Chemical constituents and in vitro antioxidant activity of Phyllanthus wightianus

The whole plant of Phyllanthus wightianus (PW) was investigated for the antioxidant effects of three successive extracts: hexane (PWHE), chloroform (PWCE) and methanol (PWME), using standard in vitro models. The PWME exhibited a strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radicals and nitric oxide radical inhibition activity, due to possessing the highest content of tannins. The free radical scavenging effect of PWME was comparable with that of reference antioxidants. The extracts were subjected to isolation of their compounds: isomeric sterol mixture (1) [stigmasterol (1a), compesterol (1b) and β-sitosterol (1c)], fredilin (2), lupeol (3), gallic acid (4), bergenin (5), geraniin (6), corilagin (7) and ellagic acid (8) were established through the use of column chromatographic methods and spectral data. The percentage of tannins was also determined and estimated using the HPLC method. The data suggest that tannins are the active antioxidant compounds of P. wightianus. This study provides proof for the ethnomedical claims and reported biological activities of this plant. The plant therefore has very good therapeutic potential.     

Effects of Pre-Harvest Supplemental UV-A Light on Growth and Quality of Chinese Kale

The effects of supplemental UV-A (385 nm) period and UV-A intensity for 5 days before harvest (DBH) on growth, antioxidants, antioxidant capacity, and glucosinolates contents in Chinese kale (Brassica oleracea var. alboglabra Bailey) were studied in plant factory. In the experiment of the UV-A period, three treatments were designed with 10 W·m⁻² UV-A supplement, T1(5 DBH), T2 (10 DBH), and no supplemental UV-A as control. In the experiment of UV-A intensity, four treatments were designed with 5 DBH, control (0 W·m⁻²), 5 w (5 W·m⁻²), 10 w (10 W·m⁻²), and 15 w (15 W·m⁻²). The growth light is as follows: 250 μmol·m⁻²·s⁻¹; red light: white light = 2:3; photoperiod: 12/12. The growth and quality of Chinese kale were improved by supplemental UV-A LED. The plant height, stem diameter, and biomass of Chinese kale were the highest in the 5 W·m⁻² treatment for 5 DBH. The contents of chlorophyll a, chlorophyll b, and total chlorophyll were only highly increased by 5 W·m⁻² UV-A for 5 DBH, while there was no significant difference in the content of carotenoid among all treatments. The contents of soluble sugar and free amino acid were higher only under 10 DBH treatments than in control. The contents of total phenolic and total antioxidant capacity were the highest in 5 W·m⁻² treatment for 5 DBH. There was a significant positive correlation between total phenolic content and DPPH and FRAP value. After 5 DBH treatments, the percentages and contents of total aliphatic glucosinolates, sinigrin (SIN), gluconapin (GNA), and glucobrassicanapin (GBN) were highly increased, while the percentages and contents of glucobrassicin (GBS), 4-methoxyglucobrassicin (4-MGBS), and Progoitrin (PRO) were significantly decreased, especially under 10 W·m⁻² treatment. Our results show that UV-A LED supplements could improve the growth and quality of Chinese kale, and 5 W·m⁻² UV-A LED with 5 DBH might be feasible for Chinese kale growth, and 10 W·m⁻² UV-A LED with 5 DBH was better for aliphatic glucosinolates accumulation in Chinese kale.     

Effects of Supplementary Blue and UV-A LED Lights on Morphology and Phytochemicals of Brassicaceae Baby-Leaves

Brassicaceae baby-leaves are good source of functional phytochemicals. To investigate how Chinese kale and pak-choi baby-leaves in response to different wavebands of blue (430 nm and 465 nm) and UV-A (380 nm and 400 nm) LED, the plant growth, glucosinolates, antioxidants, and minerals were determined. Both agronomy traits and phytochemical contents were significantly affected. Blue and UV-A light played a predominant role in increasing the plant biomass and morphology, as well as the contents of antioxidant compounds (vitamin C, vitamin E, phenolics, and individual flavonols), the antioxidant activity (DPPH and FRAP), and the total glucosinolates accumulation. In particular, four light wavebands significantly decreased the content of progoitrin, while 400 nm UV-A light and 430 nm blue light were efficient in elevating the contents of sinigrin and glucobrassicin in Chinese kale. Meanwhile, 400 nm UV-A light was able to increase the contents of glucoraphanin, sinigrin, and glucobrassicin in pak-choi. From the global view of heatmap, blue lights were more efficient in increasing the yield and phytochemical levels of two baby-leaves.     

