Rat Liver Hepatocytes Research Articles

Collagen-rich liver-derived extracellular matrix hydrogels augment survival and function of primary rat liver sinusoidal endothelial cells and hepatocytes

Liver sinusoidal endothelial cells (LSECs) are key targets for addressing metabolic dysfunction-associated steatotic liver disease (MASLD). However, isolating and culturing primary LSECs is challenging due to rapid dedifferentiation, resulting in loss of function. The extracellular matrix (ECM) likely plays a crucial role in maintaining the fate and function of LSECs. In this study, we explored the influence of liver-ECM (L-ECM) on liver cells and developed culture conditions that maintain the differentiated function of liver cells in vitro for prolonged periods. Porcine liver-derived L-ECM, containing 34.9 % protein, 0.045 % glycosaminoglycans, and negligible residual DNA (41.2 ng/mg), was utilized to culture primary rat liver cells in generated hydrogels. Proteomic analyses and molecular weight distribution of proteins of solubilized L-ECM revealed the typical diverse ECM core matrisome, with abundant collagens. L-ECM hydrogels showed suitable stiffness and stress relaxation properties. Furthermore, we demonstrated that collagen-rich L-ECM hydrogels enhanced LSECs' and hepatocytes' viability, and reduced the dedifferentiation rate of LSECs. In addition, hepatocyte function was maintained longer by culture on L-ECM hydrogels compared to traditional culturing. These beneficial effects are likely attributed to the bioactive macromolecules including collagens, and mechanical and microarchitectural properties of the L-ECM hydrogels.

Parallelized Droplet Vitrification Enables Single-Run Vitrification of the Whole Rat Liver Hepatocyte Yield.

Drug discovery pipelines rely on the availability of isolated primary hepatocytes for investigating potential hepatotoxicity prior to clinical application. These hepatocytes are typically isolated from livers rejected for transplantation and subsequently cryopreserved for later usage. The gold-standard cryopreservation technique, slow-freezing, is a labor-intensive process, with significant post-storage viability loss. In this work, we introduce parallelized droplet vitrification, a technique for high-volumetric, rapid vitrification of suspended cells. We show the utility of this technique through the single-run vitrification of the whole-rate liver hepatocyte yield, resulting in a 1600% increase in single-batch vitrification and a 500% increase in droplet generation rate compared to previous droplet vitrification approaches. Additionally, we showed that these implementations maintained improved post-preservation outcomes in primary rat hepatocytes.

ДОСЛІДЖЕННЯ АНТИОКСИДАНТНИХ ВЛАСТИВОСТЕЙ ЕКСТРАКТІВ ВІДКАСНИКА БЕЗСТЕБЛОВОГО (Carlina acaulis L.), АРНІКИ ГІРСЬКОЇ (Arnica montana L.) ТА КАЛЕНДУЛИ ЛІКАРСЬКОЇ (Calendula officinalis L.)

The intensity of lipid peroxidation (LPO) and oxidative modification of proteins (OMP) was investigated under the influence of 40 % and 70 % aqueous-ethanol plant extracts (PE) of the root of Carlina acaulis L., flowers of Arnica montana L. and Calendula officinalis L. on rat liver hepatocytes under conditions of free radical oxidation initiation in vitro. Investigated plant extracts reduce the formation of free radicals in proteins and lipids, which is evidently associated with the presence of phenolic compounds, flavonoids, and polyphenols in the extracts. The best results were demonstrated by the plant extracts of arnica and calendula. Arnica extract at a concentration of 40 % exhibited better antioxidant properties than its 70 % extract.

