Biliary proteins from hepatic bile of rats fed curcumin or capsaicin inhibit cholesterol crystal nucleation in supersaturated model bile.

Following our earlier observations that curcumin and capsaicin are antilithogenic in mice and hamsters, attempts were now made to understand the manner in which these spice principles were acting. For this purpose, the hepatic biles of rats fed a control, lithogenic, and lithogenic diet supplemented with curcumin or capsaicin were subjected to gel filtration chromatography (sepharose-4B-Cl) and the LMW protein fractions were tested for their ability to influence cholesterol crystal growth in model bile. The LMW protein fraction from the lithogenic group bile shortened the nucleation time and increased the crystal growth rate and final crystal concentration. But with the LMW protein fractions from the biles of rats on the lithogenic group supplemented with curcumin or capsaicin, the nucleation times were prolonged and the crystal growth rates and final crystal concentrations were decreased. The LMW fractions were further purified into three different sugar specific proteins by affinity chromatography. A higher proportion of LMW proteins from the lithogenic group bile was bound to Con-A whereas higher proportions of LMW proteins from the groups fed with curcumin and capsaicin were respectively bound to wheat germ agglutinin and Helix pomatia lectin. The Con-A bound fraction obtained from the lithogenic group showed a pro-nucleating effect. In contrast, the WGA-bound fraction obtained from curcumin group or the Helix pomatia lectin bound fraction obtained from capsaicin group showed a potent antinucleating activity.

Effect on curcumin on cholesterol gall-stone induction in mice.

A study was carried out on the efficacy of curcumin in reducing the incidence of cholesterol gall-stones (CGS), induced by feeding a lithogenic diet in young male mice. Feeding a lithogenic diet supplemented with 0.5 per cent curcumin for 10 wk reduced the incidence of gall-stone formation to 26 per cent, as compared to 100 per cent incidence in the group fed with lithogenic diet alone. Biliary cholesterol concentration was also significantly reduced by curcumin feeding. The lithogenic index which was 1.09 in the cholesterol fed group was reduced to 0.43 in the 0.5 per cent curcumin supplemented group. Further, the cholesterol: phospholipid (C/PL) ratio of bile was also reduced significantly when 0.5 per cent curcumin supplemented diet was fed. A dose-response study with 0.2, 0.5 and 1.0 per cent curcumin supplemented lithogenic diets showed that 0.5 per cent curcumin was more effective than a diet with 0.2 or 1 per cent curcumin.

Comparative influence of vanillin & capsaicin on liver & blood lipids in the rat.

The influence of vanillin (0.15 mg %) and capsaicin (0.3 mg %) on liver and blood lipids in growing female Wistar rats was determined, when fed along with a normal diet. Vanillin, like capsaicin, decreased the serum triglycerides and the triglycerides associated with lipoproteins viz., VLDL+LDL and HDL with a tendency to lower liver triglycerides as well. Cholesterol and phospholipids in liver and serum were unaffected. The accumulation of triglycerides in serum after Triton WR-1339 blocking was higher in animals fed a vanillin supplemented normal or hypertriglyceridemic diet than in the corresponding controls. The accumulation of triglycerides in serum with capsaicin supplementation with either diet was much higher than with vanillin supplementation.

Effect of oryzanol on cholesterol absorption & biliary & fecal bile acids in rats.

Effect of oryzanol on biliary secretion of cholesterol, phospholipid and bile acids and fecal excretion of cholesterol and bile acids was examined in male albino rats. Feeding oryzanol at 0.5 per cent level with the control diet did not cause any change in bile flow and composition. On feeding oryzanol with high cholesterol diet, the bile flow and total bile acid output were increased by 12 and 18 per cent respectively, while biliary cholesterol and phospholipids remained unchanged. The increased bile acid secretion was mainly due to taurocholic acid. In rats fed oryzanol along with high cholesterol diet, there was a significant increase in the fecal excretion of cholesterol (28%) and of bile acids (29%), whereas cholesterol absorption was lowered by 20 per cent.

Influence of oryzanol on platelet aggregation in rats.

Platelet aggregation was measured using platelet rich plasma prepared from rats fed oryzanol in the control diet and those fed oryzanol in a 1 per cent cholesterol diet (HCD). Oryzanol with the control diet did not alter platelet aggregation induced by ADP and collagen. On the other hand, oryzanol fed along with HCD significantly inhibited platelet aggregation induced by ADP and totally inhibited aggregation induced by collagen.

Studies on hypocholesterolemic activity of rice bran oil.

The serum total, free esterified and (LDL + VLDL)-cholesterol levels of rats maintained on a 10% refined rice bran oil diet were significantly lower than those on a 10% groundnut oil diet; HDL-cholesterol showed a tendency to be higher. Addition of oryzanol at 0.5% level to the diet containing rice bran oil showed a further significant decrease in serum total cholesterol. Liver lipids of rats fed bran oil were also markedly lower than their groundnut oil fed counterparts. Though the liver lipids of rats fed oryzanol along with RBO showed a further lowering, it was not statistically significant. The cholesterol lowering ability of rice bran oil appears to be due to oryzanol and to some other components of the unsaponifiable matter.

Interaction of piperine with rat liver microsomes.

Pretreatment of adult male albino rats with piperine did not induce cytochrome P-450 whereas safrole did cause such induction. Piperine and safrole interacted with different forms of cytochrome P-450 as indicated by their in vivo effect on drug metabolising enzymes and mixed function oxidases and electrophoretic patterns. Incubation of piperine or safrole with dithionite- or NADPH-reduced microsomes from untreated rats resulted in a modified type II difference spectrum with an absorption maximum at 427 nm and a trough at 408 nm.

Metabolic disposition of piperine in the rat.

