Drug interaction potential of Ankaferd blood stopper® in human hepatocarcinoma cells.

BACKGROUND: Ankaferd blood stopper® (ABS) is an herbal extract consisting of mixtures of Alpinia officinarum, Gycyrrhiza glabra, Vitis vinifera, Thymus vulgaris, and Urtica dioica plants and has been used in recent years in Turkish medicine as a hemostatic agent. Despite its extensive usage, there is no information available about the drug interaction in HepG2 cells. The current work evaluated the effect of ABS on the expression of CYP1A1-1A2, CYP2E1, and CYP3A4 isozymes that are primarily involved in drug and carcinogen metabolism. METHODS: We selected HepG2 cells as in vitro cellular models of the human liver. The cells were treated with different concentrations of ABS [0.25%-40% (v/v)]. A crystal violet staining assay was used to determine the cytotoxicity of ABS. We examined drug-metabolizing enzymes, including 7-ethoxyresorufin O-deethylase (CYP1A1), 7-methoxyresorufin O-demethylase (CYP1A2), aniline 4-hydroxylase (CYP2E1), and erythromycin N-demethylase (CYP3A4), in vitro in HepG2 cells. The expression (mRNA, protein) levels of drug-metabolizing enzymes were analyzed by qPCR and Western blotting, respectively. RESULTS: The EC05 and EC10 values for ABS were 0.37% and 0.52% (v/v), respectively. Therefore, 0.37% and 0.52% (v/v) doses were used for the remaining portion of this study. Investigation of the expression and activity levels revealed that CYP1A1-1A2, CYP2E1, and CYP3A4 activities were not affected by ABS significantly, with qPCR and Western blot results corroborating this result. DISCUSSION: Our study found that the activity, mRNA, and protein expression levels of CYP isozymes did not change with the application of ABS, suggesting that when humans are exposed to ABS, there may not be any risk associated with clinical drug toxicity, cancer formation, and drug metabolism disorders in humans.

Role of AHR, NF-kB and CYP1A1 crosstalk with the X protein of Hepatitis B virus in hepatocellular carcinoma cells.

In this study, it was aimed to elucidate the interaction between aryl hydrocarbon receptor (AHR), nuclear factor-kappa B (NF-kB), and cytochrome P4501A1 (CYP1A1) with hepatitis B virus X protein (HBX) in a human liver cancer cell line (HepG2) transfected with HBX. First, AHR, NF-kB, and CYP1A1 genes were cloned into the appropriate region of the CheckMate mammalian two-hybrid recipient plasmids using a flexi vector system. Renilla and firefly luciferases were quantified using the dual-luciferase reporter assay system to measure the interactions. Secondly, transient transfections of CYP1A1 and NF-kB (RelA) were performed into HBX-positive and HBX-negative HepG2 cells. The mRNA expression of CYP1A1 and NF-kB genes were confirmed with RT-PCR, and cell viability was measured by WST-1. Further verification was assessed by measuring the activity and protein level of CYP1A1. Additionally, CYP1A1/HBX protein-protein interactions were performed with co-immunoprecipitation, which demonstrated no interaction. These results have clearly shown that the NF-kB and AHR genes interact with HBX without involving CYP1A1 and HBX protein-protein interactions. The present study confirms that AHR and NF-kB interaction plays a role in the HBV mechanism mediated via HBX and coordinating the carcinogenic or inflammatory responses; still, the CYP1A1 gene has no effect on this interaction.

Suppression of Inflammatory Cytokines Expression with Bitter Melon (Momordica Charantia) in TNBS-instigated Ulcerative Colitis.

