extract, the time-dependent depletion of 30 mM NO was monitored in two solutions: 1) phosphate solution (pH 7.4) containing 5 mg/ml UG extract, and 2) phosphate solution without UG extract. The microelectrode experiments were performed at 25oC. The saturated stock solution contained 160 mM NO 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 2 x 106 cells/well were seeded in six-well
tissue culture plates and incubated with LPS (1 mg/ml) and/or UG (50-200
mg/ml). After 12 h, total RNA was isolated from cells by the acid
guanidine thiocyanate-phenol-chloroform extraction method.23 Integrity
of RNA was assessed on a 1.2 % agarose gel and RNA was visualized by ethidium
bromide. First-strand complementary DNAs were synthesized from 1
mg of total RNA using oligo dT and Superscript II Reverse Transcriptase
(Gibco BRL, Grand Island, NY). The first-strand complementary DNA
templates were amplified for glyceraldehyde-3-phosphate
Indomethacin-induced intestinal inflammation. Animal models of nonsteroidal antiinflammatory drug (NSAID) induced enteropathy are associated with changes in the morphology, microvascular injury and changes in epithelial permeability. 24, 25 To evaluate the antiinflammatory
activity of cat's claw against the indomethacin-induced
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 -77oC until assayed.
Determination of intestinal damage. A group of rats was used for comparison of morphologic studies. At the end of seven days, rats were anesthetized, and samples of the midjejunum were taken. Tissue was fixed in phosphate-buffered formaldehyde, embedded in paraffin, and 5-µm sections were prepared. Tissue was routinely stained with hematoxylin and eosin and evaluated by light microscopy. Metallothionein protein assay. After 7 d in the study, animals were sacrificed and samples of liver and intestinal mucosal cells were collected for metallothionein (MT) protein. The MT concentration of cytosolic fractions of liver and intestinal cells was determined by 109Cd-Hb affinity assay as previously described. 27 Statistical Analysis. Each experiment was performed at least three times and results are presented as the mean" SEM. Statistical analyses were performed using one-way ANOVA. Post hoc comparison of means was done by Least Significant Difference test. A probability of < 0.05 was considered significant. RESULTS Assessment of viability and apoptosis
in cell lines. Experiments to examine the cytotoxic effects of peroxynitrite
(300 mM, for 4 h), with and without cat's claw (UG, 100 mg/ml) were conducted
to delineate the protective effect of UG extract. Cell viability
was not affected by the experimental conditions (Table 1).
Levels of Nitrite/Nitrate. HT29 cells treated with LPS produced higher (P < 0.05) levels of NO2-/NO3- than cells simultaneously treated with LPS and UG extract (Figure 3). In another set of experiments with RAW 264.7 cells, 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 (1 mg/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 mg/ml) caused an inhibition of NF-kB. Regulation of iNOS mRNA in HT29 cells induced by LPS. Figure 6 shows levels of iNOS mRNA expression from HT29 cells treated with LPS (1 mg/ml) and/or UG extract (50-200 mg/ml). Expression of iNOS mRNA was increased in LPS treated cells as evident after 12 h incubation. However, 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 mg/ml) to peroxynitrite-containing
5 mM KOH resulted in a significant (P < 0.05) decrease in peroxynitrite
concentration (Figure 7). The decomposition of peroxynitrite was
evaluated at pH 12 because PN 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.
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 INDO (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, MT was increased (P < 0.05) in rats that received INDO after 7 d compared to CTRL rats. Administration of UG (5 mg/ml) in the drinking water to rats treated with INDO resulted in lower (P < 0.05) liver MT. In contrast to liver MT, either INDO or the UG extract did not affect the content of intestinal MT. The induction of MT 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.30 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
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 what 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 NO, 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 NO while directly degrading peroxynitrite. This is a pattern of effects that we have seen 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.
The suppression of the indomethacin?induced
acute phase response protein, metallothionein, in the liver by cat's claw
demonstrates that these transcription-dependent responses are registered
in vivo as well as in vitro.
37 Glucocorticoids can negate the induction of iNOS expression through transcriptional mechanisms, e.g. inhibition of NF-kB, 38 as seen in this study with cat's claw. As NF-kB controls the expression of a wide range of pro-inflammatory signals, including 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 pro-inflammatory 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 identical to the ethnomedical use of cat's claw in Peru and neighboring regions. This oral administration of cat's claw "tea" had an impressive protective effect on INDO-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.15 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 novel chemical entities. The possibility that novel chemical structures participate in these anti-inflammatory 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 basis 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 dollars are stretched, herbal medicines like cat's claw deserve serious consideration. 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 Diseases 1996;2:133-147. 2. Dallegri F, Ottonello LL, Ballestero A, Bogliolo F, Ferrando, Patrone F. Cytoprotection against neutrophil derived hypochlorous acid: a potential mechanism for the therapeutic action of 5-aminosalicylic acid in ulcerative colitis. Gut 1990;31:184-186. 3. Sastre J, Millán A, García de la Asunción J, et al. A ginkgo biloba extract (Egb 761) prevents mitochondrial aging by protecting against oxidative stress. 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Laus G, Brossner D, Keplinger K. Alkaloids of Peruvian Uncaria tomentosa.
Phytochemistry
41. Wagner H, Krentzkamp B, Jurcic K. Die alkaloide von Uncaria tomentosa und ihre phagozytose-steigernde wirkung. Planta Med 1985;44:419-423. 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,
Supported by grant RO1 HD 31885 and PO1 CA 28842 from National Institutes of Health, Bethesda, MD (to MJS Miller). |
