Synthesis of a cysteine functional covalent organic framework via facile click reaction for the efficient solid phase extraction of substituted p-phenylenediamine-derived quinones.

N,N'-Substituted p-phenylenediamine quinones (PPD-Qs) are the emerging toxicant, which transform from the rubber tire antioxidant N,N'-substituted p-phenylenediamines (PPDs). Because of their potential toxic and widespread occurrence in the environment, PPD-Qs have received great attention. However, efficiently extracting PPD-Qs from complex samples is still a challenge. Herein, a cysteine functional covalent organic framework (Cys-COF) designed according to the "donor-acceptor" sites of hydrogen bonding of PPD-Qs was synthesized via click reaction and then used as solid-phase extraction (SPE) adsorbent. Cys-COF can form the seven-member ring adsorption structure with PPD-Qs via hydrogen bonding. The adsorption mechanism was tentatively revealed by density functional theory (DFT). After optimizing the Cys-COF-SPE parameters, PPD-Qs were efficiently extracted from water, soil, sediment, and fish, followed by detection using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The Cys-COF-SPE-UHPLC-MS/MS method exhibited ideal linearity (R2 ≥ 0.9932), high relative recoveries (80.4-111 %), and low limits of detection (0.0001-0.0013 ng mL-1). In addition, the bioconcentration kinetics in goldfish provides a feasible platform to investigate the toxicity and accumulated ability of PPD-Qs.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway.

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.

Skin irritation assessment and potential mechanism of Capparis spinosa L. fruits.

ETHNOPHARMACOLOGICAL RELEVANCE: In China, Capparis spinosa L. fruits (CSF) are often used topically in Uyghur folk medicine in treating rheumatic diseases with remarkable efficacy. However, it has noticed severe skin irritation after a short time application with high dose of CSF, which limited long-term clinical use. To date, there is almost no research related to skin irritation of CSF. AIM OF THE STUDY: This study was intended to perform the first systematic assessment of morphological and histological changes in skin after stimulation with CSF. Furthermore, potential irritant components in CSF and related mechanisms were explored by in vitro transdermal techniques, network pharmacology, molecular docking, and experimental validation. MATERIALS AND METHODS: Skin changes after single and multiple stimulations with CSF were observed and subjected to skin irritation response scoring, irritation strength assessment, and histopathological analysis. In addition, in vitro transdermal technology, liquid chromatography-mass spectrometry (LC-MS) method, network pharmacology, molecular docking, and experimental validation were used to further exploit underlying skin irritant components and possible mechanisms of action. RESULTS: CSF induced significant morphological (erythema and edema) and histological (epidermal thickening and inflammatory infiltration) changes in skin of mice, which were similar to the clinical presentation of irritation contact dermatitis (ICD). The ethyl acetate fraction of CSF (CFEAF) was the main source of CSF-induced skin irritation. Kaempferol, flazin, and gallic acid were potential major irritant compounds. Moreover, CFEAF, kaempferol, flazin, and gallic acid could increase the levels of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and interleukin-17A (IL-17A) to promote skin inflammation. The potential mechanism of CSF-induced skin irritation may be activation of the nuclear factor kappa-B (NF-κB) signaling pathway, including phosphorylation of NF-κB p65 (p65) and nuclear factor-kappa B inhibitor alpha (IκBα). CONCLUSION: Kaempferol, flazin, and gallic acid are potential skin irritant components from CSF. Altogether, they induce skin irritation responses through promoting the release of the inflammatory factors TNF-α and ICAM-1, as well as activating the NF-κB signaling pathway. In addition, IL-17A may be an important pro-inflammatory factor in skin irritation.

Mechanism of optical limiting in metalloporphyrins under visible continuous radiation.