BACKGROUND AND OBJECTIVE: This study was aimed to elucidate the molecular mechanism of Momordica charantia (MCh), along with a standard drug prednisolone, in a rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS). METHODS: After the induction of the experimental colitis, the animals were treated with MCh (4 g/kg/day) for 14 consecutive days by intragastric gavage. The colonic tissue expression levels of C-C motif chemokine ligand 17 (CCL-17), interleukin (IL)-1ß, IL-6, IL-23, interferon-γ (IFN-γ), nuclear factor kappa B (NF-kB), and tumor necrosis factor-α (TNF-α), were determined at both mRNA and protein levels to estimate the effect of MCh. Besides, colonic specimens were analyzed histopathologically after staining with hematoxylin and eosin. RESULTS: The body weights from TNBS-instigated colitis rats were found to be significantly lower than untreated animals. Also, the IFN-γ, IL-1ß, IL-6, Il-23, TNF-α, CCL-17, and NF-kB mRNA and protein levels were increased significantly from 1.86-4.91-fold and 1.46-5.50-fold, respectively, in the TNBS-instigated colitis group as compared to the control. Both the MCh and prednisolone treatment significantly reduced the bodyweight loss. It also restored the induced colonic tissue levels of IL-1ß, IL-6, IFN-γ, and TNF-α to normal levels seen in untreated animals. These results were also supported with the histochemical staining of the colonic tissues from both control and treated animals. CONCLUSION: The presented data strongly suggests that MCh has the anti-inflammatory effect that might be modulated through vitamin D metabolism. It is the right candidate for the treatment of UC as an alternative and complementary therapeutics.

Essential oil composition, antioxidant activity and phenolic content of endemic Teucrium alyssifolium Staph. (Lamiaceae).

The present study was designed to examine the chemical composition of the essential oil, in vitro antioxidant activity and total phenolic and flavonoid content of extracts from plant parts (leaf, flower and stem) of Teucrium alyssifolium. The principle components of the essential oil were trans-ß-caryophyllene (16.87%), ar-curcumene (11.43%) and bisabolene (11.06%), representing 39.36% of the oil. The total phenolic contents ranged between 13.99 and 41.54 mg of GAE/g of extract. The concentrations of flavonoids varied from 16.82 to 49.52 mg of Ru/g of extract. Antioxidant activity was determined in vitro using DPPH reagent and expressed as concentration of each extract required to inhibit radical by 50% (IC50) values that ranged from 13.52 to 132.55 µg/ml. Our results have indicated that water extract of T. alyssifolium (part leaf) with a total content of polyphenols (41.54 mg of GAE/g) and an IC50 of 13.52 µg/ml is more antioxidant.

Contribution of ellagic acid on the antioxidant potential of medicinal plant Epilobium hirsutum.

In the present study, the possible role of ellagic acid (EA) on antioxidant potential of Epilobium hirsutum (EH) in rat liver was investigated. Wistar rats were intraperitoneally treated with 37.5 mg/kg of EH and 10 mg/kg of EA for 9 days. Effects of EH and EA on antioxidant [glutathione peroxidase (GPx) and superoxide dismutases (SOD)] and Phase II [NADPH quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTs)] enzyme activities, as well as protein and mRNA expressions of those, were investigated. Polyphenolic content of EH was determined by LC-MS/MS analysis. EH and EA injection to rats resulted in a significant increase of NQO1 (3.6-fold and 4.7-fold), GPx (1.45-fold), and SOD (1.34-fold and 1.27-fold) enzyme activities, whereas total GST (46% and 57%) and its isoforms,and GST mu (57% and 72%), and GST theta (60% and 68%) activities were significantly decreased. Western-blot and qRT-PCR analysis showed that NQO1 and GPx protein and mRNA expressions were increased significantly (P < 0.0001), whereas GST mu and GST theta were significantly decreased (P < 0.0001).

Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20 mg/kg, respectively, for 9 days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21 % in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43 %, and 57 and 57 % in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82 % as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37 % with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content.

Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

A comparative study for the evaluation of two doses of ellagic acid on hepatic drug metabolizing and antioxidant enzymes in the rat.

The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities, mRNA and protein levels in rats.

