Fabrication of food-grade Pickering high internal phase emulsions stabilized by the mixture of ß-cyclodextrin and sugar beet pectin.

Food-grade Pickering high internal phase emulsions (HIPEs) stabilized by a mixture of ß-cyclodextrin (ß-CD) and sugar beet pectin (SBP) were fabricated for the first time. The factors affecting the microstructures, mechanical properties, and stabilities of the Pickering HIPEs were systematically investigated. The corresponding hybrid particles were also separated and characterized to reveal the formation mechanism. The results indicated that the mixture could induce the formation of HIPEs with an oil phase volume fraction (φ) of 75% using a one-step high-speed shearing process at room temperature. The composition (the mass ratio of ß-CD to SBP, Rc/s) and concentration (W) of the mixture had significant effects on the formation and properties of HIPEs. When W ≥ 1.0% and Rc/s = 2:2 or 3:1, HIPEs had smaller oil droplets, higher gel strengths, better centrifugation stabilities and lutein protection effects. The spectral analysis suggested that SBP could adhere to the surface of ß-CD particles to form hybrid particles during the homogenization. Compared with native ß-CD particles, these hybrid particles had higher ζ-potential absolute values, and the SBP could also increase the viscosity of the aqueous phase, which contributed to the formation and properties of these HIPEs.

Mangiferin Accelerates Glycolysis and Enhances Mitochondrial Bioenergetics.

One of the main causes of hyperglycemia is inefficient or impaired glucose utilization by skeletal muscle, which can be exacerbated by chronic high caloric intake. Previously, we identified a natural compound, mangiferin (MGF) that improved glucose utilization in high fat diet (HFD)-induced insulin resistant mice. To further identify the molecular mechanisms of MGF action on glucose metabolism, we conducted targeted metabolomics and transcriptomics studies of glycolyic and mitochondrial bioenergetics pathways in skeletal muscle. These data revealed that MGF increased glycolytic metabolites that were further augmented as glycolysis proceeded from the early to the late steps. Consistent with an MGF-stimulation of glycolytic flux there was a concomitant increase in the expression of enzymes catalyzing glycolysis. MGF also increased important metabolites in the tricarboxylic acid (TCA) cycle, such as α-ketoglutarate and fumarate. Interestingly however, there was a reduction in succinate, a metabolite that also feeds into the electron transport chain to produce energy. MGF increased succinate clearance by enhancing the expression and activity of succinate dehydrogenase, leading to increased ATP production. At the transcriptional level, MGF induced mRNAs of mitochondrial genes and their transcriptional factors. Together, these data suggest that MGF upregulates mitochondrial oxidative capacity that likely drives the acceleration of glycolysis flux.

Systematic screening and characterization of multiple constituents in Guizhi Fuling capsule and metabolic profiling of bioactive components in rats using ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Guizhi Fuling capsule (GFC), a prestigious traditional Chinese medicinal (TCM) prescription, is efficiently used to treat primary dysmenorrhea in the clinical practice. It's significant to explore the metabolic fate of multiple components in vivo which are responsible for the pharmacological effects but not fully investigated. A rapid and high-throughput method using ultra performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was established for systematic investigation on GFC, including GFC chemical compositions, and their absorption and metabolism in rat plasma, urine, uterus and brain after oral administration of GFC. A total of 102 nonvolatile GFC phytochemistry components were identified based on the accurately measured mass value, fragmentation pattern and retention behavior. Compared to the previous GFC study, additional 47 different GFC components were detected. Furthermore 21, 9, 4 and 3 prototype compounds were separately observed in plasma, urine, uterus and brain samples with the support of in vitro GFC study. While 29, 33, 10 and 8 metabolites were also identified with the assistance of the MetaboLynx tool in these biological samples. The result indicated that the developed method was suitable for the components identification even in the complex matrix. The chemical and metabolic profiling of GFC provided an abundant substance foundation for the extensive GFC research, especially for the pharmacodynamic mechanisms research.

Metabolites identification of berberine in rats using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Berberine (BBR), the principle component for many medicinal plants such as Coptis chinensis Franch., Phellodendron chinense Schneid., and Mahonia bealei (Fort.) Carr., possesses diverse pharmacological activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic and antidiabetic activities. In this study, a rapid and reliable method using a five-step strategy based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), and metabolynx™ software with mass defect filter (MDF) technique was developed to investigate the metabolism of BBR. Plasma, bile, urine and feces samples were collected from rats after oral administration of BBR with a dose of 100mg/kg/day for three consecutive days and analyzed to characterize the metabolic profile of BBR. By comparing the molecular weights and MS fragmentations of the metabolites with those of the parent drug and reference standards, a total of 97 metabolites were identified, including 68 metabolites in urine, 45 metabolites in plasma, 44 metabolites in bile and 41 metabolites in feces. Demethylation, demethylenation, reduction, hydroxylation, and subsequent glucuronidation, sulfation and methylation were the major metabolic pathways of BBR in vivo.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Monitoring and evaluating the quality consistency of Compound Bismuth Aluminate tablets by a simple quantified ratio fingerprint method combined with simultaneous determination of five compounds and correlated with antioxidant activities.

