Spectral-luminescent analysis of sugar cane juice

A spectral-luminescent analysis has been made of the low-, medium-, and high-molecular mass fractions of sugar cane juice. The presence of pigments was detected in all the fractions. The medium-molecular-mass fraction was distinguished by the most considerable and most diverse composition of the pigments, a substantial contribution to which was made by the products of the alkaline decomposition of sugars. The amounts of pigments in all the fractions of the juice depended on the age of the plant. A pronounced dependence on the age of the plant was characteristic for the medium-molecular-mass fraction.B. I. Stepanov Institute of Physics, Belorussian Academy of Sciences, Minsk. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 456–459, May–June, 1993.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

STUDIES ON THE PROTECTIVE FUNCTION OF THE CAROTENOID PIGMENTS OF SARCINA LUTEA

Abstract— An investigation was made of both the composition of and mechanism of photo-protection by the carotenoid pigments of Sarcina lutea ATCC 9341a and three induced mutants.
The wild-type and mutants 2a and 4b were each found to contain three major pigment fractions, each fraction consisting of a single pigment having identical absorption maxima but differing from each other in chromatographic mobility. Although the mutants contain the same kinds of pigments as does the wild-type, the mutant cells contain less pigment per cell than does the wild-type. The third mutant, 93a, contains no colored carotenoids.
It was found that there were changes in both the absolute and relative amounts of the various pigment fractions when cultures of wild-type, mutants 2a and 4b, grown in nutrient broth in the dark, were examined during the logarithmic and stationary phases of the growth curve. In addition, changes were observed in the pigments when the cells were exposed to light in buffer. These changes were similar in the wild-type and in mutant 2a, but were quite different in mutant 4b. Studies of photokilling curves suggested that these changes in amounts of the various pigment fractions were not related to photoprotection, but that the important factor may be the total amount of pigment per cell.     

BIOCHEMICAL ISOLATION AND SPECTROSCOPIC CHARACTERIZATION OF POSSIBLE PHOTORECEPTOR PIGMENTS FOR PHOTOTAXIS IN A FRESHWATER Peridinium

Photoreceptor pigments have been isolated biochemically from the freshwater dinoflagellate Peridinium gatunense, and characterized spectroscopically. At least four different chromoproteins can be detected in the crude extract and the membrane fraction isolated from the cells absorbing at 580, 638, 667 and 710 nm, which correspond with the maxima in the action spectrum for phototaxis in this organism. Light energy absorbed by shorter wavelength pigments is emitted as fluorescence at wavelengths which are absorbed by pigments with maxima at longer wavelengths. Protein separation on a MonoQ anion exchanger column using fast liquid chromatography resulted in a non-bound fraction and four bound fractions eluted by an NaCI gradient, which differed in their pigment composition.     

Potential interaction between the volatile and non-volatile fractions on the in vitro antimicrobial activity of three South African Pelargonium (Geraniaceae) species

Previous studies have reported promising antimicrobial efficacy for the essential oils and solvent extracts of several indigenous Pelargonium species. This study aimed to determine if any pharmacological interaction (e.g. synergism or antagonism) exists between the volatile and non-volatile components when the different fractions were investigated. The antimicrobial activity of the following fractions were tested; the essential oil prepared by hydrodistillation (EO), non-volatile fraction (NV), prepared by extraction of plant material remaining in the distilling apparatus (having no or negligible volatile constituents) and solvent extracts prepared from fresh (FC) and dried (DC) plant material containing both volatile and non-volatile constituents. Pelargonium quercifolium oil was dominated by p-cymene (42.1%) and viridiflorol (16.9%), while P. graveolens and P. tomentosum oil had high levels of isomenthone (84.0 and 58.8%, respectively). Menthone was noted as a major constituent in the P. tomentosum EO sample. It was evident from the results that the presence of volatile constituents in the three species; P. graveolens, P. quercifolium and P. tomentosum is generally not a pre-requisite for antimicrobial activity. The most significant variations of antimicrobial activity were noted for P. tomentosum where poorer activity was noted for the FC and EO fractions against Bacillus cereus and Candida albicans. Studies on Staphylococcus aureus, however, showed the converse, where best activity was noted for the FC fraction (3.0 mg/mL). For P. quercifolium, the DC fraction indicated a notable increase in anti-staphylococcal activity (2.0 mg/mL) when compared with the FC (8.0 mg/mL) and EO (16.0 mg/mL) fractions. For P. tomentosum, the FC fraction indicated much lower antimicrobial activity (against both B. cereus and C. albicans) when compared with all other fractions, suggesting that the essential oils may impact negatively on the antimicrobial activity when tested against these two pathogens.     

Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays

Two-dimensional electrophoresis (2-DE) is known as the most effective as well as one of the simplest methods for separating proteins. However, a few hundred plant leaf proteins out of thousands visualized on a 2-DE gel can be identified by chemical analysis due to the presence of ribulose bisphosphate carboxylase/oxygenase (Rubisco) that limits protein loading. We describe the extraction and fractionation technique with polyethylene glycol (PEG) to analyze rice leaf proteins. Rice proteins were extracted with Mg/NP-40 extraction buffer. The Mg/Nonidet P-40 (NP-40) buffer extract was further fractionated with PEG into three fractions: 10% PEG and 10-20% PEG precipitants and the final supernatant fraction that was precipitated with acetone. Rubisco, the most abundant rice leaf protein, was enriched in the 20% PEG precipitant. This fractionation technique analyzed at least 2,600 well-separated protein spots and exhibited less than 1.2% of noticeable overlapping spots. An immunological approach was used to verify the efficiency whether PEG fractionation technique can detect or enrich signal transduction components such as Galpha, ADP ribosylation factor, small GTP binding protein and 14-3-3. The ADP ribosylation factor (ARF) and Galpha were only detected in the PEG supernatant fraction not in the total protein fraction. The small GTP binding protein (Rab 7) was identified in the 10% PEG fraction and only faintly in the total protein fraction. The 14-3-3 protein was detected in all fractions but was especially prevalent in the 20% PEG fraction.     

Determination of labile inorganic and organic species of Al and Cu in river waters using the diffusive gradients in thin films technique

The diffusive gradients in thin films (DGT) technique, using a diffusive gel or a restrictive gel, was evaluated for the determination of labile inorganic and organic species of Al and Cu in model synthetic solutions and river water samples. Experiments were performed both in situ and in the laboratory. In the solutions containing Al ions, the major labile fraction consisted of inorganic species. The organic complex fractions were mainly kinetically inert. For the model Cu solutions, the most labile fraction consisted of inorganic species; however, significant amounts of labile organic complexes of Cu were also present. A comparison was made between the results obtained using restrictive gel DGT and tangential flow ultrafiltration (TF-UF). The Cu fraction determined by restrictive gel DGT (corresponding to the "free" ions plus the labile fraction of small molecular size complexes) was larger than that determined by TF-UF (corresponding to all small molecular size ions), suggesting that the techniques exhibited different porosities for discrimination of inorganic species. For the river water samples analyzed in the laboratory, less than 45% of the analytes were present in labile forms, with most being organic species. For the in situ measurements, the labile inorganic and organic fractions were larger than those obtained in the laboratory analyses. These differences could have been due to errors incurred during sample collection and storage. All results were consistent with those found using two different methods, namely, solid-phase extraction and the DGT technique employing the apparent diffusion coefficient.     

A new luminol chemiluminescence reaction using a tetravalent nickel-periodate complex as the oxidant

A modified sequential extraction procedure was developed and applied to characterize the species of metals in fly ash. Two fly ash samples were collected from a coal-fired thermal power plant located in the north of China. A sample was collected from the conventional pulverized coal (PC) combustor and another from the circulating fluidized bed (CFB) combustor. After extraction by the proposed sequential extraction procedure, the elements in the fly ash samples were divided into a water-extractable fraction (F1), an acid-soluble fraction (F2), a reducible fraction (F3), an oxidizable fraction (F4) and a residual fraction (F5). Except for Cu, V, Cd in the PC sample and V, Zn in the CFB sample, most of the other metals were present in the residual fraction (F5), which was very difficult to release into the environment. The fraction distribution patterns in the two samples were also compared. The results indicated that the distribution of metals in different fractions in fly ash samples were probably impacted by different combustion processes.     

Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography

The first preparative fractionation of betalain pigments by means of ion-pair high-speed counter-current chromatography (IP-HSCCC) from berry extracts of Phytolacca americana (Phytolaccaceae) is presented. A novel HSCCC solvent system consisting of 1-butanol-acetonitrile-water (5:1:6, v/v/v) was applied using ion-pair forming trifluoroacetic acid at low concentration (0.7%, v/v). Affinity of polar betacyanins and betaxanthins to the organic stationary phase of the biphasic HSCCC solvent mixture was considerably improved. Partitioning coefficient values and influence of increasing trifluoroacetic acid additions to the biphasic solvent mixture were measured for all identified betacyanins and betaxanthins. Gentle separation by IP-HSCCC of the injected pigment extract (900 mg) yielded sufficient amounts of the principal pigments 15S-betanin/15R-isobetanin. The pure epimers separated by C18-HPLC were immediately studied by one- and two-dimensional NMR. In the recovered fractions, minor concentrated betacyanins and betaxanthins were significantly enriched by IP-HSCCC and were detected for the first time in the extracts of P. americana. IP-HSCCC and C18-HPLC were shown to be complementary techniques in the isolation procedure of recovering minor concentrated, highly polar and chemically instable betacyanins and betaxanthin from complex plant matrices. Altogether, identification of 17 betalains was achieved by HPLC-diode array detection-electrospray ionization MS/MS in the HSCCC fractions with their respective isomers, also resulting in the tentative elucidation of betacyanins with novel salicylic acid substitution pattern in the berry extracts of P. americana.     

Innovation in a Continuous System of Distillation by Steam to Obtain Essential Oil from Persian Lime Juice (Citrus latifolia Tanaka)

The citrus industry is one of the most important economic areas within the global agricultural sector. Persian lime is commonly used to produce lime juice and essential oil, which are usually obtained by batch distillation. The aim of this work was to validate a patented continuous steam distillation process and to both physically and chemically characterize the volatile fractions of essential Persian lime oil. Prior to distillation, lime juice was obtained by pressing the lime fruit. Afterwards, the juice was subjected to a continuous steam distillation process by varying the ratio of distillate flow to feed flow (0.2, 0.4, and 0.6). The distillate oil fractions were characterized by measuring their density, optical rotation, and refractive index. Gas chromatography GC-FID was used to analyze the chemical compositions of the oil fractions. The process of continuous steam distillation presented high oil recovery efficiencies (up to 90%) and lower steam consumption compared to traditional batch process distillation since steam consumption ranged from 32 to 60% for different steam levels. Moreover, a reduction in process time was observed (from 8 to 4 h). The oil fractions obtained via continuous steam distillation differed significantly in their composition from the parent compounds and the fractions.     

Development of a procedure for the sequential extraction of substances binding trace elements in plant biomass

