Reidar Bredo Sund

Antioxidant and radical scavenging effects of anthraquinones and anthrones

The activity of seven anthraquinones and four anthrones against nonenzymatic and enzymatic lipid peroxidation in vitro and their ability to scavenge free radicals have been studied. In nonenzymatic peroxidation in rat hepatocytes induced by t-butyl hydroperoxide, dithranol and anthrone were the strongest antioxidants, having IC50 values of 8 +/- 1 and 24 +/- 5 mumol/l, respectively. Rhein (IC50 64 +/- 2 mumol/l) and aloe-emodin (IC50 65 +/- 3 mumol/l) showed the highest inhibitory activity against peroxidation of linoleic acid catalyzed by soybean 15-lipoxygenase. Anthrone (IC50 62 +/- 2 mumol/l), dithranol (IC50 72 +/- 1 mumol/l) and rhein anthrone (IC50 76 +/- 6 mumol/l) were the most effective radical scavengers of the diphenylpicrylhydrazyl radical. The antioxidant activities in hepatocytes and the radical scavenging activities were correlated, whereas the inhibition of enzymatic lipid peroxidation showed no correlation with the two other effects.

Inhibition of lipid peroxidation in low-density lipoprotein by the flavonoid myrigalone B and ascorbic acid☆

Lipid peroxidation in human LDL (0.05 mg protein/mL) incubated with Cu(2+)-ions (5 microM) in vitro was dose-dependently inhibited by the flavonoid myrigalone B (MyB) and by ascorbic acid. MyB at 6 microM increased the oxidation lag time by 135 +/- 24 min (approximately 5-fold compared to controls) and reduced the maximum oxidation rate by 46 +/- 5%. Ascorbic acid, at 9 microM, increased the lag time by 179 +/- 29 min (6-fold compared to controls) but did not affect the maximum oxidation rate. The increase in lag time induced by MyB was enhanced in the presence of ascorbic acid. Their effects were additive, except when both were present at the highest concentration tested, when a significant potentiation, giving an increase in lag time of approximately 2 hr more than the sum of separate effects, occurred. Concentration-time curves for MyB in the absence and presence of ascorbic acid showed that the vitamin protected MyB against deterioration during incubation, and indicated that the net consumption of MyB in the oxidation process was reduced. No differences were observed when ordinary ascorbic acid and Ester-C, a commercial vitamin C product, were compared. In conclusion, MyB and ascorbic acid seem to interact in a way that further improves the antioxidant status of the LDL particle relative to each substance separately.

Morphological and functional recovery following exposure to deoxycholic acid

Whereas many studies deal with the deleterious effects of unconjugated deoxycholic acid on epithelial morphology, few are concerned with the reversibility of these effects, the subject of the present study. Tied jejunal loops in the rat were incubated for 30 minutes with deoxycholic acid (2.5–20 mmol/litre) in isotonic solution. Immediately after this treatment, or after a subsequent recovery period of 15 or 150 minutes following wash out of the bile acid, the loops were excised, fixed and examined by light microscopy and scanning electron microscopy. Deoxycholic acid produced epithelial lesions whose severity and reversibility depended on the concentration applied. However, even the severely affected epithelium obtained by treatment at 10–20 mmol/litre was reverted to normal within 150 minutes, and a substantial normalisation was observed already after 15 minutes. An exception to this rapid restoration of epithelial morphology and integrity was noted in villi which had suffered necrosis of lamina propria. The revertion of epithelial pathology was paralleled with a normalisation of glucose absorption, of the potassium ion and protein content of the loop fluid, and of the paracellular epithelial permeability as measured with 3‐H‐poly‐ethylenglycol. Morphometry showed that deoxycholic acid caused villous atrophy without affecting the crypt length. The extent and reversibility of this atrophy depended on dose and recovery time as above. It is suggested that the remarkably fast morphological restitution proceeds mainly by processes involving cellular remodelling and migration.

Uncoupling of respiration and inhibition of ATP synthesis in mitochondria by C-methylated flavonoids from Myrica gale L.

Abstract The uncoupling activity of seven C-methylated flavonoids from the fruits of Myrica gale L. was studied. Myrigalone A, B and G (MyA, MyB and MyG) uncoupled the oxidative phosphorylation in rat liver mitochondria. MyA was most potent, at 45 μM causing an increase of 87±8 natoms O/min/mg in the state 4 respiration rate, which is more than twice the effect of 2,4-dinitrophenol (DNP). MyG was slightly less active than MyA, but its activity decreased from 45 to 90 μM. MyB was approximately equipotent with DNP. Myrigalone D and H were weak uncouplers, whereas myrigalone E and angoletin were inactive. The uncoupling activity of MyA, MyB and MyG was accompanied by an inhibitory effect on the ATP synthesis. The inhibition caused by MyA at 45 μM was about 70%. Whereas the effect of MyA or MyB was nearly constant throughout the incubation period, the effect of MyG had almost vanished after 15 min. The uncoupling activity of the myrigalones does not seem to be related to their antioxidative properties.

