Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.     

Puerariafuran, a new inhibitor of advanced glycation end products (AGEs) isolated from the roots of Pueraria lobata

A new 2-arylbenzofuran, puerariafuran (1), as well as three known compounds, coumestrol (2), daidzein (3), and genistein (4), were isolated from a MeOH extract of the roots of Pueraria lobata as active constituents, using an in vitro bioassay based on the inhibition of advanced glycation end products (AGEs) to monitor chromatographic fractionation. The structure of 1 was determined by spectroscopic data interpretation, particularly by extensive 1D and 2D NMR studies. All the isolates (1-4) were evaluated for the inhibitory activity on AGEs formation in vitro.     

Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.     

Isolation and identification of two new flavanones and a chalcone from Citrus kinokuni

Two new flavanones and one chalcone were isolated from the peel of Citrus kinokuni Hort. ex Tanaka and identified as (2S)-5,6,7,8,4'-pentamethoxyflavanone (1), (2S)-5,6,7,3',4'-pentamethoxyflavanone (2) and 2'-hydroxy-3,4,3',4',6'-pentamethoxychalcone (3). The structures of new compounds were elucidated by spectroscopic analysis.     

A quantum mechanics study on the reaction mechanism of chalcone formation from p-coumaroyl-CoA and malonyl-CoA catalyzed by chalcone synthase

Chalcone synthase catalyzes formation of phenylpropanoid chalcone from one p-coumaroyl-coenzyme A (CoA) and three malonyl-CoA molecules. In order to elucidate structural and energetic features of the reaction mechanism, we performed the quantum mechanics calculations and obtained the following results. In loading step, only a tetrahedral intermediate is located without transition state (TS). Our results indicate that His303 acts as a H₃₁ donor, but not a hydrogen bond donor, to stabilize the intermediate formation. In decarboxylation step, the reaction proceeds via a TS and is sensitive to the environment. In elongation step, a tetrahedral TS is located. All of the results above support the reaction mechanism and further complement the proposal of Noel JP et al.     

Balanochalcone,a new chalcone from Balanophora laxiflora Hemsl.

A new chalcone named as balanochalcone (1) together with eight known compounds, methyl caffeate (2), β-hydroxydihydrochalcone (3), methyl gallate (4), dimethyl-6,9,10-trihydroxybenzo[kl]xanthene-1,2-dicarboxylate (5), p-coumaric acid (6), quercetin (7), scopoletin (8) and pinoresinol (9) have been isolated from the ethyl acetate extract of Vietnamese Balanophora laxiflora Hemsl. Their structures were characterised by IR, UV, HR-ESI-MS, 1D and 2D NMR and CD spectroscopies. Compounds 2 and 5 showed moderate cytotoxicity against four cancer cell lines, KB (a human epidermal carcinoma), MCF7 (human breast carcinoma), SK-LU-1 (human lung carcinoma) and HepG2 (hepatocellular carcinoma). In addition, compounds 1 and 5 showed moderate antioxidant activity.     

Study on the bioavailability of puerarin from Pueraria lobata isoflavone self-microemulsifying drug-delivery systems and tablets in rabbits by liquid chromatography-mass spectrometry

To evaluate the bioavailability of puerarin from Pueraria lobata isoflavone self-microemulsifying drug delivery systems (SMEDDS) and Yufengningxin tablets, a rapid and specific liquid chromatography--mass spectrometric method was developed and validated to determine puerarin in rabbit serum. The analyte was extracted from serum samples by precipitating the serum proteins, separated on a Diamonsil C(18) column and detected by mass spectrometry with an electrospray ionization interface. 4-Hydroxybenzaldehyde was used as the internal standard. The method has a limit of quantitation of 10 ng/mL using 200 microL serum. The intra-day relative standard deviations (RSDs) ranged from 3.7 to 6.9% and inter-day RSDs were within 6.5%. After administration of SMEDDS and tablets to rabbits, a significant difference was observed in main pharmacokinetic parameters of t(max), C(max) and AUC(0--infinity) between SMEDDS and tablets, and a 2.2-fold increase in the relative bioavailability of puerarin was observed with the SMEDDS compared with Yufengningxin tablets. It was concluded that the absorption of puerarin from Pueraria lobata isoflavone SMEDDS was enhanced.     

Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase

A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.     

Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection was developed for the determination of puerarin, daidzein and rutin. Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The working electrode was a 300-microm diameter carbon disc electrode positioned opposite the outlet of capillary. The three analytes could be well separated within 12 min in a 40 cm length capillary at a separation voltage of 9 kV in a 50 mmol/l borate buffer (pH 9.0). The relationship between peak currents and analyte concentrations was linear over about three orders of magnitude with detection limits (SIN=3) ranging from 0.241 x 10(-6) to 0.511 x 10(-6) mol/l for all compounds. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied for the determination of puerarin, daidzein and rutin in Chinese traditional drugs, the vines of Pueraria lobata (Wild.) Ohwi and Puerariae Radix.     

Enzymatic formation of unnatural novel polyketides from alternate starter and nonphysiological extension substrate by chalcone synthase

[reaction: see text] In the chalcone synthase (CHS) enzyme reaction, both the starter molecule and the extension unit of the polyketide chain elongation reaction were simultaneously replaced with nonphysiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from Scutellaria baicalensis afforded an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.     

Total synthesis of munchiwarin, a triprenylated chalcone from Crotalaria medicagenia

An efficient method is developed for the synthesis of the modified triprenylated chalcone, munchiwarin (1), isolated from the roots of Crotalaria medicagenia. The synthesis of 1 utilizes a Claisen-Schmidt condensation between 2,4-dihydroxy-3,5-C-diprenyl acetophenone and 4-methoxy benzaldehyde in the presence of Ba(OH)2 to yield the unusual chalcone 5 that contains a nine-membered ether ring. Further prenylation of 5 with 1-bromo-3-methylbut-2-ene and its subsequent demethylation with BBr3 gave munchiwarin (1).     

Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2'-diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.     

Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis

Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and cycloartenol synthases, respectively. These results indicated the presence of distinct OSCs for cycloartenol and cucurbitadienol synthesis in this plant.     

Antimitotic rhizoxin derivatives from a cultured bacterial endosymbiont of the rice pathogenic fungus Rhizopus microsporus

The potent antimitotic polyketide macrolide rhizoxin, the causal agent of rice seedling blight, is not produced by the fungus Rhizopus microsporus, as has been believed for over two decades, but by endosymbiotic bacteria that reside within the fungal mycelium. Here we report the successful isolation and large-scale fermentation of the bacterial endosymbiont ("Burkholderia rhizoxina") in pure culture, which resulted in a significantly elevated (10x higher) production of antimitotics. In addition to several known rhizoxin derivatives, numerous novel natural and semisynthetic variants were isolated, and their structures were fully elucidated. Cell-based assays as well as tubulin binding experiments revealed that methylated seco-rhizoxin derivatives are 1000-10000 times more active than rhizoxin and thus rank among the most potent antiproliferative agents known to date. Furthermore, more stable didesepoxy rhizoxin analogues were obtained by efficiently inhibiting a putative P-450 monooxygenase involved in macrolide tailoring.     

Isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.     

Corallistin A,a second example of a free porphyrin from a living organism. Isolation from the demosponge Corallistes sp. of the coral see and inhibition of abnormal cells

It is shown that the demosponge Corallistes sp. (Tetractinomorpha, Lithistida, Corallistidae) collected in the Coral Sea, contains corallistin A ( 1 ), the second example, of a free porphyrin from a living organism. The compound proved to be active against the Kb cell line. In contrast with the geoporphyrins which do not bear any O-atom corallistin A ( 1 ) carries two carboxylic groups.     

Isolation and characterization of a novel feather-degrading bacterial strain

Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Feather-degrading bacteria were isolated from a Brazilian poultry industrial waste. Among these isolates, a strain identified as kr2 was the best feather-degrading organism when grown on basal medium containing 10 g/L of native feather as a source of energy, carbon, and nitrogen. The isolate was characterized according to morphological characteristics and biochemical tests belonging to the Vibrionaceae family. Keratinolytic activity of this isolate was monitored throughout the cultivation of the bacterium on raw feather at different temperatures. The optimum temperature for growth was about 30°C, at which maximum enzyme and soluble protein production were achieved. The enzyme had a pH and temperature optima of 8.0 and 55°C, respectively.