This work investigates how the amounts of some important substances in a plant, and their behaviour inside the plant, depend on the levels of stress placed on the plant. To this end, model plant spinach (Spinacia oleracea L.) was cultivated on soil treated with sewage sludge. The sewage sludge contained various trace elements (As, Cd, Cu, Zn), and the uptake of these trace elements placed the plant under stress. Following this, a sequential extraction procedure was employed to determine the levels and distributions of trace elements within the most important groups of compounds present in the spinach plants. Since the usual five-step sequential extraction procedure provides only general information on the distributions of elements within individual groups of organic compounds, due to the wide range of organic compounds within the individual fractions, this scheme was extended and improved through the addition of two solvent extraction steps—a butanol step (between the ethyl acetate and methanol solvent steps) and an H₂O step (after the methanol+H₂O solvent step). The distributions and levels of the trace elements within the main groups of compounds in spinach biomass was investigated using this new seven step sequential extraction (water free solvents: petroleum ether (A) ethyl acetate (B) butanol (C) methanol (D) water solvents: methanol+H₂O (1+1; v/v) (E) H₂O (F) methanol+H₂O+HCl (49.3+49.3+1.4; v/v/v) (G)). The isolated fractions were characterized using IR spectroscopy and the trace element contents were determined in the individual fractions. Lipophilic compounds with low contents of Cd, Cu and Zn were separated in the first two fractions (A, B). Compounds with higher As contents (11.5–12.8% of total content) were also extracted in the second fraction, B. These two fractions formed the smallest portion of the isolated fractions. Low molecular compounds from secondary metabolism and polar lipids were separated in the third (C) and fourth (D) fractions, and high molecular compounds (mainly polypeptides and proteins) separated in the fifth and sixth fractions (E, F). The addition of the H₂O solvent step was particularly useful for separating compounds that have a significant impact on trace element bounds. The methanol fraction was dominant for all treatments, and a significant decrease in the spinach biomass separated in this fraction was observed when the soil was treated with sewage sludge. Most of the As (35.5–38.8% of total content), Cu (45.0–51.6%) and Zn (39.8–47.2%) was also determined in this fraction. The G fraction (obtained after acid hydrolysis) contained polar compounds. Most of the Cd was also found in this fraction, as was a significant amount of Zn. Non-extractable residues formed the last fraction (polysaccharides, proteins).     

Pyrolysis and gas chromatographic examination of phytol and isoprenoid hydrocarbons: Pre-calculation of retention indices by computer

For the identification of isoprenoid-type compounds and other degradation products derived from the pyrolysis of phytol and plant pigments, a calculation method was developed to the evaluate the chromatograms, determining the retention indices or net retention volumes of different compounds. Calculations were made by means of computer for different stationary phases and temperatures. Gas chromatographic and derivatographic measurements were made with natural plant pigments and phytol, including the thermal degradation of the compounds mentioned above. Close agreement was found between the measured and calculated data and with literature data.     

Identification and determination of triterpenoids in Hieracium pilosella L

Fractions of triterpenoids have been isolated from herb, inflorescences, and rhizomes with roots of Hieracium pilosella by typical extraction with petroleum ether. The fraction from inflorescences was investigated using GC-MS techniques. The occurrence of alpha- and beta-amyrin, taraxerol, taraxasterol, and fern-7en-3beta-ol has been observed. Quantitative analysis was also performed and taraxasterol is distinctly predominant in this triterpenoid fraction. Thin-layer chromatography on silica gel was performed on all the investigated fractions and additionally revealed the occurrence of lupeol and psi-taraxasterol in vestigial quantities. All of the triterpenoid components are reported for the first time in the investigated plant.     

Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.     

Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots

The aim of this study was to explore the extract/fractions and compounds of Diospyros lotus against various Gram-positive and Gram-negative bacteria strain. The results showed marked susceptibility of extract and its fractions against test pathogens. Among them, chloroform fraction was most dominant and effective against all tested bacteria. The chloroform fraction was subjected to column chromatography which led to the isolation of lupeol (1), 7-methyljuglone (2), β-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin (6) and 8-hydroxyisodiospyrin (7). Among the isolated compounds, betulinic acid (5) showed significant activity against most of the tested pathogen. In conclusion, our study validated the traditional uses of the plant in the treatment of infectious diseases which was also strongly supported by the isolated compound, betulinic acid (5).     

Phytochemical analysis and in vitro cytotoxic activity of volatiles from Astragalus corniculatus

The volatile fractions from Astragalus corniculatus Bieb., cultivated and collected wild, were analyzed at three different phenological phases for the first time. GC/MS analysis showed that the volatile fractions contain hydrocarbons, butyl ethers, acids, alcohols, esters, aldehydes, ketones, terpenes. These fractions were tested for cytotoxic activity in a panel of human tumor cell lines after 48 h, using the MTT-dye reduction assay. Throughout the cytotoxicity evaluation the fraction derived from the flowering phase of wild type plant was found to exert the most prominent cytotoxic activity, which could be ascribed to the high content of hydrocarbons and squalene in particular. Furthermore, the mechanistic elucidation of the mode of action of this volatile fraction in SKW-3 cells revealed that the observed activity is mediated by induction of necrotic type cell death as evidenced by the smear patterns of DNA following a 24 h exposure period.     