АНТИОКСИДАНТНІ ВЛАСТИВОСТІ ЕКСТРАКТІВ КАВОВИХ ЗЕРЕН

The antioxidant properties (AP) of 5 % aqueous extracts of roasted coffee and its spent grounds were investigated. The antioxidant activity (AA) of these extracts acting on rat liver hepatocytes in vitro was determined using two markers of oxidative stress (OS): the levels of thiobarbituric acid reactive substances and carbonyl groups in proteins. It was demonstrated that all examined extracts exhibited AA, characterized by a reduction in non-enzymatic OS markers. The formation of products of free radical damage to lipids and proteins was inhibited by both 5 % aqueous extracts of roasted coffee and 5 % aqueous extracts of used coffee grounds, which holds promise for the development of antioxidant agents based on them.

Natural Pyrethrin-Induced Oxidative Damage in Human Liver Cells through Nrf-2 Signaling Pathway.

Natural pyrethrins (NPs), one kind of bio-pesticide, have been widely used in organic agriculture and ecological environment studies. Studies have shown that NPs may affect the metabolism of rat liver and human hepatocytes; nevertheless, the toxic effects of NPs on the liver and the related mechanisms are still incompletely understood. In this research, we utilized three types of human liver cells to investigate the mechanism of NPs' induction of oxidative stress. The results showed that NPs exhibit noteworthy cytotoxic effects on human liver cells. These effects are characterized by the induction of LDH release, mitochondrial collapse, and an increased production of ROS and MDA content, subsequently activating the Kelch-like ECH-associated protein 1/Nuclear factor erythroid 2- related factor 2 (Keap1/Nrf-2) pathway. The ROS inhibitor N-acetyl-L-cysteine (NAC) can alleviate ROS/Nrf2-mediated oxidative stress. In addition, the siRNA knockdown of Nrf-2 exacerbated the injury, including ROS production, and inhibited cell viability. In summary, the ROS-mediated Keap1/Nrf-2 pathway could be an important regulator of NP-induced damage in human liver cells, which further illustrates the hepatotoxicity of NPs and thereby contributes to the scientific basis for further exploration.

Simvastatin: In Vitro Metabolic Profiling of a Potent Competitive HMG-CoA Reductase Inhibitor

Simvastatin (SV) is a semisynthetic derivative of lovastatin (LV), which is biosynthetically produced from the fungus Aspergillus terreus and has a high log p value (log p = 4.39)and thus high hepatic extraction and high efficacy in controlling cholesterol synthesis. The current study was undertaken to investigate the metabolic profile of SV using various mass spectrometry (MS) platforms. Metabolic profiling was studied in in vitro models, rat liver microsomes (RLMs), and isolated perfused rat liver hepatocytes (RLHs) using both ion trap and triple quadruple LC–MS/MS systems. A total of 29 metabolites were identified. Among them, three types of SV-related phase-I metabolites, namely exomethylene simvastatin acid (exomethylene SVA), monohydroxy SVA, and dihydrodiol SVA, were identified as new in RLMs. No phase-II metabolites were identified while incubating with RLHs.

Rosmarinic acid exerts anti-inflammatory effect and relieves oxidative stress via Nrf2 activation in carbon tetrachloride-induced liver damage.

