Suppression of the tonoplast sugar transporter, StTST3.1, affects transitory starch turnover and plant growth in potato.

Transitory starch and vacuolar sugars function as highly dynamic pools of instantly accessible metabolites in plant leaf cells. Their metabolic regulation is critical for plant survival. The tonoplast sugar transporters (TSTs), responsible for sugar uptake into vacuoles, regulate cellular sugar partitioning and vacuolar sugar accumulation. However, whether TSTs are involved in leaf transient starch turnover and plant growth is unclear. Here, we found that suppressing StTST3.1 resulted in growth retardation and pale green leaves in potato plants. StTST3.1-silenced plants displayed abnormal chloroplasts and impaired photosynthetic performance. The subcellular localization assay and the oscillation expression patterns revealed that StTST3.1 encoded a tonoplast-localized protein and responded to photoperiod. Moreover, RNA-seq analyses identified that starch synthase (SS2 and SS6) and glucan water, dikinase (GWD), were downregulated in StTST3.1-silenced lines. Correspondingly, the capacity for starch synthesis and degradation was decreased in StTST3.1-silenced lines. Surprisingly, StTST3.1-silenced leaves accumulated exceptionally high levels of maltose but low levels of sucrose and hexose. Additionally, chlorophyll content was reduced in StTST3.1-silenced leaves. Analysis of chlorophyll metabolic pathways found that Non-Yellow Coloring 1 (NYC1)-like (NOL), encoding a chloroplast-localized key enzyme that catalyzes the initial step of chlorophyll b degradation, was upregulated in StTST3.1-silenced leaves. Transient overexpression of StNOL accelerated chlorophyll b degradation in tobacco leaves. Our results indicated that StTST3.1 is involved in transitory starch turnover and chlorophyll metabolism, thereby playing a critical role in normal potato plant growth.

Cytosolic disproportionating enzyme2 is essential for pollen germination and pollen tube elongation in rice.

Degradation of starch accumulated in pollen provides energy and cellular materials for pollen germination and pollen tube elongation. Little is known about the function of cytosolic disproportionating enzyme2 (DPE2) in rice (Oryza sativa). Here, we obtained several DPE2 knockout mutant (dpe2) lines via genomic editing and found that the mutants grew and developed normally but with greatly reduced seed-setting rates. Reciprocal crosses between dpe2 and wild-type plants demonstrated that the mutant was male sterile. In vitro and in vivo examinations revealed that the pollen of the dpe2 mutant developed and matured normally but was defective in germination and elongation. DPE2 deficiency increased maltose content in pollen, whereas it reduced the levels of starch, glucose, fructose, and adenosine triphosphate (ATP). Exogenous supply of glucose or ATP to the germination medium partially rescued the pollen germination defects of dpe2. The expression of cytosolic phosphorylase2 (Pho2) increased significantly in dpe2 pollen. Knockout of Pho2 resulted in a semi-sterile phenotype. We failed to obtain homozygous dpe2 pho2 double mutant lines. Our results demonstrate that maltose catalyzed by DPE2 to glucose is the main energy source for pollen germination and pollen tube elongation, while Pho2 might partially compensate for deficiency of DPE2.

Microencapsulation by spray drying of coffee epiphytic yeasts Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684.

The objective of this work was to evaluate the microencapsulation feasibility of Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684 in three different compositions of wall material by spray-dryer. The yeasts (109 CFU mL-1) were microencapsulated separately using maltodextrin (15%), maltodextrin (15%) with sucrose (2%), or maltose (2%) as wall material. The viability was evaluated for 6 months at two different temperatures (7 and 25 °C). The yield, cell viability after spray drying, and characterization of the microcapsules were performed. Results indicate that cell viability ranged between 94.06 and 97.97%. After 6 months, both yeasts stored at 7 °C and 25 °C presented 107 and 102 CFU mL-1, respectively. Regarding Fourier-transform infrared spectroscopy analysis, all microencapsulated yeasts presented typical spectra footprints of maltodextrin. After 6 months of storage, S. cerevisiae CCMA 0543 obtained a 10.8% increase in cell viability using maltodextrin with maltose as wall material compared to maltodextrin and maltodextrin with sucrose. However, T. delbrueckii CCMA 0684 obtained a 13.5% increase in cell viability using only maltodextrin. The study showed that maltodextrin as a wall material was efficient in the microencapsulation of yeasts. It is possible to assume that maltose incorporation increased the cell viability of S. cerevisiae CCMA 0543 during storage.

