Investigation of adverse effects of interactions between herbal drugs and natural blood clotting mechanism.

Throughout the world, herbal medicines are consumed by most of the patients without considering their adverse effects. Many herbal medicines/plant extracts have been reported to interact with the natural blood clotting system. In continuation to this effort, thirty medicinal plant extracts were allowed to interact with citrated human blood and the clotting time was measured after re-calcification in vitro using Lee and White method. The aq. leaf ext. of Syzygium cumini and Camellia sinensis significantly prolonged the clotting time. In response to the prothrombin time and activated partial thromboplastin time tests, the ext. of C. sinensis showed normal APTT and marginally prolonged the PT to 16.7 s (control-15.2 s) while S. cumini showed normal PT but significantly prolonged the APTT to 66.9 s (control-20.7 s). This suggests that, C. sinensis acts on the extrinsic pathway while S. cumini on the intrinsic pathway. There are some common herbal formulations that are frequently used by the patients which contain above plant materials, like, Syzygium cumin in anti-diabetic formulations, while the ext. of C. sinensis is consumed frequently as beverage in many part of the world. Hence, patients having known bleeding tendency or haemophilia disease should take into account the interaction potential of these plants with the natural blood clotting system while taking herbal formulations containing above plants; specially, the patients suffering from intrinsic pathway factor deficiency should keep a limit on the consumption of S. cumini while extrinsic pathway factor deficiency patients should limit C. sinensis. Also, the medical practitioners should consider the patient's food consumption history before doing any major surgical procedures.

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450.

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome P-450 and cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.

Amylase inhibitors of pigeonpea (Cajanus cajan) seeds.

Pigeonpea (Cajanus cajan L) seeds were analysed quantitatively for amylase inhibitor (AI) activity and qualitatively, by an in-gel-detection method on polyacrylamide gels. At least four AI isoforms were identified in pigeonpea seeds. The AIs inhibit human salivary and bovine pancreatic amylase but fail to inhibit bacterial, fungal and endogenous amylase. Pigeonpea AIs were found to be active over a pH range of 4.5 to 9.5 and were heat labile. The isoelectric point of a major inhibitor is 6.2 AIs were tolerant to proteolysis by trypsin, chymotrypsin, bromelain and endogenous pigeonpea proteases. Pigeonpea AIs were synthesized during late seed development and also degraded during late germination. Addition of AIs or protease inhibitors (PIs) alone to a diet of Helicoverpa armigera larvae did not increase mortality. However, the larvae reared on a diet containing AIs and PIs in combination, showed increased mortality and adverse effects on larval growth and development. In vitro inhibition of Helicoverpa gut amylase revealed that only 22% activity is sensitive to inhibitors. Further investigations on interactions of pigeonpea AIs and PIs with Helicoverpa gut enzymes is necessary to develop strategies to strengthen defense mechanisms in pigeonpea against H. armigera.

Effect of caffeine on the hepatic microsomal mixed function oxidase system during phenobarbital and benzo[a]pyrene treatment in rats.

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.

Alterations in drug metabolising enzymes and lipid peroxidation in different rat tissues by fluoride.

Sodium fluoride at a dose level of 5.0 mg/kg enhanced aminopyrine N-demethylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in rat liver, kidney, lung, intestine and testis, whereas acetanilide hydroxylase activity remained unchanged in kidney and lung and was increased in liver, intestine and testis. Sodium fluoride at 20.0 mg/kg caused a decrease in aminopyrine N-demethylase, acetanilide hydroxylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in all tissues, except for an increase in NADPH cytochrome c reductase activity in the intestine and testis. Fluoride at both dose levels produced only marginal changes in glutathione-S-transferase activity except for a 4-fold increase in the testis at 5.0 mg/kg. Sodium fluoride at 5.0 mg/kg increased lipid peroxidation in all tissues studied. At 20.0 mg/kg there was a decrease in lipid peroxidation in liver, lung and testis and an increase in kidney and intestine.

Effect of ampicillin on hepatic microsomal mixed-function oxidase system in male mice.

