Methyl Jasmonate Induce Enhanced Production of Soluble Biophenols in PANAX GINSENG Adventitious Roots from Commercial Scale Bioreactors

The contents of soluble biophenols such as protocatechuic, gentisic, vanillic, caffeic, syringic, p-coumaric, ferulic, salicylic, and cinnamic acids were screened and quantified from the adventitious roots of Panax ginseng by HPLC-MS. Control adventitious roots which showed the greatest accumulation of ferulic acid (0.09 mg/g DW) were observed in our experiments. An increase in the total soluble biophenol content of adventitious roots was observed 5 days after treatment with elicitor 200 μM/L methyl jasmonate (MeJa) i.e., 35 to 40 days of inoculation. Among the biophenols investigated, the salicylic acid content was higher (0.44 mg/g DW) in MeJa treated adventitious root culture in the bioreactor. MeJa might apparently increase soluble biophenols production in adventitious roots by enhancing the biosynthesis pathway from phenylalanine to salicylic acid and other simple biophenols. __________ Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 551–554, November–December, 2005     

Bioprocess Optimization of Furanocoumarin Elicitation by Medium Renewal and Re-elicitation: A Perfusion-Based Approach

Effect of various abiotic (methyl jasmonate, salicylic acid) and biotic (yeast extract, Aspergillus niger) elicitors on furanocoumarin production and in situ product removal was studied using shoot cultures of Ruta graveolens L. Elicitation by yeast extract (1% w/v) on day 15 was most effective. It led to 7.8-fold higher furanocoumarin production that was attained 24 h after elicitation and 43% of the product was released into the medium. Changes in the relative concentration of furanocoumarins produced depend on the elicitor used. Molar ratio of bergapten increased to 93% in response to yeast extract. With the perspective of developing a commercially feasible process, an approach for preserving viability of biomass and its reuse needs to be developed. For this, medium renewal strategy was investigated. Removal of the spent medium 48 h after elicitation allowed in situ product removal and proved effective in revival of cultures, allowing reuse of biomass. A week after medium renewal, the revived biomass was re-elicited and a second furanocoumarin production peak was obtained. A perfusion-based bioprocess optimization approach, employing elicitation coupled with medium renewal with subsequent re-elicitation, as a new strategy for improved furanocoumarin production, has been suggested.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Toxicity of naturally occurring Bio-fly and chitosan compounds to control the Mediterranean fruit fly Ceratitis capitata (Wiedemann)

The efficacy of five compounds of a biopolymer chitosan and Bio-fly (Beauveria bassiana fungus) as biopesticide was evaluated on Ceratitis capitata under laboratory conditions. The inhibitory effects on acetylcholinesterase (AChE) and adenosinetriphosphatase (ATPase) as biochemical indicators were also determined in vivo. The results indicated that B. bassiana based Bio-fly exhibited significant toxicity against C. capitata (LC₅₀ = 3008 and 3126 mg/L after 48 h in females and males, respectively) followed by the derivatives of chitosan, N-(4-propylbenzyl)chitosan and N-(2-nitrobenzyl)chitosan. Bio-fly displayed remarkable inhibition of AChE activity (IC₅₀ = 2220 mg/L) while N-(2-chloro,6-flourobenzyl)chitosan, N-(4-propylbenzyl)chitosan and N-(3,4-methylenedioxybenzyl) chitosan had no significant difference in inhibitory action. In adult males, N-(2-nitrobenzyl)chitosan exhibited the highest inhibitory action (IC₅₀ = 6569 mg/L). In addition, the toxic effects of the tested compounds on the activity of ATPase indicated that highly significant inhibition was found with N-(4-propylbenzyl)chitosan with an IC₅₀ of 8194 and 8035 mg/L, in females and males, respectively.     

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.     

