Mucuna pruriens seed extract reduces oxidative stress in nigrostriatal tissue and improves neurobehavioral activity in paraquat-induced Parkinsonian mouse model

Parkinson’s disease (PD) is a neurodegenerative disease which causes rigidity, resting tremor and postural instability. Treatment for this disease is still under investigation. Mucuna pruriens (L.), is a traditional herbal medicine, used in India since 1500 B.C., as a neuroprotective agent. In this present study, we evaluated the therapeutic effects of aqueous extract of M. pruriens (Mp) seed in Parkinsonian mouse model developed by chronic exposure to paraquat (PQ). Results of our study revealed that the nigrostriatal portion of Parkinsonian mouse brain showed significantly increased levels of nitrite, malondialdehyde (MDA) and reduced levels of catalase compared to the control. In the Parkinsonian mice hanging time was decreased, whereas narrow beam walk time and foot printing errors were increased.     

Stimulation of total phenolics, L-DOPA and antioxidant activity through proline-linked pentose phosphate pathway in response to proline and its analogue in germinating fava beans (Vicia faba)

The rationale behind the study was to enhance azetedine-2-carboxylate (A2C)-linked stress in the germinating seeds to which it responds by increased proline synthesis to overcome inhibition of proline dehydrogenase (PDH). A2C is a competitive inhibitor of proline that inhibits its transport from cytosol to the mitochondria for further metabolic recycling by binding on to the active sites of PDH. The enhanced synthesis of proline would increase the ratio of the cofactors NADP/NADPH₂. The increase in the cofactors would result in the over-expression of the phenypropanoid pathway required for the phenolic acids and L-DOPA synthesis via pentose phosphate pathway through the activity of glucose-6-phosphate dehydrogenase (G6PDH). Fava bean were chosen since it has high phenolic and L-DOPA levels and could be an important part of the diet especially for patients suffering from Parkinson's Disease. The objectives were investigated by assaying for total phenolic content, the corresponding antioxidant activity by β-carotene oxidation method, proline levels and enzymes such as G6PDH and guaiacol peroxidase (GPX) using spectrophotometric methods. L-DOPA was quantified using HPLC. The fava bean seeds were primed with water, 200 μM A2C, 0.25 mM proline and a combination of A2C and proline treatments. After the priming stage, seeds were dark germinated and grown for a period of 8 days, for biochemical analysis. L-DOPA levels did not change in comparison to the control treatments while total phenolic content, proline and G6PDH were all enhanced by the treatments. During the early stages of germination the phenolic acids were antioxidant in nature, which later was reduced as they become polymerized to lignins and lignans via the GPX activity. Total phenolic synthesis was both coupled and uncoupled to PLPPP depending on the treatments. The three treatments over-expressed PLPPP since large difference between control and the treatments were observed for all parameters, except L-DOPA content.     

Microwave-induced stimulation of l-DOPA,phenolics and antioxidant activity in fava bean (Vicia faba) for Parkinson’s diet

Fava bean sprouts are rich in levo-dihydroxy phenylalanine (l-DOPA), the precursor of dopamine, and are being investigated for use in the management of Parkinson’s disease. The phytopharmaceutical value was improved during germination by a microwave treatment of the seeds, the phenolic content of the germinated sprouts increasing 700% and l-DOPA content by 59% compared to control. A higher antioxidant activity that was observed correlated with total phenolics and l-DOPA contents. The glucose-6-phosphate dehydrogenase activity peaked on the seventh day of germination with a concurrent increase in phenolics indicating enhanced mobilization of carbohydrates. A higher guaiacol peroxidase activity was observed indicating an increased polymerization of phenolics. The elevated superoxide dismutase activity was proportional to the stimulation of antioxidant activity. The major implication from this study is that microwave treatment can significantly stimulate the phenolic antioxidant activity and Parkinson’s relevant l-DOPA content of fava beans sprouts.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Change in the reaction of the antioxidant system of wheat sprouts after UV-irradiation of seeds