Anthraquinone laxatives: metabolism and transport of danthron and rhein in the rat small and large intestine in vitro.

A previous in vitro study by Sund and Hillestad in 1982 showed that dihydroxy-diphenylmethane laxatives undergo intestinal metabolism, and suggested a regionally dependent transport asymmetry of gut glucuronides. The present study was initiated since such experiments on anthraquinone diphenols are lacking. Everted sacs of rat jejunum and stripped colon were filled with Krebs-Henseleit solution (K-H) on the serosal (BL) side, and bathed at the mucosal (LU) side with K-H containing either danthron (3-4 nmol/ml) or rhein (10 nmol/ml). After 60 min incubation at 37 degrees C, LU and BL solutions and gut tissue were analysed for parent diphenol and metabolites by reverse-phase high-pressure liquid chromatography. Reference metabolites were isolated and purified from urine and bile of rats infused with danthron or rhein. The studies showed: (1) only small amounts of unchanged drug were present on the contraluminal side; (2) in both tissues, danthron was transformed into its monoglucuronide (G) and monosulphate (S); the ratio G:S was 6-8:1 in jejunum, and even greater in colon; (3) in jejunum, G and S were mainly secreted (LU:BL distribution ratios greater than 10:1); (4) in the colon, however, the main G fraction was absorbed (BL:LU ratios of 3:1), whereas a slight net secretion of S seemed to take place; (5) residuals (%) in gut tissue were small; (6) rhein was more slowly taken up and metabolized, but seemed otherwise to behave as danthron. The results are in principle similar to those obtained by indirect conjugate assay in the study on diphenylmethanes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of infrastructure and of cytoplasmic filaments in remodelling rat jejunal epithelial cells during recovery from deoxycholate

Structural features associated with reversibility of lesions induced by deoxycholic acid (DOC) were studied by electron microscopy and immunofluorescence techniques. Tied jejunal loops were incubated in vivo with 2.5–20 mmol/1 DOC in isotonic solution. Immediately after this treatment, or after a recovery period of 15 or 150 minutes following washout of the bile acid, the loops were excised and processed. DOC produced epithelial lesions whose severity and reversibility were concentration‐dependent. Ultrastructural features associated with the reversibility of the lesions were particularly apparent in specimens exposed to 10–20 mmol/1 DOC. These features included cell flattening with the formation of thin, veil‐like structures into the eroded area by cells at the edges of the erosions. Immunofluorescence studies showed a redistribution of actin and cytokeratin filaments to the margins and leading edges of the flattened cells. It is suggested that cell flattening and migration are responsible for the rapid morphological recovery of the injured epithelium. Actin and cytokeratin appear to be instrumental in the remodelling and migration.

A Note on the Complex Metabolism of Danthron Infused into the Rat

Danthron infused intravenously in rats shows a complex dose-dependent pattern of metabolism and excretion. The metabolites, particularly the more polar ones, are in general excreted predominantly in bile, to a lesser extent in urine. They can be separated as metabolite groups according to polarity and molecular size on a Sephadex LH 20 column. The present paper describes a further study within a bile-derived metabolite group, which proved to be particularly heterogeneous. It contained more than a dozen metabolites, which were conjugates of four different aglycons including the parent danthron. 1H NMR spectral data for danthron monosulfate and monoglucuronide are also presented.

Enzyme changes in remodelling epithelial cells : a histochemical study of the rat jejunum in vivo during and following exposure to deoxycholic acid

Loops of rat jejunum were exposed in vivo to different concentrations of deoxycholic acid (DOC; 0, 2.5, 5, 10 and 20 mM). Following a 30 min exposure period, DOC was washed out of the loops and the intestines were allowed to recover for 15 or 150 min. Frozen tissue for enzyme histochemistry was collected during exposure and following the recovery periods. As shown previously, exposure to DOC caused a dose‐dependent loss of epithelial cells at the villous tips and denudation of the lamina propria. Flattened epithelial cells bordering the denuded areas were, however, responsible for a rapid restoration of epithelial continuity, which was completed within 15 min. In the present study, these flattened cells showed normal reactivity for non‐specific esterase and succinate dehydrogenase. In contrast, following a prolonged recovery period (150 min), a subpopulation of enterocytes at the villous tips that otherwise appeared normal showed decreased reactivity for brush border enzymes and non‐specific esterase, and a positive reaction for mucin. A shutdown in the synthesis of cytoplasmic enzymes and redistribution of cell surface enzymes could be responsible for these late occuring enzyme changes, that were consistently observed after 150 min of recovery from DOC at 20 mM. Alternatively, retention of goblet cells and/or a modification in enzyme synthesis may explain the presence of mucin that was demonstrated in the epithelial cells which had low enzyme reactivity.