Cytochrome P450 3A4-Mediated Bioactivation and Its Role in Nomilin-Induced Hepatotoxicity.

Nomilin is a furan-containing triterpenoid isolated from the medicinal plants of citrus. The aim of this study was to investigate the in vitro and in vivo bioactivation of nomilin and the role in nomilin-induced hepatotoxicity. Microsomal incubations of nomilin supplemented with NADPH and GSH or NAL resulted in the detection of six conjugates (M1-M6). The structures of the metabolites were characterized based on LC-HRMS and NMR. Nomilin was bioactivated to a reactive cis-butene-dial (BDA) intermediate dependent on NADPH, and this intermediate suffered from the reaction with the nucleophiles (GSH and NAL) to form stable adducts. M1-M4 were identified as pyrrole derivatives, and M5 and M6 were pyrrolinone derivatives. M1 was further chemically synthesized and characterized by 13C NMR spectroscopy. M1 was the major metabolite detected in mice bile. Pretreatment with ketoconazole significantly reduced the formation of M1 in mice bile, while pretreatment with rifampicin significantly increased the formation of M1. Chemical inhibition together with recombinant human CYP450 phenotyping demonstrated that CYP3A4 was the major enzyme contributing to the bioactivation of nomilin. Toxicity study suggested that nomilin displayed dose-dependent liver injury in mice, while tetrahydro-nomilin was found to be nonhepatotoxic. Pretreatment with ketoconazole prevented mice from nomilin-induced liver injury. The liver injury induced by nomilin was deteriorated when the mice were pretreated with rifampicin. These findings provide evidence that CYP3A4-mediated bioactivation was indispensable in nomilin-induced hepatotoxicity.

Subchronic Toxicity Studies of Cortex Dictamni Extracts in Mice and Its Potential Hepatotoxicity Mechanisms in Vitro.

Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages (2.3, 4.6, or 9.2 g/kg/day, p.o.) in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines.

In vitro antitumor activities of the lichen compounds olivetoric, physodic and psoromic acid in rat neuron and glioblastoma cells.

Context Since methods utilised in the treatment of glioblastoma multiforme (GBM) are inadequate and have too many side effects, usage of herbal products in the treatment process comes into prominence. Lichens are symbiotic organisms used for medicinal purposes for many years. There are various anticancer treatments about components of two lichen species used in the present study. Objective Antitumor potential of three lichen secondary metabolites including olivetoric acid (OLA) and physodic acid (PHA) isolated from Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) and psoromic acid (PSA) isolated from Rhizoplaca melanophthalma (DC.) Leuckert (Lecanoraceae) were investigated on human U87MG-GBM cell lines and primary rat cerebral cortex (PRCC) cells for the first time. Materials and methods PRCC cells used as healthy brain cells were obtained from Sprague-Dawley rats. The treatments were carried out on the cells cultured for 48 h. Cytotoxic effects of different concentrations (2.5, 5, 10, 20 and 40 mg/L) of metabolites on the cells were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) analyses. Total antioxidant capacity (TAC) and total oxidant status (TOS) parameters were used for assessing oxidative alterations. Oxidative DNA damage potentials of metabolites were investigated via evaluating 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels. Results Median inhibitory concentration (IC50) values of OLA, PHA and PSA were 125.71, 698.19 and 79.40 mg/L for PRCC cells and 17.55, 410.72 and 56.22 mg/L for U87MG cells, respectively. It was revealed that cytotoxic effects of these metabolites showed positive correlation with concentration, LDH activity and oxidative DNA damage. Discussion and conclusion The present findings obtained in this study revealed that primarily OLA and then PSA had high potential for use in the treatment of GBM.

Antiviral activity of seed extract from Citrus bergamia towards human retroviruses.