Rosmarinic acid (RA) has biological and pharmaceutical properties and shows hepatoprotective potential. However, the hepatoprotective mechanism of RA needs to be further elucidated in vivo and in vitro. This study was aimed to evaluate the protective effect of RA on carbon tetrachloride (CCl4)-induced liver injury and elucidate the hepatoprotective mechanism of RA in vivo and in vitro. In vivo, the mice were orally administrated with RA (10, 20, and 40 mg/kg bw) daily for 28 consecutive days, and 1% CCl4 (5 mL/kg bw, dissolved in peanut oil) was used to induce liver injury. In vitro, the big rat liver (BRL) hepatocytes were pretreated with RA (0.2, 0.4, and 0.8 mg/mL) for 3 h, and then the hepatocytes were treated with CC14 (final concentration, 14 mM) for 3 h to induce cell injury. The related indexes, including hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential, were evaluated. Oral administration of RA to mice considerably decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triacylglycerols (TG), total cholesterol (TC), total bilirubin (TBIL), hepatic reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). RA also increased the levels of hepatic glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and the protein expressions of Nrf2, quinine oxidoreductase (NQO1), and heme oxygenease-1 (HO-1). Histopathological examinations indicated that RA (20 and 40 mg/kg bw) alleviated the liver tissue injury induced by CCl4. Moreover, RA inhibited the hepatocyte apoptosis caused by CCl4 based on TUNEL assay. In vitro, RA pretreatment remarkably recovered the cell viability and reduced the CCl4-induced elevation of AST, ALT, lactate dehydrogenase (LDH), ROS, and 8-OHdG. Immunohistochemistry staining demonstrated that pretreatment with RA markedly inhibited the expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and Caspase-3 in CCl4-treated hepatocytes. Additionally, RA pretreatment significantly decreased the elevation of mitochondrial membrane potential in CCl4-treated hepatocytes. RA exerted a protective effect against CCl4-induced liver injury in mice through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl4 exposure.

A high-resolution accurate mass approach to identification of graveoline metabolites using ultra-high-performance liquid chromatography combined with a photo diode array detector andquadrupole/time-of-flight tandem mass spectrometry.

Graveoline is a biologically active ingredient extracted from Ruta graveolens. Current work aimed at investigating in vitro metabolism of graveoline using rat or human liver microsomes and hepatocytes. Graveoline (20 μM) was incubated with nicotinamide adenine dinucleotide phosphate-supplemented rat and human liver microsomes as well as hepatocytes. LC coupled to a photo diode array detector and quadrupole/time-of-flight tandem mass spectrometry was used to detect and identify the metabolites. The structures of the metabolites were identified by accurate mass, elemental composition, and indicative fragment ions. A total of 12 metabolites, comprising 6 phase I and 6 phase II metabolites, were obtained. The metabolic pathways included demethylenation, demethylation, hydroxylation, glucuronidation, and glutathion conjugation. The metabolite (M10) produced by opening the ring of the methylenedioxyphenyl moiety was detected as the most abundant in both liver microsomes and hepatocytes, mainly catalyzed by CYP1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. This study provides valuable information on the in vitro metabolism of graveoline, which is indispensable for further development and safety evaluation of this compound.

Effects of Artificial Sweeteners Aspartame and Sucralose on the Size of Hepatocytes in Rat Liver

Background: The excessive use of artificial sweeteners now a days just to lose weight and to remain fit and due to other reasons without knowing its effects on liver gave me a thought to conduct this study to see whether they are safe or not for liver health. Objectives: The objectives of the study were to see the effects of artificial sweeteners Aspartame and Sucralose on the size of rat hepatocytes and to compare their effects to choose relatively safe artificial sweetener for routine use. Methods: Fifty adult Wistar albino rats used in this study were randomly divided into five groups. Group I (control group) animals were given drinking water by oral gavage. The animals of group II and III were given low (40mg/kg body weight) and high doses (1000mg/kg body weight) of Aspartame respectively. Similarly, animals of group IV and V were given low (5mg/kg body weight) and high doses (1000mg/kg body weight) of Sucralose respectively. Doses were given by oral gavage once daily, six days a week for a total duration of eight weeks. At the end of the experiment histological observation of liver in all animal groups was made. Results: Microscopic examination revealed that hepatocytes of group III animals (high dose group of Aspartame) had largest size with the mean value of 20.86±2.00µm as compared to the control group hepatocytes having mean value of 16.46±1.74µm. Conclusion: The increase in the size of hepatocytes was seen in high dose groups of Aspartame and Sucralose whereas changes seen in low dose groups of Aspartame and Sucralose were not significant. Keywords: Artificial sweeteners, Histology of rat liver, Size of hepatocytes.

