Rat Small Intestinal Homogenates Research Articles

Enzymatic Formation of Novel Ginsenoside Rg1-α-Glucosides by Rat Intestinal Homogenates.

The variation of linkage positions in ginsenosides leads to diverse pharmacological efficiencies. The hydrolysis and transglycosylation properties of glycosyl hydrolase family enzymes have a great impact on the synthesis of novel and structurally diversified compounds. In this study, six ginsenoside Rg1-α-glucosides were found to be synthesized from the reaction mixture of maltose as a donor and ginsenoside Rg1 as a sugar acceptor in the presence of rat small intestinal homogenates, which exhibit high α-glucosidase activities. The individual compounds were purified and were identified by spectroscopy (HPLC-MS, (1)H-NMR, and (13)C-NMR) as 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-(β-glcp)-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, and 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-(β-D-glcp)]-20(S)-protopanaxatriol. Among these six, 6-O-β-D-glcp-20-O-α-D-glcp-(1→6)-(β-D-glcp)-20(S)-protopanaxatriol and 6-O-α-D-glcp-(1→6)-β-D-glcp-20-O-(β-D-glcp)-20(S)-protopanaxatriol are considered to be novel compounds of alpha-ginsenosidal saponins which pharmacological activities should be further characterized. This is the first report on the enzymatic elaboration of ginsenoside Rg1 derivatives using rat intestinal homogenates. To the best of our knowledge, it is also the first to reveal the sixth and 20th positions of an unusual α-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl sugar chain with 20(S)-protopanaxatriol saponins in Panax ginseng Mayer.

Improvement of Solubility and Oral Bioavailability of Rutin by Complexation with 2-Hydroxypropyl-β-cyclodextrin

The object of this study was to enhance the solubility, dissolution rate, and oral bioavailability of rutin by complexation with 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD). The interaction of rutin with cyclodextrins (CyDs) was evaluated by the solubility, and ultraviolet (UV) and circular dichroism (CD) spectrophotometries. The chemical and enzymatic stability of rutin was examined in an alkaline buffer solution and in rat small intestinal homogenates, respectively. Dissolution rates of rutin and its CyD complexes were measured by the dispersed amount method. In vivo absorption studies of rutin after oral administration via conventional tablet containing rutin alone or its β-CyD complexes was performed on beagle dogs. The stability constants calculated from the phase solubility method increased in the order of HP-γ-CyD < G2-β-CyD < β-CyD < HP-β-CyD. Spectroscopic studies also revealed that HP-β-CyD and β-CyD formed a relatively more stable inclusion complex with rutin. The dissolution rates of rutin increased by the complexation with CyDs in the order of rutin alone < HP-β-CyD ≤ β-CyD. HP-β-CyD inhibited the hydrolysis of rutin in the alkaline buffer solution and the small intestinal homogenates of rats, suggesting that HP-β-CyD may stabilize rutin in a gastrointestinal tract after oral administration. When the tablet containing rutin or its β-CyD complexes was administered to beagle dogs, the plasma levels of homovanillic acid (HVA) (a major stable metabolite of rutin) after oral administration of HP-β-CyD complex were much higher than in either that of rutin alone or in its β-CyD complex. The in vivo absorption study suggests that HP-β-CyD increased the oral bioavailability of rutin from the gastrointestinal tracts of beagle dogs because of the increase in solubility, faster dissolution rate, and gastrointestinal stability. HP-β-CyD has a significant advantage with respect to providing high aqueous solubility while maintaining a lack of toxicity in oral pharmaceutical preparations of rutin.

Enzymatic Degradation of Leuprolide in Rat Intestinal Mucosal Homogenates

The purpose of this study was to evaluate in vitro enzymatic degradation and protection of leuprolide acetate in the mucosal homogenates of rat small intestine. When leuprolide was incubated at 37°C with the homogenates, it was degraded quickly. The apparent Michaelis–Menten constant, Km, and the maximal reaction velocity, Vmax, for leuprolide were 898 mM and 3.4 nmol/min/mg protein, respectively. At least four metabolites of leuprolide were observed in HPLC chromatograms, which were related to cleavages by some serine proteases. In the presence of protease inhibitors in the incubation medium, degradation of leuprolide was significantly suppressed by antipain and 3,4-dichloroisocoumarin (DCI), whereas bestatin and p-hydroxymercuribenzoic acid (PCMB) showed weaker protection than antipain and DCI, and α2-macroglobulin (MG) exhibited no protection. When a w/o/w emulsion formulation was used, rapid degradation of the drug in intestinal homogenates was also inhibited. Therefore, the present study with representative protease inhibitors and a w/o/w formulation revealed that the enzymatic degradation of leuprolide is preventable in the rat intestinal mucosal homogenates.

Improvement of intestinal absorption of thyrotropin-releasing hormone by chemical modification with lauric acid.

