A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry.

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN(3)), 3% of hydrogen peroxide (H(2)O(2)), NaN(3)/H(2)O(2) combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN(3)/H(2)O(2) is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN(3) and H(2)O(2) as potent peroxidase inhibitors.

Anti-inflammatory and antioxidant activities of cat's claw (Uncaria tomentosa and Uncaria guianensis) are independent of their alkaloid content.

Cat's claw is an herbal medicine from the Amazon that is used widely to treat inflammatory disorders. The purpose of this study was to characterize the antioxidative and antiinflammatory properties of cat's claw, Uncaria tomentosa (UT) and Uncaria guianensis (UG). Alkaloids and flavanols were determined using reversed-phase HPLC; scavenging of 1,1-diphenyl-2-picrilhydrazyl (DPPH), hydroxyl radicals, and lipid peroxidation by spectrophotometry; and TNFalpha production by ELISA. Anti-inflammatory activity was assessed in vitro by inhibition of TNFalpha and nitrite production from RAW 264.7 cells exposed to LPS (50 ng/ml) and in vivo using the indomethacin-induced gastritis model. Apoptosis was assessed using the TUNEL technique and TNFalpha mRNA by in situ RT-PCR. In each of the antioxidant assays tested, UG was more potent than UT (P < 0.01). The total oxindole and pentacyclic alkaloid content of UT was 35-fold > UG. The IC50 value for inhibition of TNFalpha production was significantly (P < 0.01) higher for UT (14.1 ng/ml) vs UG (9.5 ng/ml), yet at concentrations that were considerable lower than that required for antioxidant activity. Non-alkaloid HPLC fractions from UT decreased LPS-induced TNFalpha and nitrite production in RAW 264.7 cells (P < 0.01) at a concentration range comparable to the parent botanical. Oral pretreatment for 3 d with UT protected against indomethacin-induced gastritis, and prevented TNFalpha mRNA expression and apoptosis. These results indicate that while both species of cat's claw provide effective antioxidant and anti-inflammatory activities, U. guianensis is more potent. In conclusion, the presence of oxindole or pentacyclic alkaloids did not influence the antioxidant and anti-inflammatory properties of cat's claw.

Efficacy and safety of freeze-dried cat's claw in osteoarthritis of the knee: mechanisms of action of the species Uncaria guianensis.

AIM: The purpose of this investigation was to evaluate the ability of cat's claw, an Amazonian medicinal plant, to treat osteoarthritis of the knee, collect safety and tolerance information and compare the antioxidant, and anti-inflammatory actions of Uncaria guianensis and Uncaria tomentosa in vitro. MATERIALS AND METHODS: Forty-five patients with osteoarthritis of the knee were recruited, 30 were treated with freeze-dried U guianensis, and 15 with placebo. Hematological parameters were assessed on entry and exit of the four-week trial. Pain, medical and subject assessment scores and adverse effects were collected at weeks 1, 2 and 4. The antioxidant and anti-inflammatory activity of the cat's claw species was determined by the alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) free radical scavenging method. Inhibition of TNFalpha and prostaglandin E2 (PGE2) production was determined in RAW 264.7 cells by ELISA. RESULTS: Cat's claw had no deleterious effects on blood or liver function or other significant side-effects compared to placebo. Pain associated with activity, medical and patient assessment scores were all significantly reduced, with benefits occurring within the first week of therapy. Knee pain at rest or at night, and knee circumference were not significantly reduced by cat's claw during this brief trial. In vitro tests indicated that U guianensis and U. tomentosa were equivalent at quenching DPPH radicals (EC50, 13.6-21.7 microg/ml) as well as inhibiting TNFalpha production. However, the latter action was registered at much lower concentrations (EC50, 10.2-10.9 ng/ml). Cat's claw (10 microg/ml) had no effect on basal PGE2 production, but reduced LPS-induced PGE2 release (P < 0.05), but at higher concentrations than that required for TNFalpha inhibition. CONCLUSION: Cat's claw is an effective treatment for osteoarthritis. The species, U guianensis and U tomentosa are equiactive. They are effective antioxidants, but their anti-inflammatory properties may result from their ability to inhibit TNFalpha and to a lesser extent PGE2 production.

Cat's claw inhibits TNFalpha production and scavenges free radicals: role in cytoprotection.

Cat's claw (Uncaria tomentosa) is a medicinal plant from the Amazon River basin that is widely used for inflammatory disorders and was previously described as an inhibitor of NF-kappaB. Cat's claw was prepared as a decoction (water extraction) of micropulverized bark with and without concentration by freeze-drying. Murine macrophages (RAW 264.7 cells) were used in cytotoxicity assays (trypan blue exclusion) in response to the free radical 1, 1-diphenyl-2-picrilhydrazyl (DPPH, 0.3 microM) and ultraviolet light (UV) light. TNFalpha production was induced by lipopolysaccharide (LPS 0.5 microg/ml). Cat's claw was an effective scavenger of DPPH; the EC(50) value for freeze-dried concentrates was significantly less than micropulverized (18 vs. 150 microg/ml, p <.05). Cat's claw (10 microg/ml freeze-dried) was fully protective against DPPH and UV irradiation-induced cytotoxicity. LPS increased TNFalpha media levels from 3 to 97 ng/ml. Cat's claw suppressed TNFalpha production by approximately 65-85% (p <.01) but at concentrations considerably lower than its antioxidant activity: freeze-dried EC(50) = 1.2 ng/ml, micropulverized EC(50) = 28 ng/ml. In conclusion, cat's claw is an effective antioxidant, but perhaps more importantly a remarkably potent inhibitor of TNFalpha production. The primary mechanism for cat's claw anti-inflammatory actions appears to be immunomodulation via suppression of TNFalpha synthesis.