Effects of Artificial Sweeteners Aspartame and Sucralose on the Size of Hepatocytes in Rat Liver

Background: The use of artificial sweeteners in excess these days to lose weight and to remain fit without knowing its effects on our health, gave me a thought to conduct this study to see whether they are safe or not for liver health. Objectives: The objectives of this study were to see the effects of Aspartame and Sucralose (Artificial sweeteners) on rat hepatocytes and also to compare their effects to choose relatively safe artificial sweetener for routine use. Method: 50 adult Wistar albino rats were used in this study. Animals were divided into five groups by random lottery method. Control group I animals were given distilled water by oral gavage. The group II and III animals were given 40mg and 1000mg/kg body weight of Aspartame respectively. Whereas animals of group IV and V were given 5mg and 1000mg/kg body weight of Sucralose respectively. Doses to experimental animals were given by oral gavage once daily for a total duration of eight weeks. At the end of the experiment rats were sacrificed, livers were dissected out and after specimen preservation the tissue was passed through stages of slide preparation and later H and E staining was performed. Results: Light microscopic examination of liver slides revealed that largest sizes of hepatocytes were observed in high dose groups of Aspartame and Sucralose. Conclusion: The increase in the size of hepatocytes was observed in high dose groups of Aspartame and Sucralose whereas changes seen in low dose groups were not significant. Keywords: Artificial sweeteners, Histology of rat liver, Size of hepatocytes.

Effects of Artificial Sweeteners Aspartame and Sucralose on the Size of Hepatocytes in Rat Liver

Background: The excessive use of artificial sweeteners now a days just to lose weight and to remain fit and due to other reasons without knowing its effects on liver gave me a thought to conduct this study to see whether they are safe or not for liver health. Aim: To see the effects of artificial sweeteners Aspartame and Sucralose on the size of rat hepatocytes and to compare their effects to choose relatively safe artificial sweetener for routine use. Methods: Fifty adult Wistar albino rats used in this study were randomly divided into five groups. Group I (control group) animals were given drinking water by oral gavage. The animals of group II and III were given low (40mg/kg body weight) and high doses (1000mg/kg body weight) of Aspartame respectively. Similarly, animals of group IV and V were given low (5mg/kg body weight) and high doses (1000mg/kg body weight) of Sucralose respectively. Doses were given by oral gavage once daily, six days a week for a total duration of eight weeks. At the end of the experiment histological observation of liver in all animal groups was made. Results: Microscopic examination revealed that hepatocytes of group III animals (high dose group of Aspartame) had largest size with the mean value of 20.86±2.00µm as compared to the control group hepatocytes having mean value of 16.46±1.74µm. Conclusion: The increase in the size of hepatocytes was seen in high dose groups of Aspartame and Sucralose whereas changes seen in low dose groups of Aspartame and Sucralose were not significant. Keywords: Artificial sweeteners, Histology of rat liver, Size of hepatocytes.

Identification and characterization of the in vitro metabolites of batatasin III by liquid chromatography in combination with Orbitrap mass spectrometry.

Batatasin III is a biologically active ingredient extracted from Dendrobium scabrilingue, which has been demonstrated to have anticancer activity. To fully understand its action, the present study was performed to investigate the in vitro metabolism of batatasin III using rat and human liver microsomes and hepatocytes. Batatasin III (20 μM) was incubated in the presence of NADPH-supplemented rat and human liver microsomes (0.5mg protein/mL) and hepatocytes (1 million cells/mL) followed by liquid chromatography in combination with hybrid quadrupole Orbitrap tandem mass spectrometric analysis to detect and identify the generated metabolites. The structures of the metabolites were characterized by comparing the accurate masses, elemental compositions as well as indicative fragment ions with those of the parent. A total of 15 metabolites were detected and identified, including 4 phase I and 11 phase II metabolites. Batatasin III is subjected to bioactivation to form reactive quinoid intermediates, which subsequently react with glutathione (GSH) via Michael addition. Glucuronidation and GSH conjugation appear to be the primary elimination pathways in rat hepatocytes, while in human hepatocytes, GSH conjugates are formed to a lesser extent. Phase I metabolic pathways include hydroxylation and demethylation. The present study sheds light on the in vitro metabolic fates of batatasin III, which is indispensable for an understanding of its efficacy and safety.

