A mannitol-modified emodin nano-drug restores the intestinal barrier function and alleviates inflammation in a mouse model of DSS-induced ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease of the colon that is characterized by mucosal ulcers. Given its increasing prevalence worldwide, it is imperative to develop safe and effective drugs for treating UC. Emodin, a natural anthraquinone derivative present in various medicinal herbs, has demonstrated therapeutic effects against UC. However, low bioavailability due to poor water solubility limits its clinical applications. METHODS: Emodin-borate nanoparticles (EmB) were synthesized to improve drug solubility, and they modified with oligomeric mannitol into microgels (EmB-MO) for targeted delivery to intestinal macrophages that express mannose receptors. UC was induced in a mouse model using dextran sulfate sodium (DSS), and different drug formulations were administered to the mice via drinking water. The levels of inflammation-related factors in the colon tissues and fecal matter were measured using enzyme-linked immunosorbent assay. Intestinal permeability was evaluated using fluorescein isothiocyanate dextran. HE staining, in vivo imaging, real-time PCR, and western blotting were performed to assess intestinal barrier dysfunction. RESULTS: Both EmB and EmB-MO markedly alleviated the symptoms of UC, including body weight loss, stool inconsistency, and bloody stools and restored the levels of pro- and anti-inflammatory cytokines. However, the therapeutic effects of EmB-MO on the macroscopic and immunological indices were stronger than those of EmB and similar to those of 5-aminosalicylic acid. Furthermore, EmB-MO selectively accumulated in the inflamed colon epithelium and restored the levels of the gut barrier proteins such as ZO-1 and Occludin. CONCLUSIONS: EmB-MO encapsulation significantly improved water solubility, which translated to greater therapeutic effects on the immune balance and gut barrier function in mice with DSS-induced UC. Our findings provide novel insights into developing emodin-derived drugs for the management of UC.

Ginseng polysaccharides enhanced ginsenoside Rb1 and microbial metabolites exposure through enhancing intestinal absorption and affecting gut microbial metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Polysaccharides and small molecules commonly co-exist in decoctions of traditional Chinese medicines (TCMs). Our previous study outlined that ginseng polysaccharides (GP) could interact with co-existing ginsenosides to produce synergistic effect in an over-fatigue and acute cold stress model via gut microbiota involved mechanisms. AIM OF THE STUDY: This study aimed to verify the interactions by examining the impact of GP on oral pharmacokinetics of ginsenoside Rb1 (Rb1), the dominant protopanoxadiol (PPD)-type ginsenoside in Ginseng, on a dextran sulphate sodium (DSS) induced experimental colitis model which was characterized by gut dysbiosis, and to delineate the underlying mechanisms in vitro. MATERIALS AND METHODS: Rats received drinking water (normal group), 5% DSS (UC group), or 5% DSS plus daily oral administration of GP (GP group) for 7 days and fecal samples were collected on day -3, 0 and 6. On day 7 all animals received an oral dosage of Rb1 and blood samples were withdrawn for pharmacokinetic study. The in vitro metabolism study of Rb1 in gut microbiota from normal and UC rats and the transport study of Rb1 across Caco-2 cell monolayer were carried out in presence/absence of GP. Rb1 and its bacterial metabolites ginsenoside Rd (Rd), ginsenoside F2 (F2), Compound K (CK) and PPD were determined using LC-MS/MS. Total and target bacteria in fecal samples were determined by using 16S rRNA-based RT-PCR. ß-Glucosidase activity was determined by measuring 4-nitrophenol formed from 4-nitrophenyl-ß-D-glucopyranoside hydrolysis. RESULTS: DSS induction did not alter AUC0-t and Cmax of Rb1, which, however, were doubled together with elevated AUC0-t of the metabolites, in particular Rd and CK, in GP group. GP influenced the microbial composition and showed a prebiotic-like effect. Accordingly, GP treatment could partially restore the ß-glucosidase activity which was reduced by DSS induction. The presence of GP resulted in quicker microbial metabolism of Rb1 and higher Rd formation in first 8 h of incubation, while the impact on F2 and CK formation/conversion became obvious after 8 h. More interestingly, GP slightly stimulated Caco-2 cell growth and facilitated Rb1 transport across the Caco-2 monolayer in both directions, increasing the Papp of Rb1 from 10-7 cm/s to 10-6 cm/s. CONCLUSIONS: GP alleviated DSS-induced colitis-like symptoms and enhanced the systemic exposure of Rb1 through enhancing microbial deglycosylation and intestinal epithelial absorption of Rb1. These findings further demonstrated the important role of gut microbiota in the multifaceted action of polysaccharides in the holistic actions of traditional decoction of TCMs.

