The Yariv reagent: behaviour in different solvents and interaction with a gum arabic arabinogalactan-protein.

The ß-D-Glc Yariv reagent is frequently used to isolate and to study the structure of arabinogalactan-proteins with the arabinogalactan type II structure. The present paper describes the aggregation features of the Yariv reagent in water, salt solutions and in organic solvents as determined by NMR, absorption spectroscopy and light scattering experiments. The results indicate that in water the Yariv reagent forms aggregates of up to 300 units and in 1% aqueous NaCl the degree of aggregation is approx. 150. The aggregates are formed both by H-bonds and hydrophobic interactions, the former appearing to be of most importance in water. The interaction between the Yariv reagent and an AGP fraction from gum arabic, showed a degree of aggregation of the Yariv reagent when using 1% NaCl to be of approx. 150 units, whereas disruption of the aggregate took place in 10% NaCl with an aggregation number of approx. 100. Partial acid hydrolysis of an AGP from gum Arabic (Acacia Senegal) and analyses of the linkage types remaining indicated that a certain length of (1→3)-ß-linked galactose units was necessary for binding between the Yariv reagent and the AGP. This is in accordance to what also was recently observed by Kitazawa et al. (2013).

Pectic polysaccharides isolated from Malian medicinal plants protect against Streptococcus pneumoniae in a mouse pneumococcal infection model.

The aim of this study was to investigate whether the pectic polysaccharides BP-II, Oc50A1.I.A and CC1P1 isolated from the Malian medicinal plants Biophytum petersianum, Opilia celtidifolia and Cola cordifolia, respectively, were able to protect against Streptococcus pneumoniae infection in mice. The pectin preparations were administered intraperitoneally 3 h before challenge with S. pneumoniae serotype 6B. Blood samples were obtained from all animals before and at 3 h, 24 h and 72 h after challenge with the pneumococci. The number of bacteria in blood was recorded and the blood concentration of a range of cytokines measured. The pretreatment with BP-II, Oc50A1.I.A and CC1P1 demonstrated a protective activity against S. pneumoniae serotype 6B infection, albeit at different range of concentrations. The pectins showed no direct antibacterial effects towards S. pneumonia; however, they induced the production of a range of cytokines and chemokines. We have previously shown that BP-II, Oc50A1.I.A and CC1P1 exhibit complement fixation activity and also that BP-II and Oc50A1.I.A stimulate macrophages to produce NO. The observed clinical effect might therefore be linked to the ability of the pectic polysaccharides to stimulate the innate immune system.

Structural characterisation of a complex heteroglycan from the cyanobacterium Nostoc commune.

An alkali-extractable O-methylated ribofuranose-containing heteroglycan, Nc-5-s, was isolated from wild-growing field colonies of the cyanobacterium Nostoc commune collected in Iceland, using ethanol fractionation and anion-exchange chromatography. The average molecular weight was estimated to be 1500 kDa. Structural characterisation of the heteroglycan was performed by high-field NMR spectroscopy (1D proton, 2D-COSY, 2D-NOESY, 2D-TOCSY, (1)H (13)C-HSQC, HMBC, H2BC and HSQC-NOESY) as well as monosaccharide analysis after methanolysis by GC and supported by linkage analysis by GC-MS. According to the data obtained, the structure of Nc-5-s is composed of repeating units of 1, 1a, 1b and 2 and 2a in approximate molar ratio of (10:25:50:5:10).

Chemical and biological characterization of polysaccharides from wild and cultivated roots of Vernonia kotschyana.

ETHNOPHARMACOLOGICAL RELEVANCE: In Malian traditional medicine the roots of Vernonia kotschyana are used for treating gastric ulcer and gastritis. In 2006, 9000kg of roots from Vernonia kotschyana were used to produce Gastrosedal, an ameliorated traditional medicine in Mali. Harvesting from the wild, the main source of raw material, is causing a growing concern of diminishing populations of the plant, and Vernonia kotschyana is now being cultivated in several areas around Mali. In the current study the structures and bioactive properties of isolated polysaccharides from wild and cultivated Vernonia kotschyana were compared. MATERIALS AND METHODS: Pectin- and inulin-type polysaccharides were isolated from the roots of cultivated and wild Vernonia kotschyana. The isolated polysaccharides were investigated regarding their chemical compositions, and for their abilities to fixate human complement and activate macrophages from a mouse macrophage cell line. RESULTS: No significant differences in the carbohydrate composition of the fractions isolated from the cultivated versus the wild roots were observed. A previously reported pectic arabinogalactan Vk2a was found in both the cultivated and the wild roots in this study, and exhibited potent complement fixation activity, and a moderate activation of macrophages. CONCLUSIONS: The present study has shown that the cultivated roots of Vernonia kotschyana contain the same types of bioactive polysaccharides as the wild roots. It is therefore preliminarily feasible for the cultivated roots of Vernonia kotschyana to be used as a herbal medicine to replace the wild roots.

