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. 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Duncan joined Insight Investment in October 2003 as managing director of the property division, overseeing the management of more than £4.5 billion of UK commercial and residential property assets and setting strategy for thedivision’s growth. Previously, he was a founding partner of specialist boutique,Gatehouse Investment Management, being instrumental in establishing andmana
HIGHLIGHTS OF PRESCRIBING INFORMATION ----------------------- WARNINGS AND PRECAUTIONS ---------------- These highlights do not include all the information needed to use • Colitis: Clindamycin can cause severe colitis, which may result in death. VELTIN Gel safely and effectively. See full prescribing information for Diarrhea, bloody diarrhea, and colitis (including pseudomembra