The in vitro metabolism and in vivo pharmacokinetics of the bacterial β-glucuronidase inhibitor UNC10201652

In vitro incubation of the bacterial β-glucuronidase inhibitor UNC10201652 (4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine) with mouse, rat, and human liver microsomes and hepatocytes generated metabolites at multiple sites via deethylations, oxidations and glucuronidation. Two UNC10201652 metabolites were detected in human, and four in mouse and rat liver microsomal incubations. Intrinsic clearances of UNC10201652 in human, mouse, and rat liver microsomes were 48.1, 115, and 194 µL/min/mg respectively. Intrinsic clearances for human, mouse, and rat hepatocytes were 20.9, 116, and 140 µL/min/106 cells respectively and 24 metabolites were characterised: 9 for human and 11 for both rodent species. Plasma clearance was 324.8 mL/min/kg with an elimination half-life of 0.66 h following IV administration of UNC10201652 to Swiss Albino mice (3 mg/kg). Pre-treatment with 1-aminobenzotriazole (ABT) decreased clearance to 127.43 mL/min/kg, increasing the t 1/2 to 3.66 h. Comparison of profiles after oral administration of UNC10201652 to control and pre-treated mice demonstrated a large increase in C max (from 15.2 ng/mL to 184.0 ng/mL), a delay in Tmax from 0.25 to 1 h and increased AUC from 20.1 to 253 h ng/ml. ABT pre-treatment increased oral bioavailability from 15% to >100% suggesting that CYP450's contributed significantly to UNC10201652 clearance in mice.

Metabolism of Speciociliatine, an Overlooked Kratom Alkaloid for its Potential Pharmacological Effects.

Speciociliatine, a diastereomer of mitragynine, is an indole-based alkaloid found in kratom (Mitragyna speciosa). Kratom has been widely used for the mitigation of pain and opioid dependence, as a mood enhancer, and/or as an energy booster. Speciociliatine is a partial µ-opioid agonist with a 3-fold higher binding affinity than mitragynine. Speciociliatine has been found to be a major circulating alkaloid in humans following oral administration of a kratom product. In this report, we have characterized the metabolism of speciociliatine in human and preclinical species (mouse, rat, dog, and cynomolgus monkey) liver microsomes and hepatocytes. Speciociliatine metabolized rapidly in monkey, rat, and mouse hepatocytes (in vitro half-life was 6.6 ± 0.2, 8.3 ± 1.1, 11.2 ± 0.7min, respectively), while a slower metabolism was observed in human and dog hepatocytes (91.7 ± 12.8 and > 120min, respectively). Speciociliatine underwent extensive metabolism, primarily through monooxidation and O-demethylation metabolic pathways in liver microsomes and hepatocytes across species. No human-specific or disproportionate metabolites of speciociliatine were found in human liver microsomes. The metabolism of speciociliatine was predominantly mediated by CYP3A4 with minor contributions by CYP2D6.

Characterization and identification of the metabolites of dihydromethysticin by ultra-high-performance liquid chromatography orbitrap high-resolution mass spectrometry.

