Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

A rumen isolate, Coprococcus, sp. Pe₁5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K_m values of 3.0 × 10⁻⁵ M and 29.0 × 10⁻⁵ M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line,RAW 264.7, activated by lipopolysaccharide and interferon-γ

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC₅₀ of 10.7 μM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-γ for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.     

Comparative metabolic activity related to flavonoid synthesis in leaves and flowers of Chrysanthemum morifolium in response to K deficiency

K limitation could decrease the flavonoids content in many Chinese traditional herbs. These results may be caused by the influence of K deficiency on the synthesis pathway of flavonoids. In this paper, we aim to study the influence of K deficiency on secondary metabolites and activities of phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H) enzymes in the process of flavonoid synthesis in Chrysanthemum morifolium Ramat. The results show that K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium while the same effect was obtained on C4H and PAL. Total flavonoids content increased with the content of cinnamic acid and p-coumaric acid in the plant under K deficiency, respectively. The regression equations between content of flavonoids and cinnamic acid/ p-coumaric acid should be expressed in term of linear effects (R²?=?0.9809, P?<?0.001 for cinnamic acid and R²?=?0.9929, P?<?0.0001 for p-coumaric acid, respectively). But the effect of phenylalanine on flavonoids should be expressed in term of quadratic pattern (R²?=?0.9375, P?<?0.05). There were two kinds principal components from chorismate to coumaryl CoA synthesis process, principal 1 was the substrate (phenylalanine, cinnamic acid, p-coumaric acid and PAL) and principal 2 was the enzyme (4CL, C4H), the principal 1 was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.17% and 9.64%), however, the factor loading (correlation coefficient of measured targets and the corresponding principal component) in principal 1 was opposite under different K application.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Biosynthetic Pathway for Veratryl Alcohol in the Ligninolytic Fungus Phanerochaete chrysosporium

Veratryl alcohol (VA) is a secondary metabolite of white-rot fungi that produce the ligninolytic enzyme lignin peroxidase. VA stabilizes lignin peroxidase, promotes the ability of this enzyme to oxidize a variety of physiological substrates, and is accordingly thought to play a significant role in fungal ligninolysis. Pulse-labeling and isotope-trapping experiments have now clarified the pathway for VA biosynthesis in the white-rot basidiomycete Phanerochaete chrysosporium. The pulse-labeling data, obtained with ¹⁴C-labeled phenylalanine, cinnamic acid, benzoic acid, and benzaldehyde, showed that radiocarbon labeling followed a reproducible sequence: it peaked first in cinnamate, then in benzoate and benzaldehyde, and finally in VA. Phenylalanine, cinnamate, benzoate, and benzaldehyde were all efficient precursors of VA in vivo. The isotope-trapping experiments showed that exogenous, unlabeled benzoate and benzaldehyde were effective traps of phenylalanine-derived ¹⁴C. These results support a pathway in which VA biosynthesis proceeds as follows: phenylalanine → cinnamate → benzoate and/or benzaldehyde → VA.     

Hypericum maculatum CRANTZ subsp.Maculatum XH. perforatum L. (Hypericaceae): Corroboration of natural hybridization by secondary metabolite analysis

The presence of various flavonoids, naphthodianthrones and phloroglucinol derivatives was studied in the natural pentaploid hybridH. maculatum Crantz subsp.maculatum × H. perforatum L. The hybrid taxon was shown to have secondary metabolites in common with both putative parents thus confirming its parentage. Morphological and nomenclatural questions are briefly discussed.     

Studies on flavonoid metabolism. Biosynthesis of (+)-[14C]catechin by the plant Uncaria gambir Roxb

1. The formation of (+)-[¹⁴C]catechin has been demonstrated in Uncaria gambir after the administration of ¹⁴CO₂ and [1-¹⁴C]acetate. 2. By alkaline degradation to phloroglucinol and protocatechuic acid it has been shown that administration of ¹⁴CO₂ resulted in equal labelling of the A and B rings of catechin, whereas [1-¹⁴C]-acetate gave rise to labelling largely in the A ring. 3. Incorporation of ¹⁴C from both ¹⁴CO₂ and [1-¹⁴C]acetate into (+)-catechin was greater in young than in older leaves.     