Here, we present an experimental investigation on the nonlinear optical (NLO) and optical limiting properties of metalloporphyrin compounds (Cu-1-OH, Zn-1-OH, Cu-1-E and Zn-1-E) using spatial self-phase modulation (SSPM) method in the visible range. It is found that all of the samples show a large self-defocusing effect at 532 nm, which is attributed to the thermal nonlinear optical effects with negative nonlinear refractive index coefficient n2 due to the relatively high absorption at 532 nm. In contrast, at 780 nm where absorption is weak for both Zn- and Cu-porphyrins, Zn-porphyrins still exhibit visible self-defocusing while Cu-porphyrins do not show any nonlinear diffraction pattern. Such a phenomenon can be explained by the Kerr effect of Cu-porphyrins at 780 nm. As the thermal nonlinear optical effects (of negative n2) at 780 nm are reduced due to the low absorption, the Kerr effect with positive n2 becomes comparable and the overall nonlinearity is reduced. The Kerr effect of Cu-porphyrins is stronger than that of Zn-porphyrins because of the enhanced π-electron delocalization effect as Cu(II) has a variable number of valence electrons and incompletely filled d atomic orbitals. Finally, the optical limiting performance of Zn-porphyrins is demonstrated as a representative and its dependence on sample position is examined. This work not only enriches the understanding of the physical mechanism of optical limiting in porphyrin materials, but also provides a significant reference to improve the third-order NLO coefficient by adjusting the structure of compounds.

The in vitro/in vivo metabolic pathways analysis of lobetyol, lobetyolin, and lobetyolinin, three polyacetylenes from Codonopsis Radix, by UHPLC-Q/TOF-MS and UHPLC-MS/MS.

Polyacetylenes, lobetyol, lobetyolin and lobetyolinin, are responsible for antitumor, antioxidant, anti-inflammatory, immunomodulatory activities of Codonopsis Radix. However, their metabolic pathways are still unknown. The study was purposed to investigate the metabolism of three polyacetylenes in vitro and in vivo by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Moreover, a rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantitative and semi-quantitative determination of lobetyol and its 12 metabolites to investigate the metabolic stability and metabolic phenotypes. A total of 47, 30 and 34 metabolites of lobetyol, lobetyolin and lobetyolinin were found in all samples. These metabolites are produced through extensive pathways, mainly involving oxidation, glucuronidation and glutathione conjugation. Lobetyol showed good metabolic stability in liver microsomes. The results of both recombinant human CYP enzymes and chemical inhibition experiments confirmed that CYP2C19, 1A1, 2C9, and 1A2 are the major isozymes mediating lobetyol metabolism.

Simultaneous determination of cordycepin and its metabolite 3'-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies.

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3'-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3'-deoxyinosine showed excellent linearity within the concentration range of 1.05-10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3'-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.

Dynamic Tuning of Viscoelastic Hydrogels with Carbonyl Iron Microparticles Reveals the Rapid Response of Cells to Three-Dimensional Substrate Mechanics.

Current methods to dynamically tune three-dimensional hydrogel mechanics require specific chemistries and substrates that make modest, slow, and often irreversible changes in their mechanical properties, exclude the use of protein-based scaffolds, or alter the hydrogel microstructure and pore size. Here, we rapidly and reversibly alter the mechanical properties of hydrogels consisting of extracellular matrix proteins and proteoglycans by adding carbonyl iron microparticles (MPs) and applying external magnetic fields. This approach drastically alters hydrogel mechanics: rheology reveals that application of a 4000 Oe magnetic field to a 5 mg/mL collagen hydrogel containing 10 wt % MPs increases the storage modulus from approximately 1.5 to 30 kPa. Cell morphology experiments show that cells embedded within these hydrogels rapidly sense the magnetically induced changes in ECM stiffness. Ca2+ transients are altered within seconds of stiffening or subsequent softening, and slower but still dynamic changes occur in YAP nuclear translocation in response to time-dependent application of a magnetic field. The near instantaneous change in hydrogel mechanics provides new insight into the effect of changing extracellular stiffness on both acute and chronic changes in diverse cell types embedded in protein-based scaffolds. Due to its flexibility, this method is broadly applicable to future studies interrogating cell mechanotransduction in three-dimensional substrates.

Pharmacokinetic studies of ginsenosides Rk1 and Rg5 in rats by UFLC-MS/MS.