Oral CoQ10 attenuates high salt-induced hypertension by restoring neurotransmitters and cytokines in the hypothalamic paraventricular nucleus.

High salt intake leads to an increase in some proinflammatory cytokines and neurotransmitters involved in the pathogenesis of hypertension. The purpose of this work was to know if oral administration of anti-oxidant and free-radical scavenger CoQ10 may attenuate high salt-induced hypertension via regulating neurotransmitters and cytokines in the hypothalamic paraventricular nucleus (PVN). Adult male Sprague-Dawley (SD) rats were fed with a normal salt diet (NS, 0.3% NaCl) or a high salt diet (HS, 8% NaCl) for 15 weeks to induce hypertension. These rats received CoQ10 (10 mg/kg/day) dissolved in olive oil was given by gavage (10 mg/kg/day) for 15 weeks. HS resulted in higher mean arterial pressure (MAP) and the sympathetic nerve activity (RSNA). These HS rats had higher PVN levels of norepinephrine (NE), tyrosine hydroxylase (TH), interleukin (IL)-1ß, NOX2 and NOX4, lower PVN levels of gamma-aminobutyric acid (GABA), IL-10, copper/zinc superoxide dismutase (Cu/Zn-SOD) and the 67-kDa isoform of glutamate decarboxylase (GAD67), as compared with NS group. CoQ10 supplementation reduced NE, TH, IL-1ß, NOX2 and NOX4 in the PVN, and induced IL-10, Cu/Zn-SOD and GAD67 in the PVN. These findings suggest that CoQ10 supplementation restores neurotransmitters and cytokines in the PVN, thereby attenuating high salt-induced hypertension.

ß-Cyclodextrin anchoring onto pericarpium granati-derived magnetic mesoporous carbon for selective capture of lopid in human serum and pharmaceutical wastewater samples.

Functionalized magnetic carbonaceous nanomaterials, which are important materials with many practical and research applications in biomedical, pharmaceutical and biological fields, have recently attracted much attention. In this study, a magnetic mesoporous carbon coated with ß-cyclodextrin (MMC@ß-CD) was synthesized for the first time from natural pericarpium granati (PG). The as-obtained MMC@ß-CD has high surface areas (203 m(2)g(-1)), large pore volumes (0.16 cm(3)g(-1)), relatively broad mesoporous sizes (6.8 nm) and a high saturation magnetization of 26.2 emu g(-1), which is sufficient for magnetic separation by an external magnetic field. The MMC@ß-CD was used as an innovative adsorbent for magnetic solid-phase extraction of lopid via host-guest interaction prior to spectrofluorometric analysis. The proposed method was successfully applied to analyze lopid in human serum and pharmaceutical wastewater samples with recoveries in the range of 85.0-103.5% for the spiked samples. Overall, this work not only provides an inexpensive and eco-friendly method to fabricate MMC@ß-CD (or MMC) from PG, but also develops a highly selective approach for capture of lopid in biological samples and environmental substances.

Enantiomeric separation of angular-type pyranocoumarins from Peucedani Radix using AD-RH chiral column.

