Comparison of methods for determination of total antioxidant status: application to analysis of medicinal plant essential oils

The free radical scavenging capacity of a wide range of plant oil extracts, principally those used in traditional European herbal medicine (with novel therapeutic potential for patients with degenerative disorders of the CNS), has been compared in vitro. The antioxidant capacity of individual plant extracts was determined via three complementary assay procedures, based on: (i) attenuation of the generation of ABTS√⁺ radical (quantitated colorimetrically), by a metmyoglobin catalyst/hydrogen peroxide system; (ii) inhibition of iodophenol enhanced chemiluminescence by a horseradish peroxidase/perborate/luminol system; (iii) protection of a target enzyme (human brain alanyl aminopeptidase, activity quantitated via fluorimetric assay) against oxidative damage by √OH or O√⁻₂ generated by Co⁶⁰γ radiolysis. In assays (i) and (ii), only three plant extracts (cinnamon, pimento, bay) showed substantial antioxidant activity, although the two assays yielded quantitatively different values of antioxidant activity (Trolox equivalent values of 16–25 M (method ii) and 0.25–2.1 M (method (i)). None of the plant extracts investigated showed significant antioxidant protective activity against √OH or O√⁻₂ species in assay (iii). The data obtained thus demonstrate that the apparent antioxidant capacity of putative free radical scavenging agents depends entirely on the assay method utilized and particular free radical species generated. We therefore suggest that antioxidant capacity determined by a single assay method (particularly via competitive assay with ABTS√⁺) should be interpreted with some caution. This conclusion may be of particular potential importance in clinical chemistry, in view of the current interest in the assessment of the antioxidant status of tissues of patients with a variety of disorders.     

Nasal administration of arthritis-related T cell epitopes of heat shock protein 60 as a promising way for immunotherapy in chronic arthritis

Adjuvant Arthritis (AA) can be induced in Lewis rats by immunisation with mycobacterial antigens. The disease can be passively transferred with T cell clone A2b, which recognises the 180–188 amino acid sequence in mycobacterial heat shock protein 60 (hsp60) and which crossreacts with crude cartilage proteoglycans. We succeeded to induce peripheral tolerance to this AA-associated T cell epitope following nasal administration of a peptide containing this epitope (mycobacterial hsp60 176–190). In rats treated nasally with 176–190 and immunised with mycobacterial hsp60, proliferative responses to 176–190 were reduced. AA was inhibited nasally with 176–190 treated rats and not in rats nasally treated with a control mycobacterial hsp60 peptide (211–225). Moreover, nasal 176–190 led to similar arthritis protective effects in a non-microbially induced experimental arthritis (avridine induced arthritis). In a subsequent study we tried to prevent and to treat AA through nasal administration of mycobacterial hsp60 peptide 180–188 and a peptide analogue of 180–188, 180–188_L183->A (Alanine 183), which has been shown to have an increased MHC-binding affinity for rat RT1 B¹ and an increased capacity to inhibit the proliferative A2b responsein vitro. We found that nasal administration of 180–188 had a moderate arthritis suppressive effect in AA, whereas its analogue peptide Alanine 183, had a strong suppressive effect. This strong arthritis suppressive effect was only partly due to the higher MHC-binding affinity for rat RT1 B¹. Furthermore, it was possible to passively transfer nasal Alanine 183 induced disease protection. The present findings may in our view offer novel prospects for immunotherapy through nasal administration of (analogue) peptides, with a mimicry relationship with joint specific cartilage proteoglycan epitopes.