Personal Glucose Meter for α-Glucosidase Inhibitor Screening Based on the Hydrolysis of Maltose.

As a key enzyme regulating postprandial blood glucose, α-Glucosidase is considered to be an effective target for the treatment of diabetes mellitus. In this study, a simple, rapid, and effective method for enzyme inhibitors screening assay was established based on α-glucosidase catalyzes reactions in a personal glucose meter (PGM). α-glucosidase catalyzes the hydrolysis of maltose to produce glucose, which triggers the reduction of ferricyanide (K3[Fe(CN)6]) to ferrocyanide (K4[Fe(CN)6]) and generates the PGM detectable signals. When the α-glucosidase inhibitor (such as acarbose) is added, the yield of glucose and the readout of PGM decreased accordingly. This method can achieve the direct determination of α-glucosidase activity by the PGM as simple as the blood glucose tests. Under the optimal experimental conditions, the developed method was applied to evaluate the inhibitory activity of thirty-four small-molecule compounds and eighteen medicinal plants extracts on α-glucosidase. The results exhibit that lithospermic acid (52.5 ± 3.0%) and protocatechualdehyde (36.8 ± 2.8%) have higher inhibitory activity than that of positive control acarbose (31.5 ± 2.5%) at the same final concentration of 5.0 mM. Besides, the lemon extract has a good inhibitory effect on α-glucosidase with a percentage of inhibition of 43.3 ± 3.5%. Finally, the binding sites and modes of four active small-molecule compounds to α-glucosidase were investigated by molecular docking analysis. These results indicate that the PGM method is feasible to screening inhibitors from natural products with simple and rapid operations.

Three flavanols delay starch digestion by inhibiting α-amylase and binding with starch.

The study aimed to reveal the different mechanisms of delaying starch digestion by ECG, EGCG and Procyanidin based on the perspective of α-amylase-flavanol interaction and starch-flavanol interaction. The interaction characteristics of flavanols with α-amylase were studied from five aspects: enzyme inhibition, kinetics, fluorescence quenching, circular dichroism (CD) and computer simulation. The IC50 of flavanols (ECG, EGCG and Procyanidin) against α-amylase were 172.21 ± 0.22, 732.15 ± 0.13 and 504.45 ± 0.19 µg/mL according to the results of α-amylase inhibition experiment, respectively. ECG and Procyanidin showed mixed inhibition against α-amylase, while EGCG showed non-competition against α-amylase. However, thermodynamic parameters，computer-based docking and dynamic simulation proved that ECG and EGCG-α-amylase complexs were mainly driven by van der Waals and hydrogen bonds, while Procyanidin-α-amylase complexs was driven by hydrophobic interaction. In addition, it was indicated, by means of starch­iodine complex spectroscopy, that flavanols inhibited the digestion of starch not only through bind with α-amylase but also through bind with starch. Thus, flavanols as a starch-based food additive have the potential to be employed as adjuvant therapy for diabetes.

Antioxidant Function and Metabolomics Study in Mice after Dietary Supplementation with Methionine.

The antioxidant function and metabolic profiles in mice after dietary supplementation with methionine were investigated. The results showed that methionine supplementation enhanced liver GSH-Px activity and upregulated Gpx1 expression in the liver and SOD1 and Gpx4 expressions in the jejunum. Nrf2/Keap1 is involved in oxidative stress, and the western blotting data exhibited that dietary methionine markedly increased Keap1 abundance, while failed to influence the Nrf2 signal. Metabolomics investigation showed that methionine administration increased 2-hydroxypyridine, salicin, and asparagine and reduced D-Talose, maltose, aminoisobutyric acid, and inosine 5'-monophosphate in the liver, which are widely reported to involve in oxidative stress, lipid metabolism, and nucleotides generation. In conclusion, our study provides insights into antioxidant function and liver metabolic profiles in response to dietary supplementation with methionine.

Applicability and reliability of the glucose oxidase method in assessing α-amylase activity.

