Further researches upon the inhibiting action of lycorine on ascorbic acid biosynthesis.

Lycorine, an alkaloid extracted from Amarillidaceae, strongly inhibits the "in vivo" conversion of galactono-gamma-lactone to ascorbic acid. Lycorine seems to act as a non-competitive inhibitor on galactono-gamma-lactone oxidase, because the alkaloid rapidly forms a stable bound with the enzyme. In fact, a short incubation period with 50 microM lycorine gets a high inhibitory effect that persists when the alkaloid is removed from the incubation medium. Considering that lycorine induces scurvy-like symptoms in ascorbic acid-synthesising animals, it is reasonable to suppose that in both plants and animals lycorine inhibits the last step in the biosynthetic pathway leading from sugar to ascorbate.

Sorbinil prevents the hypergalactosemic-induced reduction in [3H]-myo-inositol uptake and decreased [3H]-myo-inositol incorporation into the phosphoinositide cycle in bovine lens epithelial cells in vitro.

The synthesis of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4-5-bisphosphate was studied using 3H-myo-inositol (3H-MI) as precursor in cultured bovine lens epithelial cells (BLECs) maintained in galactose-free, physiological medium or 40 mM galactose (Gal) +/- sorbinil for six days. The formation of inositol polyphosphates from phosphoinositides was also shown. Galactitol did not exceed 2mM in Gal-incubated cells after six days of exposure; no galactitol was observed in BLECs maintained in galactose-free, physiological medium or Gal supplemented with sorbinil. Uptake of 3H-myo-inositol(3H-MI) into BLECs was significantly reduced in cells exposed to Gal. A concomitant reduction in 3H-MI incorporation was observed in the phosphoinositides, as well as with the released inositol phosphates. The simultaneous addition of sorbinil to the Gal medium corrected the drop in 3H-MI uptake and normalized 3H-MI incorporation into the phosphoinositides and inositol phosphates. While an apparent decrease in the three inositol-containing lipids was observed with the Gal-incubated cells, based on 3H-MI incorporation, there was no change in total membrane phosphatidylinositol content when compared to cells maintained in physiological medium as determined by the microgram Pl PO4 per microgram total membrane PO4. The apparent loss of radiolabeled phosphoinositides was attributed to the decreased specific activity resulting from the lower internal pool of 3H-MI in the Gal-exposed cells available for incorporation into the phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoliquiritigenin: a new aldose reductase inhibitor from glycyrrhizae radix.

Traditionally in Japan, some kampo medicines (traditional oriental herbal prescriptions) have long been used for the treatment of diabetic neuropathy. We have found that some aldose reductase inhibitors are included among these drugs. We further investigated the components of glycyrrhizae radix, a constituent of some kampo medicines, and isolated six compounds (GUs 9-17). Among these, GU-17, identified as isoliquiritigenin, had the most potent aldose reductase inhibiting activity. Isoliquiritigenin inhibited rat lens aldose reductase with an IC50 of 3.2 x 10(-7) M, using DL-glyceraldehyde as a substrate. It inhibited sorbitol accumulation in human red blood cells in vitro, with an IC50 of 2.0 x 10(-6) M. Isoliquiritigenin, when administered via an intragastric tube to diabetic rats, suppressed sorbitol accumulation in the red blood cells, the sciatic nerve, and the lens as effectively as ONO-2235. These results suggest that isoliquiritigenin may be effective in preventing diabetic complications.

Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity.

The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.

Antidiabetic and aldose reductase activities of biflavanones of Garcinia kola.

Kolaviron, a mixture of C-3/C-8 linked biflavonoids obtained from Garcinia kola produces significant hypoglycaemic effects when administered intraperitoneally to normal and alloxan diabetic rabbits at a dose of 100 mg kg-1. The fasting blood sugar in normoglycaemic rabbits was reduced from 115 mg/100 mL to 65 mg/100 mL after 4 h. In alloxan diabetic rabbits, the blood sugar was lowered from 506 mg/100 mL to 285 mg/100 mL at 12 h. The hypoglycaemic effects have been compared with those of tolbutamide. Kolaviron inhibited rat lens aldose reductase (RLAR) activity, with an IC50 value of 5.4 x 10(-6). The significance of these findings in the potential use of kolaviron as an antidiabetic agent is discussed.