CONTEXT: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes. OBJECTIVE: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver. MATERIALS AND METHODS: Male Wistar Albino rats were injected with EH at a dose of 37.5 mg/kg i.p. daily for 9 d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively. RESULTS: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54 ± 1.21 and 4.48 ± 1.67 nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537 ± 0.011 and 0.109 ± 0.01 nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069 ± 0.015 to 0.107 ± 0.026 nmol/min/mg and from 163.34 ± 92 to 588.3 ± 14 nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment. DISCUSSION AND CONCLUSION: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.

Alteration of drug metabolizing enzymes in sulphite oxidase deficiency.

The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments.

Drug interaction potential of the seed extract of Urtica urens L. (dwarf nettle).

Dwarf nettle (Urtica urens) seed extract was examined in vivo in the rat for its potential to modulate drug metabolizing enzymes including aminopyrine N-demethylase (APND; CYP2C6), aniline 4-hydroxylase (A4H; CYP2E1), nitrosodimethylamine N-demethylase (NDMA-ND; CYP2E1) erythromycin N-demethylase (ERND; CYP3A1) CYP2D1/2 and glutathione S-transferase (GST). RT-PCR data and western blotting studies clearly demonstrated that CYP2C6 and CYP2E1 mRNA levels were substantially increased after Urtica treatment, while the level of CYP3A1 mRNA decreased and that of CYP2D1/2 remained unchanged. Urtica treatment significantly induced GST activity in the liver, lung and kidney (66-, 46- and 31-fold, respectively) while decreasing that of APND (35-, 61- and 94-fold) and NDMA-ND (23, 28 and 54-fold). ERND activity in liver was reduced 45-fold, but increased in the lung and kidney (78- and 144-fold) after Urtica treatment. These results indicate that Urtica seed extract may have the potential to inhibit and/or induce the metabolism of certain co-administered drugs.

Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet (Liza saliens).

In this study, feral leaping mullet (Liza saliens) liver microsomal 7-ethoxyresorufin O-deethylase (EROD), and cytosolic glutathione S-transferases (GSTs) activities were investigated using 7-ethoxyresorufin, 1-chloro-2,4-dinitrobenzene (CDNB), and ethacrynic acid (EA) as substrates, respectively. The average EROD activity was found as 1139+/-175 pmol resorufin/min/mg protein. The average GST activities towards CDNB and EA were found as 1364+/-41 and 140+/-19 nmol/min/mg protein, respectively. We have, then, investigated the in vitro effects of some metals and detergents on CYP1A and GST activities in leaping mullet liver. Leaping mullet liver microsomal EROD activity was significantly inhibited by Hg (0.1 mM), Ni (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Sb (0.1 mM), Fe2+ (1 mM), Co (1 mM), Al (1 mM), and Fe3+ (1 mM), with the percent inhibition of 80, 80, 77, 75, 70, 69, 56, 53, 46, and 44, respectively. Similarly, conjugation of CDNB catalyzed by GST was inhibited significantly to lesser extend by Hg (0.1mM), Sb (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Fe3+ (1 mM), Co (1 mM), and Fe2+ (1 mM), with the percent inhibition of 70, 69, 65, 61, 54, 51, 47, and 43, respectively. The degrees of inhibition observed on GST catalyzed EA conjugation by Hg (0.1 mM), Cd (0.1 mM), Sb (0.1 mM), Cu (0.1 mM), and Zn (0.1 mM) were 86, 78, 69, 51, and 42, respectively. In addition to metals, the effect of various detergents on leaping mullet liver EROD, GST-CDNB, and GST-EA activities were studied. It was found that ionic detergents strongly inhibited the EROD activity, whereas much less inhibitions were observed with GST catalyzed activities. Therefore, the CYP1A inhibition potencies of metals and detergents suggest that their contribution to the overall CYP1A induction in polycyclic aromatic hydrocarbons contaminated environmental samples has to be taken into account for better interpretation of environmental studies.