Tissue Distribution of Berberine and Its Metabolites after Oral Administration in Rats

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR’s plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MSⁿ-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR’s level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR’s metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC _(0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR’s pharmacological effects on human diseases in clinic.     

Orally Administered Glucosylceramide Improves the Skin Barrier Function by Upregulating Genes Associated with the Tight Junction and Cornified Envelope Formation

Dietary glucosylceramide improves the skin barrier function. We used a microarray system to analyze the mRNA expression in SDS-treated dorsal skin of the hairless mouse to elucidate the molecular mechanisms involved. The transepidermal water loss of mouse skin was increased by the SDS treatment, this increase being significantly reduced by a prior oral administration of glucosylceramides. The microarray-evaluated mRNA expression ratio showed a statistically significant increase in the expression of genes related to the cornified envelope and tight junction formation when compared with all genes in the glucosylceramide-fed/SDS-treated mouse skin. We then examined the contribution of glucosylceramide metabolites to the tight junction formation of cultured keratinocytes. The SDS treatment of cultured keratinocytes significantly decreased the transepidermal electrical resistance, this decrease being significantly ameliorated in the presence of sphingosine or phytosphingosine, the major metabolites of glucosylceramide. These results suggest that an oral administration of glucosylceramide improved the skin barrier function by up-regulating genes associated with both the cornified envelope and tight junction formation.     

Orally administered glucosylceramide improves the skin barrier function by upregulating genes associated with the tight junction and cornified envelope formation

Dietary glucosylceramide improves the skin barrier function. We used a microarray system to analyze the mRNA expression in SDS-treated dorsal skin of the hairless mouse to elucidate the molecular mechanisms involved. The transepidermal water loss of mouse skin was increased by the SDS treatment, this increase being significantly reduced by a prior oral administration of glucosylceramides. The microarray-evaluated mRNA expression ratio showed a statistically significant increase in the expression of genes related to the cornified envelope and tight junction formation when compared with all genes in the glucosylceramide-fed/SDS-treated mouse skin. We then examined the contribution of glucosylceramide metabolites to the tight junction formation of cultured keratinocytes. The SDS treatment of cultured keratinocytes significantly decreased the transepidermal electrical resistance, this decrease being significantly ameliorated in the presence of sphingosine or phytosphingosine, the major metabolites of glucosylceramide. These results suggest that an oral administration of glucosylceramide improved the skin barrier function by up-regulating genes associated with both the cornified envelope and tight junction formation.     

Anti-initiating activity of 3beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34) from the stem bark of Picea jezoensis Carr. var. jezoensis

Ultraviolet light is the major cause of skin cancers in human, and several effects of ultraviolet light B (UVB) are thought to contribute to skin photocarcinogenesis. 3Beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34; 1) isolated from Picea jezoensis Carr. var. jezoensis showed the strongest antitumor-promoting activity among naturally occurring triterpenoids in the in vivo two-stage mouse skin-carcinogenesis test. To investigate the anti-initiating activity, we further studied mouse models initiated with ultraviolet-B (UVB) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Oral administration of the PJJ-34 (1) during a period before and after the three times of UVB irradiation led to a remarkable effect: oral administration of a 0.0025% solution of 1 only to the test group, which started one week before and ended one week after the irradiation, showed less than half papillomas, and inhibition of tumor incidence and tumor multiplicity in comparison to the control group. Therefore, it was recognized that PJJ-34 (1) showed strong anti-initiating activity as well as anti-promoting activity. After all, 1 seems to be useful as cancer-chemopreventive agent.     

Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice

Resveratrol (RESV) is a polyphenol with pleiotropic effects that include reduction of oxidative stress and increased vascular nitric oxide (NO) production. However, whether or not RESV can prevent rises in blood pressure (BP) is controversial and remains to be firmly established. The purpose of this study was to determine whether RESV attenuates elevated BP and subsequent adaptive cardiac hypertrophy and to better understand the mechanisms involved. The spontaneously hypertensive rat (SHR) and the angiotensin (Ang)-II infused mouse were used as hypertensive models. Compared to a standard control diet, consumption of diets containing RESV by SHRs and Ang-II hypertensive mice, markedly prevented rises in systolic BP. In addition, flow-mediated vasodilation was significantly improved by RESV in SHRs. RESV also reduced serum and cardiac levels of the lipid peroxidation by-product, 4-hydroxy-2-nonenal in the hypertensive rodents and inhibited the production of superoxide in human-derived endothelial cells. Analysis of mesenteric arteries from SHRs and Ang-II infused mice demonstrated that RESV increased endothelial NO synthase (eNOS) phosphorylation by enhancing the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signal transduction pathway. Moreover, RESV reduced hypertrophic growth of the myocardium through reduced hemodynamic load and inhibition of the p70 S6 kinase pro-hypertrophic signaling cascade. Overall, we show that high dose RESV reduces oxidative stress, improves vascular function, attenuates high BP and prevents cardiac hypertrophy through the preservation of the LKB1–AMPK–eNOS signaling axis.     