Glucose oxidase (GOD) is an enzyme widely used in glucose monitoring systems owing to its high specificity towards glucose. However, in our previous work maltose was found to show significant interaction with GOD and based on this observation, a novel microplate-based method was developed to assess α-amylase inhibitory activity (GOD method). Concerns regarding the interaction of GOD with maltose has limited the widespread use of the GOD method in assessing α-amylase activity. The present paper provides answers to concerns regarding the interaction of GOD with maltose using HPLC studies and application of the GOD method in assessing α-amylase activity. According to the results, the newly developed GOD method can be considered as a well-suited method for the determination of α-amylase activity and as an easy method to do kinetic studies compared to other available methods.

Enzymatic Glucosylation of Salidroside from Starch by α-Amylase.

α-Amylases are among the most important and widely used industrial enzymes for starch processing. In this work, an α-amylase from Bacillus subtilis XL8 was purified and found to possess both hydrolysis and transglycosylation activities. The optimal pH and temperature for starch hydrolysis were pH 5.0 and 70 °C, respectively. The enzyme could degrade soluble starch into beneficial malto-oligosaccharides ranging from dimer to hexamer. More importantly, it was able to catalyze α-glycosyl transfer from the soluble starch to salidroside, a medicinal plant-derived component with broad pharmacological properties. The transglycosylation reaction catalyzed by the enzyme generated six derivatives in a total high yield of 73.4% when incubating with 100 mg/mL soluble starch and 50 mM salidroside (pH 7.5) at 50 °C for 2 h. These derivatives were identified as α-1,4-glucosyl, maltosyl, maltotriosyl, maltotetraosyl, maltopentaosyl, and maltohexaosyl salidrosides, respectively. They were novel promising compounds that might integrate the bioactive functions of malto-oligosaccharides and salidroside.

Lectin Isolated from Japanese Red Sword Beans (Canavalia gladiata) as a Potential Cancer Chemopreventive Agent.

In this study, we investigated the chemical and biological profile of lectin isolated from Japanese red sword beans (Canavalia gladiata; RSBs). RSB lectin was purified using maltamyl-Sepharose 4B and subjected to amino acid composition and partial amino acid sequencing analyses, and evaluated for blood and carbohydrate specificity, mitogenic activity, splenic natural killer (NK) cell activity, and its effect on B16 melanoma cell proliferation, compared with Concanavalin A (Con A). The amino acid composition and sequences of RSB lectin were similar to those of Con A. RSB lectin showed specificity to mannose, glucose, maltose, methyl-D-mannoside, and thyroglobulin, but not rhamnose, using mouse, sheep, and rabbit erythrocytes. Compared with Con A, RSB lectin showed low resistance to proteases and to temperatures greater than 70 °C, but high mitogenic activity for mouse splenic cells. Notably, while treatment with RSB lectin and Con A (0.01 and 0.1 µg/mL) promoted similar levels of splenic NK cell activity, which were higher than that observed in the control (0 µg/mL) and interleukin 2 (IL-2) (25 U)-treated populations, RBS lectin exerted a significantly stronger anti-proliferative effect than Con A at a concentration of 125.0 µg per well. Overall, our results show that RSB lectin might exert immunological effects on mouse splenic cells and could thus be used as a potential cancer chemopreventive agent. PRACTICAL APPLICATION: Japanese red sword bean (RSB) is a tropical perennial legume consumed in many Asian countries. RSB lectin shows specificity to mannose, glucose, maltose, methyl-d-mannoside, and thyroglobulin, but not to rhamnose, using mouse, sheep, and rabbit erythrocytes. RSB lectin exhibits similarities to Concanavalin A in amino acid composition and sequence, shows mitogenic activity for mouse splenic cells and strong anti-proliferative activity for B16 melanoma cells, and also enhances the activity of splenic natural killer (NK) cells against YAC-1 cells. Thus, RSB lectin has the potential to be used as a bioactive protein in medical research.

Changes in physicochemical properties and in vitro starch digestion of native and extruded maize flours subjected to branching enzyme and maltogenic α-amylase treatment.

