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The antiinflammatory activity of five structurally related binaphthaquinones and plumbagin has been studied using a blood platelet aggregation assay. The results suggest that for maximum activity a free hydroxyl peri to the quinoid ring is important. Antiplatelet activity does not correlate with other previously determined biological activity, such as antitumour action, but is consistent with the theory of free radical generation as a mechanism for cytotoxicity and enzyme inhibition but not antiinflammatory activity. © 1998 John Wiley & Sons, Ltd.

The extraction of natural antifungal compounds from the peels of yam (Dioscorea alata) and the effect of these compounds on both the vegetative and reproductive structures of some yam not pathogens were studied. Four prominent antifungal components were obtained; one of the components was fully characterized and identified as beta-sitosterol. The antifungal activity of the compounds toward the germination of spores of two yam pathogens showed an inhibition of less than 57% at a concentration of 50 mg/L while inhibition on the elongation of germ-tubes of Fusarium moniliforme was as high as 82% at the same concentration. However, the ED50 for inhibition of germ-tube elongation in the yam compounds for the same organism was below 32 mg/L. The role of the yam compounds at high concentrations in disease resistance is discussed.

Anthranilate synthase (AS) and chorismate mutase (CM) were monitored throughout the growth cycle of cells from Ailanthus altissima . Two isoenzymes were found, both of which were inhibited by 20 mM tryptophan: AS-a (by 66%) and AS-b (by 97%). Because AS-a was not always present, the purification and properties relate to AS-b. For this isoenzyme the K m and V max for chorismate were 73 μ m and 93 pkat for l -glutamine were 50 μ m and 35 pkat and for MgCl 2 were 180 μ m and 45 pkat AS-b occurred as a dimer with M r for the monomers, by SDS-PAGE, of 60(±1.3) × 10 3 and 37(±1) × 10 3 .

The enzymology of chorismate mutase in plants and microorganisms is reviewed to include occurrence, gene-enzyme relationships, reaction mechanisms and details of active site studies.

Diosquinone and plumbagin isolated from the root of Diospyros mespiliformis (Hostch), a common ingredient in several folk medicines and foods, have been shown to have antibacterial activity against a wide range of organisms. The minimum inhibitory concentrations (MICs) of diosquinone against Staphylococcus aureus NCTC 6571 and S. aureus E3T ranged from 3 to 30μg/mL, while those against Escherichia coli KL16 and Pseudomonas aeruginosa NCTC 6750 ranged from 15 to 16μg/mL. MICs were found to increase with the concentration of cells used in the inoculum. Bacterial studies showed that S. aureus NCTC 6571 exhibited a paradoxical biphasic response to dioquinone in nutrient broth, whereas bacterial activity against E. coli KL16 increased with concentration up to the highest concentration of dioquinone tested. Activity against E. coli KL16 was more pronounced in phosphate-buffered saline than in nutrient broth. The other active compound isolated, the naphthoquinone plumbagin, gave MIC values between 400 and 600μg/mL.

Twenty-six tropane alkaloids were identified from the root-bark of Erythroxylum monogynum by GC and GC-MS and twenty of these alkaloids had not previously been reported as constituents of the root-bark. 3α-(3′,4′,5′-Trimethoxybenzoyloxy) tropane was the main base and in addition, three new alkaloids were identified as 3α-isobutyryloxynortropane, 3α-(4-methylvaleroyloxy)tropane and tentatively, 3β-phenylacetoxytropane.

Cell suspension cultures of Brucea javanica were grown in media with a variety of plant growth regulators. Although quassinoid production could not be initiated, high yields of canthin-6-one alkaloids were found. The plant growth regulator regimes used, IAA + KIN, 2,4-D + KIN, NAA + KIN, NAA + BAP, 2,4-D + BAP and IAA + BAP affected cell growth and overall alkaloid production. Variations in the yields of the individual alkaloids were also observed. The most efficient production of canthin-6-one, 11-hydroxycanthin-6-one, 11-methoxycanthin-6-one and 5-methoxycanthin-6-one was found in media containing NAA+ BAP.

