Natural Products as Lead Compounds for Sodium Glucose Cotransporter (SGLT) Inhibitors.

Glucose homeostasis is maintained by antagonistic hormones such as insulin and glucagon as well as by regulation of glucose absorption, gluconeogenesis, biosynthesis and mobilization of glycogen, glucose consumption in all tissues and glomerular filtration, and reabsorption of glucose in the kidneys. Glucose enters or leaves cells mainly with the help of two membrane integrated transporters belonging either to the family of facilitative glucose transporters (GLUTs) or to the family of sodium glucose cotransporters (SGLTs). The intestinal glucose absorption by endothelial cells is managed by SGLT1, the transfer from them to the blood by GLUT2. In the kidney SGLT2 and SGLT1 are responsible for reabsorption of filtered glucose from the primary urine, and GLUT2 and GLUT1 enable the transport of glucose from epithelial cells back into the blood stream.The flavonoid phlorizin was isolated from the bark of apple trees and shown to cause glucosuria. Phlorizin is an inhibitor of SGLT1 and SGLT2. With phlorizin as lead compound, specific inhibitors of SGLT2 were developed in the last decade and some of them have been approved for treatment mainly of type 2 diabetes. Inhibition of SGLT2 eliminates excess glucose via the urine. In recent times, the dual SGLT1/SGLT2 inhibitory activity of phlorizin has served as a model for the development and testing of new drugs exhibiting both activities.Besides phlorizin, also some other flavonoids and especially flavonoid enriched plant extracts have been investigated for their potency to reduce postprandial blood glucose levels which can be helpful in the prevention and supplementary treatment especially of type 2 diabetes.

Arabinogalactan-proteins stimulate somatic embryogenesis and plant propagation of Pelargonium sidoides.

Root extracts of the medicinal plant Pelargonium sidoides, native to South Africa, are used globally for the treatment of common cold and cough. Due to an increasing economic commercialization of P. sidoides remedies, wild collections of root material should be accompanied by effective methods for plant propagation like somatic embryogenesis. Based on this, the influence of arabinogalactan-proteins (AGPs) on somatic embryogenesis and plant propagation of P. sidoides has been investigated. High-molecular weight AGPs have been isolated from dried roots as well as from cell cultures of P. sidoides with yields between 0.1% and 0.9%, respectively. AGPs are characterized by a 1,3-linked Galp backbone, branched at C6 to 1,6-linked Galp side chains terminated by Araf and to a minor extent by GlcpA, Galp or Rhap. Treatment of explants of P. sidoides with AGPs from roots or suspension culture over 5.5 weeks resulted in effective stimulation of somatic embryo development and plant regeneration.

Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis.

The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure.

A sulfated cyanobacterial polysaccharide proven as a strong inhibitor of human complement activity in an in vitro assay.

Cyanobacterial exopolysaccharides are a rich source of, so far, widely unexplored polysaccharides. One of these exopolysaccharides is a highly sulfated, linear polysaccharide from Synechocystis aquatilis containing the amino sugar N-acetyl-fucosamine. Some sulfated polysaccharides and glycosaminoglycans are known to be inhibitors of the human complement system, which is an important part of the innate immune system. Defects in this system or misregulation can cause serious diseases. Therefore, new compounds with complement inhibiting activity and simple test assays are of great interest. Exopolysaccharides from S. aquatilis (arabinofucans) were compared to those from Synechocystis pevalekii (complex heteropolysaccharides) and the well-known complement inhibitor heparin. Investigations were performed with a modified ELISA test system based on a commercially available test kit quantifying the membrane attack complex. Hereby the testing becomes more stable, robust, reproducible, easier to handle and, for the first time, the effect of exopolysaccharides and heparin on the lectin pathway could be tested. The exopolysaccharides from S. aquatilis could be shown to be a 30 times stronger inhibitor of the classical pathway of the complement system compared to heparin (IC50 = 0.3 µg/mL vs. 9.2 µg/mL). These exopolysaccharides are also inhibitors of the lectin pathway (IC50 = 10.8 µg/mL) in which, however, heparin is more potent (IC50 = 2.0 µg/mL). Interestingly, these exopolysaccharides do not inhibit the alternative pathway. The exopolysaccharides from S. pevalekii are inactive in all pathways. Furthermore, partially hydrolyzed and desulfated exopolysaccharides from S. aquatilis were tested showing that a minimum molecular size and degree of sulfation are important for the inhibitory effects, whereas unspecific influences by complex formation of exopolysaccharides with calcium could be excluded.