Absorption and distribution of sodium [2-14C]barbital in tissues of normal and dystrophic mice

The absorption and distribution of [2-14C]barbital after oral administration was studied in various tissues, including skeletal muscle, of normal and dystrophic mice. There appeared to be a more rapid gastric emptying in the mutant homozygote as reflected in lower levels of the drug recuperated from the gastrointestinal tract. This resulted in initially higher plasma and tissue concentrations of barbital in the dystrophic mice. Two hours after oral administration, this kinetic profile was reversed so that less barbital remained in the tissues of the dystrophic mouse. The tissue:plasma concentration ratios were consistently, but not significantly, higher in all tissues of the dystrophic animals. Analysis of the half-life of the drug in both groups suggests that there is an increase in the distribution volume of barbital in the dystrophic mice. The phenomenon of more rapid absorption of the barbiturate seems to be more consistent as the symptoms of the disease progress. The altered absorption and disposition of barbital in various tissues of the dystrophic mouse support the concept that a generalized multisystemic disorder may be crucial to the pathogenesis of murine muscular dystrophy, in contradistinction to a purely myogenic origin.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Oral administration of whole dihomo-γ-linolenic acid-producing Saccharomyces cerevisiae suppresses cutaneous inflammatory responses induced by croton oil application in mice

Polyunsaturated fatty acids have been attracting considerable interest because of their many biological activities and important roles in human health and nutrition. Dihomo-γ-linolenic acid (DGLA; C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. Taking advantage of genetic tractability and increasing wealth of accessible data of Saccharomyces cerevisiae, we have previously constructed a DGLA-producing yeast strain by introducing two types of desaturase and one elongase genes to convert endogenous oleic acid (C18:1n-9) to DGLA. In this study, we investigated the efficacy of oral intake of heat-killed whole DGLA-producing yeast cells in the absence of lipid purification on cutaneous inflammation. Topical application of croton oil to mouse ears induces ear swelling in parallel with the increased production of chemokines and accumulation of infiltrating cells into the skin sites. These inflammatory reactions were significantly suppressed in a dose-dependent manner by oral intake of the DGLA-producing yeast cells for only 7 days. This suppression was not observed by the intake of the γ-linolenic acid-producing (C18:3n-6, an immediate precursor of DGLA) yeast, indicating DGLA itself suppressed the inflammation. Further analysis demonstrated that DGLA exerted an anti-inflammatory effect via prostaglandin E1 formation because naproxen, a cyclooxygenase inhibitor, attenuated the suppression. Since 25-fold of purified DGLA compared with that provided as a form of yeast was not effective, oral administration of the whole DGLA-producing yeast is considered to be a simple but efficient method to suppress inflammatory responses.     

Uptake of [14C]oxamniquine by Schistosoma mansoni

The uptake and retention of drug-related material by Schistosoma mansoni was studied in the mouse host following a single oral or intramuscular dose (50 mg/kg) of [14C]oxamniquine. Male worms took up more labelled material than did female worms but the amount in each particular sex of worm was found to be similar after both routes of administration. Exposure of worms was therefore independent of the route of administration. Six days after drug administration, at the time of an hepatic shift, significantly more drug-related material was present in male worms than in female worms. Examination of worms recovered from mice 4 h after treatment showed that metabolites of oxamniquine constituted 70--90% of the drug-related material present in the worms. Both sexes of worms were able to take up metabolites of oxamniquine in vitro.     

Intrinsic Differences between Oral and Skin Keratinocytes

Keratinocytes cover both the skin and some oral mucosa, but the morphology of each tissue and the behavior of the keratinocytes from these two sites are different. One significant dissimilarity between the two sites is the response to injury. Oral mucosal wounds heal faster and with less inflammation than equivalent cutaneous wounds. We hypothesized that oral and skin keratinocytes might have intrinsic differences at baseline as well as in the response to injury, and that such differences would be reflected in gene expression profiles.     