Structure and morphology of the 60/40 poly(p-oxybenzoate-co-ethylene terephthalate) copolymer

The copolyester composed of 60 mol% p-hydroxybenzoic acid and 40 mol% ethylene terephthalate has been studied extensively by earlier workers. However, some confusion exists particularly with respect to the nature of the thermal transitions and the possible presence of additional phases. In the present study we have examined this copolyester and found that it is possible to separate the material into soluble and insoluble fractions with significantly different ratios of p-hydroxybenzoic acid to ethylene terephthalate. In fact, the soluble PET-rich fraction is blocky in nature, while the PHBA-rich fraction is more random. The multiple thermal transitions can now be readily interpreted in terms of a heterogeneous composition which tends to segregate and phase separate.     

CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

An improved procedure for phosphorous fractionation in plant materials exploiting sample preparation and monosegmented flow analysis

Sample treatment procedures were evaluated for fractionation of phosphorous in plant materials (determination of organic and inorganic, soluble and insoluble fractions). The procedures aimed the conversion of different species into orthophosphate, minimizing time, reagent amounts and waste generation. A monosegmented flow system with multicommutation was developed for the spectrophotometric determination of orthophosphate by the molybdenum blue method. Linear response within 0.5 and 25.0 mg L^− 1 P, detection limit of 24 μg L^− 1 P (99.7% confidence level), coefficient of variation of 3.5% (n = 10) and sampling rate of 38 measurements per hour were estimated. Each determination consumes 5.0 mg ascorbic acid and 0.60 mg of ammonium molybdate. Total phosphorous determination can be carried out after microwave-assisted acid digestion by employing 100 mg of plant material and 500 μL of concentrated HNO₃. Extraction of soluble phosphorous can be carried out with water by stirring for 10 min and organic soluble phosphorous can be determined either after microwave-assisted acid digestion or photodegradation in the presence of ammonium persulfate in acid medium. The results for the different fractions agreed with those obtained by ICP OES at the 95% confidence level.     

Chitosan Elicitation Impacts Flavonolignan Biosynthesis in Silybum marianum (L.) Gaertn Cell Suspension and Enhances Antioxidant and Anti-Inflammatory Activities of Cell Extracts

Silybum marianum (L.) Gaertn is a rich source of antioxidants and anti-inflammatory flavonolignans with great potential for use in pharmaceutical and cosmetic products. Its biotechnological production using in vitro culture system has been proposed. Chitosan is a well-known elicitor that strongly affects both secondary metabolites and biomass production by plants. The effect of chitosan on S. marianum cell suspension is not known yet. In the present study, suspension cultures of S. marianum were exploited for their in vitro potential to produce bioactive flavonolignans in the presence of chitosan. Established cell suspension cultures were maintained on the same hormonal media supplemented with 0.5 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L NAA (α-naphthalene acetic acid) under photoperiod 16/8 h (light/dark) and exposed to various treatments of chitosan (ranging from 0.5 to 50.0 mg/L). The highest biomass production was observed for cell suspension treated with 5.0 mg/L chitosan, resulting in 123.3 ± 1.7 g/L fresh weight (FW) and 17.7 ± 0.5 g/L dry weight (DW) productions. All chitosan treatments resulted in an overall increase in the accumulation of total flavonoids (5.0 ± 0.1 mg/g DW for 5.0 mg/L chitosan), total phenolic compounds (11.0 ± 0.2 mg/g DW for 0.5 mg/L chitosan) and silymarin (9.9 ± 0.5 mg/g DW for 0.5 mg/L chitosan). In particular, higher accumulation levels of silybin B (6.3 ± 0.2 mg/g DW), silybin A (1.2 ± 0.1 mg/g DW) and silydianin (1.0 ± 0.0 mg/g DW) were recorded for 0.5 mg/L chitosan. The corresponding extracts displayed enhanced antioxidant and anti-inflammatory capacities: in particular, high ABTS antioxidant activity (741.5 ± 4.4 μM Trolox C equivalent antioxidant capacity) was recorded in extracts obtained in presence of 0.5 mg/L of chitosan, whereas highest inhibitions of cyclooxygenase 2 (COX-2, 30.5 ± 1.3 %), secretory phospholipase A2 (sPLA2, 33.9 ± 1.3 %) and 15-lipoxygenase (15-LOX-2, 31.6 ± 1.2 %) enzymes involved in inflammation process were measured in extracts obtained in the presence of 5.0 mg/L of chitosan. Taken together, these results highlight the high potential of the chitosan elicitation in the S. marianum cell suspension for enhanced production of antioxidant and anti-inflammatory silymarin-rich extracts.     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

Production of 2,3- butanediol from pretreated corn cob byKlebsiella oxytoca in the presence of fungal cellulase

A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol byKlebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100°C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced byK. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.     