Inhibition of the intestinal sodium-coupled glucose transporter 1 (SGLT1) by extracts and polyphenols from apple reduces postprandial blood glucose levels in mice and humans.

SCOPE: There is a growing interest in food constituents that could reduce intestinal glucose absorption to prevent overshooting plasma glucose and insulin levels in patients with prediabetes and diabetes mellitus type 2. METHODS AND RESULTS: We here demonstrate that an extract and individual polyphenols from apple diminish sodium-coupled glucose transporter 1 (SGLT1) mediated glucose uptake in vitro and in vivo. Inhibition of transport of sugars by SGLT1 was shown in Xenopus oocytes and in mice jejunal segments. Strongest inhibition was observed for phlorizin with IC50 values for transport inhibition of 0.46 ± 0.19 and 4.1 ± 0.6 µM in oocytes and intestinal segments, respectively. An oral glucose tolerance test performed in volunteers with prior administration of the apple extract reduced venous blood glucose and plasma insulin levels, similar to findings obtained in C57BL/6N mice. Analysis of human urine samples revealed that the extract increased modestly renal glucose loss that is most likely a result of inhibition of renal glucose reabsorption by phloretin derivatives found in plasma of the volunteers. CONCLUSION: Although the apple extract substantially decreased intestinal glucose absorption in all test systems, the finding that there are systemic effects that relate to inhibition of glucose transport processes beyond the intestine addresses safety issues that need further exploitation.

Exopolysaccharides of Synechocystis aquatilis are sulfated arabinofucans containing N-acetyl-fucosamine.

Cyanobacteria are known to be a rather diverse group of organisms regarding e.g. morphology, metabolism and composition of excreted exopolysaccharides (EPS). Considering the high number of known cyanobacterial species the EPS from only a small percentage are investigated in detail. This work examined EPS from the unicellular strains of Synechocystis aquatilis and S. pevalekii with various methods. The results emphasize the heterogeneity of cyanobacterial EPS. S. pevalekii secrets complex hetero-polysaccharides and acidic proteins as proteoglycan-complexes whereas the protein-free but highly sulfated EPS from S. aquatilis only consist of 4 dominant monosaccharides. Especially remarkable is the composition of these EPS: an arabinofucan with higher amounts of N-acetyl-fucosamine (FucNAc) and only minor quantities of glucose. Both EPS and the newly found component FucNAc in EPS from S. aquatilis extend the possible components of cyanobacterial EPS and the knowledge of heterogeneity of cyanobacterial metabolites.

Structural investigations on arabinogalactan-protein from wheat, isolated with Yariv reagent.

For the first time a pure arabinogalactan-protein (AGP) could be isolated from whole grain of wheat ( Triticum aestivum L.) by performing a double precipitation with beta-glucosyl Yariv reagent. The putative bioactive AGP has been characterized with regard to its polysaccharide and protein parts. Analytical investigations by GLC-MS and (13)C NMR revealed a carbohydrate moiety consisting of a 1,3-Galp backbone, linked in position 6 to short 1,6-Galp-chains, terminating in Araf. In the protein part, a high content of hydroxyproline has been found, probably responsible for linkage between protein and polysaccharide moieties. The molecular mass of AGP has been determined by size exclusion chromatography with laser light scattering detection and found to be 125 kDa. Alkaline hydrolysis of the protein resulted in single carbohydrate moieties with a molecular mass of about 20 kDa, indicating that AGP from whole grain of wheat belongs to the wattle blossom type of AGPs.

