Lipoxygenase activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.).

In this study we investigated the influence of biotic elicitor (phytopathogenic fungus Botrytis cinerea) and abiotic elicitors (methyljasmonate [MJ] and salicylic acid [SA]) on lipoxygenase (LOX) activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.). We have observed different time effects of elicitors (10, 24, 48 and 72 h) on LOX activity and production of sanguinarine in in vitro cultures. All elicitors used in the experiments evidently increased the LOX activity and sanguinarine production in contrast to control samples. The highest LOX activities were determined in samples elicitated by MJ after 48 h and 72 h and the lowest LOX activities (in contrast to control samples) were detected after biotic elicitation by Botrytis cinerea. These activities showed about 50% lower level against the activities after MJ elicitation. The maximal amount of sanguinarine was observed after 48 h in MJ treated cultures (429.91 mg/g DCW) in comparision with control samples. Although all elicitors affect the sanguinarine production, effect of SA and biotic elicitor on sanguinarine accumulation in in vitrocultures was not so significant than after MJ elicitation.

Influence of lipid imbalance on butyrylcholinesterase activity and biotransformation efficiency.

Butyrylcholinesterase (EC 3.1.1.8, BChE) is highly active in plasma, skin and lung, the tissues that first contact xenobiotics, supporting a role for BChE in detoxication of xenobiotics including medicaments. A possible involvement of BChE in lipid metabolism has been suggested. Elevated BChE activity in obese individuals correlates with some parameters of lipid metabolism including increased levels of triacylglycerols (TAG) and cholesterol. The aim of this study was to estimate the BChE activity in rats on subcellular and inter-organ levels under the conditions of untreated and treated primary hypertriacylglycerolemia with the TAG lowering agent fenofibrate. No changes in BChE activity were observed in obese animals. However fenofibrate administration led to significant increase of BChE activity in all examined tissues (plasma, liver, white adipose tissue). The impact of lipid metabolic imbalance on BChE biotransformation ability was tested by measuring the rate of hydrolysis of 0,1 to 8 mM concentrations of the antimicrobial agent N-(2-benzoyloxyethyl)-ethyldimethylammonium bromide (BCH2). The results revealed a complete shift in the BChE kinetics in all studied models. In animals with hypertriacylglycerolemia the Km value of liver BChE rised 4,6-fold, but the total enzyme efficiency expressed as Vmax/Km dropped 40% comparing to control. In contrast, in animals treated with fenofibrate the BChE efficiency increased in liver 1,6-fold. We conclude here that BChE detoxification capacity is essentially altered under conditions of disturbed lipid metabolism. Clinically, this knowledge could be important in a view of xenobiotic elimination, especially when routinely prescribed medicaments are concerned.

[Characterization of polyphenoloxidase from the latex of greater celandine (Chelidonium majus L.)]. / Charakterizácia polyfenoloxidázy z latexu lastovidníka väcseho (Chelidonium majus L.).

Greater celandine, similarly as other plants of the family Papaveraceae, produces benzylisoquinoline alkaloids, primarily benzophenanthridines. Polyphenoloxidase (PPO) is most probably involved in the formation of dopamine, which is one of the precursors of norcoclaurine, the first intermediate with the benzylisoquinoline structure. This study has revealed that PPO present in the latex of greater celandine is localized in the organelles, which serve to store alkaloids (the so-called 1000 g organelles). The enzyme was purified by means of affinity chromatography into electrophoretic homogeneity. It possesses a relative molecular mass of approximately 65 kDa and exerts two activities, the monophenolase and diphenolase ones. With the use of a polymerase chain reaction, it was possible to amplify a part of the PPO gene from the region of the active site.

[Effect of a fungal elicitor on levels of sanguinarine and polyphenoloxidase activity in a suspension culture of Papaver somniferum L]. / Zmeny obsahu sanguinarínu a aktivity polyfenoloxidázy vplyvom fungálneho elicitora v suspenzných kultúrach maku siateho Papaver somniferum L.

The opium poppy (Papaver somniferum L.) is still a source for isolation of codeine and morphine. Cell cultures from this plant lose their ability to produce morphinans. Their major alkaloid is sanguinarine. The elicitation of the opium poppy cell cultures by fungal preparation lead to a nine-fold increase in the content of sanguinarine. The specific activity of polyphenoloxidase (PPO) was three-times higher in the elicited compared to the nonelicited cells. Two isoforms of PPO (Mr 63 kDa, 41 kDa) were identified in opium poppy cell cultures by PAGE. The number of PPO isoforms was not affected by elicitation. Phenyl-Sepharose CL-4B was used for affinity purification of PPO. In a single purification step the specific activity of PPO was enriched 14-fold.