The response of the antioxidant system of sprouts of wheat Triticum aestivum L. to preliminary irradiation of seeds with UV light was studied. The dependence of lipid peroxidation and the extent of antioxidant activity on the duration of irradiation was studied. It was shown that low doses of UV radiation (5-15 min) stimulate the antioxidant protection of green wheat sprouts grown for eight days. Increasing the irradiation time to 30-60 min leads to the inhibition of lipid peroxidation by the antioxidant system. A more prolonged irradiation of seeds with UV light (for 1-6 h) led to an increase in the level of lipid peroxidation in sprouts. However, 1-2-day-old sprouts from seeds irradiated for 5-6 h, adapted themselves to the influence due to the compensatory mechanisms. By the 8th day of germination of preliminarily irradiated seeds, the content of antioxidants and malone dialdehyde returned to the norm. The dynamics of activity of peroxidase in seeds irradiated with low doses of UV light for 30 min was studied. It was found that on the third day of seed germination, a decrease in peroxidase activity followed by its slight increase occurred. The maximum activity of the enzyme in the endosperm was observed on day 5-6, and in roots and green sprouts, on day 3-5 of germination. It was concluded that antioxidants and peroxidase are involved in the compensatory mechanisms of inhibition of free radicals formed upon UV irradiation of seeds.     

Threonine 53 in α-synuclein is conserved in long-living non-primate animals

α-Synuclein is the main constituent of Lewy bodies in familial and sporadic cases of Parkinson’s disease (PD). Autosomal dominant point mutations, gene duplications or triplications in the α-synuclein (SNCA) gene cause hereditary forms of PD. One of the α-synuclein point mutations, Ala53Thr, is associated with increased oligomerization toxicity leading to familial early-onset PD in humans. The amino acid in position 53 in α-synuclein is an alanine in humans, great apes and Old World primates. However, this amino acid is a threonine in the α-synuclein of all other examined species, including New World monkeys. Here, we present DNA sequence analysis of SNCA and the deduced amino acid sequences of α-synuclein cloned from various different species, ranging from fish to mammals, which are known for their long-living potential. In all these investigated species the 53Thr is found. We conclude that 53Thr is not a molecular adaptation for long-living animals to minimize the risk of developing PD.     

β-Sheet-breaking peptides inhibit the fibrillation of human α-synuclein

α-Synuclein is the major components of the intracellular protein-aggregates, found in the dopaminergic neurons of Parkinson’s disease patients. Previously, we screened for α-synuclein substitution mutants that prevent fibril formation of both wild-type and Parkinson’s disease-linked α-synuclein variants. In the present study, we show that short synthetic peptides derived from these mutant sequences not only prevented α-synuclein fibrillation but also dissolved preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV, which was the shortest peptide that retained the ability to block α-synuclein fibrillation, may serve as a lead compound for the development of therapeutics for Parkinson’s disease.     

Estimation of genetic diversity in varieties of Mucuna pruriens using RAPD

Genetic diversity was estimated in 13 accessions of the otherwise self pollinated Mucuna pruriens (L.) DC. (velvetbean) comprising varieties pruriens and utilis collected from tropical humid forest using 15 RAPD primers. Similarity index value of 0.68 based on Nei and Li's similarity coefficient indicated high degree of genetic variability. Analysis of various genetic diversity indices like total heterozygosity, Nei's gene diversity, percentage of polymorphic loci, expected and observed number of alleles and Shannon index strongly suggests that variety pruriens is genetically more diverse than variety utilis. Chemical analysis with respect to 3,4-dihydroxy-L-phenylalanine (L-DOPA) content showed uniform distribution. Cluster analysis showed grouping of accessions into two major clusters and tendency of accessions of variety pruriens to group according to their geographical locations. Bootstrap analysis confirmed the robustness of the phenogram. The putative hybrid MMP6 with relatively low similarity value index and low L-DOPA content was promising as food or fodder.     

