Enzymic reduction of codeinone in vitro cell-free systems from Papaver somniferum and P. Bracteatum

Cell-free extracts have been prepared from Papaver somniferum which catalyze the reduction of codeinone-[16-³H] to codeine. The methodology for examining this conversion has pointed to conditions for exploring the preparation of suitable enzyme extracts. P. bracteatum also yielded a cell-free system which reduced codeinone to codeine, both of which are foreign to this species.     

The alkaloidal constituents of Papaver bracteatum arya II

Seven alkaloids were isolated from Papaver bracteatum Arya II, six of which: thebaine, 14β-hydroxycodeine, codeine, neopine, alpinigenine and protopine, have been previously found to be present in other types of this species. It is the first report of the isolation of O-methylflavinantine from P. bracteatum.     

A rapid quantitative method for the determination of thebaine in Papaver bracteatum

A rapid method enabling a quantitative analysis of thebaine in capsules and latex of Papaver bracteatum has been devised, based on a TLC technique. This method was compared to the commonly used GLC procedure, and highly significant correlation coefficient (r = 0.90) and linear regression were found between the two methods. Values for concentrations to the nearest ±0.25% of the standard spots can be reached by this simple and rapid thebaine determination.     

Biosynthesis of thalicarpine in Thalictrum minus

Feeding experiments with ¹⁴C-labelled reticuline, protosinomenine, orientaline, their N-nor-analogues and ³H-labelled isoboldine have shown reticuline and isoboldine to be the most efficient precursors of thalicarpine in Thalictrum minus. A biosynthetic pathway for thalicarpine with reticuline and isoboldine at the benzylisoquinoline and aporphine stages respectively has been suggested. Support for this proposal has been provided by the demonstration by radioisotopic dilution that reticuline and isoboldine are minor constituents of the plant.     

Peroxidases of Papaver somniferum

Extracts of Papaver somniferum that had peroxidase activity were ineffective in catalysing oxidation of reticuline. Two peroxidases were purified from young seedlings and their properties examined. Only one of them was active toward indole-3-acetic acid (IAA).     

Bound forms of alkaloids in Papaver somniferum and P. Bracteatum

The polysaccharide fraction of the pericarp and seed of Papaver somniferum were shown to contain bound forms of morphine which were derived from radioactive morphine fed to living plants. Bound forms of codeine, thebaine and some unidentified alkaloid-like compounds were also detected in the pericarp and bound thebaine occurred in the pericarp of Papaver bracteatum. The complexity and molecular weight of the bound alkaloids seemed to increase during ripening, and it is suggested that these substances represent transitional forms in the metabolism and transiocation of morphine from latex to seed.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

Biosynthesis of Camptotheca acuminata alkaloids

Tracer feeding experiments with Camptotheca acuminata plants show that [1′-¹⁴C]L-tryptophan, [Ar-³H₄]L-tryptophan, [Ar-³H₄,1′-¹⁴C]tryptophan, [1′-¹⁴C]-tryptamine, [2-¹⁴C]DL-mevalonate, and [2-¹⁴C]geraniol-[2-¹⁴C]nerol are incorporated into camptothecin. Direct stem injection of the labeled precursors into C. acuminata plants resulted in a substantial increase in the activity of isolated Camptotheca alkaloids as compared to root feeding of the same tracer.     

Morphine metabolism and normorphine in Papaver somniferum

Normorphine has been established as an active metabolite of morphine in P. somniferum. This was done by, (a) demonstrating the presence of normorphine throughout the life cycle of the plant, (b) finding normorphine-¹⁴C after feeding morphine-¹⁴C via the roots, and (c) exposing opium poppies to ¹⁴CO₂ under steady state conditions which led to morphine and normorphine of the same specific activity. Feeding normorphine-¹⁴C showed that the N-demethylation step is irreversible. A sensitive procedure was developed for the detection of normorphine in the presence of large amounts of morphine; using this procedure, normorphine was found in raw opium. These results indicate that the major, if not the sole, morphine degradative pathway involves an initial demethylation to normorphine, which is subsequently degraded to non-alkaloidal metabolites. The high rates of turnover observed led to the conclusion that the morphine alkaloids do play an active metabolic role, perhaps as specific methylating agents.     

Biosynthesis of tropane ester alkaloids in Datura

Datura meteloides plants were fed via the roots with [1″,2′-¹⁴C]tigloyl hygroline and as a control, [2′-¹⁴C]hygrine. After a week the alkaloids were isolated and degraded. Despite hydrolysis of the putative precursor it was possible, by label ratio, to show that esterification occurs after, and not before, the tropane ring has been synthesized. Hygroline is proposed as a possible intermediate.     