Extrusion is an increasingly used type of processing which combined with enzymatic action could open extended possibilities for obtaining clean label modified flours. In this study, native and extruded maize flours were modified using branching enzyme (B) and a combination of branching enzyme and maltogenic α-amylase (BMA) in order to modulate their hydrolysis properties. The microstructure, pasting properties, in vitro starch hydrolysis and resistant starch content of the flours were investigated. Whereas BMA treatment led to greater number of holes on the granule surface in native samples, B and BMA extruded samples showed rougher surfaces with cavities. A reduction in the retrogradation trend was observed for B and BMA native flours, in opposition to the flat pasting profile of their extruded counterparts. The glucose release increased gradually for native flours as the time of reaction did, whereas for extruded flours a fast increase of glucose release was observed during the first minutes of reaction, and kept till the end, indicating a greater accessibility to their porous structure. These results suggested that, in enzymatically treated extruded samples, changes produced at larger hierarchical levels in their starch structure could have masked a slowdown in the starch digestion properties.

Functional characterization of a novel disaccharide transporter in lobster hepatopancreas.

In animals, the accepted model of carbohydrate digestion and absorption involves reduction of disaccharides into the monosaccharides glucose, fructose, and galactose followed by their individual transmembrane transport into cells. In 2011, a gene for a distinct disaccharide sucrose transporter (SCRT) was found in Drosophila melanogaster and characterized in a yeast expression system. The purpose of the present investigation was to functionally identify and characterize a putative disaccharide transporter analog in the hepatopancreas of the American lobster, Homarus americanus. Purified hepatopancreatic brush-border membrane vesicles (BBMV) were used in transport experiments using 14C-sucrose and a Millipore filter isolation technique. In the absence of sodium, an external pH of 4 significantly stimulated the uptake of 14C-sucrose compared to that occurring at pH 5, 6, or 7. At pH 7, increasing external concentrations of sodium increased 14C-sucrose uptake by BBMV in a hyperbolic fashion and this stimulation was significantly reduced when the pH was changed to 4, suggesting that both protons and sodium ions were each capable of driving the uptake of the sugar. In experiments with a variety of monosaccharides, disaccharides, and trisaccharides, used as potential inhibitors of 14C-sucrose uptake, only maltose and trehalose inhibited carrier-mediated 14C-sucrose transport. An additional experiment showed that 20 mM maltose was a competitive inhibitor of 14C-sucrose uptake. The use of a putative lobster SCRT by both maltose and trehalose is nutritionally appropriate for lobsters as they commonly digest glycogen and chitin, polymers of maltose and trehalose, respectively. These findings suggest there is a brush-border proton- or sodium-dependent, hepatopancreatic carrier process, shared by sucrose, maltose, and trehalose, that may function to absorb disaccharides that are produced from digestion of naturally occurring dietary constituents.

Maltitol inhibits small intestinal glucose absorption and increases insulin mediated muscle glucose uptake ex vivo but not in normal and type 2 diabetic rats.

This study investigated the effects of maltitol on intestinal glucose absorption and muscle glucose uptake using ex vivo and in vivo experimental models. The ex vivo experiment was conducted in isolated jejunum and psoas muscle from normal rats. The in vivo study investigated the effects of a single bolus dose of maltitol on gastric emptying, intestinal glucose absorption and digesta transit in normal and type 2 diabetic rats. Maltitol inhibited glucose absorption in isolated rat jejunum and increased glucose uptake in isolated rat psoas muscle in the presence of insulin but not in the absence of insulin. In contrast, maltitol did not significantly (p > 0.05) alter small intestinal glucose absorption or blood glucose levels as well as gastric emptying and digesta transit in normal or type 2 diabetic rats. The results suggest that maltitol may not be a suitable dietary supplement for anti-diabetic food and food products to improve glycemic control.

A new thermostable and organic solvent-tolerant lipase from Staphylococcus warneri; optimization of media and production conditions using statistical methods.

A new thermostable and solvent-tolerant lipase was isolated from newly isolated Staphylococcus warneri from oil-contaminated soil. Optimization of the fermentation media for production of thermostable and organic solvent-tolerant lipase was carried out using two statistical methods, i.e., Plackett-Burman design (PBD) and central composite design (CCD) were used for the optimization of the media components. PBD was used to efficiently select important medium components affecting the lipase production. Out of 15 medium components screened, four components, i.e., olive oil, peptone, maltose, and K2HPO4 were found to contribute positively to lipase production. CCD and response surface methodology (RSM) were used to determine the optimum levels of the selected components using Design-Expert 8.0 software. Production medium with olive oil (1.45 %), peptone (0.28 %), maltose (0.054 %), and K2HPO4 (0.091 %) was optimized with a maximum lipase production of 10.43 IU/ml/min. Similarly, production conditions for the lipase production were optimized by using CCD and RSM. Optimized conditions were found to have an incubation temperature of 55 °C, medium pH of 8.0, agitation of 120 rpm, and inoculum volume of 2 %. RSM revealed the maximum lipase production of 17.21 IU/ml using these optimized production conditions. Crude lipase showed enhanced activity in organic solvents such as diethyl ether, hexane, and cyclohexane.