Characterization of human papillomavirus by capillary isoelectric focusing with whole‐column imaging detection

CIEF with whole‐column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF‐WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF‐WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF‐WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single‐point detection. In the example, the five HPVs displayed pI values of 8.43 ± 0.06 (n = 10; HPV 6L1), 8.70 ± 0.04 (n = 10; HPV 11L1), 7.94 ± 0.05 (n = 18; HPV 16L1), 7.57 ± 0.04 (n = 18; HPV 18L1), and 8.45 ± 0.05 (n = 10; HPV 58L1). This CIEF‐WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry.     

Synthesis and characterization of chitosan‐modified polymethyl methacrylate latex particles

Stable chitosan‐modified polymethyl methacrylate (PMMA) latex particles were prepared by using 2,2′‐azobis(2‐amidinopropane) dihydrochloride (V‐50) as the cationic initiator. The polymerization rate (R_p) is controlled by the V‐50 concentration ([V‐50]) and R_p is less sensitive to the chitosan concentration ([C]) used in the synthesis work. The reaction system follows Smith–Ewart Case III kinetics due to the relatively large particles produced. The zeta potential data show that the isoelectric point (pI) of the latex particles is 10.7. The amounts of V‐50 (C_V‐50) and chitosan (C_c) ultimately incorporated into the particles correlate reasonably well with [V‐50] and [C], respectively. At pH 7, the quantity of the negatively charged bovine serum albumin (BSA, pI = 4.8) adsorbed on the positively charged chitosan‐free particles (Q) via the electrostatic interaction increases with increasing C_V‐50. However, Q is relatively insensitive to changes in C_c. This result implies that only the outermost region of the hairy chitosan‐modified particles is available for adsorption of the relatively large protein species. Colloidal stability shows a significant influence on the BSA adsorption process. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1489–1499, 1999     

Studies on N-methylene phosphonic chitosan

N-methylene phosphonic (NMPC) chitosan was studied by several techniques to determine their properties in aqueous solution and its capacity to emulsify edible oils. The phosphonic groups are all equivalent and their ionization constants were estimated (pK _a1=6.45 and pK _a2=11.75). The chitosan derivative in pure water starts to aggregate at 0.09% w/v, and the aggregates’ structure at 0.5% w/v. Unlike pure chitosan, NMPC chitosan is strongly surface active. Its hydrophile-lipophile balance (HLB) value was estimated in 37, very similar to that of chitosan. As a consequence, it favors the formation of oil in water (o/w) emulsions with scarce water/oil/water (w/o/w) droplets.     

Synthesis,Characterization, and Antimicrobial Activity of Carboxymethyl Chitosan-Graft-Poly(N-acryloyl,N′-cyanoacetohydrazide) Copolymers

Carboxymethyl chitosan was grafted with N-acryloyl,N′-cyanoacetohydrazide in homogenous aqueous phase using potassium persulfate initiator. The maximum grafting yield achieved was 448% at 0.03 mol/L potassium persulfate, 0.75 mol/L N-acryloyl,N′-cyanoacetohydrazide, and 60°C within 2 h. The grafted copolymers showed better thermal stability than that of carboxymethyl chitosan. The samples with percent grafting values up to 98% were soluble in water, but a higher grafting extent resulted in insoluble copolymers. The grafted copolymers are nontoxic materials and showed an inhibition effect on both Escherichia coli and Staphylococcus aureus bacteria and Aspergillus flavus and Candida albicans fungi better than those of chitosan and carboxymethyl chitosan themselves.     

