Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.).

Opium poppy (Papaver somniferum L.) is an ancient medicinal plant producing pharmaceutically important benzylisoquinoline alkaloids. In the present work we focused on the study of enzyme lipoxygenase (LOX, EC 1.13.11.12) from opium poppy cultures. LOX is involved in lipid peroxidation and lipoxygenase oxidation products of polyunsaturated fatty acids have a significant role in regulation of growth, development and plant defense responses to biotic or abiotic stress. The purpose of this study was to isolate and characterize LOX enzyme from opium poppy callus cultures. LOX was purified by ammonium sulfate precipitation and then followed by hydrophobic chromatography using Phenyl-Sepharose CL-4B and hydroxyapatite chromatography using HA Ultrogel sorbent. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting revealed that LOX from opium poppy cultures was a single monomeric protein showing the relative molecular weight of 83 kDa. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid and the products were analyzed by high-performance liquid chromatography in two steps, firstly with reverse phase (120-5 Nucleosil C18 column) and secondly with normal phase (Zorbax Rx-SIL column). LOX converted linoleic acid primarily to 13-hydroperoxy-(9Z,11E)-octadecadienoic acids (78%) and to a lesser extent 9-hydroperoxy-(10E,12Z)-octadecadienoic acids (22%). Characterization of LOX from opium poppy cultures provided valuable information in understanding LOX involvement in regulation of signaling pathways leading to biosynthesis of secondary metabolites with significant biological activity.

Derivatization of Rosmarinic Acid Enhances its in vitro Antitumor, Antimicrobial and Antiprotozoal Properties.

On its own, rosmarinic acid possesses multiple biological activities such as anti-inflammatory, antimicrobial, cardioprotective and antitumor properties, and these are the consequence of its ROS scavenging and inhibitory effect on inflammation. In this study, two quaternary phosphonium salts of rosmarinic acid were prepared for the purpose of increasing its penetration into biological systems with the aim of improving its antimicrobial, antifungal, antiprotozoal and antitumor activity. The synthetized molecules, the triphenylphosphonium and tricyclohexylphosphonium salts of rosmarinic acid, exhibited significantly stronger inhibitory effects on the growth of HCT116 cells with IC50 values of 7.28 or 8.13 µM in comparison to the initial substance, rosmarinic acid (>300 µM). For the synthesized derivatives, we detected a greater than three-fold increase of activity against Acanthamoeba quina, and a greater than eight-fold increase of activity against A. lugdunensis in comparison to rosmarinic acid. Furthermore, we recorded significantly higher antimicrobial activity of the synthetized derivatives when compared to rosmarinic acid itself. Both synthetized quaternary phosphonium salts of rosmarinic acid appear to be promising antitumor and antimicrobial agents, as well as impressive molecules for further research.

Evaluation of Manganese Chloride's Effect on Biosynthetic Properties of In Vitro Cultures of Eschscholzia californica Cham.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4'-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

HPLC Analysis and Biochemical Characterization of LOX from Eschscholtzia californica Cham.

BACKGROUND: Plant lipoxygenases (LOXs, EC 1.13.11.12) are involved in lipid degradation, regulation of growth and development, senescence, and defence reactions. LOX represents the starting enzyme of the octadecanoid pathway. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. METHODS: LOX from California poppy seedlings was purified by hydrophobic chromatography (Phenyl-Sepharose CL-4B) and by ion-exchange chromatography (Q-Sepharose). The isolated LOX was incubated with linoleic acid used as a substrate. The HPLC experiments were performed with the Agilent Technologies 1050 series HPLC system. For the preparative separation of a mixture of hydroxy fatty acids from the sample matrix, the RP-HPLC method was used (column 120-5 Nucleosil C18). Then, the NP-HPLC analysis (separation, identification, and determination) of hydroxy fatty acid isomers was carried out on a Zorbax Rx-SIL column. RESULTS: The purified LOX indicates the presence of a nontraditional plant enzyme with dual positional specificity (a ratio of 9- and 13-hydroperoxide products 1:1), a relative molecular mass of 85 kDa, a pH optimum of 6.5, an increasing activity stimulation by CaCl2 till 2 mM, and a high substrate reactivity to linoleic acid with kinetic values of KM 2.6 mM and Vmax 3.14 µM/min/mg. CONCLUSIONS: For the first time, the LOX from California poppy seedlings was partially purified and the biochemical properties of the enzyme were analyzed. A dual positional specificity of the LOX found from California poppy seedlings is in agreement with the results obtained for LOXs isolated from other Papaveraceaes. A 1:1 ratio of 9-/13-HODE is attractive for the simultaneous investigation of both biotic stress responses (indicated by the 9-HODE marker) and the biosynthesis of jasmonic acid and jasmonates (indicated by the 13-HODE marker).

Inhibition of 12/15 lipoxygenase by curcumin and an extract from Curcuma longa L.