Production of L-DOPA by cell suspension cultures of Mucuna pruriens

The endogenous synthesis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) by cell suspension cultures of Mucuna pruriens was found to be influenced by several environmental parameters. The nature of the nitrogen source as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to affect the biosynthesis of L-DOPA. Addition of 2, 4-dichlorophenoxyacetic acid to the medium suppressed L-DOPA production; continuous illumination of the cultures had a strong beneficial effect on L-DOPA production. L-DOPA was accumulated intracellularly by the cell suspension cultures. These observations further demonstrate that for certain products of plant cell suspensions product synthesis can be manipulated by a proper selection of specified nutrients.     

Distribution of the hordatines in barley

Concentrations of agmatine, coumarylagmatine and the antifungal hordatines in the shoots of barley seedlings have been determined at various stages of growth. Coumarylagmatine declined with age on a fresh weight basis, both in diurnal illumination and in continuous darkness. Hordatines A and B (estimated together) declined in the light to the 30th day after germination but their concentrations were stable in the dark to the 12th day. Hordatine M declined in the light to the 30th day and in the dark to the 12th day from germination. Agmatine declined in both light and dark to the 12th day. On the 30th day from germination potassium deficiency caused an increase in hordatines A + B ( × 6), hordatine M ( × 2) and agmatine ( × 13). Infection of the 11-day-old seedlings with mildew (Erysiphe graminis) caused an increase in the content of hordatine A + B ( × 6), hordatine M ( × 2) and agmatine ( × 2) 13 days later. Hordatines occurred in seedlings of H. bulbosum, H. distichon, H. murinum and H. spontaneum, though not in seedlings of H. jubatum, maize, millet, oats, rice, rye or wheat. Arginine decarboxylase activity declined with age in barley seedlings grown in the light or dark from the 3rd to the 12th day.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

Enzymatic synthesis of l-DOPA α-glycosides by reaction with sucrose catalyzed by four different glucansucrases from four strains of Leuconostoc mesenteroides

l-DOPA α-glycosides were synthesized by reaction of l-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of l-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by ¹H NMR spectroscopy. The major glycoside was 4-O-α-d-glucopyranosyl l-DOPA, and the minor glycoside was 3-O-α-d-glucopyranosyl l-DOPA. The two glycosides were formed by all four of the glucansucrases. The ratio of the 4-O-α-glycoside to the 3-O-α-glycoside produced by the B-512FMC dextransucrase was higher than that for the other three glucansucrases. The glycosylation of l-DOPA significantly reduced the oxidation of the phenolic hydroxyl groups, which prevents their methylation, potentially increasing the use of l-DOPA in the treatment of Parkinson’s disease. The use of one enzyme, glucansucrase, and sucrose as the d-glucosyl donor makes the synthesis considerably simpler and cheaper than the formerly published procedure using cyclomaltodextrin and cyclomaltodextrin glucanyltransferase, followed by glucoamylase, and β-amylase hydrolysis.     