Effects of UVR and UVR-induced cytokines on production of extracellular matrix proteins and proteases by dermal fibroblasts cultured in collagen gels%

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV-B or UV-A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free-floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV-B (2.5-50 mJ/cm2) nor UV-A (2-12 J/cm2) altered synthesis of ECM proteins, but UV-A increased MMP-1 and -3 production. Tropoelastin synthesis increased in response to transforming growth factor-beta1 (5 ng/mL) treatment. Both interleukin-1beta and tumor necrosis factor-alpha (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP-1, -3 and -9 synthesis markedly. Collagen synthesis was not modulated by UV-B, UV-A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV-B and UV-A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.     

Effects of freeze-drying of samples on metabolite levels in metabolome analyses

Freeze-drying (FD) is a useful technique for removing water from biological tissues, such as food samples. Cellular components freeze at once, and the ice sublimates under conditions of high vacuum and low temperatures. Because biological activity is restricted during FD, the degradation of cellular metabolites is often believed to be limited. However, the cellular structure is damaged by several factors, such as the increase in cell volume during freezing, and this has serious effects on the levels of some cellular metabolites. We studied these effects of FD on metabolite levels when using it as a sample preparation step in metabolome analysis. We observed significant decreases in the levels of some metabolites, such as succinate and choline, in Arabidopsis and pear, respectively. We also found that the effects of FD on certain metabolite levels differed between Arabidopsis plants and pear fruits. These results suggest that it is necessary to confirm the metabolite recovery in each sample species when FD is used for sample preparation.     

Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimate metabolite production, and PKS genes

Lichens are composite and symbiotic organisms. Biologically, they often have been interpreted as one organism (fungi and algae associated within a common thallus), but taxonomically as a life form of ascomycetous fungi; as the lichen-forming fungus or "mycobiont" has been, in most cases, classified as the dominant symbiotic partner. About 46% of the ascomycota are lichen-forming, however, about 2-3% of the lichen fungi are basidiomycota. Lichen-forming fungi produce a great variety of secondary metabolites, biosynthetically derived from the acetyl polymalonyl, mevalonic and shikimate pathways. Thus, secondary metabolites comprise a significant proportion of the lichen thallus dry weight (0.1-5% or even more). The majority of secondary lichen products are aromatic polyketides, and a number of them has been shown to exhibit marked biological activity.     

Effect of the medium pH on the release of secondary metabolites from roots ofDatura stramonium,Catharanthus roseus,andTagetes patula cultured in vitro

The release of alkaloids from root culturesDatura stramonium andCatharanthus roseus and thiophenes from root cultures ofTagetes patula was found to increase when the pH of the culture media (ranging from 4.8 to 7.0) was reduced to 3.5. The extent of the effect was different in each type of culture. Increases ranged from 4- to 20-fold, which in some cases accounted for 75% of the total secondary metabolite pool produced per flask. When the release of individual metabolites was measured, even larger increases, were observed (nearly 400-fold for ajmalicine). Increased release of alkaloids fromC. roseus roots were also observed in cultures growing in a 14-L fermentor, when the medium pH was reduced. Reduction of the pH of the media did not affect growth of the root cultures in subsequent subcultures. The importance of this treatment as a stategy to improve the recovery of secondary metabolites from producing cultures is discussed.     

Effects of water,methanol, and ethanol on the production of starch-g-polystyrene copolymers by cobalt-60 irradiation

Starch-g-polystyrene copolymers were made by simultaneous ⁶⁰Co irradiation of mixtures of wheat starch and styrene at room temperature. The extent of conversion of monomer to polymer is increased drastically with increasing water content up to a level of about 26 wt % on starch. Methanol had approximately the same effect as water at equivalent concentrations but ethanol was clearly less effective as a promoter of homo- and graft copolymerizations. Drying the starch reduced the conversion to polymer with all promoters but caused the greatest deterioration in the ability of ethanol to promote polymerization. The effects of physically swelling the starch by freeze drying the gelatinized material were also studied, as were preirradiation graft copolymerizations. The effects of promoters on graft polymerization parallel their abilities to scavenge stable radicals in irradiated starch. Water and methanol are better radical scavengers and polymerization promoters because they are better able to disrupt hydrogen bonds and permeate the starch structure. Radiolysis of the promoter is important in the grafting reaction.     

The production and characterisation of an antibody to detect the coccidiostat toltrazuril and its metabolite ponazuril

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins (HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses (1 mg and 0.1 mg) were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC50 of 18 ng ml(-1) using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites (toltrazuril sulfoxide and toltrazuril sulfone) but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.     

