The results of the present paper clearly indicates that topical application of the extracts (chemotype 1 and chemotype 2) and fractions F-1 (mixture of pseudopterosins) F-2 (mixture of pseudopterosins, seco -pseudopterosins and amphilectosins) and F-3 (mixture non-glycosylated diterpenes) isolated from P. elisabethae (Figure 1), and the anti-inflammatory drug indomethacin resulted in a significant inhibition of two important events related to the topical inflammatory response induced by TPA, oedema formation, and PMNs infiltration and degranulation, events that modulate MPO levels at inflammation site. Therefore, these results consistently support that the compounds present in the mentioned fractions possess excellent topical anti-inflammatory properties, similar to as was previously reported for other pseudopterosins as PsA-D  and PsM, PsN and PsO .
The MPO decrease level even down to basal levels (Figure 3) confirms that the compounds present in the assayed extracts and fractions can reduce the leukocyte infiltration. This was verified on ear homogenates. Based on these results we evaluated in vitro whether the pure pseudopterosins and seco -pseudopterosin (isolated from F-1, F-2) and IMNGD (isolated from F-3) could have inhibition actions on cellular functions in human PMNs.
Initially, we evaluated leukocyte degranulation of calcium ionophore A23187 stimulated cells (Figure 4). The biomarker used was MPO since this is a recognized granular enzyme engaged in events of activation of PMNs and is associated with tissue injury. Therefore, this is necessary to form the strong oxidant hypoclorous acid, which by reaction with superoxide can in turn generate the reactive hydroxyl radical. In these assays PsQ, PsS, PsT and PsU inhibited significantly the release of MPO in a similar way as the positive controls indomethacin and dexamethasone. In contrast, the IMNGD showed superior inhibition as compared to the positive controls suggesting that glycosylated conditions could reduce the inhibitory activity of these molecules. These results confirm the potential of these molecules and the possibility that they contribute to the inhibition of neutrophil-mediated tissue injury.
Additionally, by comparing the different MPO inhibition values (Figure 4) for the tested compounds in terms of chemical structure, interesting structure-activity relationships arise. First of all, the comparison of the activity of pseudopterosins with different sugar moiety linked to diterpene may indicate that activity depends on: 1) kind of sugar moiety, 2) whether sugar moiety is in a free form or acetylated, 3) acetylation position within the sugar moiety and 4) glycosylation position. For example, PsT glycosylated with non-acetylated arabinopyranose has more activity than PsP which is glycosylated with non-acetylated fucopyranose. Likewise, PsQ and PsS (acetylated fucose as sugar moiety) have more activity than PsP. With regards to the acetylation position, the results showed that acetylation in C-4' of fucose moiety could improve the activity – MPO inhibition value of PsQ (acetylated in C-4') compared to that shown by PsS (acetylated in C-2').On the other hand the glycosylation position might affect the inhibitory activity profile. For example, all pseudopterosins glycosylated in C-10 (PsQ, PsS, PsT and PsU), except PsP, showed more activity than PsG and PsK which are glycosylated in C-9. In the same way, the stereochemistry could be a determinant factor in the inhibition of MPO and leukocyte degranulation, since the activity of PsG and PsK, both glycosylated with fucopyranose but with different stereochemistry in the aglycone (Figure 1), showed different activity. More experiments in relation to this theme should be done to confirm the above discussion.
Regarding NO release in J-774 cell-based assay (Figure 5), we found that the activity of IMNGD and all pure compounds is concentration-dependent. Additionally, IMNGD showed a major activity than the pseudopterosins and seco -pseudopterosin. Again as in the MPO assay, the non-glycosylation improves the inhibition of NO release.
By comparing the different NO inhibition values for tested compounds (Figure 5), we also observed structure-activity relationships as with the MPO assay. In general, in this assay the inhibitory activity apparently depends on the glycosylation position (i. e. activity of PsP versus PsG). As to the stereochemistry of the aglycone, it does not seem to be a determinant factor to improve the inhibition (i. e. activity of PsG versus PsK). In contrast the skeleton type might influence the activity. For example, the amphilectane skeleton (PsP) has more inhibitory activity than the serrulatane skeleton (seco -PsK). As was mentioned before, more experiments have to be performed to support structure-activity relationships among these kinds of compounds.
In aiming to understanding the behavior of these compounds with respect to their potential as inhibitors of the NO release, we carried out NO scavenger activity assay (Figure 6) to determine whether the inhibition of NO liberation within J-774 cells is produced by inhibition of some molecular process in the cellular machinery (such us inhibition of expression and activity of Inducible Nitric Oxide Synthase (iNOS)), or whether the inhibition is due to scavenger activity . According to the results of these assays, PsG, PsP and seco -PsK did not exhibit any scavenger activity, suggesting the possibility that these compounds may inhibit iNOS or other routes that influence this enzyme.
PsQ, PsS, and PsU showed scavenger activity (Figure 6) which let us to confirm that these compounds inhibit NO release in macrophage cells by scavenger activity. However, it is important to carry out more studies in order to confirm if these compounds might inhibit some molecular routes upstream from NO production in cells.