A rapid ultra-fast liquid chromatography tandem mass spectrometry method was developed and validated to determine ginsenosides Rk1 and Rg5, a pair of isomers, in rat plasma, which was successfully applied to their pharmacokinetic studies. Two ginsenosides were given to male Sprague-Dawley rats via intragastrical and intravenous routes, respectively, and the impact of double bond position on the pharmacokinetic features of the two ginsenosides was elucidated in rats. Ginsenoside Rg3 was used as internal standard and ethyl acetate was applied to extract analytes and internal standard. Chromatographic separation was carried out on a reverse-phase UPLC HSS T3 column (100 × 2.1 mm, 1.8 µm). The flow rate was set to 0.4 ml/min. The fragmentation transition was m/z 765.4 → m/z 101.1 for two ginsenosides. The mobile phases were composed of 0.1% formic acid aqueous solution and acetonitrile. The linear range was 2-1,000 ng/ml for the two ginsenosides. Intra- and inter-day precisions were <11.67%, and accuracy fluctuated from -7.44 to 6.78%. The extraction recovery, matrix effect and stability were within acceptable levels. After treatment with ginsenosides Rk1 and Rg5, some differences were found in their pharmacokinetic profiles in rats. The maximum plasma drug concentration and the area under the plasma drug concentration-time curve of ginsenoside Rg5 were about 5 times bigger than those of ginsenoside Rk1 after oral administration, and 3 times higher after intravenous administration. The oral bioavailabilities of ginsenosides Rk1 and Rg5 were 0.67 and 0.97%, respectively. The results indicated that ∆20(22) -ginsenosides showed better pharmacokinetic features than ∆20(21) -ginsenosides with the same glycosylation.

Biotransformation patterns of dictamnine in vitro/in vivo and its relative molecular mechanism of dictamnine-induced acute liver injury in mice.

Dictamnine (DIC), a typical furan-quinoline alkaloid, has a wide range of pharmacological and toxicological effects, such as anti-bacterial, antifungal, anti-cancer, and hepatoxicity. But the molecular mechanism of DIC-induced hepatoxicity in mice remains unclear. This study aimed to clarify the biotransformation patterns of DIC in vitro/in vivo and the relative molecular mechanism of DIC-induced hepatoxicity in mice. All metabolites of DIC were identified by comparing the blank and drug-containing urine, feces, plasma, and liver samples. The structure of epoxide intermediate derived from DIC was confirmed by trapping assay. Oxidative stress injury and inflammation have been confirmed to be involved in the toxicological process of DIC-induced hepatoxicity in mice by detecting the relative biochemical indexes. The results will help to develop a deeper understanding about the biotransformation patterns of DIC, structure of the epoxide intermediate, and the molecular mechanism of DIC-induced hepatoxicity in mice.

Antifatigue and antihypoxia activities of oligosaccharides and polysaccharides from Codonopsis pilosula in mice.

Codonopsis pilosula is a traditional Chinese medicine and food supplement that is widely used in China. This study aimed to investigate the antifatigue and antihypoxia activities of different extracts and fractions from C. pilosula, including ethanol extract (ETH), water extract (WAT), polysaccharides (POL), inulin (INU) and oligosaccharides (OLI). Different extracts and fractions were orally administered to mice at the doses of 0.25, 0.5 and 1.0 g kg-1 once a day for 21 days. Antifatigue activity was assessed through the weight-loaded swimming test on the 21st day, and antihypoxia activity was evaluated through the normobarie hypoxia test on the following day. Finally, biochemical parameters, such as liver glycogen (LG), muscle glycogen (MG), blood urea nitrogen (BUN), lactic dehydrogenase (LDH), malondialdehyde (MDA), and glutathione (GSH) levels, were determined. The results showed that, compared with the control treatment, only POL treatment significantly prolonged the swimming time of the mice. POL groups had the strongest hypoxia tolerance, followed by the OLI and WAT groups. The levels of LG and MG were significantly increased by treatment with POL at the doses of 0.5 and 1.0 g kg-1, whereas BUN and LDH levels in POL groups were significantly lower than those in the control group. MDA under POL and OLI treatment was significantly lower than that under the control treatment. In addition, treatments with POL and OLI, except for treatment with a low dose of OLI, significantly increased GSH levels. In conclusion, POL could efficiently enhance antifatigue and antihypoxia abilities by increasing energy resources, decreasing detrimental metabolite accumulation, and enhancing antioxidant activity. OLI could improve antihypoxia activity by preventing lipid peroxidation and enhancing antioxidant activity.