Genotoxic effects of cadmium chloride on human amniotic fluid cells cultured in vitro

In this study we report the results of cytogenetic tests, namely a search for chromosome aberrations (CA) and sister chromatid exchanges (SCEs), performed on human amniotic fluid cells cultured and treated with Cadmium chloride. The cells from primary cultures were exposed to CdCl2 at 1 microM and 10 microM for 24 h. At the higher dose, no metaphases were scored and at the lower dose (1 microM) no effects were evident on cell proliferation, and no chromosome aberrations were found. In the subsequent experiments we used cells from subcultures exposed to 1 microM and 5 microM CdCl2. At the 5 microM dose was evident the induction of chromatid breaks, while the frequency of sister chromatid exchanges shows a small increase, not statistically significant at the dose of 1 microM. In this study we positively demonstrated that amniotic fluid cells grown in vitro are reliable for testing various mutagenic or teratogenic substances. With regard to cadmium treatment results, it is evident a clastogenic effect of cadmium chloride but not a significant induction of SCEs.     

Immune modulation properties of herbal plant leaves: Phyllanthus niruri aqueous extract on immune cells of tuberculosis patient - in vitro study

Disease progression in Tuberculosis (TB) is dependent on host’s immune system. Phyllanthus niruri, a traditional herb, has long been used to boost immune system in Indonesian society. This study aimed to observe the potential role of P. niruri in inducing immune cells activity in TB patients by in vitro approach. Peripheral blood mononuclear cells (PBMCs) and macrophages were collected from active pulmonary TB patients. After stimulation with graded doses of P. niruri aqueous extract, cell proliferation, phagocytic activity and nitric oxide (NO) release were analysed. P. niruri aqueous extract induced proliferation of PBMCs, increased NO release, and improved macrophages phagocytic activity. These effects were observed in a dose-dependent manner. This may lead to further research for the potential role of P. niruri as immunomodulatory adjuvant therapy for TB patients.     

Isolation and evaluation of cytogenetic effect of Brahmi saponins on cultured human lymphocytes exposed in vitro

Major saponins of Brahmi (Bacopa monniera, Fam: Scrophulariaceae) – bacosides A and B – were isolated from the total methanol extract and characterised based on melting point, TLC, IR, ¹H NMR and ¹³C NMR. They were evaluated for their in vitro cytogenetic effects on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The frequency of chromatid type aberrations and reciprocal interchanges between sister chromatids in the treated cells was scored in comparison to the untreated control. At 30 μg/mL dose, bacoside A showed a statistically significant increase in the frequency of both CA and SCE and bacoside B showed an increase only in SCE. Our report of the genotoxicity of the saponins is significant in view of the reports of anticancer activity of Brahmi extracts.     

Enhancement of saccharothriolide production and discovery of a new metabolite,saccharothriolide C2, by combined-culture of Saccharothrix sp. and Tsukamurella pulmonis

A combined-culture method was applied to saccharothriolide-producing strain Saccharothrix sp. A1506 with the mycolic acid-containing bacterium (MACB) Tsukamurella pulmonis TP-B0596. The combined-culture not only enhanced saccharothriolide production, but also produced saccharothriolide C₂, a new C-2 epimer of saccharothriolide C, thereby indicating its potential for increasing the chemical diversity of microbial metabolites.     

In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic, and synthetic approach

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A(1) are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL, novL, and couL, respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and (1)H-NMR spectroscopy.     

Isolation from pig liver microsomes, identification by electrospray tandem mass spectrometry and in vitro immunosuppressive activity of a rapamycin tris-epoxide metabolite

It was demonstrated that rapamycin is metabolized in vitro by pig liver microsomes under the influence of the cytochrome P450-dependent mixed function oxygenase system to a rapamycin tris-epoxide metabolite, as demonstrated by electrospray tandem mass spectrometry. The in vitro immunosuppressive activity of this metabolite was found to be lower than that of rapamycin, probably because the rapamycin effector sector was structurally modified. The effector region of rapamycin was recognized to include the conjugated double bonds of this compound and metabolic reactions affecting this region may change the binding affinity of the rapamycin-FKBP binary complex towards another pharmacological receptor bound to the binary complex. Moreover, metabolic modifications in the effector region are probably able to induce a change in the binding affinities of the rapamycin-FKBP binary complex, including the pipecolic acid moiety and the lactone function of the parent drug.