光周期对苦荞芽菜生长与品质的影响

苦荞(Fagopyrum tataricum)芽菜是一种新兴健康食品,该研究通过不同光周期(0、4、8、12、16和20 h·d~(-1))处理苦荞芽菜,测定其生物量、叶绿素和主要营养成分的含量以及黄酮合成相关基因的表达水平,以明确不同光周期对苦荞芽菜品质的影响机理。结果表明:(1)随着苦荞芽菜生长时间的增加,芽菜生物量总体呈上升趋势,16 h·d~(-1)光周期时芽菜生长状态最好,鲜重最大。(2)不同光周期对苦荞芽菜各营养成分含量的影响有所差异,可溶性糖含量在芽菜萌发第2天开始逐渐下降,10 d后含量仅为第2天的13.5%～14.5%;花青素含量在芽菜萌发第2天光周期为12 h·d~(-1)时最高(2.16 mg/g),不同光周期处理4～10 d后均显示出降低趋势;芦丁是苦荞芽菜中主要的黄酮类化合物,芦丁含量在不同光周期处理后有所变化且适当的光照有利于其合成和积累,并在处理第4天光周期为16 h·d~(-1)时含量最高(59.60 mg/g)。(3)qRT-PCR分析表明,不同光周期处理的苦荞芽菜中各黄酮合成相关基因的表达量在第2天最高,随生长周期其表达量均不同程度降低,但黄酮醇合成支路关键酶基因FtFLS1和花青素支路基因FtDFR2表达量上升、且显示出强烈的光诱导特征。研究发现,光周期对苦荞芽菜的生物量影响较大,黑暗有利于胚轴伸长,长光周期(16 h·d~(-1))有利于鲜重的增加;在苦荞萌发过程中,苦荞芽菜可溶性糖、花青素和芦丁含量整体呈减少趋势,可溶性糖和花青素含量均在光周期16 h·d~(-1)处理第2天时最高,芦丁含量在光周期为16 h·d~(-1)处理第4天时最高,建议苦荞芽菜在光周期为16 h·d~(-1)生长4 d时采食。     

Molsidomine,a nitric oxide donor,modulates rotational behavior and monoamine metabolism in 6-OHDA lesioned rats treated chronically with L-DOPA

Some biochemical and histological studies of Parkinson’s disease patients’ brains and 6-OHDA-lesioned rats suggest that dopaminergic dennervation of the striatum leads to the nitrergic system hypofunction in this structure. Hence, recently the modulation of nitric oxide (NO)– soluble guanylyl cyclase–cyclic GMP signaling is considered to be a new target for the treatment of Parkinson’s disease. The aim of our study was to examine the impact of chronic combined treatment with low doses of the NO donor molsidomine (2 and 4 mg/kg) and L-DOPA (12.5 and 25 mg/kg) on rotational behavior and monoamine metabolism in the striatum (STR) and substantia nigra (SN) of unilaterally 6-OHDA-lesioned rats.     

The hexapeptide PGVTAV suppresses neurotoxicity of human α-synuclein aggregates

In Parkinson’s disease patients, α-synuclein is the major component of the intracellular protein aggregates found in dopaminergic neurons. Previously, short synthetic α-synuclein-derived peptides have been shown to not only prevent α-synuclein fibrillation but also dissolve preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV was the shortest peptide that retained the ability to block α-synuclein fibrillation. For preventative or therapeutic effectiveness, a treatment must suppress the neurotoxicity of α-synuclein aggregates and remain stable in plasma. The present study shows that specific peptides can protect neuronal cells from α-synuclein aggregation-induced cell death. The β-sheet-breaking hexapeptide PGVTAV remained intact in human plasma for longer than one day, suggesting that it may be a candidate for the development of therapeutics to treat Parkinson’s disease.     

Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyl-dopamine induced neurotoxicity

Parkinson’s disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra. Recent investigations have shown that conjugates such as the 5-S-cysteinyl-dopamine, possess strong neurotoxicity and may contribute to the underlying progression of the disease pathology. Although the neuroprotective actions of flavonoids are well reported, that of hydroxycinnamates and other phenolic acids is less established. We show that the hydroxycinnamates caffeic acid and p-coumaric acid, the hydroxyphenethyl alcohol, tyrosol, and a Champagne wine extract rich in these components protect neurons against injury induced by 5-S-cysteinyl-dopamine in vitro. The protection induced by these polyphenols was equal to or greater than that observed for the flavonoids, (+)-catechin, (−)-epicatechin and quercetin. For example, p-coumaric acid evoked significantly more protection at 1 μM (64.0 ± 3.1%) than both (−)-epicatechin (46.0 ± 4.1%, p < 0.05) and (+)-catechin (13.1 ± 3.0%, p < 0.001) at the same concentration. These data indicate that hydroxycinnamates, phenolic acids and phenolic alcohol are also capable of inducing neuroprotective effects to a similar extent to that seen with flavonoids.     