Metabolites identification and reversible interconversion of chelerythrine and dihydrochelerythrine in vitro/in vivo in rats using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Chelerythrine (CHE) and dihydrochelerythrine (DHCHE), two typical benzophenanthridine alkaloids, have a wide range of pharmacological activities, such as antibacterial, anti-tumour and antiparasitic activities. To date, the biological activities of CHE and DHCHE are well reported, but the biotransformation of CHE and DHCHE in vivo remains unknown. This study aims to clarify the metabolic pathway of CHE and DHCHE in rat liver microsomes (RLMs) in vitro and in vivo. An ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) method was developed for metabolites identification of CHE and DHCHE. The urine, feces, bile, and plasma samples and RLMs samples were collected for analyzing the biotransformation pathway of CHE and DHCHE. The result showed that there is a phenomenon of mutual reversible interconversion between CHE and DHCHE in vivo and in vitro. The other biotransformation pathways of CHE and DHCHE including demethylation, hydroxylation, methylene dioxy cycle opening, and glucuronidation mainly occurred in the side chain of benzophenanthridine parent structure. Twenty-five phase I and eight phase II metabolites of CHE, twenty-two phase I and eight phase II metabolites of DHCHE were detected. The results will help to develop a deeper understanding of CHE and DHCHE in vivo process and provide some references for the biotransformation research of other benzophenanthridine alkaloids.

Rapid identification and pharmacokinetic studies of multiple active alkaloids in rat plasma through UPLC-Q-TOF-MS and UPLC-MS/MS after the oral administration of Zanthoxylum nitidum extract.

Zanthoxylum nitidum (Roxb.) DC. (ZN) belongs to the genus Zanthoxylum of Rutaceae and has various chemical ingredients and pharmacologic effects. Alkaloids are its main active constituents responsible for diverse pharmacologic effects, such as anti-tumor, anti-bacterial, anti-inflammatory, and analgesic activities. The chemical and pharmacological effects of ZN are well reported, but the in vivo pharmacokinetic profiles of its main active alkaloids are poorly investigated. This study aims to elucidate the absorbed constituents and pharmacokinetic behavior of main active ingredients in rat plasma after the oral administration of ZN extract. The absorbed constituents in rat plasma were qualitatively analyzed using ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination and pharmacokinetic studies of dihydrochelerythrine (DHCHE), nitidine chloride (NIT), chelerythrine (CHE), sanguinarine (SAN), liriodenine (LIR), skimmianine (SKI), γ-fagarine (FAG), and dictamnine (DIC) in rat plasma. Eighteen prototypes and metabolites were identified according to exact mass, characteristic diagnostic fragment ions, and reference standards. The established UPLC-MS/MS quantitative method met the requirements of FDA for biological analysis methods. Method validation showed that this method has good linearity (r ≥ 0.9910), precision (RSD ≤ 18.63 %), accuracy (88.11 %-117.50 %), and stability. The limit of detection (LOD) could reach 1 ng/mL, and the limit of quantitation could reach 2 ng/mL. The plasma drug concentration of benzophenanthridine alkaloids, such as NIT, CHE, and DHCHE, were still low even after dose differences were deducted. For the furan quinoline alkaloids (such as SKI, FAG, and DIC), only SKI showed high plasma drug concentration, although SKI content comprised only approximately 1/6 of benzophenanthridine alkaloids. This study is the first to simultaneously determine the above-mentioned active alkaloids in rat plasma and would contribute to the comprehensive understanding of in vivo pharmacokinetic behavior on active alkaloids in ZN extract.

The transdermal performance, pharmacokinetics, and anti-inflammatory pharmacodynamics evaluation of harmine-loaded ethosomes.