The relationship between dose-dependent antitussive and bronchodilatory effects of Opilia celtidifolia polysaccharide and nitric oxide in guinea pigs.

A crude polysaccharide composed of uronic acids (32%), arabinose (26%), glucose (15%), galactose (11%), rhamnose (7%), mannose (5%), xylose (4%) and small amount of fucose residues has been isolated from the leaves of Opilia celtidifolia by boiled water extraction. Chemical analyses of Opilia polysaccharide revealed the prevalence of a pectin material with high arabinose and galacturonic acid contents. Opilia polysaccharide showed significant biological effects on chemically induced cough reflex and reactivity of airways smooth muscle in vitro and in vivo conditions in guinea pigs test system. Tests confirmed the dose-dependent cough-suppressive effect of Opilia polysaccharide comparable with activity of centrally acting codeine. Further, the bronchodilatory tests resulted in significant decrease in the values of specific airway resistance, which is very sensitive predictor of airway smooth muscle reactivity in vivo conditions regardless of bronchoconstricting mechanism. The results of in vitro experiments confirmed not only the bronchodilatory effect Opilia polysaccharide but revealed that its bronchodilatory mechanism is partially accompanied with enhanced NO production.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system.

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Antitussive activity of polysaccharides isolated from the Malian medicinal plants.

From the leaves of popular Malian medicinal plants Trichilia emetica (TE) and Opilia celtidifolia (OC), and fruits of Crossopteryx febrifuga (CF) water and water-ethanol soluble polysaccharide materials were isolated. The results of chemical analysis of the crude polysaccharides showed the dominance of the arabinogalactan ( approximately 54%) and the rhamnogalacturonan ( approximately 30%) in T. emetica leaves, the arabinogalactan ( approximately 60%), the rhamnogalacturonan ( approximately 14%) and the glucuronoxylan ( approximately 14%) in O. celtidifolia leaves, and pectic type of polysaccharides ( approximately 75%) with a lower content of the arabinogalactan ( approximately 17%) in C. febrifuga fruits. The plant polysaccharides showed various biological effects on the citric acid-induced cough reflex and reactivity of airways smooth muscle in vivo conditions. T. emetica and O. celtidifolia polysaccharides possessed significant cough-suppressive effect on chemically induced cough. Furthermore, values of specific airways resistance pointed on bronchodilatory property of polysaccharides isolated from O. celtidifolia. However, the crude extract from C. febrifuga in the same dose as T. emetica and O. celtidifolia did not influence the experimentally induced cough as well as reactivity of airways smooth muscle despite of the fact that the water-ethanol extract is recommended for cough therapy in Mali in the form of syrup.

Screening of Malian medicinal plants for antifungal, larvicidal, molluscicidal, antioxidant and radical scavenging activities.

A total of 78 different extracts from 20 medicinal plants belonging to 14 plant families from Mali were tested for their antifungal, larvicidal, molluscicidal, antioxidant and radical scavenging activities. Dichloromethane, methanol, water and ethanol extracts were used. TLC autobiography for antifungal activity was run with Cladosporium cucumerinum and Candida albicans. Extracts were also tested on the larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Molluscicidal activities were established with the snails Biomphalaria glabrata, Biomphalaria pfeifferi and Bulinus truncatus. beta-Carotene and DPPH solutions sprayed on TLC plates were used for antioxidant and radical scavenging assays. Of the extracts investigated, 20% were antioxidant and radical scavengers, 19% fungicidal, 30% were larvicidal and 11% were molluscicidal. Three of the plant extracts, from Cussonia barteri (Araliaceae), Glinus oppositifolius (Aïzoaceae) and Lannea velutina (Anacardiaceae) gave positive responses in all four tests.

Polysaccharides from the roots of Entada africana Guill. et Perr., Mimosaceae, with complement fixing activity.