Characterization and immunolocalization of arabinogalactan-proteins in roots of Echinacea purpurea.

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.

Ochratoxin a lowers mRNA levels of genes encoding for key proteins of liver cell metabolism.

Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 micromol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 micromol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 micromol/l OTA. A particularly strong down-regulation due to 2.5 micromol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism.

Effect of drying on extruded pellets based on kappa-carrageenan.

kappa-Carrageenan is a new pelletisation aid for the wet extrusion/spheronisation process. Its promising behavior during pelletisation offers a potential replacement of the commonly used microcrystalline cellulose in several formulations. Due to its high water binding, the drying step of the pelletisation has gained increasing attention. Therefore, a systematic investigation of the effect of drying conditions on pellet properties was the aim of this study. Pellets with different formulations were dried in fluid bed, circulation-, and vacuum oven and then characterized by mean dissolution time, tensile strength and disintegration time. Increasing the temperature and duration of the drying process decreased the mean dissolution time, tensile strength and disintegration time of the pellets. The observed effects were attributed to a cautious decomposition of the kappa-carrageenan above 70 degrees C. Furthermore an ionic interaction between calcium ions of dicalciumphosphate and sulfate ester groups of kappa-carrageenan affected the pellet properties like drug release.

Complement modulating and anticoagulant effects of a sulfated exopolysaccharide released by the cyanobacterium Synechocystis aquatilis.

During batch cultivation, the cyanobacterial strain Synechocystis aquatilis Sauvageau B90.79 was found to release a fucose-rich sulfated polysaccharide, which was mainly composed of arabinose (45%) and fucose (47%) and had a degree of sulfation of 0.43. The released polysaccharide showed merely an anticoagulant activity of less than 10% compared to the reference compound, unfractionated heparin. However, distinctive effects on the complement activation were observed: its inhibitory effect on the classical pathway of complement activation was 600-fold stronger than that of unfractionated heparin, whereas that on the alternative pathway of complement activation was 2- to 3-fold weaker. The results indicate that this biotechnologically producible, released polysaccharide represents a specifically acting complement modulator.

Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots.

Highly water soluble fructans have been isolated from Echinacea purpurea (L.) Moench. roots by hot water extraction and precipitation at three different ethanol concentrations (80% v/v, 60% v/v and 40% v/v). The structure of the fructans has been characterised by three analytical methods: GC of silylated oxime derivatives and partially methylated alditol acetates, respectively, as well as 13C NMR analysis. The mean degree of polymerisation (mean DP) of each fructan has been determined by the glucose/fructose ratio. E. purpurea fructans represent linear inulin-type fructans with almost exclusively beta-(2-->1)-linked fructosyl units, terminal glucose and terminal fructose. Small proportions of beta-(2-->1,2-->6)-linked branch point residues were detected. The mean DP of the fructan fractions depends on the ethanol concentration used for precipitation: the lower the ethanol concentration the higher the mean DP. Corresponding results were found with all of the three analytical methods: 80% ethanol-insoluble fructan from E. purpurea shows an average mean DP of 35, 60% ethanol-insoluble fructan of 44 and 40% ethanol-insoluble fructan of 55. The applied methods provide sufficient sensitivity to determine not only the composition and structure but also the mean degree of polymerisation of fructans.

An acidic arabinogalactan-protein from the roots of Baptisia tinctoria.

