Theo M. Malingré

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens.

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.

PURIFICATION AND PROPERTIES OF A PHENOL OXIDASE DERIVED FROM SUSPENSION-CULTURES OF MUCUNA-PRURIENS

From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90000±5000 dalton, and consisted of two subunits, each of 42000±1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5–6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono-and o-dihydroxysubstrates were determined.

Cyclodextrin-facilitated bioconversion of 17β-estradiol by a phenoloxidase from Mucuna pruriens cell cultures

After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased from almost insoluble to 660 microM. The bioconversion efficiency after 72 hr increased in the following order: freely suspended cells (0%), immobilized cells (1%), cell homogenate (6%), phenoloxidase preparation (40%). Mushroom tyrosinase converted 17 beta-estradiol, as a complex with beta-cyclodextrin, solely into 2-hydroxyestradiol, with a maximal yield of 30% after 6-8 hr. Uncomplexed 17 beta-estradiol was not converted at all in any of these systems.

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.

ALKALOIDS OF SOME EUROPEAN AND MACARONESIAN SEDOIDEAE AND SEMPERVIVOIDEAE (CRASSULACEAE)

Some 22 pyrrolidine and piperdine alkaloids were detected in the leafy parts of Sedum acre, S. aetnense, S. anglicum, S. brissemoreti, S. farinosum, S. fusiforme, S. lancerottense, S. melanantherum, and S. nudum. In addition to the alkaloids known from S. acre, 1-(2-pyrrolidyl)-propan-2-one and 2-monosubstituted piperidine alkaloids bearing butan-2-one, butan-2-ol, pentan-2-one and pentan-2-ol sidechains were identified. Phenylethylamine was isolated from the vegetative parts of S. album. In S. lydium, S. meyeri-johannis, and 16 species of S. series Rupestria, Aeonium, Greenovia, Jovibarba and Sempervivum no alkaloids could be detected. The results indicate a correlation between the presence of alkaloids and the major evolutionary trends in the European and Macaronesian Crassulaceae.

Improvement of the production of 5-methoxypodophyllotoxin using a new selected root culture of Linum flavum L.

A new, morphologically root-like cell line of Linum flavum was selected and used for the optimization of the production of 5-methoxypodophyllotoxin. The omittance of naphthaleneacetic acid from the medium resulted in further root formation, which was accompanied with a 2.6 fold increase of 5-methoxypodophyllotoxin levels. The feeding of the phenylpropanoids L-tyrosine and L-phenylalanine resulted in enhanced 5-methoxypodophyllotoxin contents. After the removal of vitamins together with the phytohormone from the medium, 5-methoxypodophyllotoxin levels were enhanced 6 times. The maximal content ever found using this production medium was 1.01% on a dry weight basis. The application of higher sucrose concentrations led to proportional biomass increase, cells grown on 6% sucrose resulted in the highest yield, 121.4 mg l-1 5-methoxypodophyllotoxin.

BIOCONVERSION OF PARA-SUBSTITUTED MONOPHENOLIC COMPOUNDS INTO CORRESPONDING CATECHOLS BY ALGINATE ENTRAPPED CELLS OF MUCUNA-PRURIENS

Alginate-entrapped cells ofM. pruriens were able to convert a number of parasubstituted monophenolic compounds into the corresponding catechols. All catechols produced were released into the medium, which offered the opportunity to isolate these products via a relatively simple procedure. Prepurification was performed on a Sephadex G10 gel and catechols were concentrated on Affigel 601. The identity of all products was confirmed with combined liquid chromatography/mass spectrometry (LC/MS) or MS using the desorption chemical ionization technique, depending on the catechol. For the entrapped cells and for a cell homogenate prepared of the same cell line ofM. pruriens the substrate specificities were qualitatively identical when judged on initial rates of synthesis calculated on protein basis.

Composition of the essential oil of Mentha aquatica

Abstract The overall composition of the essential oil of a botanically described Mentha aquatica (2 n = 96) has been determined. Limonene, caryophyllene and germacrene-D are the major hydrocarbons and bicyclogermacrene and viridiflorol, an oxygenated compound, are present in substantial amounts. The presence of small amounts of menthones, mentholes and menthyl acetates is indicated.

High Peroxidase Activity In Cell Cultures of Artemisia Annua With Minute Artemisinin Contents

Abstract In callus and cell suspension cultures of Artemisia annua minute amounts of artemisinin were found. In these cultures, a high peroxidase activity was measured, intracellularly and, especially, in the medium. In vitro, artemisinin rapidly decomposed when incubated with a cell homogenate of A. annua or with spent culture medium, as well as in a solution of commercially obtained horseradish peroxidase (EC 1.11.1.7). In contrast, the peroxidase activity in the intact plant, containing considerable amounts of artemisinin, was low. We suggest that the high peroxidase activity in A. annua cell cultures contributes to their very low artemisinin contents.

A chemotaxonomical study of someBoraginaceae: pyrrolizidine alkaloids and phenolic compounds

By means of thin layer chromatography pyrrolizidine alkaloids and phenolic compounds in some members of the familyBoraginaceae, subfamiliesHeliotropioideae andBoraginoideae, were studied. FromOmphalodes verna a main alkaloid was isolated with an isoretronecanol (or stereoisomeric) nucleus. The chemotaxonomical model ofTétényi forBoraginaceae based on fatty acids is generally supported, but relationships betweenHeliotropioideae andCynoglosseae appear to be closer, suggesting parallel developments from common ancesters.