Heats of immersion of titanium dioxide pigments in alcohol—water mixtures

The heats of immersion of partially dried anatase and rutile pigments in mixtures of water with methanol, ethanol, and n-propanol were measured by a differential calorimetric method. The anatase heats of immersion could best be explained by assuming preferential adsorption of the alcohols, the effect being greatest for n-propanol The rutile pigment, however, appeared to adsorb water preferentially in methanol—water and ethanol—water mixtures over the whole concentration range. In propanol—water mixtures the rutile pigment preferentially adsorbed water below an alcohol mol fraction of 0.25, and preferentially adsorbed propanol at mol fractions of alcohol greater than 0.25. The differences in behaviour between the two pigments may be explained qualitatively from the point of view of their surface morphology.     

Changes in free and conjugated polyamines during starvation of sugarcane juices as analyzed by high-performance liquid chromatography

Changes of the polyamines (PAs) titer in high- and mid-molecular-mass carbohydrates (HMMCs and MMMCs, respectively) obtained from sugarcane juices stored for 72 h at pH 5.2 or clarified at pH 8.0 have been studied. Cadaverine (CAD) is the most abundant free (S) PA in the MMMC fraction from juices at pH 5.2, whereas putrescine (PUT) was revealed as the main PA at pH 8.0. A slight increase in the free PUT titer can be noted at pH 5.2 for 72 h of juice starvation. PAs from MMMC were mainly conjugated to acid-insoluble (PH) molecules. Accumulation of PH-PAs with the time of starvation was especially significant for PUT and CAD. However, CAD has also been detected in the acid-soluble (SH) fraction and its concentration increases with the time of starvation at pH 5.2. The accumulation pattern of free and conjugated PAs from HMMCs is similar to that found for MMMCs although some differences can be observed. For instance, the increase in free PUT with the time at pH 8.0 was 2.7-times higher in the HMMC fraction than in the MMMC fraction. Conjugated PAs associated to acid-soluble macromolecules (SH fraction) achieved a level in HMMC fractions higher than that observed in the MMMC fraction. Moreover, the reported increase with time that was observed in PH-CAD from the MMMC fraction was not observed in the HMMC fraction, and, finally, the increase in PH-PUT with the time was lower for the HMMC fraction than for the MMMC fraction.     

Isolation and purification of digalactosyldiacylglycerols

Summary A convenient method for the isolation of digalactosyldiacylglycerols from plant material has been developed using solid phase extraction. Commerical cartridges were utilized to prepare 100 mg fractions of galactolipids containing more than 97% of digalactosyldiacylglycerol. The purity of the fractions was monitored by an isocratic normal phase analytical HPLC procedure, which was optimized using factorial design. The optimized system was successfully scaled up to a semi-preparative HPLC system with a capacity of about 10 mg/injection which was used to further purify the digalactosyldiacylglycerol fraction.     

Separation of pyranoanthocyanins from red wine by column chromatography

With the aim of monitoring the formation of anthocyanin-derived pigments and contributing to the study of their chromatic properties, stability and relative contribution to the colour of red wines, a method for fractionation of the colouring material was set up. The method was based on the distinct reactivity of the different pigment families towards bisulfite (hydrogen sulfite). The wine, acidified and bleached with NaHSO₃, was placed in a Toyopearl^® HW-40(s) gel column and submitted to elution with ethanol. Two fractions with different pigment compositions were collected and analysed by liquid chromatographay diode array detection-mass spectrometry. Compounds present in each fraction were identified according to their UV-visible and MSⁿ mass spectra, showing that the first one was mostly constituted of pyranoanthocyanins, whereas the second basically contained anthocyanins and anthocyanin-flavanol condensation products. A large variety of new pigments were detected, some of which had not been previously reported in red wines, as far as we know. Characteristic MS² and MS³ fragmentation patterns were observed within each family of compounds, which could be further applied for characterisation of unknown pigments in other wines.