Alkaloids from Papaver fugax of Turkish origin

Thebaine, narcotine, rhoeadine and armepavine were identified in the aerial parts of Papaver fugax of Turkish origin.     

Enzymatic transformations of morphinane alkaloids

Horseradish peroxidase transforms morphinane alkaloids into N-oxides and morphine to pseudomorphine in the presence of hydrogen peroxide. The crude poppy enzyme fraction shows the same activities. The rates of reactions were influenced by phenolic compounds and their relation controlled by the concentration of hydrogen peroxide and the presence of ascorbic acid.     

Biotransformation of (RS)-reticuline and morphinan alkaloids by cell cultures of Papaver somniferum

(RS)-Reticuline was stereospecifically converted to (—)-(S)-scoulerine and (—)-(S)-cheilanthifoline by cell cultures of Papaver somniferum and (—)-(R)-reticuline was recovered as an optical pure compound by racemic resolution. (—)-Codeinone was converted in high yield to (—)-codeine in both cell culture and enzyme preparation, but the other morphinans, thebaine, codeine and morphine, were not metabolized.     

Synthesis of [3-14C]-2′-methylreticuline and its incorporation into alkaloid fractions of Papaver somniferum

[3-¹⁴C]-2′-Methylreticuline has been synthesized by standard methods. This modified opium alkaloid precursor is efficiently incorporated by aberrant biosynthesis into alkaloid fractions of Papaver somniferum, particularly into a highly purified codeine fraction.     

Biosynthesis of limonoids in Citrus

Analyses of citrus leaves and fruits, and radioisotope tracer work showed that the leaves could synthesize limonoids and that the trees could translocate limonoids from leaves to the fruit. These results suggest that limonoids in citrus fruit tissues are synthesized in leaves and translocated to the fruit. No evidence was found to support the presence of limonoid biosynthetic systems in the fruit tissues.     

Biosynthesis of radiolabelled alkaloids from 14C-tyrosine in Erythrina crista-galli

Alkaloid biosynthesis has been demonstrated in the young expanding tissues of Erythrina crista-galli shoots, the stem part of which elaborated, from [2-^14C]-tyrosine, alkaloid of specific radioactivity 2μuCi mmol^?1, 10-fold greater than previously reported.     

In vitro conversion of morphine to its N-oxide in Papaver somniferum latex

Radio active morphine (¹⁴C- and ³H-labelled) was fed in vitro to freshly collected samples of capsules and stem latex of Papaver somniferum and it was shown that some of it was converted to radioactive N-oxide. Although metabolic activity and variation between samples of latex collected at different times were much less marked than those previously found using in vivo methods, the results do confirm that the isolated latex is a metabolically viable tissue.     

Morphinane alkaloids in cultured tissues and redifferentiated organs of Papaver somniferum

The capacity of alkaloid synthesis was examined in cultured tissues of Papaver somniferum. Callus, derived meristemoids, redifferentiated roots and     

Radioimmunoassay for quantitative determination of morphine in capsules of Papaver somniferum

A radioimmunoassay (RIA) procedure for the determination of pmol quantitites of morphine in capsule samples of Papaver somniferum was developed. An antiserum developed against a conjugate of morphine-3-hemisuccinate-BSA was relatively specific for morphine and possessed moderated cross-reactivity with codeine and mild cross-reactivity with thebaine, but none with narceine, papaverine, or noscapine. The standard curve was linear over a range of 0.01–0.20 ng. This assay allows for the rapid, sensitive and precise determination of morphine in unpurified aqueous extracts of capsule samples. The amounts of morphine in the aqueous extracts determined by radioimmunoassay were validated by high performance liquid chromatography (HPLC). The two methods show a high correlation coefficient (r = 0.98) with no significant difference in determinations of morphine content by RIA and HPLC.     

Biosynthetic and metabolic activities of some organelles in Papaver somniferum latex

The biosynthesis of the five major alkaloids of the opium poppy (Papaver somniferum L. from radioactive dihydroxyphenylalanine has been studied in the 1000 g, 10 000 g, 100 000 g pellets and the 100 000 g supernatant fractions of the capsule latex. A normal poppy variety as well as one which produces only traces of alkaloids were used. Definite evidence of biosynthesis was obtained for both varieties but only in the 1 000 g pellet (as previously reported . None was found in the other fractions although electron microscopy showed that organelles, including vesicles, were present. The amounts of alkaloid biosynthesized however were very small relative to the amounts involved in the rapid changes already reported for the developing capsules. In contrast, all fractions of the latex were able to metabolize T-morphine in vitro, with the 100 000 g supernatant showing the highest activity and the amounts involved were also consistent with the changes found in the living plant.