Dihydromethysticin, a natural component from Piper methysticum Forst, has been reported to display pharmacological effects in mental disorders and some malignant tumors. However, the metabolism of this component remained unknown. The goal of this work was conducted to discover the metabolic profiles of dihydromethysticin. The in vitro incubation was performed by incubating dihydromethysticin with rat, monkey, and human liver microsomes and hepatocytes. An analytical assay of ultra-high performance liquid chromatography combined with Orbitrap high-resolution mass spectrometry was utilized to detect and identify the metabolites. With high resolution mass spectrometric determination, the accurate mass, elemental composition, and product ions of the metabolites were determined, which enabled structural characterization to become easy. Under the present conditions, four phase-I metabolites, as well as six phase-II metabolites, were detected and their tentative structures were characterized by mass spectra. M4 was found as the most abundant metabolite both in liver microsomes and hepatocytes. Cytochrome P450 1A2, 2C9, and 3A4 contributed to the formation of this metabolite by using human recombinant P450 enzymes. M4 can be oxidized into reactive ortho-quinone intermediate followed by conjugating with glutathione. M4 was also subject to glucuronidation (M1 and M2) and methylation (M5). Demethylenation, oxidation, hydroxylation, glucuronidation, glutathionylation, and methylation were the primary metabolic pathways of dihydromethysticin. This study provides in vitro metabolism data of dihydromethysticin, which is indispensable for understanding the disposition of this compound.

Identification of the metabolites of methylophiopogonanone A by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry.

Methylophiopogonanone A (MOA) is a naturally occurring homoisoflavonoid from the Chinese herb Ophiopogon japonicus, which has been demonstrated to attenuate myocardial apoptosis. However, the metabolism of MOA remains unknown. The goal of the present work was to investigate the in vitro metabolism of MOA using liver microsomes and hepatocytes. The metabolites were generated by incubating MOA with rat, monkey and human liver microsomes or hepatocytes. The resulting samples were analyzed by using a quadrupole-orbitrap high-resolution mass spectrometer. The metabolites were identified through the measurements of the exact mass, elemental composition and product ions. A total of 15 metabolites were detected and identified. Among these metabolites, M7 (demethylenation) was the most abundant metabolite in liver microsomes, while M6 (hydroxylation) was the predominant metabolite in hepatocytes, and glucuronidation metabolites (M9 and M10) were also the main metabolites in hepatocytes. The metabolic pathways of MOA included hydroxylation, demethylenation, glucuronidation, methylation, sulfation and glutathione conjugation. This study for the first time provides valuable data on the metabolites of MOA, which will be of great importance for a better understanding of its disposition and to predict human pharmacokinetics.

RTICBM-74 Is a Brain-Penetrant Cannabinoid Receptor Subtype 1 Allosteric Modulator that Reduces Alcohol Intake in Rats.

The endocannabinoid system is implicated in the neuronal mechanisms of alcohol use disorder (AUD), with the cannabinoid receptor subtype 1 (CB1) representing a promising target for AUD therapeutic interventions. We have previously shown negative allosteric modulators (NAMs) of the CB1 receptor attenuated the reinstatement of other drugs of abuse including cocaine and methamphetamine in rats; however, their effects on alcohol-related behaviors have not been investigated. Here, we tested the pharmacokinetic properties of one such CB1 NAM, RTICBM-74, and its effects on alcohol self-administration in rats. RTICBM-74 showed low aqueous solubility and high protein binding but had excellent half-life and low clearance against rat liver microsomes and hepatocytes, and excellent brain penetrance in rats. RTICBM-74 pretreatment specifically reduced alcohol intake across a range of doses in male or female Wistar or Long-Evans rats that were trained to self-administer alcohol. These effects were similar to the CB1 antagonist/inverse agonist rimonabant, which was tested as a positive control. Importantly, RTICBM-74 was effective at reducing alcohol intake at doses that did not affect locomotion or sucrose self-administration. Our findings suggest that CB1 NAMs such as RTICBM-74 have promising therapeutic potential in treatment of AUD. SIGNIFICANCE STATEMENT: The present work shows that a metabolically stable and brain-penetrant cannabinoid receptor subtype 1 negative allosteric modulator reduces alcohol self-administration in rats without affecting locomotion or sucrose self-administration, suggesting potential therapeutic relevance for the treatment of alcohol use disorder.