The effects of an extract from Citrus bergamia (BSext) and those of two products purified from the same extract, that is, nomilin and limonin, and reference compounds, towards HTLV-1 have been reported. Moreover, they were also compared with those obtained towards HIV-1. Results showed that the efficacy of both BSext and limonin in inhibiting HTLV-1 as well as HIV-1 expression in infected cells, as evaluated by comparable quantitative assays, was close to that of the effective, reference compounds, respectively. The protective effect of BSext and of the purified products was associated with the inhibition of both HTLV-1 and HIV-1 RT activities in conceptually similar, cell-free assays. The cytotoxicity of the assayed compounds of natural origin was substantially less pronounced than that of the reference compounds, thus showing a favourable selectivity index for the novel BSext product.

Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity.

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.

Use of in vitro testing to identify an unexpected skin sensitizing impurity in a commercial product: a case study.

Due to regulatory constraints and ethical considerations, the quest for alternatives to animal testing has gained a new momentum. In general, animal welfare considerations and compliance with regulations are the key drivers for this research. Mechanistically based in vitro tests addressing specific toxicological questions can yield new information, for example on reactive components, and thus in certain cases the in vitro tests are not only second choice replacements of a 'gold standard' animal test but can also be used to develop safer products. Here we report a case study from the in vitro investigation on the commercial fragrance chemical Azurone. This compound was found to be a moderate skin sensitizer in the LLNA, whereas the structurally closely similar compound Calone is a non-sensitizer. A peptide reactivity assay indicated, that indeed Azurone yields peptide depletion, thus the in vitro assays confirmed the animal test result. LC-MS analysis of the peptide reactivity sample showed the presence of peptide adducts of unexpected molecular weight. They were consistent with the reaction of the peptide with a catechol related to Azurone. Detailed analytics indicated that indeed this catechol is present in the original batches as an impurity, but it has escaped quality control analysis, as it is not detectable in routine GC-analysis. A new purified batch was prepared, re-tested in the in vitro assays and predicted by the tests to be a non-sensitizer. A confirmatory LLNA test indeed yielded a significantly (10-fold) higher EC3 value of the new batch, but the LLNA was still positive. A dose-response study in the EpiSkin assay indicated that this molecule still has a significant skin irritation potential, which may generate the weak positive signal in the LLNA. This case study illustrates how the mechanistically based in vitro LC-MS peptide reactivity assay can be used to contribute to the understanding of the sensitization mechanism of a commercial product and help to define a safer product specification.

Studies in hypotensive agents. Synthesis and hypotensive activity of 3,4-dihydro-1(2H)-benzoxepine oxime ethers and their derivatives.

Substituted oximino-ethers of 3,4-dihydro-1(2H)-benzoxepines were synthezised from 3,4-dihydro-1(2H)benzoxepin-5-ones. The hypotensive activity of these compounds was evaluated on anaesthetized cats.

Synthesis and anti-inflammatory properties of benzoyl, halogenobenzoyl or cinnamoyl substituted 1-benzoxepanes and 2-methyl-1-benzoxolanes.

A series of new 7-acyl substituted 1-benzoxepanes and 5-acyl substituted 2-methyl-1-benzoxolanes were synthesized and studied for anti-inflammatory properties. The benzoyl derivatives were more active than the corresponding halogenobenzoyl derivatives and previously reported structural analogues containing less methylene groups in their heterocyclic ring. The introduction of a methyl group at the 2-position of 5-cinnamoyl-1-benzoxolane heterocyclic ring significantly potentiated the activity. Anti-inflammatory and analgesic effects of 7-benzoyl-1-benzoxepane and 5-cinnamoyl-2-methyl-I-benzoxolane were less pronounced than those of indomethacin and diclofenac but greater than those of acetylsalicylic acid (ASA). Both compounds were less toxic and 5-cinnamoyl-2-methyl-1-benzoxolane was also less gastrotoxic than all the reference drugs mentioned above. Therapeutic indices of 5-cinnamoyl-2-methyl-1-benzoxolane were greater than those of diclofenac and significantly greater than those of ASA and indomethacin.