A novel chemiluminescent method for determination of phloroglucinol

It was found that the inhibition and enhancement by phloroglucinol of the chemiluminescence from the luminol–K₃Fe(CN)₆ system were dependent on the pH of luminol solution and the concentration of phloroglucinol. In Na₂CO₃–NaHCO₃ buffer, phloroglucinol exhibited strong chemiluminescent enhancement at pH 9.4. On this basis, a flow injection method was developed for the determination of phloroglucinol. The method was simple, rapid, convenient and sensitive, with a detection limit of 2.0 × 10^?9 mol/L. It is effective for determining phloroglucinol in the range of 1.0 × 10^?5–5.0 × 10^?9 mol/L. The relative standard deviation is 1.3% within one day and 3.2% between days for the determination of 5.0 × 10^?7 mol/L phloroglucinol. The method has been successfully used to determine phloroglucinol in environmental water, with satisfactory results. Copyright © 2003 John Wiley & Sons, Ltd.     

Incorporation of [14C]cinnamate into hydrolase-resistant components of the primary cell wall of spinach

[¹⁴C]Cinnamate was taken up very rapidly by cultured spinach cells and completely incorporated into low-MW conjugates within 20 min. The ¹⁴C-labelled products were similar whether the [¹⁴C]cinnamate was supplied continuously over a period of hours via a peristaltic pump or instantaneously. Radioactivity was slowly recruited from the low-MW pool into aromatic components of the cell-wall fraction. Saponification of the radioactive wall fraction yielded, in addition to radioactive ferulate and p-coumarate, large amounts of ethyl acetate-soluble radioactive material with the properties of oxidatively coupled phenols. The coupled material was associated with the most highly ‘Driselase’-resistant fractions of the cell wall. In contrast, ‘Driselase’ released most of the wall's ferulate and p-coumarate on disaccharide fragments. It is suggested that the oxidatively coupled phenols are formed from simpler phenols by peroxidase and that they cross-link the polysaccharides to which they are attached, making these polysaccharides relatively ‘Driselase’-resistant.     

Mevalonate decarboxylation in lemon grass leaves

The activity of mevalonate-5-pyrophosphate (MVAPP) decarboxylase was assayed in the extracts of green leaves of lemon grass. The enzyme was found to be exclusively cytosolic, had a pH optimum of 6.0 and had a specific requirement for ATP; Mg²⁺ was required and Mn²⁺ could replace it partially. The phenolic compounds, p-coumaric acid, protocatechuic acid, ferulic acid and phloroglucinol carboxylic acid inhibited the activity.     

Inhibition of plant phytases by phloroglucinol

Plant phytases (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) were inhibited by phloroglucinol (1,3,5-benzenetriol) in vitro. The inhibition of the Cucurbita maxima phytase was found to be non-competitive and pH dependent with an apparent inhibition constant (K_i) value of 2.3 × 10^?1 M at optimum pH (4.8) and temperature (50°). The apparent number of inhibitor molecules (n) bound per enzyme molecule was found to be 2.2 suggesting that the positive cooperativity phenomenon may be present.     

Acaricidal potential of active components derived from Alpinia galanga rhizome oils and their derivatives against Haemaphysalis longicornis (Acari: Ixodidae)

Acaricidal activities and acetylcholinesterase (AChE) inhibitory activities were evaluated of active constituents of the essential oil extracted from Alpinia galanga rhizomes cultivated from India and their derivatives against Haemaphysalis longicornis nymphs. In addition, the effect was investigated of active components of A. galanga oil on egg laying of adult females of H. longicornis and egg hatchability. Of the volatile components identified in A. galanga oil, ethyl cinnamate, ethyl methoxycinnamate, and methyl cinnamate at 0.32 mg/cm² resulted in 100% mortality, respectively, indicating that the acaricidal activity of the A. galanga oil against H. longicornis nymphs could be attributed to these compounds. To evaluate the structure–activity relationship between cinnamate derivatives and their acaricidal activities, allyl cinnamate, benzyl cinnamate, isopropyl cinnamate, isobutyl cinnamate, and isoamyl cinnamate were selected. Among cinnamate derivatives tested, allyl cinnamate exhibited the most potent toxicity (LC₅₀?=?0.055 mg/cm²) against H. longicornis nymphs. The allyl cinnamate was also tested for AChE activity in vivo in H. longicornis nymphs and was found to affect the AChE activity. Allyl cinnamate at 10–50 mg/mL inhibited egg laying of adult females of H. longicornis by 10–43%. Egg hatching was suppressed completely by treatment with allyl cinnamate at 50 mg/mL, whereas allyl cinnamate was minimally toxic against non-target earthworms, Eisenia fetida. These results suggest that allyl cinnamate can be used as an active ingredient for the development of eco-friendly tick acaricides against H. longicornis, a vector for Sever fever with thrombocytopenia syndrome (SFTS) virus.