Intestinal absorption of 125I-labelled lauryl thyrotropin-releasing hormone (Lau-TRH), a novel lipophilic derivative of TRH, was examined by rat in-situ closed intestinal loops. At a dose of 1 mumol per rat into the small intestine, a significant increase in percent of dose in plasma radioactivity of Lau-TRH was observed in comparison with that of TRH. A dose-dependent decrease in percent of dose in plasma radioactivity of TRH was noted, suggesting a saturable process of TRH transport. In contrast, the percent of dose in plasma radioactivity of Lau-TRH increased with increasing dose of Lau-TRH. The stability of TRH and Lau-TRH was studied in plasma and rat small intestinal homogenates. Lau-TRH was more stable than TRH in rat plasma. These results suggest that chemical modification of TRH with lauric acid may not only increase the lipophilicity of TRH but also reduce the degradation of TRH, resulting in the increased plasma radioactivity of TRH. On the other hand, Lau-TRH was gradually converted to TRH in the intestinal mucosal homogenate. These findings indicate that chemical modification of TRH with lauric acid might be a useful approach for improving the intestinal absorption of this peptide.

Rat intestinal peroxidase: Inhibition by endogenous xanthine and xanthine oxidase

The high-speed supernatant from homogenates of rat small intestine contains a heat-stable, dialyzable factor which showed a time-dependent inhibition of peroxidase activity in salt extracts of the tissue. The inhibitor was purified by chromatography on Dowex 50W-X8 and identified as xanthine. The inhibition of peroxidase by xanthine was prevented by allopurinol, an inhibitor of xanthine oxidase, and hypoxanthine was also found to be inhibitory. H 2O 2, produced in the reaction catalyzed by xanthine oxidase, was shown to be directly responsible for the observed inhibition. The time-dependent loss of peroxidase activity in the presence of xanthine or hypoxanthine occurred more rapidly in NH 4Cl than in CaCl 2 extracts of small intestine and was due to the difference in the initial concentration of H 2O 2 in these two extracts. The possible relationship between peroxidase and xanthine oxidase in the rat small intestine is discussed.

Postnatal Development of Peptidase Enzymes in Rat Small Intestine

SummaryThe development and distribution of peptidase activity in mucosal homogenates of rat small intestine has been investigated. Substrates used were glycyl‐L‐leucine (GL), L‐seryl‐L‐methionine (SM), and L‐leucyl‐glycyl‐glycine (LGG). During the first 2 weeks of life there was high peptidase activity toward GL and SM in the distal regions of the small intestine. In the third postnatal week, activity in the distal small intestine toward GL and SM decreased, while activity in the proximal small intestine increased. In contrast, there was no difference in activity toward LGG along the length of the small intestine, nor was there a developmental change. Activity toward all three substrates was not affected by cortisone acetate treatment. However, the classical effect of glucocorticoids on sucrase activity and body weight was observed. All peptidases studied showed maximal activities at neutral pH, indicating that they were not lysosomal in origin. Activity towards GL and SM was predominantly located in the cytosol. It is suggested that these dipeptidase activities play a role in the terminal steps of protein digestion following pinocytosis and lysosomal hydrolysis.

Postnatal development of peptidase enzymes in rat small intestine.

The development and distribution of peptidase activity in mucosal homogenates of rat small intestine has been investigated. Substrates used were glycyl-L-leucine (GL), L-seryl-L-methionine (SM), and L-leucyl-glycyl-glycine (LGG). During the first 2 weeks of life there was high peptidase activity toward GL and SM in the distal regions of the small intestine. In the third postnatal week, activity in the distal small intestine toward GL and SM decreased, while activity in the proximal small intestine increased. In contrast, there was no difference in activity toward LGG along the length of the small intestine, nor was there a developmental change. Activity toward all three substrates was not affected by cortisone acetate treatment. However, the classical effect of glucocorticoids on sucrase activity and body weight was observed. All peptidases studied showed maximal activities at neutral pH, indicating that they were not lysosomal in origin. Activity towards GL and SM was predominantly located in the cytosol. It is suggested that these dipeptidase activities play a role in the terminal steps of protein digestion following pinocytosis and lysosomal hydrolysis.

Requirement of Zn to demonstrate HCO3-stimulated ATPase activity of rat small intestinal brush border.

The existence of a membrane-bound HCO3-stimulated ATPase in intestinal mucosa is controversial. A crude brush border fraction of rat small intestinal homogenates contained HCO3-ATPase activity which was inhibited by preincubation with 3 mM EDTA. Alkaline phosphatase activity of this preparation was also inhibited in a parallel, time-dependent fashion by preincubation with EDTA. When 5 mM ZnSO4 accompanied 3 mM EDTA in the preincubation mix, preservation of both enzyme activities occurred, demonstrating a requirement of Zn for the activity of both these phosphatases. These studies support the earlier contention that HCO3-ATPase and alkaline phosphatase activities may be different properties of the same enzyme, and raise the possibility that the ATPase could play a role in intestinal ion transport. The failure to identify a membrane-bound HCO3-ATPase by other workers could be due to the exposure of EDTA which occurred in their tissue preparation.

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine.

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.

Anion-stimulated ATPase activity of brush border from rat small intestine.

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.