An S -adenosyl- l -methionine (SAM) requiring methyltransferase responsible for the methylation of 1-hydroxycanthin-6-one has been isolated from a series of cell culture lines of Ailanthus altissima . This soluble enzyme is separable from various coumarin methyltransferases and has been shown to be specific for 1-hydroxycanthin-6-one. The enzyme was purified 73-fold and characterized; it had a pH optimum of 7.3, a temperature optimum of 35° and a M r of 63 000 ± 3%. The K m for 1 -hydroxycanthin-6-one was determined to be 22 μM and for SAM, 1 8 μM. S -Adenosyl- l -homocysteine afforded competitive inhibition with a K i of 5 μM.

The enzymology of anthranilate synthase (EC 5.4.99.6) in microorganisms and plants is reviewed. Aminoacid sequences of the enzyme subunits in different species are compared, and the mechanism of reaction is discussed.

The enzymology of chorismate mutase in plants and microorganisms is reviewed to include occurrence, gene-enzyme relationships, reaction mechanisms and details of active site studies.

The uptake and sequestration of alkaloids by latex vacuoles of P. somniferum sedimenting at 900 x g and 1100 x g were studied; both populations take up morphine effectively. Morphine uptake by the 1100 x g vacuoles was stimulated by exogenous ATP and, after uptake of large amounts of alkaloid, both populations were stimulated by exogenous ATP to take up further morphine; this effect was no longer present in 900 x g vacuoles isolated from the latex of more mature capsules. Uptake by 1100 x g vacuoles was more sensitive to temperature than that of 900 x g vacuoles. Determination of the proton gradient across the vacuolar membrane (△pH), controlled dissipation of △pH through the use of NH4Cl, and correlation with morphine uptake demonstrated that morphine uptake was strongly dependant on the maintenance of △pH. Inhibitors designed to produce loss of protons from the vacuole had, however, little effect on the system. Nitrate, as an inhibitor of tonoplast ATPase, had an inhibitory effect on morphine uptake by the 1100 x g vacuoles. Uptake of meconate, [35S]sulphate and L-[U14C]malate, important acid constituents of the 900 x g vacuoles was investigated. Uptake of sulphate and malate was continuous and saturation was not reached; the rate of uptake of sulphate and malate was much lower than that of morphine and other alkaloids, and uptake of meconate by these vacuoles was not detected. Studies on specificity of alkaloid uptake with the 900 x g vacuoles indicated no absolute preference for either the ( + )- or the (-)-isomer of codeine. Noscapine uptake was stimulated by ATP under conditions where ATP had no effect on morphine uptake. Nicotine was not taken up, but low levels of caffeine and l-m ethoxycanthin-6-one were taken up, but less effectively than morphine or noscapine. Uptake of alkaloids by P. somniferum latex vacuoles is dependant on the maintenance of tonoplast △pH and an ATPase generates this △pH. Sequestration appears to involve protonation and anion-cation stabilization involving meconate and sulphate. The specificity of alkaloid uptake suggested no clear correlation with pK or lipophilicity, and some sort of channel mechanism, more related to alkaloid shape is suggested

Papaver somniferum latex vacuoles were separated into two discrete fractions–-the 900 Xg and the 1100 Xg vacuoles. The two vacuolar populations were assayed over the three-week period of capsule maturation. Estimations of the anions sulphate, meconate and chloride, as well as the inorganic cations K+,Na+, Mg2+ and Ca2+, were made, together with measurements of the alkaloids morphine, codeine, thebaine, papaverine and noscapine. A correlation between alkaloids and organic acids has been established which supports the proposed “ion trap” mechanism proposed for alkaloid sequestration in poppy latex vacuoles.

The blood-red sap of Croton lechleri was found to contain proanthocyanidins as major constituents which accounted for up to 90% of the dried weight. In addition to (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin and dimeric procyanidins B-1 and B-4, five novel dimers and trimers were isolated and characterized as catechin-(4α→8)- epigallocatechin, gallocatechin-(4α→8)-epicatechin, gallocatechin-(4α→6)-epigallocatechin, catechin-(4α→8)- gallocatechin-(4α→8)-gallocatechin and gallocatechin-(4α→8)-gallocatechin-(4α→8)-epigallocatechin. Higher oligomers were also obtained. A new procedure combining chemical degradation with 1H NMR spectroscopy has been developed for determination of the composition and molecular size of oligomeric/polymeric proanthocyanidins. The oligomers of the sap were shown to have the mean degree of polymerization of 4,5–6 and 6–7, respectively, and Mr up to 2130. The heterogeneity of the oligomers was clearly indicated by the presence of a variety of flavan-3-ols as extension and terminal units. An exceptionally high content of gallocatechin and epigallocatechin in the oligomers was observed.