Harmine (HAR) is a ß-carboline alkaloid with anti-inflammatory and antipruritic effect. However, the low bioavailability and side effects of HAR severely limited its clinical application. The main objective of this study was to develop harmine-loaded ethosomes (HLE) drug delivery system for topical application to treat inflammation. HLE were obtained by ethanol injection method and characterized. The morphology of HLE was evaluated by transmission electron microscopy (TEM). HLE exhibited a good biocompatibility with human embryonic skin fibroblasts and rat skin. The in vitro skin penetration studies showed that HLE had the greatest skin deposition than harmine-loaded liposomes (HLL) and harmine solution (HS). In vivo pharmacokinetic study demonstrated that AUC(0-∞) and Cmax of HLE in subcutaneous tissues were much higher than that of in blood. Moreover, for convenience of fixing on skin, HLE were mixed with gel. HLE gel significantly inhibited the overexpression of inflammation cytokines prostaglandin E2, interleuking (IL)-1ß, nitric oxide, and tumor necrosis factor-alpha (TNF-α) in the inflammation model of rat paw edema compared with HS gel. In short, HLE was promising formulation for topical delivery in treatment of inflammatory diseases.

Improvement of Oxazolone-Induced Ulcerative Colitis in Rats Using Andrographolide.

Ulcerative colitis (UC) is usually accompanied with symptoms of abdominal pain, diarrhea, and bloody stool, which impair the quality of life of patients. Previous studies have shown that Andrographis paniculata extracts, which have andrographolide (AND) as their main compound, can relieve UC symptoms in patients. The aim of the study was to investigate the alleviating effect of AND on UC using the oxazolone (OXZ)-induced UC rat model. A total of 66 healthy male Sprague Dawley rats were used to evaluate the efficacy and mechanism of AND on UC (n = 11 per group) and grouped into control, model, SASP (sulfasalazine, positive control group, 500 mg/kg), AND-L (40 mg/kg), AND-M (80 mg/kg), and AND-H (120 mg/kg). The colonic disease activity index (DAI), colon length, spleen coefficient, pathological damage, and inflammation-related cytokine and protein expression levels were used as indices for evaluation. Results showed that the AND groups had reduced DAI and mortality, and significantly improved colon length and spleen coefficient compared with the model group. Furthermore, OXZ-induced histological injury was relieved significantly after AND treatment due to an improved crypt structure and reduced infiltration of inflammatory cells. Moreover, AND inhibited myeloperoxidase (MPO) activity and the secretion of interleukin-4 (IL-4), IL-13, and tumor necrosis factor α (TNF-α). The results of the anti-inflammatory mechanism revealed that AND blocked the signal transduction by reducing IL-4/IL-13 specific binding to IL-4 receptor (IL-4R) and inhibiting the phosphorylation of the signal transducer and activator of transcription 6 (p-STAT6). In conclusion, aside from natural plants, AND may be a candidate ingredient for UC therapy.

Developing and validating utility parameters to establish patient-reported outcome-based perioperative symptom management in patients with lung cancer: a multicentre, prospective, observational cohort study protocol.

INTRODUCTION: Patient-reported outcome-based symptom monitoring and alerting have been attractive for patient care after a tumour-removal surgery. However, the implementation parameters of this patient-centred symptom management system in perioperative patients with lung cancer are still lacking. We aim to develop a perioperative symptom scale (PSS) for monitoring, to determine the optimal time points for symptom assessment and to define the alert thresholds for medical intervention. METHODS AND ANALYSIS: This study will prospectively recruit 300 patients undergoing lung cancer surgery in six hospitals. The MD Anderson Symptom Inventory-Lung Cancer Module (MDASI-LC) is used to collect longitudinal symptom data preoperatively, daily postoperatively during in-hospital stay and weekly after discharge until 4 weeks or the start of postoperative oncological therapy. Symptoms that change significantly over time will be generated as the PSS. We will determine the optimal time points for follow-up using the generalised linear mixed-effects models. The MDASI-LC interference-measured functional status will be used as the anchor for the alert thresholds. ETHICS AND DISSEMINATION: Ethics Committee of Sichuan Cancer Hospital approved this study on 16 October 2017 (No. SCCHEC-02-2017-042). The manuscript is based on the latest protocol of Version 3.0, 15 September 2019. The results of this study will be presented at medical conferences and published in peer-reviewed journals. TRIALS REGISTRATION NUMBER: NCT03341377.