Enantiomers and diastereoisomers of angular-type pyranocoumarins (APs) are abundant in Peucedani Radix (Chinese name: Qian-hu), eliciting distinct activities in vitro and in vivo. Our ongoing investigation on APs yielded eight pairs of enantiomers (1a and 1b, 2a and 2b, 3a and 3b, 4a and 4b, 5a and 5b, 6a and 6b, 7a and 7b and 8a and 8b) via enantiomeric separation of trans-3'-angeloylkhellactone (1), trans-3'-acetyl-4'-isobutyrylkhellactone (2), trans-3'-acetyl-4'-angeloyl-khellactone (3), 3'-angeloyloxy-4'-oxo-3',4'-dihydroseselin (4), cis-3'-acetyl-4'-angeloylkhellactone (5), cis-3'-isovaleryl-4'-acetylkhellactone (6), cis-3'-angeloyl-4'-isovalerylkhellactone (7) and cis-3',4'-diisovalerylkhellactone (8), respectively, using semi-preparative AD-RH chiral column. All the compounds (1-8) were enantioseparated for the first time, while the absolute configurations of 2a, 2b, 6a and 8b were reported first.

¹H nuclear magnetic resonance based-metabolomic characterization of Peucedani Radix and simultaneous determination of praeruptorin A and praeruptorin B.

As a widely used traditional herbal medicine, it is crucial to characterize the holistic metabolic profile of Peucedani Radix (Chinese name: Qian-hu). However, it is quite arduous to obtain the whole picture of chemical constituents appropriately with the existing analytical techniques that were based on HPLC-UV or LC-MS/MS system. In present investigation, nuclear magnetic resonance (NMR) spectroscopy coupled with principal components analysis (PCA) was introduced to metabolomic characterization of Qian-hu crude extracts without any chromatographic separation. In addition, the contents of praeruptorin A (PA) and proaeruptorin B (PB) in Qian-hu were simultaneously determined using quantitative (1)H NMR (q(1)H NMR) spectroscopy. Eighteen reference compounds (1-18), which were purified from this herbal drug extract previously, were recruited for the assignment of the protonic signals in the (1)H NMR spectra. Following PCA, 15 batches of Peucedani Radix were divided into two groups (I and II), and angular-type pyranocoumarins, in particular PA and PB, as well as 5-methoxycoumarin were demonstrated as the predominant markers being responsible for the distinguishment of Qian-hu from different districts. The contents of the two analytes (PA & PB) were calculated by the relative ratio of the integral values of the target peak for each compound to the known amount of the internal standard, formononetin (IS). The lower limits of quantitation were determined as 19.5µg/mL for both PA and PB. The quantitative results indicated that the contents of PA and PB showed quite variable qualities among different extract samples. Above all, (1)H NMR spectroscopy, that could not only provide comprehensive profiles of the metabolites but also achieve convenient determination of praeruptorin A and praeruptorin B, is a promising means for evaluating the medicinal samples of Peucedani Radix.

Metabolic characterization of (±)-praeruptorin A in vitro and in vivo by high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry.

(±)-Praeruptorin A (PA) is the major bioactive component in Peucedani Radix (Chinese name: Qian-hu), and exhibits dramatically anti-hypertensive effect typically through acting as a calcium channel blocker. The current study aims on the characterization of the metabolic profiles of PA in vitro and in vivo using high performance liquid chromatography (HPLC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). A total of 12 phase I metabolites (M1-12) in rat liver microsomes (RLMs), 9 phase I metabolites (M1-3, M5-6 and M9-12) in human liver microsomes (HLMs), 2 hydrolyzed products in rat plasma (M11 and M12), none metabolite in human plasma, none metabolite in rat intestinal bacteria, 7 metabolites (M1, M4-7, M13 and M15) in PA-treated rat urine and 6 metabolites (M1, M4-7 and M15) in PA-treated feces were detected and tentatively identified using predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (predictive MRM-IDA-EPI) mode in combination with enhanced mass spectrum-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode in the mass spectrometer domain, respectively, while TOF-MS was adopted to confirm the identification. Further, 2 glucuronidated metabolites (M13-14) in RLMs and none metabolite in HLMs of cis-khellactone (CKL), which was the main actual form of PA in vivo, were generated, while its sulfated product was not observed in either rat liver S9 fractions (RS9) or human liver S9 fractions (HS9). Oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation were demonstrated to be the predominant metabolic types for PA in vitro and in vivo. Judging from the decrement of peak areas, PA was metabolized quickly in both RLMs and HLMs, indicating extensively hepatic first-pass elimination. Taken together, the metabolic fates of (±)-praeruptorin A in vitro and in vivo were elucidated in current study, and Q-trap-MS coupled with LightSight™ software can be adopted as a useful tool for quick detection and identification of metabolites in complex biological matrices.