Comparison of the bioavailability of quercetin and catechin in rats

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.     

Resveratrol Improved Flow-Mediated Outward Arterial Remodeling in Ovariectomized Rats with Hypertrophic Effect at High Dose

Objectives

Chronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats.

Methods

Blood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat.

Results

Arterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups.

Conclusion

Resveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.     

Antioxidant,antiapoptotic, and antifibrotic effects of the combination of liposomal resveratrol and carvedilol against doxorubicin‐induced cardiomyopathy in rats

The use of the cytotoxic antibiotic doxorubicin (DOXR) is limited by its dose‐dependent cardiotoxicity. The aim of this study was to evaluate the cardioprotective effect of the combination of carvedilol (CARD) and liposomal resveratrol (LIPO RESV) against DOXR‐induced cardiomyopathy in rats. The results of the present study showed that DOXR administration significantly increased heart weight/body weight ratio by 35.6%, creatine kinase‐MB (CK‐MB) by 40.6%, troponin‐I levels by 85%, and decreased reduced glutathione level and superoxide dismutase activity by 47% and 52%, respectively compared to the control group. Moreover, cardiac caspase‐3 protein expression was upregulated by 51.6% vs the control group. In contrast, treatment of DOXR‐administered rats with CARD, RESV, or LIPO RESV and their combination for 6 weeks improved all the above‐mentioned measured parameters. In conclusion, concomitant administration of CARD and LIPO RESV exerted additive pharmacological effects in some measured parameters against DOXR‐induced cardiomyopathy and this may be a useful cardioprotective strategy.     

Inhibition of leukocyte adhesion by the in vivo and in vitro administration of cannabinoids

Cannabinoids have been shown to affect various aspects of arachidonic acid metabolism both in vivo and in vitro. Eicosanoid metabolites of arachidonate and related octadecanoate are believed to be involved in cell adhesion processes as agonists in some instances and as antagonists in other cases. This report shows data in which cannabinoids exhibit marked inhibitory effects on the adhesion of mouse peritoneal cells to polystyrene culture dishes. The effects could be seen by in vivo administration of the drugs as well as by direct exposure of the cells in vitro. The data suggest that this inhibition of adhesion is mediated by one or more products generated by stimulation of a lipoxygenase pathway.     

Alpha-MSH peptides inhibit acute inflammation and contact sensitivity

Alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs within the CNS, skin, circulation and in other body sites. This tridecapeptide and its COOH-terminal tripeptide, alpha-MSH (11-13), have antipyretic and anti-inflammatory actions. Studies of the anti-inflammatory effects of these molecules have been confined mainly to tests of inhibition of histamine and endogenous pyrogen-induced increases in capillary permeability in rabbits and acute inflammation of ear tissue in mice. The aim in the present experiments was to learn if alpha-MSH peptides also antagonize inflammation in two additional models: acute edema induced in the mouse paw and contact sensitivity. Significant anti-inflammatory effects were observed with MSH peptides in both models. These findings converge with previous results to indicate that alpha-MSH peptides modulate inflammation. Because circulating alpha-MSH increases after treatment of animals with endogenous pyrogen or endotoxin, administration of the peptides may simply mimic a naturally occurring modulation of host defense reactions.     

Influence of footshock stress on pharmacokinetics of nicorandil in rats

The influence of footshock stress on the pharmacokinetics of nicorandil was examined in rats. In the group exposed to a 30-min period of footshock immediately after the oral administration of nicorandil (10 mg/kg), plasma nicorandil levels were markedly lower than those in the control group 30-120 min after administration. Plasma levels after the subcutaneous injection of nicorandil (5 mg/kg) were also slightly but significantly lower in stressed rats than in control rats. When footshock was applied from 60 min after oral administration or 30 min after subcutaneous injection (the time when the plasma nicorandil level was maximum), it also significantly decreased the plasma levels thereafter. Furthermore, footshock applied immediately after intravenous injection of nicorandil (3 mg/kg) significantly decreased the plasma levels 30-60 min after injection. Plasma levels of N-(2-hydroxyethyl) nicotinamide, one of the main metabolites of nicorandil, were slightly increased 30 min after the intravenous injection of nicorandil (10 mg/kg) by footshock. Nicorandil levels in the heart, kidney, and skin were significantly lower in the stressed rats similar to the change in the plasma level, but levels in the muscle, liver, and thymus showed no significant difference. The urinary excretion of nicorandil tended to be higher in the stressed rats. These results suggest that footshock stress affects not only the absorption of nicorandil but also its distribution, metabolism, and excretion.     