Ring-opening polymerization of 1-(p-substituted phenyl)-2-vinylcyclopropanes by Ziegler–Natta catalysts

1-(p-Substituted phenyl)-2-vinylcyclopropanes such as 1-phenyl-2-vinylcyclopropane (I_a), 1-(p-chlorophenyl)-2-vinylcyclopropane (I_b), 1-(p-anisyl)-2-vinylcyclopropane (I_c), and 1-(p-tolyl)-2-vinylcyclopropane (I_d) were prepared and polymerized radically, cationically and with Ziegler–Natta catalysts. I_a and I_b polymerized exclusively in 1,5-fashion with radical initiators. However, I_c and I_d polymerized in 1,5-fashion only with Ziegler–Natta catalysts. All polymers were soluble in ordinary organic solvent and solution-cast films were clear and flexible, showing T_g values in the range of 39–71°C. Spectral data indicated that the double bonds of the polymer chains were in trans form in all cases. The difference between the polymerizabilities of different monomers are interpreted in terms of electronic properties of substituents.     

Effect of sialic acid content on glycoprotein pI analyzed by two‐dimensional electrophoresis

2‐DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2‐DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2‐DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2‐DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2‐DE pattern of three serum glycoproteins (haptoglobin, α1‐antitrypsin and ribonuclease 1), which N‐glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule.     

Chemical profile and antihyperlipidemic effect of Portulaca oleracea L. seeds in streptozotocin-induced diabetic rats

Hypolipidemic effect of Portulaca oleracea L. seed extract and its fractions have been studied on streptozotocin (STZ) at dose 75 mg/kg b.wt. After fractionation of the alcoholic extract; petroleum ether fraction was the most active fraction that decreased different hyperlipidemia biochemical parameters. After chromatographic analysis; oleamide, ethylpalmitate, β-amyrin, stigmasterol and β-sitosterol were identified. The GLC analysis of unsaponifiable matter revealed the presence of; lignoceric acid as a major constituent in the most bioactive fraction. In conclusion, petroleum ether fraction possessed a hypolipidemic effect in STZ-induced diabetic rats, which may be attributed to its phytosterols, fatty acid and amide compounds. The finding of the present investigation strongly demonstrates the potential of non-polar fraction of P. oleracea L. seed in combating hyperlipidemia in diabetic condition. So the petroleum ether fractions and its constituents can be used as hypolipdemic supplement in the developing countries towards the development of new therapeutic agents.     

Antibacterial activity of extract and fractions from branches of Protium spruceanum and cytotoxicity on fibroblasts

The crude ethanol extract (CEE) and fractions from branches of Protium spruceanum were subjected to antibacterial and cytotoxicity assays. Compounds of the most active fraction were identified by GC-MS and LC-MS. CEE was active against 19 bacteria and the ethyl acetate fraction (EAF) showed the lowest minimum bactericidal concentration (MBC 0.3–80.0 mg/mL). Through time-kill assay was observed that EAF induced rapid bactericidal effect against Staphylococcus saprophyticus. The cytotoxicity tests against L929 fibroblasts showed great potential of EAF on the treatment of infections caused by five bacteria (MBC < IC₅₀). The results provide in vitro scientific support to the possible application of branches of P. spruceanum as antimicrobial agent that may contribute for treatment of infections.     

Improved postharvest quality in patagonian squash (Cucurbita moschata) coated with radiation depolymerized chitosan

Different molecular weight chitosans were evaluated on the decay of coated Anquito squashes (Cucurbita moschata) as well as the maintenance of the fruit quality along five storage months. The original chitosan (Mw=391 kDa, 83% DD), was depolymerized by gamma radiation. Apart from chain scission, other chemical changes were not detected by FTIR or UV–vis analyses. The molecular weight characterization of chitosans was done by size exclusion chromatography with dual light scattering and concentration detection (SEC-MALLS-RI). The coating effectiveness was evaluated on the following parameters: fungal decay incidence, weight loss, firmness, total reducing sugar, soluble solid, flesh color, carotene content, pH and titratable acidity. No sign of fungal decay was observed in squashes coated with 122 and 56 kDa chitosans, which were also the most effective treatments in reducing the weight loss. The chitosan with Mw=122 kDa was also the best treatment considering firmness, internal aspect, sugar and carotene content. Then, radiation degraded chitosan was better in C. moschata preservation than the original chitosan.