Phenological variations of secondary metabolites from Hypericum triquetrifolium Turra

The essential oils and phenolic constituents from the aerial parts of Hypericum triquetrifolium Turra, were analyzed at three developmental stages (vegetative, flowering and fruiting stages). The highest content of oil (0.12% w/w) was obtained at full flowering. Whatever the analyzed stage, n-octane, α-pinene, β-caryophyllene, 2-methyloctane, n-nonane, α-longipinene, caryophyllene oxide and β-pinene were found to be the main compounds. However, their percentages varied with the phenological cycle. Analysis by RP-HPLC-DAD of the methanolic extracts enabled us to identify 14 phenolic components and rutin, hyperoside, quercitrin and quercetin were reported as the main components. With the exception of chlorogenic acid, kaempferol and amentoflavone, the content of the remaining identified phenolic components varied with the phonological cycle.     

Regeneration via somatic embryogenesis of the endangered wild arum (Arum palaestinum)

Somatic embryogenesis was obtained from callus of wild arum (Arum palaestinum). Callus was induced from sterilized corm bud sprouts cultured on basal medium containing 4.4???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Callus was maintained under dark conditions using basal medium with 4.4 or 8.8???M 6-benzyladenine and 5.4 or 10.8???M 1-naphthaleneacetic acid. The highest callus weight and most desirable callus phenotype were achieved using basal medium containing 8.8???M 6-benzyladenine and 5.4???M 1-naphthaleneacetic acid. Friable calli were cultured in the dark on basal medium containing 4.5???M 2, 4-dichlorophenoxyacetic acid, 0.46???M 6-furfurylaminopurine, 5.4???M 1-naphthaleneacetic acid, and 1.7?mM proline to induce embryogenesis before transfer to regeneration medium. Embryos that developed on regeneration medium were transferred to medium minus plant growth regulators for germination. Ninety percent of the germinating embryos developed into rooted plantlets. Rooted plants were grown in the greenhouse and acclimatized successfully with a 95?% survival rate. This is the first report of successful somatic embryogenesis and plant regeneration in A. palaestinum.     

A multi-assay comparison of seed germination inhibition by Lonicera maackii and co-occurring native shrubs

Recent work suggests that germination inhibition via allelopathy may be an important component of some species’ invasion ecology. We conducted four germination assays to test the inhibitory potential of Amur honeysuckle (Lonicera maackii), a problematic invasive shrub. The subject species in the first assay, Festuca arundinacea, exhibited a significant delay in germination when treated with extract from ground honeysuckle foliage. In the second assay, two concentrations of L. maackii extract were created by soaking foliage. F. arundinacea germination was not significantly influenced by the treatments while germination in Impatiens wallerana was substantially decreased. The third assay compared the impact of foliar extract from honeysuckle, with the native shrubs Lindera benzoin and Asimina triloba using F. arundinaceae, I. wallerana, Coreopsis lanceolata and Poa pratensis as subject species. In this assay, I. wallerana was strongly inhibited by L. maackii foliage; however, none of the other species were significantly influenced by the treatments. In the fourth assay, fruit extracts from L. maackii and L. benzoin were applied to the same four subject species. L. maackii fruit extract had an inhibitory influence on seed germination of all subject species, and inhibition from L. benzoin was also noted. Across all assays, there was a mixed reaction to extract from L. maackii and co-occurring native species that was species-specific and dependent upon the extract source. Our findings provide support for the idea that L. maackii has allelopathic activity, but further work is needed to (i) understand how broad the impact may be across the wide variety of species that are found in its invasion range and (ii) substantiate that the allelopathic effect has relevance in field environments.