Simultaneous determination of polyphenols and triterpenes in pomegranate peel based on high-performance liquid chromatography fingerprint by solvent extraction and ratio blending method in tandem with wavelength switching.

Traditionally, pomegranate (Punica granatum L.) has been consumed as fresh fruit or as pomegranate juice. Pomegranate peel, the dried husk of P· granatum, is an important herbal medicine for treating diarrhea, hemostasis and insect-induced abdominal pain in China. However, the quality control methods for pomegranate peel remain unsatisfactory. In this work, a new HPLC-based qualitative and quantitative method for quality control of pomegranate peel was developed and validated for the simultaneous determination of polyphenols and triterpenes (including punicalagins A and B, ellagic acid, oleanolic acid and ursolic acid) by solvent extraction and ratio blending method in tandem with wavelength switching. The average recoveries were 98.07-100.61% with relative standard deviation no more than 4.27%. In addition, the fingerprint analysis was conducted to interpret the consistency of the quality test. Thirteen characteristic peaks were selected to evaluate the similarities of 16 batches of pomegranate peel. The similarities of samples were all more than 0.80, indicating that the samples from different areas of China were consistent. The results demonstrated that quantitative analysis and the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation can be successfully used to assess the quality and to identify the authenticity of pomegranate peel.

Ameliorative effect of deoxyvasicine on scopolamine-induced cognitive dysfunction by restoration of cholinergic function in mice.

BACKGROUND: Aerial parts of Peganum harmala Linn is used as a traditional medical herb for treatment of amnesia in Uighur medicine in China. Deoxyvasicine (DVAS) is one of the chief active ingredients in P. harmala, it possesses strong acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities in vitro, but the therapeutic effect and mechanisms on amnesia in vivo are unclear. PURPOSE: The objective of this study was to investigate the improvement effect of DVAS from P. harmala in learning and memory deficits of scopolamine-induced mice and elucidate the underlying mechanisms involved. METHODS: Mice were pretreated with DVAS (5, 15 and 45 mg/kg) and huperzine-A (0.2 mg/kg) by gavage for 7 days, and subsequently were daily intraperitoneally injected with scopolamine (1 mg/kg) to induce learning and memory deficits and behavioral performance was assessed by Morris water maze. To further evaluate the potential mechanisms of DVAS in improving learning and memory capabilities, pathological change, levels of various biochemical markers and protein expressions related to cholinergic system, oxidative stress, and neuroinflammation were examined. RESULTS: The results showed that DVAS could alleviate learning and memory deficits in scopolamine-treated mice. DVAS could regulate cholinergic function by inhibiting AChE and activating choline acetyltransferase (ChAT) activities and protein expressions. DVAS could induce brain-derived neurotrophic factor and protect hippocampal pyramidal cells against neuronal damage. DVAS also enhanced antioxidant defense via increasing the antioxidant enzyme level and activity of glutathione peroxidase, and anti-inflammatory function through suppressing tumor necrosis factor-α. Additionally, DVAS could regulate the neurotransmitters by elevating acetylcholine, 5-hydroxytryptamine, γ-aminobutyric acid and reducing 5-hydroxyindole-3-acetic acid and glutamic acid. CONCLUSION: Results illustrated that DVAS may be a promising candidate compound against amnesia via restoration of cholinergic function, regulating neurotransmitters, attenuating neuroinflammation and oxidative stress.

Analogous ß-Carboline Alkaloids Harmaline and Harmine Ameliorate Scopolamine-Induced Cognition Dysfunction by Attenuating Acetylcholinesterase Activity, Oxidative Stress, and Inflammation in Mice.

