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.

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.

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.

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.

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting.

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.

Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.).

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).

Isolation and characterization of a glycoprotein allergen, Art v II, from pollen of mugwort (Artemisia vulgaris L.).

A glycoprotein allergen, Art v II, was isolated from pollen of mugwort by two different isolation procedures. Art v II-A was isolated by a combination of ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Con A-Sepharose and ion-exchange chromatography on Mono P. Art v II-B was isolated by a combination of preparative IEF in Ultrodex granulated gel, affinity chromatography on Con A-Sepharose and HPLC size exclusion on Ultropac TSK G2000SW. Art v II-A and Art v II-B were shown to be antigenically identical with the allergen we have formerly denoted Ag7. The MW of Art v II A/B was determined to be 34,000-38,000 by HPLC size exclusion, and 35,000 and 20,000 by SDS-PAGE under non-reducing and reducing conditions, respectively. Art v II was found to consist of 6(7) isoforms with pI 4.10, 4.20 (major component), 4.35, 4.45, 4.55, 4.65, (4.80). The glycoprotein allergen had a protein to carbohydrate ratio of 10:1 and the carbohydrate part contained mannose (70.7%), N-acetyl-glucosamine (17.0%), glucose (7.0%) and galactose (5.3%). In R(R)IE the purified allergen bound IgE from 5 (33%) of 15 sera from patients with clinical allergy against mugwort pollen and from 13 (52%) of 25 sera from patients selected only on the basis of a RAST-class 4 against mugwort pollen.

Allergens in pollen from mugwort (Artemisia vulgaris L.). II. Characterization of a crude and a partly purified extract of mugwort pollen with particular emphasis on the glycoprotein allergen Ag7.

A crude and a partly purified extract of mugwort pollen were characterized with particular emphasis on the glycoprotein allergen Ag7. Crossed immunoaffinoelectrophoresis with immobilized Con A in the intermediate gel demonstrated binding of Ag7 to this lectin, indicating that Ag7 contains terminal alpha-D-mannopyranosyl and/or alpha-D-glucopyranosyl residues. Crossed immunoaffinoelectrophoresis with free Con A in the first-dimension gel demonstrated microheterogeneity in the carbohydrate moiety of the allergen. Crossed radioimmunoelectrophoresis analysis using sera from 26 mugwort-allergic patients indicated that Ag7 is an important allergen in mugwort pollen. Heat stability experiments demonstrated the presence of at least three heat-resistant allergens, one of these being Ag7. Crossed immunoelectrofocusing indicated a pI value for Ag7 of 4.3.

Purification of a basic glycoprotein allergen from pollen of timothy by high-performance liquid chromatography.

The use of high-performance ion-exchange and size-exclusion chromatography in the purification of the basic timothy pollen allergen antigen 30 (Ag 30) was investigated. The most efficient purification was achieved when an initial purification step on a CM-Sepharose CL-6B column was followed by chromatography on Mono S and TSK G 2000 SW columns. This procedure was highly reproducible and well suited for semi-preparative scale purification of the allergen. The purified allergen gave one band on isoelectric focusing, corresponding to a pI of 9.30. On fused rocket immunoelectrophoresis a single precipitate was obtained that coincided with the allergenic activity.

Glycoprotein allergens in pollen of timothy. II. Isolation and characterization of a basic glycoprotein allergen.

A basic glycoprotein allergen has been isolated from pollen of timothy by a combination of chromatography on columns of CM-Sepharose CL-6B, Bio-Gel P-30, DEAE-Sepharose CL-6B and Ultrogel AcA 44. The allergen which is immunologically identical with the allergen formerly denoted as antigen 30 had an isoelectric point of 9.45. The MW of the glycoprotein was found to be 54,000 and 38,000 by SDS-PAGE and gel filtration, respectively. The allergen contains arabinose (3.1%), fucose (0.7%), xylose (0.7%), mannose (2.1%), galactose (2.8%) and glucose (0.2%). In rocket radioimmunoelectrophoresis the allergen bound IgE from 57 (78%) of 73 patient sera tested. The allergen did not bind Yariv artificial galactosyl- and glucosyl-carbohydrate-binding antigens or a monoclonal antibody against ryegrass pollen glycoprotein allergens.