Entada africana is a tree used in traditional medicine in Mali. The root is, for example, used for wound-healing. Since polysaccharides from other plants are thought to play a role in the wound-healing process, we wanted to study the polysaccharides present in the root of this species. The polysaccharides were extracted with water at 50 and 100 degrees C and were further separated by anion exchange chromatography. The acidic fractions were finally purified by affinity chromatography on a Con A column. The fraction denoted Ea100 acidic I had the highest activity in the complement fixation test system, while the other acidic fractions had minor activities and the neutral fractions were almost negative. Ea100 acidic I has a structure resembling the arabinogalactan-protein type II polymer, which also was demonstrated by the abilities to precipitate with the Yariv reagent. The biological activity was reduced considerably after removal of arabinofuranoside residues by weak acid hydrolysis. The main core of the other polysaccharides extracted with 100 degrees C were pectins resembling the rhamnogalacturonan type I. These fractions also contain arabinogalactan type II structures, shown by the formation of precipitates with the Yariv reagent.

Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L.

The interaction between a pectin type polysaccharide fraction, PMII, isolated from the leaves of Plantago major, and human complement was tested in two different hemolytic complement-fixation tests and in addition by two ELISA methods detecting complement-activation products. Sera were used as a complement source of 10 arbitrary human volunteers, individually and as a pool. The complement-fixation tests were designed to measure the concentration of the pectin necessary to inhibit 50% of the hemolysis (ICH(50)). The ELISA tests for complement-activation products were measured in AU/mg using a fully activated serum as a standard. We observed a more than 200-fold difference in ICH(50) activity of the PMII pectin in one of the hemolytic tests by varying the individual sera used as complement-source. On the other hand, the ELISA complement-activation tests showed no significant variation in activity of the PMII depending on the complement-serum used. The level of antibodies against PMII detected in the complement-sera did not correlate with the ICH(50) activity of PMII. The results show that PMII is a potent complement activator with an activity of the same order of magnitude on a weight basis as that of aggregated human immunoglobulin (Ig)G. This activation leads to a complement consumption probably explaining the PMII's effect in the complement-fixation tests. PMII seems to be an activator both on the classical and the alternative pathway of activation. The results might be related to the reported wound-healing effect of the leaves of Plantago major.

Protective effect of Plantago major L. Pectin polysaccharide against systemic Streptococcus pneumoniae infection in mice.

The antibacterial effect of a soluble pectin polysaccharide, PMII, isolated from the leaves of Plantago major, was examined in inbred NIH/OlaHsd and Fox Chase SCID mice experimentally infected with Streptococcus pneumoniae serotype 6B. Serotype 6B is known to give a more protracted infection when injected intraperitoneally into susceptible mice than more virulent serotypes like type 4. PMII was administered i.p. either once 3 days before challenge or once to thrice from 3 to 48 h after challenge. The number of bacteria in blood and the mouse survival rate were recorded. Pre-challenge administration of PMII and also lipopolysaccharide (LPS), included as a control, gave a dose-dependent protective effect against S. pneumoniae type 6B infection. However, injection of PMII after establishment of the infection in NIH/OlaHsd mice had no effect. The data demonstrate that, firstly, the polysaccharide fraction PMII from P. major protects against pneumococcal infection in mice when administered systemically prechallenge, and secondly that the protective effect is owing to stimulation of the innate and not the adaptive immune system.

An ethnopharmacological study from thane district, maharashtra, India: traditional knowledge compared with modern biological science.

This paper is a synthesis of local traditional knowledge of plants used as medicine and western biological science. In many areas women are responsible for curing their family members and others for various illness. Ethnomedical uses of 51 plants by women in hamlets/villages of Jawhar and Mokhada talukas in Thane district, Maharashtra, have been recorded. Information from scientific literature for each plant collected has been incorporated with traditional knowledge to explain or substantiate traditional medicinal uses.

An ethnopharmacological study from kulu district, himachal pradesh, India: traditional knowledge compared with modern biological science.

A synthesis of ethnopharmacological knowledge and western biological science has been attempted in this paper. Thirty-four species of plants used by local women in hamlets of Banjar taluka, Kulu district, Himachal Pradesh have been recorded. The knowledge of medicinal plants that local women have is important as they have a lifetime experience in using them through caring for themselves, their families and others around them. For the plants recorded, information from scientific literature has been included in order to explain or justify the traditional medical use.

Rhamnopyranosylgalactofuranan, a new immunologically active polysaccharide from Thamnolia subuliformis.