     

Interaction of α-cyano[14C]cinnamate with the mitochondrial pyruvate translocator

The binding of α-cyanocinnamate to rat-heart mitochondrial membrane was investigated using α-cyano[¹⁴C]cinnamate. The binding was correlated to the inhibition of pyruvate transport. The results obtained demonstrate that both these functions reach saturation at the same titre of the inhibitor. Quantitative parameters of α-cyano[¹⁴C]cinnamate binding have been determined. The binding can be prevented by pyruvate and other substrates of the carrier but not by acetate. Pyruvate decreases the affinity of α-cyanocinnamate binding, leaving the maximum number of binding unchanged. It is concluded that rat-heart mitochondria contain a specific site at which α-cyanocinnamate binds which is directly involved in the inhibition of pyruvate transport.     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions

Inactivation of soybean urease in aqueous solution at pH 5.4, 36°C, and high-frequency sonication (2.64 MHz, 1.0 W/cm²) is substantially reduced in the presence of seven structurally different flavonoids. A comparative kinetic study of the effect of these flavonoids on the effective first-order rate constants that characterize the total (thermal and ultrasonic) inactivation k _i , thermal inactivation k*_i, and ultrasonic inactivation k _i (US) of 25 nM enzyme solution was carried out. The dependences of the three inactivation rate constants of the urease on the concentrations of flavonoids within the range from 10^?11 to 10^?4 M were obtained. The following order of the efficiency of the flavonoids used in respect of the urease protection from ultrasonic inactivation was found: astragalin > silybin > naringin > hesperidin > quercetin > kaempferol > morin. The results confirm a significant role in the inactivation of the urease of HO^. and HO ₂ ^. free radicals, which are formed in the ultrasonic cavitation field.     

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.     

The lack of movement of [14C]-phenylalanine and [14C]-cinnamate after administration to leaves of Polygonum and wheat

U-¹⁴C-phenylalanine and 3-¹⁴C-cinnamic acid were fed to detached Polygonum leaves through the cut petioles and to the bases of detached wheat leaves. After feeding, the leaves were divided into basal, middle and terminal segments; for each treatment of each plant more than 80% of the total radioactivity incorporated remained in the basal segment. The distribution of radioactivity between ethanol-soluble and insoluble fractions in each segment was examined. The basal segments contained more insoluble radioactivity than the terminal ones; the differences were far more marked for both plants when cinnamate rather than phenylalanine was administered. In view of the gross differences in distribution of radioactivity between the basal and terminal segments of each leaf, it is concluded that basal infusion of precursors is not the most suitable technique for studies of phenolic biosynthesis.     

Phloroglucinol inhibits the bioactivities of endothelial progenitor cells and suppresses tumor angiogenesis in LLC-tumor-bearing mice

Background

There is increasing evidence that phloroglucinol, a compound from Ecklonia cava, induces the apoptosis of cancer cells, eventually suppressing tumor angiogenesis.

Methodology/Principal Findings

This is the first report on phloroglucinol''s ability to potentially inhibit the functional bioactivities of endothelial progenitor cells (EPCs) and thereby attenuate tumor growth and angiogenesis in the Lewis lung carcinoma (LLC)-tumor-bearing mouse model. Although Phloroglucinol did not affect their cell toxicity, it specifically inhibited vascular endothelial growth factor (VEGF) dependent migration and capillary-like tube formation of EPCs. Our matrigel plug assay clearly indicated that orally injected phloroglucinol effectively disrupts VEGF-induced neovessel formation. Moreover, we demonstrated that when phloroglucinol is orally administered, it significantly inhibits tumor growth and angiogenesis as well as CD45⁻/CD34⁺ progenitor mobilization into peripheral blood in vivo in the LLC-tumor-bearing mouse model.

Conclusions/Significance

These results suggest a novel role for phloroglucinol: Phloroglucinol might be a modulator of circulating EPC bioactivities, eventually suppressing tumorigenesis. Therefore, phloroglucinol might be a candidate compound for biosafe drugs that target tumor angiogenesis.