Curcumin (diferuloylmethane) is an orange-yellow secondary metabolic compound from the rhizome of turmeric (Curcuma longa L.), a spice often found in curry powder. It is one of the major curcuminoids of turmeric. For centuries, curcumin has been used in some medicinal preparations or as a food colouring agent. A variety of enzymes that are closely associated with inflammation and cancer were found to be modulated by curcumin. This paper summarized the results of the inhibitory effect of curcumin and a Curcuma longa L. ethanolic extract on lipoxygenase from the rat lung cytosolic fraction. The positional specificity determination of arachidonic acid dioxygenation by RP- and SP-HPLC methods showed that in a purified enzyme preparation from the rat lung cytosol the specific form of lipoxygenase (LOX) is present exhibiting 12/15-LOX dual specificity (with predominant 15-LOX activity). The inhibitory activity of curcumin and Curcuma longa extract on LOX from cytosolic fraction of rat lung was expressed in the percentage of inhibition and as IC50. Lineweaver-Burk plot analysis has indicated that curcumin is the competitive inhibitor of 12/15 LOX from the rat lung cytosolic fraction.

[Effect of abiotic elicitation on the sanguinarine production and polyphenol oxidase activity in the suspension culture of Eschscholtzia californica CHAM]. / Vplyv abiotickej elicitácie na produkciu sanguinarínu a aktivitu polyfenoloxidázy v suspenznej kultúre Eschscholtzia californica CHAM.

Elicitation of plant in vitro cultures represents a biotechnological tool to improve the production of secondary metabolites. In this study, the effect of AgNO3 and CdCl2 on the sanguinarine production by the suspension culture of Eschscholtzia californica CHAM. was investigated. Elicitors were added to the cultures at the 14th day of subcultivation and their effect on the sanguinarine production was evaluated after a 48 h exposure. AgNO3 at the concentration of 0.075 mmol.l-1 and CdCl2 at the concentration of 4 mmol.l-1 induced a ca. 5.2- and 5.6-multiple increase in sanguinarine synthesis, respectively. This amount represents probably the maximal production, because a further increase in the elicitors concentrations did not increase sanguinarine production. Both abiotic elicitors induced a polyphenol oxidase specific activity increase. Polyphenol oxidase is probably involved in the biosynthesis of sanguinarine at the level of dopamine formation. Dopamine is a precursor of (S)-norcoclaurine, the first intermediate with the benzylisoquinoline structure.

[Comparison of sanguinarine production in suspension cultures of the Papaveraceae plants]. / Porovnanie produkcie sanguinarínu suspenznými kultúrami rastlín celade Papaveraceae.

Intact plants of the Papaveraceae family are producers of a whole range of benzylisoquinoline alkaloids, which are used in pharmaceutical industry. In vitro cultures derived from plants of the Papaveraceae do not have the ability to produce such a broad spectrum of alkaloids, only the biosynthetic pathway leading to sanguinarine is active. This study deals with the preparation of in vitro cultures of Papaver somniferum, Eschscholtzia californica, Chelidonium majus and Macleaya cordata. Their sanguinarine production abilities were tested and compared. The lowest amounts of sanguinarine from all cultures tested were accumulated in suspension cultures of the opium poppy (0.45-0.55 µg in 1 g of fresh weight). Eschscholtzia californica, Chelidonium majus and Macleaya cordata cultures produced similar amounts of sanguinarine (18.0-22.7 µg; 20.5-26.3 µg; 15.4-20.3 µg in 1 g of fresh weight, resp.). The elicitation study used a biotic stressor, Botrytis cinerea hydrolysate. In all cultures treated, an increase in sanguinarine accumulation was observed. Of all cultures tested, the most intensive response was observed in the opium poppy cultures, although the amount of sanguinarine in the elicited poppy cultures was lower than in the non-elicited samples of the other cultures.

Synthesis of modified D-mannose core derivatives and their impact on GH38 α-mannosidases.

Nine new compounds having five- and modified six-member carbohydrate core derived from D-lyxose or D-mannose, and non-hydrolysable aglycones (benzylsulfonyl or aryl(alkyl)triazolyl) were synthesised to investigate their ability to inhibit the recombinant Drosophila melanogaster homologs of two human GH38 family enzymes: Golgi mannosidase II (dGMIIb) and lysosomal mannosidase (dLMII). Two compounds were weak selective dGMIIb inhibitors showing IC50 at mM level. Moreover, it was found that another GH38 enzyme, commercial jack bean α-mannosidase, was inhibited by triazole conjugates regardless of the carbohydrate core while the corresponding sulfones were inactive.

'Click chemistry' synthesis of 1-(α-D-mannopyranosyl)-1,2,3-triazoles for inhibition of α-mannosidases.

Three new triazole conjugates derived from d-mannose were synthesized and assayed in in vitro assays to investigate their ability to inhibit α-mannosidase enzymes from the glycoside hydrolase (GH) families 38 and 47. The triazole conjugates were more selective for a GH47 α-mannosidase (Aspergillus saitoi α1,2-mannosidase), showing inhibition at the micromolar level (IC50 values of 50-250 µM), and less potent towards GH38 mannosidases (IC50 values in the range of 0.5-6 mM towards jack bean α-mannosidase or Drosophila melanogaster lysosomal and Golgi α-mannosidases). The highest selectivity ratio [IC50(GH38)/IC50(GH47)] of 100 was exhibited by the phenyltriazole conjugate. To understand structure-activity properties of synthesized compounds, 3-D complexes of inhibitors with α-mannosidases were built using molecular docking calculations.

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures.

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 µg g⁻¹ dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 µg g⁻¹ dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.