An acidic arabinogalactan-protein (AGP) isolated from an aqueous extract of the roots of wild indigo [ Baptisia tinctoria (L.) R. Br.] by precipitation with beta-glucosyl Yariv reagent consists of L-arabinose (34.0 %) and D-galactose (58.7 %) (1 : 1.7), minor amounts of D-glucuronic acid (4.0 %) and traces of D-rhamnose (2.3 %) and D-glucose (<1 %). The protein part of the AGP mainly contains asparagine/aspartic acid (11.3 %), glutamine/glutamic acid (10.8 %), alanine (8.0 %), serine (8.0 %), leucine (7.0 %) and hydroxyproline (6.3 %). Methylation analysis revealed that the carbohydrate moiety of the glycoprotein has a highly branched structure. The core consists of 3-linked beta- D-galactopyranose units carrying side chains of 6-linked beta- D-galactopyranose in position C(O)6, partly substituted in position C(O)3 by side chains of 5- and 3-linked alpha- L-arabinofuranosyl residues and 4-linked beta- D-galactopyranose units. Galactose and arabinose as well as glucose, rhamnose and glucuronic acid occur in terminal positions. The presence of these glycosyl linkage types was confirmed by (13)C-NMR data. The AGP was separated into two fractions by SEC. The major peak corresponded to a hydrodynamic volume of 5.6 x 10 (4) Da (AGP-F2) and the minor peak to a hydrodynamic volume of 1.2 x 10 (6) Da (AGP-F1). MALLS revealed apparent weight average molecular masses of 1.4 x 10 (5) Da and >1.0 x 10 (7) Da, respectively. Both fractions show corresponding carbohydrate compositions and structural features with regard to the carbohydrate moiety. Mild acid hydrolysis of the AGP leads to loss of terminal arabinofuranosyl units. The residual galactan backbone does not react with the Yariv reagent in gel diffusion tests while the total AGP still interferes with the reagent following reduction of terminal glucuronic acid residues.

In vitro anti-Helicobacter pylori activity of Extractum liquiritiae, glycyrrhizin and its metabolites.

OBJECTIVES: To investigate the in vitro activity of Extractum liquiritiae (EL), glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and a novel lipophilic derivative of glycyrrhetinic acid monoglucuronide (GAMG), acetylated GAMG (aGAMG), against 29 Helicobacter pylori strains. METHODS: The MIC of each compound was determined by the agar dilution method, and the killing kinetics were monitored in brain heart infusion broth ( approximately 10(6)-10(7) cfu/mL) at 0, 4, 24, 48, 72 and 96 h. RESULTS: GA was the most potent compound (MIC(50 /90), 50/100 mg/L), inhibiting 79.3% of the strains at MIC < or =50 mg/L. Clarithromycin-resistant strains were susceptible at 12.5 and 25 mg/L, and metronidazole-resistant strains at 25-50 and at 200 mg/L. The MIC distribution (mg/L) of aGAMG was < or =6.25 (29.2%), 50 (4.2%), 100-200 (12.5%) and > or =400 (54.1%). EL and GL were less active (MICs >400 mg/L). GA exhibited rapid, concentration and strain-dependent bactericidal activity. CONCLUSIONS: The potent in vitro activity of GA against H. pylori provides a further explanation for its beneficial effect on peptic ulcers. Its effectiveness against clarithromycin-resistant strains provides hope that it can form the basis for an alternative therapeutic agent against H. pylori.

Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea. Abbreviations. AGP:arabinogalactan-protein AGP-hydr.:hydrolysed arabinogalactan-protein AP-CA:haemolytic complement assay for the alternative pathway CP-CA:haemolytic complement assay for the classical pathway EGTA-VB:veronal buffered saline containing EGTA and Mg 2+HPS:human pooled serum RT:room temperature LPS:lipopolysaccharide RaE:rabbit erythrocytes RT:room temperature ShE(A):(sensitised) sheep erythrocytes VB:veronal buffered saline containing Ca 2+ and Mg 2+

An arabinogalactan-protein from cell culture of Malva sylvestris.

An arabinogalactan-protein (AGP) from suspension culture medium of Malva sylvestris was isolated by precipitation with beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of AGPs: a high amount of polysaccharide with a ratio of galactose to arabinose of 1.9 : 1, some uronic acids, and a small protein moiety with the main amino acids serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.3 x 10(6) Da. Linkage analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and Galp. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule and heavily reduced its reactivity with beta-glucosyl Yariv antigen.