The ethyl acetate fraction obtained from the extraction of Artemisia annua yielded 11 flavones, four flavone glycosides and two chromene derivatives. Three new compounds were isolated: quercetagetin 4′-methyl ether, 2,2-dihydroxy-6-methoxychromene and 2,2,6-trihydroxychromene.

Canthin-6-one, 11-hydroxycanthin-6-one, 11-methoxycanthin-6-one, 5-methoxycanthin-6-one, 4-hydroxy-5-methoxycanthin-6-one and canthin-6-one-3 N -oxide have been isolated from cell suspension cultures of Brucea javanica . The total yield of alkaloid produced in the cells and the medium is in excess of 2.0 mg/g dry weight of cells.

The biogenesis of alkaloids has been the cause of much speculation and since the beginning of the century there has been a great effort directed at the elucidation of some of the pathways. The advent of isotopically labeled compounds in the 1950s heralded the beginning of research that has given an insight into many aspects of alkaloid biosynthesis and metabolism.

The formation and sequestration of secondary metabolites is often regulated in space and time and correlated with differentiation (Wiermann, 1981). The importance of this correlation has been encountered in undifferentiated plant cell cultures, which either do not produce an alkaloid that is typically present in the intact plant or they accumulate the compounds in very low yields. The comparably few systems that produce high amounts of alkaloids are unfortunately rather the exception (Chapter 7). On the other hand, differentiated organ cultures, such as root or shoot cultures, are very reliable production systems, indicating that it is not the in vivo condition that is responsible for low yields in cell suspension cultures, but rather the degree of differentiation, i.e., differential gene expression of the enzymes involved in alkaloid biosynthesis and alkaloid storage.

The enzymology of anthranilate synthase (EC 5.4.99.6) in microorganisms and plants is reviewed. Aminoacid sequences of the enzyme subunits in different species are compared, and the mechanism of reaction is discussed.

Enzymes for the methylation of 1hydroxycanthin-6-one and a series of coumarins have been isolated from Ailanthus altissima cell suspension cultures. The coumarin methyltransferases methylate aesculetin to scopoletin and isoscopoletin, but not scopoletin, to scoparone. Fraxetin was methylated to isofraxidine but not to fraxidine and only fraxidine was methylated to 6,7,8-trimethoxycoumarin. These enzymes were studied throughout the culture growth cycle with two cell lines: 1, which produced 1-methoxycanthin-6-one as the major alkaloid and 2, in which canthin-6-one was the major alkaloid.

Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have IC50 values at 2.4 - 6.5 × 10(-5)M against P. falciparum in vitro compared with an IC50 value of about 3 × 10(-8)M for purified artimisinin. At concentrations of 5 × 10(-6)M these flavonoids were not active against P. falciparum but did have a marked and selective potentiating effect on the antiplasmodial activity of artemisinin.

From the in vitro hairy root cultures of a Datura candida hybrid, 19 tropane alkaloids have been identified using capillary gas-liquid chromatography and mass spectrometry. As in the parent plants, scopolamine is the major alkaloid. Two hitherto undescribed alkaloids have been detected and their structure tentatively characterised on the basis of their mass spectral fragmentations.

Aerial parts of Ageratina deltoidea yielded 13 methoxylated flavonoids which were identified using UV, MS and 1 H NMR, and with reference to data given in the literature.

The effect of beta-sitosterol on spore germination and prevention of elongation of germ-tube of Aspergillus niger and Botryodiplodia theobromae was studied. The antifungal activity of the compound showed a percentage inhibition, of the fungal spore germination, of about 40% at a concentration of 50 micrograms/ml while inhibition on the elongation of germ-tubes was as high as 65% at the same concentration. The ED50 for inhibition of germ-tube elongation in A. niger was about 31 micrograms/ml. The role of the compound in disease resistance is discussed.