A complex polysaccharide, Ths-3, consisting mainly of rhamnopyranosyl and galactofuranosyl units, has been isolated from the water extract of the lichen Thamnolia subuliformis using ethanol fractionation, dialysis, ion-exchange chromatography, gel filtration and preparative GP-HPLC. The mean M(r) of Ths-3 was determined to be 1450 kD, and the monosaccharide composition is gal/rha/glc/xyl/man in the ratio of 40:31:13:10:6. The structure of Ths-3 was further elucidated by methylation analysis by GC-MS and NMR spectroscopy and found to be basically composed of (1-->3)-linked beta-D-galactofuranosyl units with branches on C6, and rhamnosyl units being predominantly (1-->2)-linked with branches on C3 and C4, while some units are (1-->3)-linked. Glucose, mannose and galactofuranose are found as terminal units and glucose and mannose are also (1-->4)-linked, while xylose is only present as terminal units. The trisaccharide xylglcglc was detected after partial hydrolysis of the polysaccharide. The immunomodulating activity of Ths-3 was tested in an in vitro phagocytosis assay and the classical anticomplementary assay, and proved to be active in both tests. The authors suggest the trivial name thamnolan for Ths-3.

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.

Immunologically active (1-->3)-(1-->4)-alpha-D-glucan from Cetraria islandica.

A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.

Comparison of IgE-binding antigens in horse dander and a mixture of horse hair and skin scrapings.

Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEF, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins. The most important allergen was a glycoprotein with molecular mass of 27 kDa showing IgE-binding ability to the 16 patient sera available. Another important allergen with molecular mass of 67 kDa is probably horse albumin. IEF/immunoblotting showed the presence of a glycoprotein with IgE-binding ability at pH 3.2 in both extracts. Tandem CRIE showed that both extracts contained the same four allergens as major allergens. In addition, as we observed when analyzing both extracts in CRIE against 16 different patient sera, each extract contained allergens not present in the other extract. We may conclude from this investigation that commercial allergen extracts to be used in diagnosis and immunotherapy should include both horse dander and horse hair and skin scrapings in the starting material for the preparation of extracts. If not, allergens which may be of importance for some patients will be missing.

Cross-reactivity between pollen extracts from six artemisia species.

Pollen extracts of six different ARTEMISIA species, A. VULGARIS, A. SCOPARIA, A. PRINCEPS, A. TRIDENTATA, A. ANNUA, and A. CAMPESTRIS were compared using SDS-PAGE, IEF, immunoblotting, and immunoelectrophoretic methods. The band patterns obtained after SDS-PAGE and IEF showed a large degree of similarity between the extracts. Immunoblotting of these gels using a pool of sera from patients allergic to A. VULGARIS gave essentially the same IgE-binding band pattern with all the extracts, demonstrating an extensive degree of cross-reactivity between A. VULGARIS and the other ARTEMISIA species. FRIE using a polyspecific antiserum against A. VULGARIS showed that all the extracts contained several antigens that were immunologically identical to antigens in A. VULGARIS extract. Antigens showing immunological identity to the important A. VULGARIS allergens Ag 12 and ART V II were present in all the extracts. The cross-reactivity between A. VULGARIS and A. PRINCEPS was further verified by screening of ten Korean and nine Norwegian individual patient sera against extracts of both species in SDS-PAGE or IEF immunoblotting. Both groups of patients had essentially the same pattern of reactivity towards both pollen extracts.

Increase in thebaine content of Papaver bracteatum Lindl. after colchicine treatment of seeds. Annual fluctuations in thebaine level of individual plants.

From colchicine-treated seeds of Papaver bracteatum Lindl. some poppy plants were obtained that developed capsules richer in thebaine than the controls. The individual poppies were analyzed for capsule thebaine content annually for eight successive years, the results revealing significant year-to-year differences. One of the poppies, X7, (ca 5% thebaine) developed capsules consisting partly of polyploid tissue during the first and second year. This plant was propagated vegetatively to give a series (the X7 series) of new P. bracteatum plants. The capsule thebaine content of these individuals differed markedly the first two years, whereupon the alkaloid production decreased and appeared to level out and reach a value still clearly higher than the controls (mean values 2.3% and 1.3%, respectively). From seeds of four of the thebaine-rich poppies of the X7 series, four new series of P. bracteatum plants were obtained. The capsule thebaine level of these was significantly lower than that of the mother plants.