The effect of ampicillin [(D)-alpha-aminobenzyl penicillin] administration on the hepatic mixed-function oxidase (MFO) system was studied in male mice. Ampicillin (100 mg/kg, i.p., 3 days) decreased the levels of cytochrome P-450, aminopyrine N-demethylase, acetanilide hydroxylase and cytochrome c-reductase activity significantly. In carbon tetrachloride (CCl4)-pretreated mice, ampicillin increased acetanilide hydroxylation compared with CCl4 treatment alone; however, all other parameters of the MFO system remained unchanged. Ampicillin exhibited type II binding with microsomes (trough at 388 nm, peak at 430 nm). Thus ampicillin acts as an inhibitor of the MFO system.

Detection of electrophoretically separated protease inhibitors using X-ray film.

Pigeon-pea seed extracts have been analyzed for the protease inhibitors using a new, sensitive and simple method for visualization of electrophoretically separated protease inhibitors. The visualization involves equilibrating the gel successively in the protease assay buffer, protease solution, rinsing the gel in protease assay buffer, and exposing it to an exposed, undeveloped X-ray film. Gelatin on the film in places corresponding to the inhibitor bands remains unhydrolyzed. By this method the pigeon-pea seed extract was found to contain nine trypsin and at least seven chymotrypsin inhibitors but no papain or bromelain inhibitors.

In vivo and in vitro effects of caffeine on hepatic mixed-function oxidases in rodents and chicks.

Administration of caffeine, ip 100 mg/kg/day for 1-5 days, to adult male rats resulted in a significant increase in hepatic cytochrome P-450 and b5 concentrations and in cytochrome c reductase, aminopyrine N-demethylase and acetanilide hydroxylase activities. No change was seen in relative liver weight but microsomal protein content was increased after treatment for 1 day and decreased after treatment for 3 or 5 days. In adult rats given 25, 100 or 150 mg caffeine/kg for 3 days, maximum stimulation of mixed-function oxidases was seen with the 100-mg/kg dose. Caffeine treatment (100 mg/kg for 3 days) increased relative liver weight in female guinea-pigs and decreased it in chicks and female mice, and decreased microsomal protein content in male mice, female guinea-pigs and young rats, and increased it in chicks. A significant increase in hepatic cytochrome P-450 content was seen in all species studied. Cytochrome b5 content was increased in chicks and young rats, while cytochrome c reductase activity was increased in male and female mice, young rats and chicks and decreased in female guinea-pigs. Aminopyrine N-demethylase activity was increased in young rats and female guinea-pigs, and acetanilide hydroxylase was increased in all test species except male mice. In vitro addition of 2.5 mM-caffeine to microsomal incubations from untreated rats, guinea-pigs, mice and chicks inhibited aminopyrine N-demethylase activity, although only to a significant extent in male mice; addition of caffeine to incubations containing microsomes from caffeine-treated animals produced significant inhibition of aminopyrine N-demethylase activity in microsomes from adult and young rats and female guinea-pigs. Aminopyrine N-demethylase inhibition did not increase with increasing concentration of added caffeine, although acetanilide hydroxylase activity was progressively inhibited in the microsomal incubates from both control and caffeine-treated animals.

Alterations in hepatic microsomal mixed-function oxidase system during different levels of food restriction in adult male and female rats.

Wistar strain adult male and female rats were given 25, 50 and 75% less food than an ad libitum-fed group of rats for 45 d and the effects of food restriction on hepatic drug metabolizing enzymes, microsomal electron transport components, NADPH-dependent lipid peroxidation and glutathione-S-transferase activities were studied. Compared to ad libitum-fed controls, the cytochrome P-450 levels were higher in food restricted male rats, while they were lower in food restricted females. The activities of NADPH cytochrome c reductase were lower in food restricted females than in ad libitum-fed controls. The activities of drug metabolizing enzymes, aminopyrine N-demethylase and acetanilide hydroxylase were higher in food restricted males, whereas in food restricted females these activities were lower than in respective groups fed ad libitum. Microsomal, NADPH-dependent lipid peroxidation was higher in 25 and 50% food restricted females while in 50 and 75% food restricted males it was lower than in ad libitum controls of the same sex. The cytosolic glutathione-S-transferase activities were lower in food restricted rats of both the sexes than in the same sexed controls. Another group of male and female rats were given 75% less food than the ad libitum-fed rats and refed for 3 d prior to killing. Here also, the effects of restriction were different between sexes. It is concluded that hepatic microsomal mixed-function oxidase system (MFOS) is altered due to feed restriction and food restriction followed by refeeding, in a sex-related manner.

Identification of amylase inhibitor deficient mutants in pigeonpea (Cajanus cajan (L.) Millisp.).

We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation.