Mitragyna speciosa Korth leaves extracts induced the CYP450 catalyzed aminopyrine-N-demethylase (APND) and UDP-glucuronosyl transferase (UGT) activities in male Sprague-Dawley rat livers.

BACKGROUND: Mitragyna speciosa leaves have been abused by drug addicts as some of the alkaloids (mainly mitragynine) from the plant possess opiate and cocaine-like effects. These bring to its prohibition in Malaysia in 2004 as consumption of M. speciosa leaves has been perceived to lead to the abuse of other drugs such as cannabis and heroin. METHODS: In the current study, the in vitro and in vivo effects of M. speciosa methanolic, aqueous and total alkaloid leaves extracts on drug metabolizing enzymes, namely, cytochrome P450s (CYP450s) and UDP-glucuronosyl transferase (UGT) had been evaluated in rat liver cytosolic fraction and microsomes. Aminopyrine and p-nitrophenol (pNP) were employed as probe substrates in aminopyrine N-demethylase (APND) and UGT enzyme assays, respectively. Furthermore, mitragynine was also tested in vitro for its likelihood to inhibit APND and UGT activity. The assessment of the enzyme activity was conducted using spectrophotometric methods. RESULTS: In vitro, the IC50 value could only be obtained for the methanolic extract in APND study (595.30±30.78 µg/mL) and not in other studies due to the enzyme percentage inhibitions being <70%. In contrast to the in vitro study, the oral treatment of male Sprague-Dawley rats for 14 days with 50, 100 and 200 mg/kg of methanolic and aqueous extracts and with 5, 10 and 20 mg/kg of total alkaloid extract showed a profound increment on the APND and UGT activities. CONCLUSIONS: The current findings showed that possibilities exist for herb-drug interaction with increased clearance of drugs, which are primarily metabolized by CYP450s and UGT1A6 among M. speciosa leaves extract users.

Reversal of experimental ethanol-induced liver steatosis by borage oil.

The aim of study was to evaluate the hepatoprotective effect of borage oil containing predominantly gamma-linolenic acid in rats with alcoholic steatohepatitis. Liver of ethanol-treated animals was characterized by fatty and hydropic dystrophies. Liver triglyceride contents and activitiies of serum marker enzymes were significantly increased. Ethanol increased nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-induced chemiluminescence and the contents of liver thiobarbituric acid reactive substances (TBARS). The reduced glutathione content in the liver was decreased. Ethanol enhanced liver microsomal cytochrome P-450 (CYP450) content, aniline p-hydroxylase and amydopyrine-N-demethylase activities. The treatment with borage oil improved the liver morphology, decreased triglyceride contents and normalized serum marker enzyme activities. Borage oil developed an antioxidant effect in ethanol-treated rats. The treatment with this compound decreased NADPH-induced chemiluminescence and the content of lipid peroxidation products. Borage oil normalized CYP450 content compared with the ethanol-treated group. CYPI450 2E1 isoform is a main source of free oxygen radicals in the liver of ethanol-treated rats and we propose that the antioxidant effect of borage oil is realized via the normalization of CYP450 content and activities of CYP450-related microsomal oxidases, as borage oil can improve the lipid surrounding of CYP450. In our opinion, the hepatoprotection by borage oil in alcoholic steatosis is connected with its antioxidant properties.

[Effects of ethyl acetate extract of Semen Hoveniae on liver microsomal cytochrome P450 isoenzyme in rat].

OBJECTIVE: To investigate the effects of the ethyl acetate extract of Semen Hoveniae (ESH) on liver microsomal cytochrome P450 isoenzyme in rats. METHOD: The rats were given orally the ESH in the doses of 0.14, 0.17, 0.2 g x kg (equivalent to the crude herb) for 10 days respectively. Rat liver microsomal cytochrome P450, NADPH-Cyt C reductase, erythromycin N-demethylase (ERD), Aniline hydroxylase (ANH), aminopyrine N-demethylase (ADM) activities were quantitated by UV chromatography. The levels of mRNA expression of CYP1A1, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcripatase-polymerase chain reaction (RT-PCR). RESULT: The cytochrome P450 content, NADPH-Cyt C reductase activities and erythromycin N-demethylase (ERD) activities were not affected. Aniline hydroxylase (ANH) activities in liver were decreased by up to35.1%; aminopyrine N-demethylase (ADM) activitiesin liver were increased by up to 42.4%. The mRNA expression of CYP1A1, CYP2C11 and CYP3A1 were found to be increased markedly. CONCLUSION: A specific effect of ESH on liver microsomal cytochrome P450 isoenzyme in rats was observed in this investigation. ESH had various effects on liver microsomal cytochrome P450 isoenzyme.

Zinc mediates normalization of hepatic drug metabolizing enzymes in chlorpyrifos-induced toxicity.

The present study investigated the protective effects of zinc in attenuating the altered activities of drug metabolizing enzymes in the livers of rats intoxicated with chlorpyrifos. Male Sprague-Dawley rats received oral chlorpyrifos treatment (at a dose level of 13.5 mg/kg body weight in corn oil every alternate day), zinc supplementation alone (at a dose level of 227 mg/l in drinking water), or combined chlorpyrifos plus zinc treatments for a total duration of 8 weeks. The effects of different treatments were studied on the specific activities of various drug metabolizing enzymes including cytochrome P(450), cytochrome b(5), NADPH cytochrome-c-reductase, NADH cytochrome-c-reductase, aminopyrene-N-demethylase (APD) and aromatic hydrocarbon hydroxylase (AHH). Additionally, serum zinc levels were also determined in each of the treatment groups at the end of the study. Chlorpyrifos treatment resulted in a significant decrease in the serum zinc concentrations. Analogous to these changes, we observed significant depression in the activities of majority of the drug metabolizing enzymes investigated in the present study, except for AHH, where the decrease in enzyme activity was not statistically significant. However, zinc treatment to chlorpyrifos treated animals effectively restored the depressed serum zinc levels to within normal limits. Similarly, co-administration of zinc to chlorpyrifos intoxicated animals normalized the enzymatic activities of cytochrome P(450), NADPH cytochrome-c-reductase and NADH cytochrome-c-reductase within normal range. Collectively, these findings suggest that zinc plays an important role in regulating the hepatic activities of drug metabolizing enzymes in chlorpyrifos intoxicated animals, although it remains to be determined whether such protective effects of zinc are regulated directly, or through some indirect mechanism.

Inhibition of drug metabolizing enzymes (cytochrome P450) in vitro as well as in vivo by Phyllanthus amarus SCHUM & THONN.

An alcoholic extract of Phyllanthus amarus (P. amarus) was found to inhibit cytochrome P450 (P450) enzymes both in vivo as well as in vitro. This was studied using specific resorufin derivatives, as substrate for isoenzymes in the P450 super family. Concentration needed for 50% inhibition of 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 was 4.6 microg/ml while concentration needed for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2 was 7.725 microg/ml and 7-pentoxyresorufin-O-depentylase (PROD), CYP2B1/2 was found to be 4.18 microg/ml indicating that the extract inhibited the P450 enzymes at very low concentration. Extract also inhibited the activity of aniline hydroxylase (an indicator of CYP 2E1 activity, IC(50) 50 microg/ml) and aminopyrine demethylase (an indicator of CYP 1A, 2A 2B, 2D and 3A activity, IC(50) >1000 microg/ml). Oral administration of the extract was also found to reduce the elevated P450 enzyme activities produced by phenobarbitone by 50% at 250 mg/kg body weight. The implication of these results on the inhibition of carcinogenesis produced by the extract is discussed.

Herbal monoterpene alcohols inhibit propofol metabolism and prolong anesthesia time.

2,6-Diisopropylphenol (Propofol) is a short-acting intravenous anesthetic that is rapidly metabolized by glucuronidation and ring hydroxylation catalyzed by cytochrome P450. The goal of this research was to determine whether dietary monoterpene alcohols (MAs) could be used to prolong the anesthetic effect of propofol by inhibiting propofol metabolism in animals. Mice were injected intraperitoneally (i.p.) with MAs (100-200) mg/kg followed by the administration of 100 mg/kg propofol 40 min later via an i.p. injection. The time of the anesthesia of each mouse was recorded. It was found that (+/-)-borneol, (-)-carveol, trans-sobrerol, and menthol significantly extended the anesthetic effect of propofol (>3 times). The concentration of propofol in the mouse blood over time (up to 180 min) also increased in mice pre-treated with (-)-borneol, (-)-carveol, and trans-sobrerol. The volume of distribution of propofol decreased in the (-)-borneol (p<0.05), pre-treated group as compared to the propofol control group. Moreover, the maximum blood concentration of propofol and the concentration of propofol in the blood as indicated by the area under the curve were significantly increased in (-)-borneol and (-)-carveol pre-treated groups. Additional evidence using rat hepatocytes showed that (-)-borneol inhibited propofol glucuronidation whereas trans-sobrerol and (-)-carveol inhibited cytochrome P450 dependent microsomal aminopyrine N-demethylation. These results suggest that (-)-borneol extends propofol-induced anesthesia by inhibiting its glucuronidation in the mouse whereas trans-sobrerol (-)-carveol extends propofol-induced anesthesia by inhibiting P450 catalyzed propofol metabolism.

Changes in hepatic drug metabolizing enzymes and lipid peroxidation by methanol extract and major compound of Orostachys japonicus.

The effects of methanol extract and gallic acid (3,4,5-trihydroxybenzoic acid) of Orostachys japonicus A. Berger on hepatic drug metabolizing enzymes and lipid peroxidation were investigated in rats treated with bromobenzene. The methanol extract of Orostachys japonicus reduced the activities of phase I enzymes, aminopyrine N-demethylase and aniline hydroxylase, that had been increased by i.p. injection of bromobenzene. Gallic acid isolated from Orostachys japonicus also reduced the aniline hydroxylase activity, while it did not affect the aminopyrine N-demethylase activity. The methanol extract and gallic acid restored the activity of epoxide hydrolase which had been decreased by bromobenzene. Hepatic glutathione content was lowered, along with increase in hepatic lipid peroxide, by bromobenzene administration. The hepatic lipid peroxidation induced by bromobenzene was prevented with the methanol extract and gallic acid of Orostachys japonicus. However, the decrease in glutathione was not altered by gallic acid. The present results suggest that the methanol extract and gallic acid of Orostachys japonicus may protect liver from bromobenzene toxicity through, at least in part, inhibiting the cytochrome P450-dependent monooxygenase activities and enhancing the activity of epoxide hydrolase. Antioxidant effect also may contribute to the protection of Orostachys japonicus against the bromobenzene-induced hepatotoxicity.

[Modulation of the activities and mRNA expression of cytochrome P450 isoenzymes in rat liver by Panax gingseng and coadministration with Veratrum nigrum].

OBJECTIVE: To study the modulatory effect of Panax gingseng and coadministration with Veratrum nigrum on the activity and mRNA expression of cytochrome P450 isoenzymes in rat liver. METHOD: Rat liver microsomal cytochrome P450, b5, aminopyrine N-demethylase(APND), p-nitrophenol-hydroxylase(pNPH)activities were quantitated by UV chromatography. The mRNA expression level of five CYP isoenzymes CYP1A1, CYP2B1/2, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). RESULT: P. gingseng coadministrated with V. nigrum obviously decreased the P450 contents of liver microsomes, and the b5 contents. Both single and combined used inhibited the activities of aminopyrine N-demethylase. At the mRNA level, the expression of CYP2C11 markedly induced exposure to V. nigrum, but combinative groups decreased the expression of CYP2C11. The combination of P. gingseng and V. nigrum induced the expression of CYP1A1. P. gingseng has inhibitory effect on CYP2B1/2 and inductive effect used with V. nigrum. The combination of P. gingseng with V. nigrum also induced the expression of CYP3A1. CONCLUSION: P. gingseng used singly has some different modulation effects compared with combinative used, which may occur because of drug-drug interaction based on cytochrome P450. To elucidate the drug-drug interaction, it needs further analysis and metabolism research.

Anti-hepatotoxic effects of Rosa rugosa root and its compound, rosamultin, in rats intoxicated with bromobenzene.

The effects of a methanol extract of Rosa rugosa root and its triterpenoid glycoside, rosamultin, on hepatic lipid peroxidation and drug-metabolizing enzymes were investigated in rats treated with bromobenzene. The methanol extract of R. rugosa root reduced the activities of aminopyrine N-demethylase and aniline hydroxylase, which had been increased by bromobenzene, but rosamultin did not affect the activities of the two enzymes. Both the methanol extract and rosamultin restored the activity of epoxide hydrolase, which had also been decreased by bromobenzene. Hepatic glutathione concentrations were lowered and hepatic lipid peroxides were increased in rats intoxicated with bromobenzene. The hepatic lipid peroxidation induced by bromobenzene was prevented with the methanol extract and rosamultin. However, the decrease in glutathione was not altered by the methanol extract of R. rugosa. These results suggest that the extract of R. rugosa and its compound, rosamultin, may protect against bromobenzene-induced hepatotoxicity through, at least in part, enhanced activity of epoxide hydrolase. Antioxidant properties may contribute to the protection of R. rugosa against bromobenzene-induced hepatotoxicity.

Effects of long-term tea polyphenols consumption on hepatic microsomal drug-metabolizing enzymes and liver function in Wistar rats.

AIM: To investigate the effects of long-term tea polyphenols (TPs) consumption on hepatic microsomal drug-metabolizing enzymes and liver function in rats. METHODS: TPs were administered intragastrically to rats at the doses of 833 mg.kg(-1).d(-1) (n=20) and 83.3 mg.kg(-1).d(-1) (n=20) respectively for six months. Controlled group (n=20) was given same volume of saline solution. Then the contents of cytochrome P450, b5, enzyme activities of aminopyrine N-demethylase (ADM), glutathione S-transferase (GST) and the biochemical liver function of serum were determined. RESULTS: The contents of cytochrome P450 and b5 in the livers of male rats in high dose groups (respectively 2.66 +/- 0.55, 10.43 +/- 2.78 nmol.mg MS pro(-1)) were significantly increased compared with the control group (1.08 +/- 1.04, 5.51 +/- 2.98 nmol.mg MS pro(-1); P<0.01, respectively). The enzymatic activities of ADM in the livers of female rats in high dose groups (0.91 +/- 0.08 mmol.mg MS pro(-1)min(-1)) were increased compared with the control group (0.82 +/- 0.08 mmol.mg MS pro(-1).min(-1); P<0.05). The GST activity was unchanged in all treated groups, and the function of liver was not obviously changed. CONCLUSION: The antidotal capability of rats' livers can be significantly improved after long-term consumption of TPs. There are differences in changes of drug-metabolizing enzymes between the sexes induced by TPs and normal condition.

[Effects of Angelica sinensis polysaccharides on hepatic drug metabolism enzymes activities in mice].

OBJECTIVE: To study the effects of Angelica sinensis Polysaccharides (ASP) on the hepatic drug metabolism enzymes activities in normal mice and those prednisolone (PSL)-induced liver injury. METHOD: The activities of phase II enzymes (GSH-related enzymes) and cytochrome P450 enzymes were measured by biochemical method. RESULT: ASP increased the activities of glutathione S-transferase in liver microsomes and mitochondria. The cytochrome P450 content, NADPH-cytochrome c reductase, aminopyrine N-demethylase, and aniline hydroxylase activities in liver microsomes were also increased. PSL significantly increased serum ALT levels, and decreased the liver mitochondrial glutathione content. At the same time, other enzymes activities were all increased. When mice were treated with ASP 2.0 g.kg-1, the PSL-induced changes on cytochrome P450 enzymes, glutathione S-transferase, and GSH content were restored. CONCLUSION: ASP can modulate the activities of drug metabolism enzymes.

Oestrogenicity and effect on hepatic metabolism of the aqueous extract of the leaf mixture of Aloe buettneri, Dicliptera verticillata, Hibiscus macranthus and Justicia insularis.

The aqueous extract of the leaf mixtures of Aloe buettneri, Dicliptera verticillata, Hibiscus macranthus and Justicia insularis given by oral route to immature female rats, at doses of 13, 49 and 94 mg/kg per day for 15 days induced a significant increase in ovarian and uteri weight as well as serum and ovarian oestradiol. Moreover, a significant decrease in liver of aminopyrine N-demethylase activity was noticed in treated animals.

[Some pathogenetic aspects of developing liver failure and preventing it in patients with liver cirrhosis after portosystemic shunting]. / Nekotorye patogeneticheskie aspekty razvitiia pechenochnoi nedostatochnosti i ee profilaktika u bol'nykh s tsirrozom pecheni posle portosistemnogo shuntirovaniia.

Patients with cirrhosis of the liver were found to have a considerable suppression of the system of biotransformation of the liver before operation which correlated with the data of the direct indices of monooxigenase system of hepatocytes--cytochrome P-450 and activity of N-demethylase of amidopyrine. Operative interventions on such patients independent of the type of portosystemic shunting result in considerably decreased content of metabolites of amidopyrine--4AAP and N-ac-4-APP in urine as compared with the preoperative level (p < 0.05). Hyperbaric oxygenation is the optimal stimulator of activity of the liver biotransformation system. Better indicators characterizing the increased metabolic activity of the liver were noted in patients with selective portosystemic anastomoses and hyperbaric oxygenation in the postoperative period.

Kalopanaxsaponin A from Kalopanax pictus, a potent antioxidant in the rheumatoidal rat treated with Freund's complete adjuvant reagent.

The stem bark of Kalopanax pictus is an anti-rheumatoidal arthritis drug in Oriental medicine. In the rheumatoidal rat, induced by Freund's complete adjuvant (FCA) reagent, we investigated the effects of hederagenin monodesmosides of K. pictus on oxidative stress and hepatic drug-metabolizing enzymes. Kalopanaxsaponin-A (KPS-A) significantly decreased malondialdehyde formation and the activities of xanthine oxidase and aldehyde oxidase of hepatic non-microsomal systems in FCA reagent-treated rats. In addition, increased activity levels of superoxide dismutase, catalase and glutathione peroxidase were also observed. The effects of KPS-A were more potent than the effects of KPS-I. These results suggested that KPS-A, extracted from K. pictus, could reduce rheumatoidal syndromes through antioxidative mechanisms.

Constituents of the essential oil of the Cinnamomum cassia stem bark and the biological properties.

GC-MS analysis on the essential oil (CC-oil) of Cinnamomum cassia stem bark led to the identification of cinnamaldehyde (CNA, 1), 2-hydroxycinnamaldehyde (2-CNA), coumarin (2), and cinnamyl acetate. The major volatile flavor in CC-oil was found to be 2-CNA. Coumarin was first isolated from this plant by phytochemical isolation and spectroscopic analysis. CNA and CC-oil showed potent cytotoxicity, which was effectively prevented by N-acetyl-L-cysteine (NAC) treatment. Intraperitoneal administration with CNA considerably decreased malondialdehyde (MDA) formation and glutathione S-transferase activity in rats. These results suggest that CC-oil and CNA can regulate the triggering of hepatic drug-metabolizing enzymes by the formation of a glutathione-conjugate.

Protection by Osbeckia aspera against carbon tetrachloride-mediated alterations in microsomal drug metabolizing enzyme activity.

Previous investigations have confirmed the protective effect of Osbeckia aspera leaf extract on carbon tetrachloride-mediated liver injury in rat models. It is well known that the earliest alterations in liver cell structure and function following carbon tetrachloride poisoning involve the endoplasmic reticulum and its drug metabolizing enzymes. Therefore, we investigated whether an aqueous leaf extract of O. aspera could offer protection against carbon tetrachloride-induced changes in the microsomal drug metabolizing enzymes aniline hydroxylase and p-aminopyrine N-demethylase. This enzyme activity was compared with phenobarbital-induced righting reflex and lipid peroxidation. Treatment of rats with the aqueous leaf extract of O. aspera (before or after the administration of carbon tetrachloride) resulted in a marked decrease in carbon tetrachloride-mediated alterations in aniline hydroxylase and p-aminopyrine N-demethylase activity, phenobarbital-induced loss of righting reflex and malondialdehyde formation due to lipid peroxidation. The Km value of these enzymes in control and Osbeckia-treated rats were the same. These results show that the plant extract can markedly decrease the carbon tetrachloride-mediated reduction in aniline hydroxylase and p-aminopyrine N-demethylase activity and inhibit peroxidative damage to the cell membrane. Phenobarbital-induced sleeping time in rats and kinetic enzyme studies suggested that the effects of the plant extract was neither due to an induction of the drug-metabolizing enzymes under investigation, nor due to an alteration in the Km values of these enzymes.