The uptake of L-[methylene-(14)C]-tryptophan from culture medium into root organs of Cinchona ledgeriana and the subsequent incorporation of the radiolabel into quinine and quinidine is reported. In addition, feeding unlabelled L-tryptophan at levels of 500mg/l to the cultures results in a 5-fold increase in the yields of both quinoline alkaloids.

Studies with [methyl- 14 C]- l -methionine have established that the methyl carbon of l -methionine can act as a precursor of the N -methyl group of methyl coniine in Conium maculatum .

This article focusses on the importance of the attitude of the adult(s) in terms of early language development at the pre-school stage. The adult's ability to create a reciprocal, on-going relation ship between himself and a child, a bond of trust based on a sense of mutuality through the spoken word, conveys to the child a sense of what positive human relationships are about. He in turn makes his unique contribution to the relationship as in the child's confident question to a visitor to the Nursery School: "Whose are yours?".

Phaseolus lunatus seedlings treated with aqueous cuprous chloride produced 25 isoflavonoids which were isolated and characterized by UV, mass and 1H NMR spectroscopy as kievitone, isoferreirin, 5-deoxykievitone, kievitone hydrate, cyclokievitone, cyclokievitone hydrate, daidzein, 2′-hydroxydaidzein, genistein, 2′-hydroxygenistein, 2,3-dehydrokievitone, luteone, phaseollidin, 2-(γ,γ-dimethylallyl)-phaseollidin, coumestrol, psoralidin, 7,8,2′,4′-tetrahydroxyisoflavone, 5,7,8,2′,4′-penta hydroxyisoflavone, 2,3-dehydrokievitol, lunatone, 5-deoxykievitol, kievitol, 3′-(γ,γ-dimethylallyl)-kievitone, 4-(γ,γ-dimethylallyl)-phaseollidin and 2-(γ,γ)-dimethylallyl)-6a-hydroxy-phaseollidin. The last nine compounds have novel structures. Possible biogenetic routes for these isoflavonoids and the taxonomic implications of their occurrence are discussed.

In continuing studies on the metabolic activity of Papaver somniferum , latex has been examined for its enzyme and alkaloidal metabolite content. After an initial centrifugation of latex at 1000 g , the pellet which contained a heterogeneous population of dense organelles was further resolved on sucrose gradients. Of the enzymes monitored, acid phosphatase and l -3,4-dihydroxyphenylalanine decarboxylase were found to be in the latex 1000 g supernatant, whereas catecholase (polyphenolase) was localized in two distinct organelles within the 1000 g sediment. The lighter organelles, sedimenting at 30% sucrose, contained a soluble enzyme which was readily released on organelle plasmolysis, whereas the catecholase found within the heavier organelles, sedimenting at 55–60% sucrose, was membrane bound and showed significant activity only in the presence of Triton X-100. These latter organelles also contained the alkaloids, including morphine and thebaine, and were observed to readily accumulate [ 14 CH 3 ]morphine. The alkaloid precursor, dopamine, was localized in the same dense vesicle fraction as the alkaloids. The rate of uptake of [7- 14 C]dopamine into these fractions at room temperature, however, was markedly lower than that of morphine. Electron microscopic examination of the organelles of various densities revealed that they possessed different morphology. The results are consistent with the concept that both the 1000 g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a sub-population of dense organelles found in the 1000 g sediment have at least a function as a storage compartment for both alkaloids and their catecholamine precursor.

L-Alanine:5-ketooctanal aminotransferase has been isolated from leaves of Conium maculatum and purified 369-fold. The enzyme MW 56 200 catalyse

γ-Coniceine reductase, isolated from leaves and fruits of a number of Conium maculatum cultivars, was NADPH dependent. The hydride ion was transf

The 1000 x g organelles of Papaver somniferum latex have been shown to accumulate rapidly, large quantities of alkaloids (0.05- 1 mg/mg organelle protein), particularly morphine, thebaine, codeine and papaverine and have been found to exhibit a surprising degree of specificity for the accumulation of these alkaloids. The uptake of alkaloid is independent of temperature and does not show a requirement for ATP 10-3 ᴍ, Mg2+ 10-3 ᴍ, or KCl 10-3 ᴍ. It is unaffected by known ATPase inhibitors (chlormadinone acetate 2.5 × 10-5 ᴍ, N,N-dicyclohexylcarbo- diimide 5 × 10-4 ᴍ and sodium vanadate 10-4 ᴍ) but showed considerable increase in morphine accumulation in the presence of 4-chloromercumbenzoate 5 x 10-4 ᴍ, while N-methylmaleimide 5 × 10-4 ᴍ had no effect, and carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone 5 × 10-4 ᴍ caused a reduction (ca. 30%) of morphine uptake. An acid medium, pH 4.5-5.5, also results in decreased morphine uptake.

Phytochemical studies on aerial parts of Kalanchoe gracilis resulted in the isolation of nine flavonoids and one coumarin. Among the nine flavonoids, four new flavonoids were identified as eupafolin-rhamnosides. The five known compounds were determined as luteolin, quercetin, quercitrin, haempferol, and eupafolin, based on spectroscopic analysis

A series of isoflavones, isoflavanones, pterocarpans, isofiavans and the coumestan aureol, obtained from five Phaseolus species, have been tested for fungitoxicity against Aspergillus niger and Cladosporium cucumerinum and a study made of the relationship between their structure and fungitoxicity. A high level of lipophilicity and the presence of at least one phenolic function appear important to iso-flavonoid fungitoxicity. The yield and range of structurally different isoflavonoids elicited in each of the five species in response to GuCl 2 is discussed in relation to plant/fungus interactions.

L -Alanine:5-keto-octanal aminotransferase has been separated from GOT and GPT activities by DEAE-cellulose chromatography. Two transaminase peaks A and B were observed for γ-coniceine formation and assay showed that these peaks also contained the amino acid:aldehyde aminotransferase (AAT) responsible for aliphatic amine biosynthesis. Kinetic studies showed that γ-coniceine formation with transaminase A had K m for L -alanine and 5-keto-octanal of 27 mM and 1.6 mM respectively. The V max was 1.3 nkats/mg protein and the activation energy was 3.0 kJ/mol. For transaminase B the Kinm for L -alanine and 5-keto-octanal are 55 mM and 0.14 mM respectively with a V max of 3.3 nkats/mg protein and an activation energy of 0.33 kJ/mol. Transaminase A and B had the same MW and have distinguishable pH max . γ-Coniceine formation by transaminase A was inhibited uncompetitively by glyoxalate and competitively by pyruvate, whereas with transaminase B uncompetitive inhibition was also observed with glyoxalate; no inhibition was observed with pyruvate which at low concentration (0.25 M) showed slight stimulation of activity.

Sixteen isoflavonoids have been isolated from the black gram. Phaseolus mungo L. (syn. Vigna mungo) following treatment with aqueous CuCl2. These include 12 known isoflavonoids: genistein. 2′-hydroxygenistein. 2′-hydroxydaidzein, kievitone, dalbergioidin, cyclokievitone. 5-deoxykievitone, 2′-hydroxydihydrodaidzein, isoferreirin, aureol, glycinol and demethylvestitol. In addition, kievitone hydrate, which was previously known only as a fungal metabolite of kievitone, was isolated together with 3 novel natural isoflavanones which were characterized as 4′-O-methylkievitone, cyclokievitone hydrate and 5-deoxykievitone hydrate.

After treatment with CuCl 2 , the following isoflavonoids have been isolated from the runner bean, Phaseolus coccineus : daidzein, genistein, isoprunetin, 2′-hydroxygenistein, phaseoluteone, 2′-hydroxydihydrodaidzein, isoferreirin, kievitone, cyclokievitone, glycinol, phaseollidin, phaseollin, demethylvestitol, phaseollinisoflavan, 2′-hydroxyisoprunetin and 7,4′-dihydroxy-5,2′-dimethoxyisoflavanone. The latter two compounds are novel natural products.

The presence of l -dopa decarboxylase has been demonstrated in poppy latex utilising l -dopa-1-[14C] and l -dopa-3-[14Cl] as substrates. The enzyme appeared to have maximum activity at pH 7.2 and showed both substrate and pyridoxal phosphate inhibition. The substrates l -tyrosine, l -phenylalanine and l -histidine were also decarboxylated. l -dopa decarboxylase was found to occur solely in the latex supernatant fraction. The possible involvement of this enzyme in alkaloid biosynthesis in the latex is discussed.