The analogous ß-carboline alkaloids, harmaline (HAL) and harmine (HAR), possess a variety of biological properties, including acetylcholinesterase (AChE) inhibitory activity, antioxidant, anti-inflammatory, and many others, and have great potential for treating Alzheimer's disease (AD). However, studies have showed that the two compounds have similar structures and in vitro AChE inhibitory activities but with significant difference in bioavailability. The objective of this study was to comparatively investigate the effects of HAL and HAR in memory deficits of scopolamine-induced mice. In the present study, mice were pretreated with HAL (2, 5, and 10 mg/kg), HAR (10, 20, and 30 mg/kg) and donepezil (5 mg/kg) by intragastrically for 7 days, and were daily intraperitoneal injected with scopolamine (1 mg/kg) to induce memory deficits and then subjected to behavioral evaluation by Morris water maze. To further elucidate the underlying mechanisms of HAL and HAR in improving learning and memory, the levels of various biochemical factors and protein expressions related to cholinergic function, oxidative stress, and inflammation were examined. The results showed that HAL and HAR could effectively ameliorate memory deficits in scopolamine-induced mice. Both of them exhibited an enhancement in cholinergic function by inhibiting AChE and inducing choline acetyltransferase (ChAT) activities, and antioxidant defense via increasing the antioxidant enzymes activities of superoxide dismutase and glutathione peroxidase, and reducing maleic diadehyde production, and anti-inflammatory effects through suppressing myeloperoxidase, tumor necrosis factor α, and nitric oxide as well as modulation of critical neurotransmitters such as acetylcholine (ACh), choline (Ch), L-tryptophan (L-Trp), 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (γ-GABA), and L-glutamic acid (L-Glu). Furthermore, the regulations of HAL on cholinergic function, inflammation, and neurotransmitters were more striking than those of HAR, and HAL manifested a comparable antioxidant capacity to HAR. Remarkably, the effective dosage of HAL (2 mg/kg) was far lower than that of HAR (20 mg/kg), which probably due to the evidently differences in the bioavailability and metabolic stability of the two analogs. Taken together, all these results revealed that HAL may be a promising candidate compound with better anti-amnesic effects and pharmacokinetic characteristics for the treatments of AD and related diseases.

Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines.

Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min-1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 µm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 µL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 µM for inosine), and low cost (small enzyme consumption of 0.007 unit mL-1 for ADA and substrate of 3.74 µM for AD in individual inhibition), and no matrix effects (101.64%-107.12%). Stability results showed that all analytes were stable under the investigated conditions. The developed method was successfully applied to the detection of the inhibitory activity of ADA from traditional Chinese medicines.

The inhibitory effects of compound Muniziqi granule against B16 cells and harmine induced autophagy and apoptosis by inhibiting Akt/mTOR pathway.

BACKGROUND: Compound Muniziqi granule (MNZQ) is a multi-component herbal preparation and a popular traditional Uighur medicine used in China for treating endocrine disorder-induced acne, chloasma, dysmenorrhea, menopausal syndrome, and melanoma. Harmine presented in MNZQ has been confirmed potential anticancer effect on the B16 cells among others. The purpose of this study is to explore the inhibitory effects of MNZQ against B16 cells and mechanism of autophagy and apoptosis induced by harmine in B16 cells. METHODS: The cell viability was calculated by CCK8 assay. The in vitro tyrosinase activity was determined by spectrophotometry. The harmine-induced autophagy was demonstrated by electron microscopy and MDC staining. Flow cytometry was used to measure cell death and cell cycle distribution. All proteins expression was assessed by western blot. RESULTS: MNZQ and some herb extracts contained in preparation displayed inhibitory effects on B16 cells but without inhibition on mushroom tyrosinase compared with kojic acid. The formation of autophagosome was markedly induced by harmine with the accretion of LC3-II and the degeneration of p62 in B16 cells, which indicated that harmine was an autophagy inducer. Cell death and sub-G2 population suggested that harmine could induce cell death. Particularly, 3-MA, an autophagy inhibitor, was discovered to prevent harmine-induced decrease of the cell viability and cell cycle arrest on G2 phase, indicating that autophagy was vital to the cell death. In addition, the results indicated that harmine could inhibit the phosphorylation of Akt and mTOR, which might mediate autophagy. CONCLUSION: Harmine could induce autophagy and apoptosis by inhibiting Akt/mTOR pathway in B16 cells. Harmine might be a promising therapeutic agent for treatment of melanoma in MNZQ.