Effect of vitamin E on lipid peroxidation and liver monooxigenase activity in experimental influenza virus infection.

Influenza virus infection was associated with development of oxidative stress in liver of mice, viz. increase in amount of lipid peroxidation products, decrease in cytochrome P-450 and NADP. H-cytochrome c-reductase activity, and inhibition of liver monooxygenases (aniline hydroxylase, ethylmorphine-N-demethylase, amidopyrine-N-demethylase and analgin-N-demethylase). These effects were most pronounced on the 7th day after virus inoculation as compared to the 5th one. Supplementation of mice with vitamin E before virus inoculation leads to liver protection against oxidative stress and toxicosis. A marked decrease of lipid peroxidation products and an increase of cytochrome P-450 and activities of monooxygenases was established. The stabilizing effect of vitamin E was dose-dependent and was most pronounced on the 5th day after virus inoculation as compared to the 7th one.

Hepatoprotective effect of C-phycocyanin: protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicty in rats.

Effect of C-phycocyanin (from Spirulina platensis) pretreatment on carbontetrachloride and R-(+)-pulegone-induced hepatotoxicity in rats was studied. Intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of phycocyanin to rats, one or three hours prior to R-(+)-pulegone (250 mg/kg) or carbontetrachloride (0.6 ml/kg) challenge, significantly reduced the hepatotoxicity caused by these chemicals. For instance, serum glutamate pyruvate transaminase (SGPT) activity was almost equal to control values. The losses of microsomal cytochrome P450, glucose-6-phosphatase and aminopyrine-N-demethylase were significantly reduced, suggesting that phycocyanin provides protection to liver enzymes. It was noticed that the level of menthofuran, the proximate toxin of R-(+)-pulegone was nearly 70% more in the urine samples collected from rats treated with R-(+)-pulegone alone than rats treated with the combination of phycocyanin and R-(+)-pulegone. The possible mechanism involved in the hepatoprotection is discussed.

Effects of neem flowers, Thai and Chinese bitter gourd fruits and sweet basil leaves on hepatic monooxygenases and glutathione S-transferase activities, and in vitro metabolic activation of chemical carcinogens in rats.

The objectives of this study were to determine the effects of feeding of four vegetables commonly consumed in Thailand, namely, flowers of the neem tree (Azadirachta indica var. siamensis), fruits of Thai and the Chinese bitter gourd (Momordica charantia Linn.) and leaves of sweet basil (Ocimum basilicum Linn) on the levels of phase I enzymes, which include cytochrome P450 (P450), aniline hydroxylase (ANH) and aminopyrine-N-demethylase (AMD) as well as the capacity to activate the mutagenicities of aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP), and to induce the phase II enzymes [i.e. glutathione S-transferase (GST)] in rat liver. It was found that feeding of the diets containing 12.5% neem flowers and Thai bitter gourd fruits for 2 weeks strongly enhanced GST activity, 2.7- and 1.6- fold of the pair-fed control values, respectively, while resulting in a marked reduction of the levels of most phase I reactions. Fruits of the Chinese bitter gourd, which is in the same species as Thai bitter gourd, had no effect on GST activity but decreased AMD activity and the in vitro metabolic activation of AFB1 and BaP. On the other hand, however, dietary sweet basil leaves caused a significant increase in the levels of both GST and all phase I enzymes. Results in the present study clearly demonstrate that neem flowers and Thai bitter gourd fruits contain monofunctional phase II enzyme inducers and compounds capable of repressing some monooxygenases, especially those involved in the metabolic activation of chemical carcinogens, while sweet basil leaves contain compounds, probably bifunctional inducers, capable of inducing both phase I and phase II enzymes and Chinese bitter gourd fruits contain only compounds capable of repressing some monooxygenases. These results therefore suggest that neem flowers and Thai bitter gourd fruits may possess chemopreventive potential, while those of Chinese bitter gourd fruits and sweet basil leaves are uncertain.