Inhibition of serine proteases by anti-inflammatory triterpenoids.

The lupane triterpenoid lupeol, the ursane triterpenoid alpha-amyrin and esters of these compounds are present in the bark of roots of Alstonia boonei (Apocynaceae) and have anti-inflammatory properties. alpha-Amyrin is a competitive inhibitor of bovine trypsin and chymotrypsin (Ki values 29 microM and 18 microM, respectively). Lupeol linoleate, lupeol palmitate and alpha-amyrin linoleate are non-competitive inhibitors of trypsin (Ki values 7 microM, 10 microM and 16 microM, respectively). alpha-Amyrin linoleate is also a non-competitive inhibitor of chymotrypsin (Ki value 28 microM). Lupeol is a competitive inhibitor of both trypsin and chymotrypsin (Ki values 22 and 8 microM, respectively). alpha-Amyrin palmitate is a potent non-competitive inhibitor of chymotrypsin (Ki 6 microM). Lupeol, alpha-amyrin and the palmitic and linoleic acid esters of these compounds are ineffective or very weak as inhibitors of porcine pancreatic elastase and of Lucilia cuprina and Helicoverpa punctigera leucine aminopeptidases. These hydrophobic triterpenoids represent further examples of anti-inflammatory triterpenoids that are PKA inhibitors as well as being selective protease inhibitors.

Selective inhibition of eukaryote protein kinases by anti-inflammatory triterpenoids.

The ursane triterpenoid alpha-amyrin and the lupane triterpenoid lupeol are potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA) with IC50 values of 8 and 5 microM, respectively. The palmitate and linoleate esters of alpha-amyrin and lupeol are also potent inhibitors of cAK (IC50 values in the range of 4-9 microM). alpha-Amyrin, lupeol and lupeol linoleate are much less potent as inhibitors of rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC) (IC50 values 32, 82 and 35 microM; respectively) and alpha-amyrin linoleate and the palmitate esters of lupeol and alpha-amyrin are ineffective or very poor inhibitors of this protein kinase. These compounds are very poor or ineffective as inhibitors of chicken gizzard calmodulin-dependent myosin light chain kinase (MLCK). alpha-Amyrin inhibits plant Ca(2+)-dependent protein kinase (CDPK) (IC50 52 microM) but lupeol and the triterpenoid esters tested are ineffective. alpha-Amyrin and the linoleate and palmitate esters of alpha-amyrin and lupeol inhibit cAK in a fashion that is competitive with respect to both peptide substrate and ATP (Ki values in the range 2-6 microM). However, while lupeol is competitive with respect to ATP it is apparently non-competitive with respect to peptide substrate. alpha-Amyrin also inhibits CDPK competitively and alpha-amyrin, lupeol and lupeol linoleate are competitive inhibitors of PKC. alpha-Amyrin and the palmitate esters of lupeol and alpha-amyrin are competitive inhibitors of the potato high affinity cyclic AMP-binding phosphatase (Pase) but lupeol inhibits the Pase non-competitively. These hydrophobic triterpenoids are further examples of anti-inflammatory triterpenoids that are cAK inhibitors.

A comparison of the hydroxyl radical scavenging properties of the shark bile steroid 5 beta-scymnol and plant pycnogenols.

The hydroxyl radical (OH.) quenching abilities of the following compounds were compared in the deoxyribose degradation system (initiated by the ferrous-ascorbate Fenton reaction): (a) 5 beta-scymnol, the hepatoprotective shark bile sterol, and its mono- and di-sulfate esters; (b) three marketed pycnogenol preparations (syn: proanthocyanidin--natural plant-derived polyphenolic bioflavonoids) extracted from pine tree (Pinus maritima) bark and grape (Vitis vinifera) seeds; and (c) two known hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, and the peroxyl radical scavenger Trolox (the alpha-tocopherol analogue). 5 beta-scymnol was a more potent OH. quencher than dimethyl sulfoxide, mannitol and Trolox, and markedly more potent than the pycnogenol preparations. Increased sulfation of 5 beta-scymnol progressively reduced its free radical scavenging activity, thus clearly attributing the potent OH. quenching properties to its novel tri-alcohol-substituted aliphatic side chain. The favourable interaction of these bile steroids with reactive oxygen species in an aqueous environment, makes them attractive candidates for evaluation as protective agents against disorders in which oxidative stress is implicated.

Anti-inflammatory activity of a lipid fraction (lyprinol) from the NZ green-lipped mussel.

A lipid-rich extract, preparared by supercritical fluid extraction of fresh stabilized mussel powder (Lyprinol), showed significant anti-inflammatory (AI) activity given therapeutically and prophylactically po to Wistar and Dark Agouti rats developing either (a) adjuvant-induced polyarthritis or (b) collagen(II)-induced autoallergic arthritis, with ED(50)/=25 mg/kg or various therapeutic oils (flaxseed, evening primrose, fish)>/=1800 mg/kg given orally. Lyprinol showed little or no activity in acute irritation assays (carrageenan, kaolin, histamine) indicating it is not mimicking rapid-acting NSAIDs.Incorporating Lyprinol into arthritigenic adjuvants composed of heat-killed Mycobacterium. tuberculosis suspended in olive oil or squalane, effectively prevented arthritis development at a dose of 5 mg/rat. By contrast, 'dummy adjuvants' prepared with Mycobacterium tuberculosis and flaxseed, evening primrose or fish oils were still arthritigenic in Dark Agouti rats (doses of oil=90 mg/rat).Lyprinol subfractions inhibited leukotriene-B(4) biosynthesis by stimulated human polymorphonuclear leukocytes in vitro, and prostaglandin-E(2) production by activated human macrophages in vitro. Much of this AI activity was associated with polyunsaturated fatty acids and natural antoxidants (carotenoids, etc.).In contrast to NSAIDs, Lyprinol is non-gastrotoxic in disease-stressed rats at 300 mg/kg po and does not seem to affect platelet aggregation (human, rat). These data show Lyprinol to be a reproducible, relatively stable, source of bioactive lipids with much greater potency than plant/marine oils currently used as nutritional supplements to ameliorate signs of inflammation.