Direct bioethanol production by amylolytic yeast Candida albicans.

UNLABELLED: An attempt was made to produce bioethanol using optimized fermentation parameters and mutationally improved strain of Candida albicans. The mutant strain OMC3E6 obtained by UV irradiation followed by ethidium bromide successive mutations showed 2.6 times more glucoamylase secretion and 1.5 times more bioethanol production via direct conversion of starch. Enhanced hydrolysis of insoluble starch (72%) and potato starch (70%) was achieved with glucoamylase enzyme preparation from mutant C. albicans. In fermentation medium, the use of maltose, corn steep liquor, NaH2 PO4 , NaCl + MgSO4 and Triton X-100 has increased the glucoamylase production by the microbe. Under optimized conditions, C. albicans eventually produced 437 g ethanol kg(-1) potatoes. Earlier reports mentioned the use of thrice the quantity of starch as reported by us followed by more fermentation period (3-4 days) and demanded pretreatment of starch sources with alpha-amylase as well. Here, we simplified these three steps and obtained 73% conversion of insoluble starch into ethanol via direct conversion method in a period of 2 days without the involvement of cell immobilizations or enzyme pretreatment steps. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to fast depletion of fossil fuels in the modern world, bioethanol usage as an alternate energy source is the need of the hour. For the first time, we report bioethanol production by Candida albicans via direct conversion of starchy biomass into ethanol along with enhanced starch-hydrolysing capacity and ethanol conversion ratio. So far, C. albicans was dealt in the field of clinical pathology, but here we successfully employed this organism to produce bioethanol from starchy agri-substrates. Optimizing fermentation parameters and improving the microbial strains through successive mutagenesis can improve the end product yield.

Involvement of hexokinase1 in plant growth promotion as mediated by Burkholderia phytofirmans.

Potato plantlets inoculated with strain PsJN of the bacterium Burkholderia phytofirmans exhibit consistent and significant increases in plant growth under in vitro conditions, when compared with uninoculated plants. The greatest influence on the degree and type of growth enhancement that develops has been shown to be mediated by the sugar concentration in the agar media. Bacterial growth promotion has been suggested in other studies to be regulated by the sugar sensor enzyme hexokinase1, the role of which is activation of glucose phosphorylation. In this present study, we examined the co-relationship between root and stem development in potato plants treated with PsJN and the activity of hexokinase1. Plants grown in the presence of 1.5% and 3% sucrose showed increased levels of hexokinase1 activity only in the roots of inoculated plants, suggesting that the increased enzyme levels may be associated with root growth. Analysis for mRNA using reverse transcriptase did not reveal any significant differences in transcription levels of the gene between inoculated and uninoculated plants. When PsJN-inoculated plants were grown in 1.5% and 3% concentrations of glucose and fructose, stem height and mass, leaf number, root mass, and overall biomass increased. No growth promotion occurred when PsJN-inoculated plants were grown in 3% maltose. Subsequently, a hexokinase1 activity assay showed that PsJN-induced growth of potato plants was found to only occur when plants were grown in the presence of sugars that are recognized by the plant hexokinase1. The results suggest that PsJN may enhance sugar uptake in plants by direct or indirect stimulation of hexokinase1 activity in roots and this results in enhanced overall plant growth.

[Utilization of carbon sources by Fusarium poae (Peck) Wollenw. Strains from different trophic groups].

It was shown that saprophytic (soil), endophytic and plant pathogenic strains of F. poae under cultivation conditions in the media containing carbon sources from mono- to polysaccharides had different abilities to use them and to accumulate biomass. Maltose, xylose, fructose, pectin were the most favorable carbon sources for the studied strains; less assimilated lactose, arabinose, and especially microcrystalline cellulose were less assimilated. It was found that endophytes and plant pathogens accumulated biomass equally, while soil strains had low ability for that.

Alpha amylase enzyme inhibitory and anti-inflammatory effect of Lawsonia inermis.