Studies on lens-aldose-reductase inhibitor in medicinal plants. II. Active constituents of Monochasma savatierii Franch. et Maxim.

The 70% acetone extract of Monochasma savatierii FRANCH. et MAXIM. showed very strong inhibition of rabbit lens aldose reductase (AR). From the active fraction, five iridoid glucosides along with the two phenolic glycosides, acteoside and dehydroacteoside, have been isolated. Among them, acteoside showed the highest activity, being about 2.5 times more potent than baicalein, a known natural inhibitor of AR (IC50 = 9.8 x 10(-7) M). Demethylmussaenoside and 7-O-acetyl-8-epi-loganic acid, which are iridoid glucosides, had weak inhibitory activity.

The effect of aldose reductase inhibition on the pattern of nerve conduction deficits in diabetic rats.

Conduction deficits caused by 2-4 months diabetes were examined in one sensory and six motor nerve branches of mature rats. The effect of aldose reductase inhibitor (ponalrestat) treatment was assessed in preventative and reversal studies. The efficacy of 1% dietary myoinositol supplementation was also examined in a 2 month preventative group. Diabetes suppressed a maturation-related increase in conduction velocity in the interosseus nerve supplying foot muscles. This was unaffected by any treatment. Large conduction velocity reductions (22-29%) seen for fast nerves supplying four calf muscles and sensory saphenous nerves were prevented by ponalrestat treatment. In a reversal group, which had 2 months of diabetes followed by 2 months of treatment, restoration of conduction varied between nerves, ranging from 100% in sensory saphenous to 25% in soleus motor branches. Myo-inositol supplementation had little effect. Sciatic nerves accumulated the sugar alcohol sorbitol with diabetes. This was markedly reduced by treatment, and correlated with the conduction velocity improvement. There was a 40% reduction in nerve free myo-inositol levels after 2 months diabetes. Ponalrestat normalized myo-inositol in the short term but failed to do so in 4 month preventative and reversal groups. Myo-inositol treatment did not affect nerve levels. The data implicate the polyol pathway in the diabetic conduction velocity deficits seen in normally fast conducting motor and sensory nerves and suggest that aldose reductase inhibitor action does not depend on restoration of nerve myo-inositol levels.

Studies on aldose reductase inhibitors from natural products. II. Active components of a Paraguayan crude drug "Para-parai mí," Phyllanthus niruri.

Aldose reductase (AR) inhibitory activity-directed fractionation of the 70% ethanolic extract of Para-parai mí, Phyllanthus niruri, has led to the isolation of three active components, ellagic acid (1), brevifolin carboxylic acid (4) and ethyl brevifolin carboxylate (5). Among them, 1 showed the highest inhibitory activity, being about 6 times more potent than quercitrin, which is a known natural inhibitor of AR.

Effect of aldose reductase inhibition and insulin treatment on retinal capillary basement membrane thickening in BB rats.

This study compared the effect of glycemic control with insulin to that of the aldose reductase inhibitor ponalrestat on capillary basement membrane thickening in the retinas of diabetic BB rats. Diabetic animals with or without ponalrestat treatment were compared with diabetic rats subjected to vigorous insulin therapy or nondiabetic control rats with or without ponalrestat treatment after 6 mo of follow-up. Untreated diabetic animals showed the characteristic capillary basement membrane thickening in both the superficial and deep capillary beds of the retina. Vigorous blood glucose control with insulin therapy was accompanied by a complete prevention of capillary basement membrane thickening in both capillary beds, whereas aldose reductase inhibitor treatment achieved a complete prevention of basement membrane thickening in the deep capillary bed but not in the superficial capillary bed of the diabetic retina. These findings suggest that the mechanisms responsible for capillary basement membrane thickening in diabetes may be varied and modulated by topographical peculiarities in various capillary beds.

Glucose inhibition of human fibroblast proliferation and response to growth factors is prevented by inhibitors of aldose reductase.