Identification of cytochrome P450 isoenzymes involved in metabolism of (+)-praeruptorin A, a calcium channel blocker, by human liver microsomes using ultra high-performance liquid chromatography coupled with tandem mass spectrometry.

Angular-type pyranocoumarins (APs) show attractive prospects in anti-hypertension, chemotherapy and anti-HIV treatment. Previous studies revealed extensive hepatic metabolisms of several APs following similar pathways. This study investigated the enzyme kinetics and the main CYP450 isozyme(s) involved in metabolism of (+)-praeruptorin A (dPA), an AP with significant cardio-protective activities, in human liver microsomes (HLMs) using ultra high-performance liquid chromatography coupled with a hybrid quadrupole-linear ion trap mass spectrometry (UHPLC-QT-MS/MS). dPA produced 6 metabolites via hydrolysis (M1-M3), oxidation (M4-M6), and hydrolysis followed by acyl migration (M2 or M3). Oxidation at the C-3' side chain instead of the coumarin ring was consolidated with the aromatic proton signal in NMR spectra. The major metabolite (-)-cis-khellactone (M1) followed biphasic kinetics in HLMs with high affinity (Km1 0.02µM) and intrinsic clearance (CLint1, invitro1.29mL/min/mg protein), whereas other metabolites (M2-M6) fitted typical Michaelis-Menten kinetics with lower affinity (Km 3.85-39.13µM). Recombinant human CYP3A4 showed the highest activity toward M1 and M4 formation, while it was CYP2C19 for M2/M3 and M5 and CYP2B6 for M6. Principal component analysis of the metabolite formation profile of dPA also revealed the highest similarity between CYP3A4 and HLMs. Both quercetin (CYP2C8 inhibitor) and ketoconazole (CYP3A4 inhibitor) showed 60-100% inhibition of M1-M4 and M6 formations in HLMs, while M5 formation was mainly inhibited by α-naphthoflavone (CYP1A2 inhibitor, 70-80%) and quercetin (90%). Moreover, formations of all metabolites were predominantly inhibited by CYP3A4 antibody (37-68%). These findings shed a light on main involvement of CYP3A4 in human hepatic elimination of APs, indicating potential drug interactions.

Characterization of metabolism of (+)-praeruptorin B and (+)-praeruptorin E in human and rat liver microsomes by liquid chromatography coupled with ion trap mass spectrometry and time-of-flight mass spectrometry.

Peucedani Radix is a Chinese medicinal herb noted for its effects on treatments of respiratory and pulmonary disorders. As a part of a systematic pharmacokinetic evaluation of the herb in our laboratory, the present study investigated, for the first time, the metabolic profile of (+)-praeruptorin B (dPB) and (+)-praeruptorin E (dPE), two main bioactive constituents of Peucedani Radix in pooled liver microsomes of rats (RLMs) and humans (HLMs). dPE was eliminated faster than dPB in both species. The incubation of dPB with RLMs and HLMs resulted in eight (B1-B8) and nine (B1-B9) metabolites, respectively, while both RLMs and HLMs converted dPE into 13 metabolites (E1-13). Structures of all the metabolites were proposed through comparing their mass data obtained via tandem mass spectrometry on an MSD ion trap system (IT-MS/MS) coupled with high-resolution mass measurement by time-of-flight mass spectrometry (TOF-MS) with those of the respective parent compound. B1 and E1 were unambiguously identified as (-)-cis-khellactone. The formations of all the metabolites were NADPH-dependent. Oxidation and hydrolysis were demonstrated to be two predominant metabolic pathways of dPB and dPE. Oxidation initiated at either the C-3' or C-4' substituent, while hydrolysis only started from the C-3' substituent. Fragmentation of all metabolites followed similar pathways to those of the parent pyranocoumarins. The information on metabolic properties of dPB and dPE and the mass fragmentation profiles of their metabolites obtained in the present study will aid in characterization of metabolic profiles of other angular-type pyranocoumarins and further investigation of in vivo fates of these pyranocoumarins and the herb.