Rapid detection and structural characterization of methysticin metabolites generated from rat and human liver microsomes and hepatocytes using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

Methysticin is a naturally occurring ingredient isolated from Piper methysticum Forst. The metabolic profile of methysticin is unknown. The goal of this study was to elucidate the metabolism of methysticin using rat and human liver microsomes and hepatocytes. The incubation samples were analyzed using ultra-high-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-HRMS). The structures of the metabolites were characterized based on the elemental composition, exact mass, and product ions. A total of 10 metabolites were detected and identified. Among these metabolites, M4 (ring opening of 1,3-benzodioxole) was the predominant metabolite in rat and human liver microsomes. M4 and its glucuronide conjugate (M2) were the major metabolites in rat and human hepatocytes. The metabolic pathways of methysticin are summarized as follows: (a) oxidative ring opening of 1,3-benzodioxole forms the catechol derivative (M4), which subsequently undergoes glucuronidation (M1 and M2), methylation (M8), and sulfation (M7). (b) Demethylation to yield desmethyl methysticin (M6), followed by glucuronidation (M3 and M5). (c) Hydroxylation (M9 and M10). For the first time, this study provides new information on the in vitro metabolic profiles of methysticin, which facilitates an understanding of the disposition of this bioactive ingredient.

Identification of the metabolites of XL092 in rat and human by using ultra-high performance liquid chromatography high resolution mass spectrometry

XL092 is a novel tyrosine kinase inhibitor with antitumor activity. The goal of this study was to evaluate its in vitro metabolism of XL092 using rat and human liver microsomes and hepatocytes. The metabolites were identified using ultra-high performance liquid chromatography combined with high resolution mass spectrometry. The structure of the metabolite was characterized by accurate mass, elemental composition and MS/MS spectra. The cytochrome P450 enzyme responsible for XL092 metabolism was evaluated by using recombinant human CYP450 enzymes. A total of 26 metabolites, including 21 phase I metabolites and 5 phase II metabolites, were characterized. XL092 was metabolized mainly through oxidative defluorination, hydroxylation, N-demethylation, O-demethylation, amide hydrolysis, N-dealkylation, O-dealkylation, N-oxygenation and glucuronidation. Among these metabolites, M10 (oxidative defluorination) and M17 (hydroxylation) were the most abundant metabolites. CYP3A4 and CYP2D6 were the major enzymes responsible for XL092 metabolism. Taken together, this study for the first time evaluated the in vitro metabolic profiles of XL092 in rat and human, which is of great help for us to investigate the XL092 pharmacokinetic and toxicity assessment and to predict the in vivo human metabolism.

Identification of the metabolites of rhapontigenin in rat and human by ultra-high-performance liquid chromatography-high-resolution mass spectrometry.

Rhapontigenin, a stilbene compound isolated from the medicinal plant of rhubarb rhizomes, has shown a variety of biological activities. The purpose of this study was to identify and characterize the metabolites of rhapontigenin in rat liver microsomes, hepatocytes, urine, and human liver microsomes and hepatocytes. The samples were analyzed by ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q/Orbitrap-HRMS). The structures of the metabolites were interpreted by MS, MS/MS data, and elemental compositions. A total of 11 metabolites were detected and tentatively identified. M1, identified as piceatannol, was unambiguously identified using reference standard. Our results suggested that rhapontigenin was metabolized through the following pathways: (a) demethylation to produce piceatannol (M1), which further underwent oxidation to form ortho-quinone intermediate. This intermediate was reactive and conjugated with GSH (M10 and M11), which were further converted into N-acetyl-cysteine and excreted in urine. M1 also underwent sulfation (M8) and glucuronidation (M5); (b) direct sulfation, forming M6 and M7; and (c) direct glucuronidation to form M2, M3, and M4. Glucuronidation was a major metabolic pathway in hepatocytes and urine. The current study provides an overview of the metabolism of rhapontigenin, which is of great importance for us to understand the disposition of this compound.