Previously it was reported elsewhere that Lawsonia inermis have anti-inflammatory and analgesic effect in experimental animals. The in vitro porcine alpha amylase inhibitory effect was investigated of this plant methanolic extracts and consequently hypoglycemic effect by quantitatively determining the maltose from the maltose standard curve while the anti-inflammatory effect by acetic acid induced writhing test in mice. Acarbose (10 microg mL(-1)) and Diclofenac sodium (20 mg kg(-1)) were used as reference hypoglycemic and anti-inflammatory drugs, respectively, for this study. The methanolic leaves extract of the plant significantly inhibited (60.97% compared to untreated) enzymatic activity of the amylase at 10 microg mL(-1) dose (p < 0.05) also reduced the chemically induced nociceptive pain stimuli significantly at all doses (p < 0.01). Carbohydrates, glycosides, flavonoids, saponins and tannins were found to have in phytochemical screening of the extract which are thought to bring these effects. For the conclusive purpose, it is suggesting from the result that the pharmacological properties of this Lawsonia inermis can elicit hypoglycemic effect by inhibiting alpha-amylase enzyme and can reduce neurogenic pain stimulus. It gives the notion that how this group of patient would be therapeutically benefitted by decreasing both these effects by the same agent which is easy available.

A preliminary study of the mechanism of hypoglycaemic activity of Nauclea latifolia leaf ethanolic extract.

The aim of this study was to determine the possible underlying mechanism of the hypoglycaemic activity of the ethanolic extract of Nauclea latifolia leaves in rats. The extract, glibenclamide or water was administered orally in a glucose, sucrose and maltose tolerance tests. In addition, the effect of the extract on α-glucosidase enzymes was also studied. The ethanolic extract at 200mg/kg body weight inhibited the increase in glucose level after both oral and intraperitoneal glucose loads as did glibenclamide. The extract also dose dependently inhibited both maltase and sucrase activities in vitro but not in vivo. The hypoglycaemic effect of N. latifolia leaf ethanolic extract thus appears to be most probably exerted through a mechanism similar to that of glibenclamide which is related to increased insulin release from pancreatic ß-cells.

Berberine acutely inhibits the digestion of maltose in the intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese Goldthread Rhizome has been used in the Traditional Chinese Medicine as an important ingredient of many formulas for the treatment of diabetes mellitus. Berberine, the main effective composition of Chinese Goldthread Rhizome, is also effective in treating diabetes in today's clinical practice of Traditional Chinese Medicine. AIM OF THE STUDY: To evaluate the hypoglycemic activity of berberine which treats acutely on the postprandial blood glucose, and to explore the mechanism of this activity. MATERIALS AND METHODS: 1. One-dose preprandial intragastric administrations of berberine were given to normal animals (dogs and rats), and the postprandial blood glucose concentration curves were measured. Serum insulin enzyme linked immunosorbent assay (ELISA) was only performed in rats. 2. The euglycemic clamp test was performed to evaluate the effect of one-dose berberine intragastric administration on the blood glucose transformation and utilization rate in rats. 3. In the Caco-2 cell monolayer test, the changes of glucose concentration on the apical and basolateral sides were measured when the maltose solution containing berberine was added to the apical side. 4. The inhibition ratio of berberine against α-glucosidase was measured in vitro. 5. The effect of berberine on the fluorescence emission spectrums of α-glucosidase was studied. RESULTS: One-dose preprandial intragastric administration of berberine delayed the rise of post-maltose blood glucose, did not affect postprandial blood glucose after glucose meal, and did not affect the insulin level in normal rats; reduced post-maltose blood glucose in normal dogs. 2. The result of euglycemic clamp test showed that one-dose intragastric administration of berberine had no effect on the blood glucose transformation and utilization rate in rats. 3. Berberine added to the maltose solution on the apical side of Caco-2 cell monolayer reduced the glucose concentration on the apical side. Glucose in basolateral side of all groups cannot be detected. 4. Berberine inhibited the activity of α-glucosidase in vitro. 5. Berberine significantly and concentration dependently quenched the fluorescence emission spectrum of α-glucosidase. CONCLUSION: Our findings suggest an additional mechanism of the hypoglycemic activity of berberine by demonstrating its ability to acutely inhibit the α-glucosidase, and support the traditional use of berberine and Chinese Goldthread Rhizome for the treatment of diabetes mellitus.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.