13C NMR spectra of some naturally occurring binaphthoquinones and related compounds

¹³C NMR chemical shift assignments have been shown to be diagnostic for the establishment of the dimeric linkage of some naturally occurring binaphthoquinones. The unsymmetric ¹³C and ¹H spin-spin coupled pattern observed in the ¹H coupled ¹³C NMR spectrum of plumbagin for C-6 has also been noticed earlier with the related compound juglone. The nature of these effects has been substantiated for the first time using benzene induced solvent shifts and D₂O exchange. ¹³C chemical shift assignments of plumbagin reported earlier for C-6 and C-8 have been revised.     

Monoclonal antibodies against naturally occurring bioactive compounds

The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 μg/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 μg/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO₄ solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of naturally occurring cyanogenic compounds

The literature dealing with the detection, isolation, purification and characterization of cyanogenic glycosides has been integrated with spectral and chemical data as well as other techniques from our laboratory to establish a method for the positive identification of glycosides of this type. The compounds are arranged into biosynthetically related groups (those derived from l-phenylalanine; l-tyrosine; l-leucine, l-valine; l-isoleucine; those with cyclopentene rings and pseudocyanogenic glycosides) and features of each of the above procedures are critically reviewed and spectral data for each group presented (IR, MS, UV and NMR). The NMR spectra of TMS ethers of cyanogenic glycosides have proven especially useful in chemical structure determination. This information is sufficient to permit identification of any of the 26 known glycosides as well as certain uncharacterized ones.     

Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Among naturally occurring products, sulfur-containing compounds (OSCs), especially garlic compounds (GCs) and isothiocyanates (ITCs), represent two important and promising chemopreventive families because of their potent chemopreventive effects in various in vivo and in vitro models. In recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. In the course of apoptosis induction by GCs and ITCs, multiple signal-transduction pathways and apoptosis intermediates are modulated. In particular, modulation of MAPKs and production of reactive oxygen species (ROS) seem to play pivotal roles in apoptosis induction by most GCs and ITCs. However, the role of P53 is still controversial. Based on present knowledge, GCs and ITCs may target not only the metabolism of carcinogens but also apoptosis signaling molecules. The effects of ITCs and GCs at multiple points of cancer development make these compounds highly promising candidates in cancer chemoprevention. However, the mechanisms of their anticancer effects are not fully understood, and further studies are required, especially to elucidate the role of cell-death receptors (the extrinsic pathway) and whether these agents induce apoptotic effects in non-tumor cells.     

Syntheses of naturally occurring terphenyls and related compounds

Naturally occurring terphenyls and related compounds such as terferol and its corresponding quinone and phlebiarubrone were synthesized from 2,5-diphenyl-1,4-benzoquinone. According to the proposed biosynthetic pathway, chemical conversion of phlebiarubrone to ustalic acid, a toxic compound isolated from the poisonous mushroom, Tricholoma ustale, was examined to find a low-yield conversion to the ustalic acid dimethyl ester.     

The behaviour of some naturally occurring strains of potato virus Y

Potato virus Y was obtained from field crops of potatoes in many strains which differed widely in virulence and caused diseases in the variety Majestic ranging from severe leaf-drop streak to mild mosaic. The symptoms caused by these strains in seven potato varieties and tobacco are described and compared with those caused by the serologically related potato virus C. No changes were noted in the behaviour of any of the strains over three years, during which they were transmitted to many different plants.
Potato virus C was not transmitted by Myzus persicae , the most efficient vector of other strains of virus Y. Nor was virus C transmitted by eleven other species of aphides, eight of which transmitted virus Y. The efficiency with which different species acted as vectors of virus Y varied greatly, and it is suggested that in some species only occasional individuals can transmit.
Possible mechanisms for the evolution of viruses C and Y are indicated, and the effects of changes in virus, vector, and host on the prevalence of insect-transmitted viruses are discussed.     

Cytotoxicity and superoxide anion generation by some naturally occurring quinones.

Four naturally occurring quinones, mansonone-D (MD), mansonone-H (MH), thespone (TP) and thespesone (TPE), extracted from the heartwood of Thespesia populnea have been tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma (MCF-7) cells. Toxicity of the quinones follows the order MD > TP > MH approximately TPE. EPR spectrometric and Clark electrode oximetric studies indicate that redox cycling of these quinones produce superoxide anion radical (O2*-) and H2O2 on aerobic incubation with NADH:cytochrome c reductase. Generation of superoxide radical during enzymatic reduction of quinones, was confirmed by EPR spin trapping experiment using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap. Cyclic voltammetric studies show reversible redox couples for MD and TP whereas TPE and MH show irreversible redox couple. The electrochemical results indicate that MH and TPE are more difficult to reduce than TP and MD.     

Determination of formaldehyde by the Hantzsch reaction: interference by naturally occurring compounds

The quantitation of formaldehyde, a product of many drug oxidations, can be determined by a simple and sensitive assay which employs the Hantzsch reaction. The present study demonstrates that both oxaloacetate and acetoacetate in the presence of Mg²⁺ interfere with the Hantzsch reaction by interacting with formaldehyde, making the aldehyde unavailable for the condensation reaction with acetylacetone. This can lead to errors when trying to quantitate either the disappearance or the formation of formaldehyde in metabolic studies utilizing tissue slices, homogenates, or subcellular fractions containing mitochondria.     

High-performance liquid chromatographic analysis of fucoganglioside hydrolysis by alpha-L-fucosidase

A sensitive HPLC method has been developed for monitoring fucoganglioside hydrolysis by purified alpha-L-fucosidase. The high-resolution method employs a Lichrosorb-NH2 column, a 10-min isocratic elution with potassium phosphate/acetonitrile buffer, detection of ganglioside products with a uv monitor at 195 nm, and quantification of low picomolar amounts of these gangliosides with an integrator. The usefulness of the HPLC method has been exemplified by using it to demonstrate the hydrolysis of gangliosides fucosyl-GM1 and fucosyl-GD1b by purified human liver alpha-L-fucosidase in the absence of activator proteins and/or detergents.     

Thermodynamic analysis of purified human placental alpha-L-fucosidase

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pK_m were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme.     

Total synthesis of naturally occurring moracinflavan E and related compounds

A total synthesis of four flavans with a furan ring in different positions (1–4) was achieved successfully. The flavan was synthesized from flavanone by reduction and followed by deoxygenation steps. The synthesized compound 2 was confirmed as the naturally occurring moracinflavan E based on the comparison of their NMR spectral data.