Diabetes mellitus is associated with premature senescence of cultured dermal fibroblasts. The present study investigated the effect of elevated glucose concentrations on cultured human fibroblasts from normal donors. Mean population doubling times, population doublings until senescence, saturation density at confluence (cells/cm2), tritiated thymidine incorporation, and response to platelet-derived growth factor (PDGF) were inhibited with the increasing glucose concentrations (11.0, 22, 44, or 55 mM glucose) (P less than 0.05). Replicative life span was markedly diminished by multiple passages in high glucose medium (5.5 mM glucose: 62.4 +/- 7.9 population doublings; 22 mM glucose: 22.8 +/- 3.4 population doublings: P less than 0.05). Aldose reductase activity was present in the cultured fibroblasts (3.9 +/- 0.5 nmol/min per mg protein), and inhibitors of aldose reductase, including sorbinil (10(-4) M--10(-6) M) and tolrestat (10(-6) M--10(-8) M), completely prevented glucose-mediated inhibition of fibroblast proliferation, restored the response to PDGF, and allowed a normal replicative life span. Myo-inositol (11 microM--5.5 mM) also reversed the adverse effects of glucose. These in vitro data demonstrate that elevated concentrations of glucose inhibit cell growth and promote premature senescence, effects which can be prevented with inhibitors of aldose reductase or supplemental myo-inositol. These aldose reductase-related effects may explain the impaired growth and premature senescence of cultured connective tissue from diabetic patients.

The existence of aldose reductase inhibitors in some kampo medicines (Oriental herb prescriptions).

Traditionally in Japan, some kampo medicines which contain Glycyrrhizae radix (GR) and Paeoniae radix (PR) have long been used for the treatment of diabetic neuropathy. Since we have previously shown that GR und PR have potent aldose reductase inhibitory activities, we further investigated the constituents of these two. The boiled water extract of GR was applied to Sephadex LH-20 column chromatography and 6 fractions (Frs. A, B, Cs, Cp, D, and E) were obtained. Frs. Cp and D were retreated in the same manner and 7 pure compounds (GUs 1-7) were obtained. The boiled water extract of PR was fractionated with ethyl acetate followed by n-butanol and 3 fractions (Frs. 1-3) were collected. Fr. 1 was retreated in the same manner and 2 pure compounds (PRs 1 and 2) were obtained. Among the GU compounds, GU-2 was the most potent inhibitor of rat lens aldose reductase (RLAR) by inhibiting 86% at the concentration of 1.0 microgram/ml. The IC50 of GU-2 was 7.2 x 10(-7) M. Furthermore, GU-2 markedly inhibited sorbitol accumulation in human red blood cells, having an IC50 of 2.9 x 10(-5) M. GU-5 and PR-1 also inhibited RLAR (IC50: 5.6 x 10(-7) M and 6.3 x 10(-7) M, respectively). The structures of GU-2, GU-5, and PR-1 were identified as isoliquiritin, licuraside, and 1, 2, 3, 6-tetra-O-galloyl-beta-D-glucose, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Deficiency of ascorbic acid in experimental diabetes. Relationship with collagen and polyol pathway abnormalities.

The plasma and tissue concentration of ascorbic acid (AA) is reduced in diabetes. This study was designed to investigate the mechanism and significance of this phenomenon. The low plasma AA concentration of diabetic rats can be normalized by dietary AA supplement (20-40 mg/day), a dosage approximately equal to the maximal synthetic rate of this substance in the rats. Treatment of diabetic rats with this regime prevented the decrease in activity of granulation tissue prolyl hydroxylase (PRLase), an AA-dependent enzyme required for maintaining the normal properties of collagen. The decreased plasma AA concentration and granulation tissue PRLase activity in diabetes can also be normalized by the aldose reductase inhibitor tolrestat. We conclude that in diabetic animals there is a true deficiency of AA that may be responsible for some of the changes of collagen observed in diabetes. Treatment with AA or an aldose reductase inhibitor may prevent some of the diabetic complications with underlying collagen abnormalities.

Novel inhibitors of rat lens aldose reductase: N-[[(substituted amino)phenyl]sulfonyl]glycines.

A number of N-[[(substituted amino)phenyl]sulfonyl]glycines 3a-n were synthesized as analogues of the simple (phenylsulfonyl)glycines 1a-c with increased lipophilic character and therefore greater aldose reductase inhibitory potential. The 2-benzoylamino derivative 3c was found to be less potent than the corresponding amine 1c as an inhibitor of rat lens aldose reductase, but both the 3- and 4-benzoylamino analogues, 3b and 3a, are substantially more potent than their amines 1b and 1a; compound 3a is the most effective inhibitor of this series, with an IC50 of 0.41 microM. The 4-benzoylamino derivative 3a is also significantly more active than the 4-acetylamino analogue 3d and the 4-benzylamino (3e) and 4-dimethylamino (3f) derivatives, suggesting that both the additional carbonyl moiety and aromatic ring present in this compound may bind to complementary sites present on the enzyme. Furthermore, structure-activity studies reveal that increasing the number of atoms between the carbonyl and aromatic moieties of 3a results in a decrease in inhibitory activity. Kinetic studies demonstrate that 3a, like other known inhibitors of aldose reductase, functions as an uncompetitive inhibitor with respect to the substrate and therefore may interact at the proposed common inhibitor binding site of this enzyme.