After oral administration of piperine (170 mg/kg) to rats, the metabolites in bile and urine were examined by thin-layer chromatography, high-performance liquid chromatography and combined gas chromatography-mass spectrometry. Four metabolites of piperine, viz. piperonylic acid, piperonyl alcohol, piperonal and vanillic acid were identified in the free form in 0-96 h urine whereas only piperic acid was detected in 0-6 h bile. Based on these results, a pathway for the biotransformation of piperine in rats is proposed.

Effect of black pepper and piperine on bile secretion and composition in rats.

The influence of black pepper (Piper nigrum L.) and its active principle, piperine on the secretion and composition of bile was investigated in the rat. They were administered by gavage (black pepper at 250 or 500 mg and piperine at 12.5 or 25 mg/kg body wt.) or fed in the diet for 4 weeks (black pepper at 0.2 and 0.4%, piperine at 0.01 and 0.02%). The lower dose by gavage of black pepper caused an increase in bile solids while with other treatments bile secretion or dry matter in bile was not changed. Dietary feeding of black pepper caused an increase in bile flow with a concomitant decrease in bile solids -- a hydrocholagoguic effect. Cholesterol and bile acid output were not affected by black pepper or piperine at either level irrespective of the mode of administration; in contrast, the secretion of uronic acids in bile was enhanced by both levels of pepper as also of piperine indicating possible excretion of some of the components of black pepper or of piperine as glucuronides.

Studies on the metabolism of piperine: absorption, tissue distribution and excretion of urinary conjugates in rats.

Upon administration of piperine, the major bite factor of black pepper, to male albino rats at a dose of 30 mg (170 mg/kg) by gavage or 15 mg (85 mg/kg) intraperitoneally, about 97% was absorbed irrespective of the mode of dosing. Three per cent of the administered dose was excreted as piperine in the feces. Piperine was not detectable in urine. When everted sacs of rat intestines were incubated with 200-1000 micrograms of piperine, about 47-64% of the added piperine disappeared from the mucosal side. Only piperine was present in the serosal fluid and also the intestinal tissue, indicating that piperine did not undergo any metabolic change during absorption. Examination of the passage of piperine through the gut indicated that the highest concentration in the stomach and small intestine was attained at about 6 h. Only traces (less than 0.15%) of piperine were detected in serum, kidney and spleen from 30 min to 24 h. About 1-2.5% of the intraperitoneally administered piperine was detected in the liver during 0.5-6 h after administration as contrasted with 0.1-0.25% of the orally administered dose. The increased excretion of conjugated uronic acids, conjugated sulphates and phenols indicated that scission of the methylenedioxy group of piperine, glucuronidation and sulphation appear to be the major steps in the disposition of piperine in the rat.

Effect of garlic (Allium sativum linn) on serum lipoproteins and lipoprotein cholesterol levels in albino rats rendered hypercholesteremic by feeding cholesterol.

The hypocholesteremic activity of garlic was tested by incorporation freeze-dried garlic powder at 0.5, 1.0, 2.0 and 3.0% levels in an atherogenic diet fed to rats. It was observed that 0.5 and 1.0% levels were not effective whereas the other 2 levels were. The group fed 2.0% garlic powder had much lower serum cholesterol level than the one fed 3%. The increased levels of low density lipoproteins (LDL) and LDL-cholesterol in rats fed the atherogenic diet were partly reversed in rats receiving a supplement of 2% garlic powder. On a cholesterol-containing diet, high density lipoprotein (HDL) and HDL-cholesterol levels were decreased. Inclusion of garlic powder in the atherogenic diet enhanced the percentage of HDL whereas no change was observed in HDL cholesterol levels. Commercial garlic pearls (equivalent to 0.15% garlic powder in the diet) produced a significant decrease in serum and liver cholesterol levels in rats fed the atherogenic diet. On the other hand, asafoetida at 1.5% level failed to reduce the serum cholesterol levels in the cholesterol-fed rats.

In vitro studies on the intestinal absorption of curcumin in rats.

When everted sacs of rat intestines were incubated with 50-750- micrograms of curcumin in 10 ml incubation medium, 30-80% of the added curcumin disappeared from the mucosal side. No curcumin was however detectable in the serosal fluid. Less than 3% of the added curcumin was found in the tissue at the highest concentration. In experiments with [3H[ curcumin, 5-6% of added radioactivity was found in the serosal side. TLC examination of the mucosal extract showed the presence of 2 compounds, 1 corresponding to curcumin and the other to a less polar, colourless compound. The serosal fluid had no curcumin but a compound whose RF was identical with the colourless compound present in the mucosal side. These experiments indicated that curcumin undergoes transformation during absorption from the intestine.

Metabolism of curcumin--studies with [3H]curcumin.

Radioactivity was detectable in blood, liver and kidney following dosage with 400, 80 or 10 mg of [3H]curcumin. The major route of elimination of the label was the feces; the urinary excretion of the label was very low regardless of the dose. At the lower doses of 80 mg and 10 mg of [3H]-curcumin, most of the label was excreted within 72 h, while with 400 mg, considerable amounts of the label was present in the tissues 12 days after dosage. The percentage of curcumin absorbed (60-66% of the given dose) remained constant regardless of the dose administered.

Absorption and tissue distribution of curcumin in rats.

After oral administration of 400 mg curcumin to rats, about 60% of the dose was absorbed. No curcumin was detectable in urine. The urinary excretion of conjugated glucuronides and sulfates significantly increased. No curcumin was present in heart blood. Only traces (less than 5 microgram/ml) in portal blood and negligible quantities in liver and kidney (< 20 micrograms/tissue) were observed from 15 min upto 24 h after administration of curcumin. At the end of 24 h the concentration of curcumin remaining in the lower part of the gut namely caecum and large intestine amounted to 38% of the quantity administered.