IMQ Induced K14-VEGF Mouse: A Stable and Long-Term Mouse Model of Psoriasis-Like Inflammation

An imiquimod (IMQ) induced wild type (WT) mouse can mimic some features of psoriasis, such as thickened skin, abnormal keratinocyte-related proteins, infiltration of inflammatory cells and pro-inflammatory cytokines. This model is a prevalent model that is widely used in the study of psoriasis. However, skin inflammation decreases during the eighth day when IMQ is given to WT mice, which may result in false results when evaluating the pharmacodynamics effects of a drug. To extend the timeliness and inherit the advantages of this model, we applied IMQ to the skin of 8-week-old homozygous K14-VEGF mice to investigate whether IMQ can prolong mice ear inflammation. In our experiments, we found that, compared to the IMQ induced WT mice model, the IMQ induced K14-VEGF mice have serious skin inflammation, even on the fourteenth day. We also evaluated the stability of skin inflammation at days 8, 10, and 13, and the inflammatory situation remained stable in the skin. This research intends to improve the existing model, and we hypothesize that the IMQ induced K14-VEGF mouse will become a practical mouse model in psoriasis research.     

Chronopharmacology of Oral Nitrates in Healthy Subjects

The pharmacokinetics and the hemodynamic effects (blood pressure, heart rate) of oral organic nitrates have been investigated in healthy subjects after oral single-dose application either in the morning or in the evening. Isosorbide-5-monitrate (IS-5-MN, 60 mg) was administered as an immediate-release tablet or as a slow-release formulation. Isosorbide dinitrate (ISDN, 20 mg) was ingested as an immediate-release tablet. After administration of IS-5-MN as an immediate-release tablet, the drug was more rapidly absorbed in the morning (t_max of 0.9 h) than in the evening (t_max of 2.1 h). The rapid absorption led to more pronounced effects in the morning, at which time maximum drug concentrations occurred at the same time as peak effects were observed. After evening administration, however, peak effects were in advance of the maximum drug concentrations. No chrono-kinetics were observed after application of the slow-release formulation of IS-5-MN. In accordance with the results of the immediate-release formulation, peak effects of the slow-release preparation occurred significantly earlier than peak drug concentrations after evening than after morning dosing. ISDN bioavailability was higher after morning than after evening administration and hemodynamic effects were more pronounced in the evening than in the morning. These results show that daily variations in pharmacokinetics and/or hemodynamic effects can be observed with oral nitrates. In addition, galenic formulation can influence the time-specified pharmacokinetics of IS-5-MN.     

Effects of duration of fast and animal age on the gastrointestinal absorption of plutonium

The fraction of plutonium absorbed after oral administration of Pu(VI) to 24-h-fasted mice was 19 X 10(-4), 13-fold higher than in fed mice, 1.4 X 10(-4). We have investigated the relevance of the high gastrointestinal (GI) absorption value for the 24-h-fasted animals in setting drinking water standards for humans. When fasting was initiated at the beginning of the active phase of the mouse's daily activity cycle (when they would normally eat), plutonium GI absorption rose from 2.8 X 10(-4) at zero-time to a level typical of the 24-h-fasted mouse after only 2 h of fasting. In contrast, in mice allowed to eat for 4 h into their active phase prior to initiation of the fast (meal-fed mice), 8 h of fasting were required before GI absorption rose to a level similar to that of the 24-h-fasted mouse. The fraction of plutonium retained after gavage administration of Pu(VI) to 1-day-old rats was 74 X 10(-4), 70-fold higher than the value for fed adults. Retention after GI absorption in neonates remained 30- to 70-fold higher than in adults until weaning. One week after weaning, the fraction absorbed and retained by fed weanling rats was the same as that for fed adults, 1 X 10(-4). Drinking water standards for plutonium have been set based on GI absorption values for fed adult animals. The 10- to 100-fold increases in plutonium absorption in young and fasted animals reported by ourselves and others, and the rapid rise to fasted levels of absorption at the start of the animal's active phase, indicate that consideration should be given to elevated levels of plutonium absorption in young and fasted individuals.