[Submicroscopic restructurings in the glomerular portion of the rat nephron in streptozotocin diabetes and their possible drug correction]. / Submikroskopicheskie perestroiki v klubochkovom otdele nefrona krys pri streptozototsinovom diabete i vozmozhnost' ikh medikamentoznoi korrektsii.

Experiments on Wistar male rats have shown that in 2 weeks the administration of streptozotocin results in an increase in relative kidney mass by 23%, the development of submicroscopic rearrangements indicating the hypertrophy of the main glomerular components and the acceleration of ultrafiltration. In one month a sharp widening of the lumens of capillaries and intersubosseous spaces, inflammatory symptoms, and signs of primary urine congestion are noted in parallel with hypertrophic changes. The relative kidney mass increases by 73.5% (p less than 0.01). The use of the aldose reductase blocker isodibut prevents, to a great extent, the development of ultrastructural changes in a glomerulus detected one month after administration of streptozotocin, improves ultrafiltration and inhibits a sharp increase in kidney mass, i. e. it prevents disorder of renal excretory function at the initial stages of experimental diabetes.

New therapies for the chronic complications of older diabetic patients.

Recently, four biochemical mechanisms have been implicated in the pathogenesis of certain late complications of diabetes mellitus. All of these mechanisms (altered polyol pathway activity, disrupted myo-inositol metabolism, increased vascular permeability, and increased nonenzymatic glycosylation of proteins) are activated by exposure of tissues to hyperglycemia. There is evidence to suggest that the development of retinopathy, nephropathy, and neuropathy is directly related to the level of glycemia in patients with diabetes mellitus. Whether strict glycemic control will prevent or reverse diabetic complications is the subject of the Diabetes Control and Complications Trial. Until the results of that study are reported, and until euglycemia can be achieved in all diabetic patients, the search will continue for other pharmacologic agents that might prevent the development of complications. Therapies that are currently under investigation include administration of aldose reductase inhibitors and supplementation of dietary myo-inositol. It is too early to conclude whether such therapies will prove useful in the prevention or reversal of diabetic complications.

Slow component-a of axonal transport, nerve myo-inositol, and aldose reductase inhibition in streptozocin-diabetic rats.

This study measured sugars and polyols, weight/unit length, and slow component-a of axonal transport (SCa) in dorsal root afferents of the sciatic nerves of control rats and rats with streptozocin (STZ)-induced diabetes of 4-wk duration. The effects of two treatments--aldose reductase inhibition [Statil ("Statil" is a trademark; the property of Imperial Chemical Industries PLC.) ICI 128436 at 25 mg/kg/day, p.o.] and myo-inositol supplementation (650 mg/kg/day, p.o.)--were studied in control and diabetic groups. Inclusion of untreated controls and diabetics gave a total of six groups for the study. The treatments were begun on the day after injection of STZ and were maintained throughout the protocol. The sciatic nerves of the diabetic (untreated) rats showed accumulation of sorbitol and fructose, depletion of myo-inositol, and an 8% increase in weight/unit length. All of these abnormalities were prevented by treatment with Statil. Treatment of diabetic rats with myo-inositol prevented its depletion in the sciatic nerve, but did not affect the accumulation of sorbitol and fructose nor the increase in weight/unit length. Neither treatment exerted any apparent effect on body weight, blood glucose, nerve weight, or nerve sugars and polyols in the control rats. The diabetic rats showed a retardation of the wave of transported-labeled protein (shown as increased leftward skewness of the wave) and a reduction in mean transport velocity (calculated as the mean velocity for all segments contributing to the transport wave: 0.96 +/- 0.09 mm/day in diabetics versus 1.15 +/- 0.07 mm/day in controls).(ABSTRACT TRUNCATED AT 250 WORDS)