A combination method of multi-wavelength fingerprinting and multi-component quantification by high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was developed and validated to monitor and evaluate the quality consistency of herbal medicines (HM) in the classical preparation Compound Bismuth Aluminate tablets (CBAT). The validation results demonstrated that our method met the requirements of fingerprint analysis and quantification analysis with suitable linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ). In the fingerprint assessments, rather than using conventional qualitative "Similarity" as a criterion, the simple quantified ratio fingerprint method (SQRFM) was recommended, which has an important quantified fingerprint advantage over the "Similarity" approach. SQRFM qualitatively and quantitatively offers the scientific criteria for traditional Chinese medicines (TCM)/HM quality pyramid and warning gate in terms of three parameters. In order to combine the comprehensive characterization of multi-wavelength fingerprints, an integrated fingerprint assessment strategy based on information entropy was set up involving a super-information characteristic digitized parameter of fingerprints, which reveals the total entropy value and absolute information amount about the fingerprints and, thus, offers an excellent method for fingerprint integration. The correlation results between quantified fingerprints and quantitative determination of 5 marker compounds, including glycyrrhizic acid (GLY), liquiritin (LQ), isoliquiritigenin (ILG), isoliquiritin (ILQ) and isoliquiritin apioside (ILA), indicated that multi-component quantification could be replaced by quantified fingerprints. The Fenton reaction was employed to determine the antioxidant activities of CBAT samples in vitro, and they were correlated with HPLC fingerprint components using the partial least squares regression (PLSR) method. In summary, the method of multi-wavelength fingerprints combined with antioxidant activities has been proved to be a feasible and scientific procedure for monitoring and evaluating the quality consistency of CBAT.

Capillary electrophoresis fingerprinting coupled with chemometrics to evaluate the quality consistency and predict the antioxidant activity of Sanhuang tablet as part of its quality control.

A capillary electrophoresis fingerprint was constructed for Sanhuang tablet, a Chinese traditional patent medicine, that was commonly used in clinical practice, where the isosceles trapezoid method was first applied for the optimization of background electrolyte solution, and the resolution index was performed to assess the experimental conditions; furthermore, a novel linear quantitative fingerprint method was established for accurate qualitative and quantitative discrimination of the test samples from diverse commercial brands. The fingerprint analysis coupled with quantitative determination of two components was employed to elucidate that the quality consistency of the products was relatively good within one manufactory, but poor among different companies for the 30 batches of samples. In addition, the fingerprint-efficacy relationship between chemical components and antioxidant activity in vitro was investigated using partial least squares analysis, and the calibration and prediction of the antioxidant activity of the selected samples via fingerprint data were presented with the desired results. This work illustrates that the proposed fingerprint analysis based on linear quantitative fingerprint method can be applied for the quality evaluation of traditional Chinese medicine and herbal preparations as part of their quality control, and the constructed mathematical model is particularly suitable for depicting the fingerprint-efficacy relationship.

Kava chalcone, flavokawain A, inhibits urothelial tumorigenesis in the UPII-SV40T transgenic mouse model.

Flavokawain A (FKA) is the predominant chalcone identified from the kava plant. We have previously shown that FKA preferentially inhibits the growth of p53 defective bladder cancer cell lines. Here, we examined whether FKA could inhibit bladder cancer development and progression in vivo in the UPII-SV40T transgenic model that resembles human urothelial cell carcinoma (UCC) with defects in the p53 and the retinoblastoma (Rb) protein pathways. Genotyped UPII-SV40T mice were fed orally with vehicle control (AIN-93M) or FKA (6 g/kg food; 0.6%) for 318 days starting at 28 days of age. More than 64% of the male mice fed with FKA-containing food survived beyond 318 days of age, whereas only about 38% of the male mice fed with vehicle control food survived to that age (P = 0.0383). The mean bladder weights of surviving male transgenic mice with the control diet versus the FKA diet were 234.6 ± 72.5 versus 96.1 ± 69.4 mg (P = 0.0002). FKA was excreted primarily through the urinary tract and concentrated in the urine up to 8.4 µmol/L, averaging about 38 times (males) and 15 times (females) more concentrated than in the plasma (P = 0.0001). FKA treatment inhibited the occurrence of high-grade papillary UCC, a precursor to invasive urothelial cancer, by 42.1%. A decreased expression of Ki67, survivin, and X-linked inhibitor of apoptotic proteins (XIAP) and increased expression of p27 and DR5, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive apoptotic cells were observed in the urothelial tissue of FKA-fed mice. These results suggest a potential of FKA in preventing the recurrence and progression of non-muscle-invasive UCC.

[Evaluation of Renshen Guipi Wan quality by linearly quantified fingerprint method based on three-wavelength high performance liquid chromatographic fingerprints].

The theoretical model of linearly quantified fingerprint method (LQFM) for qualitative and quantitative control traditional Chinese medicine (TCM) has been developed based on the relationship of linear function between a sample and the reference fingerprint vector, as the concepts of correlation coefficient r, slope a, intercept b, linearly qualitative similarity S(L), linearly quantitative similarity M(L) and so on were introduced into the study. The high performance liquid chromatography coupled with diode array detector (DAD) was used to simultaneously obtain chromatograms of ten batches of Renshen Guipi Wan (RSGPW) at 203, 228 and 326 nm, separately. The model of LQFM was applied to evaluate the three-wavelength fingerprints for the quality control of RSGPW, it could obviously distinguish RSGPW from different resources and the results showed the quality was almost consistent from the same manufacture. The results of LQFM were compared with those of the systematically quantified fingerprint method (SQFM), a known method for quality control of TCM. It proved that LQFM can evaluate qualitatively and quantitatively the complex system of TCM which is composed of diverse chemical components. The quality control can be better and more reliable by multiple-wavelength fingerprints which give full consideration of the ultraviolet absorption characteristics of different components.

Study on the digitized and quantified evaluating method for the super information cluster of traditional Chinese medicine ultraviolet spectral fingerprints.

The theories of ultraviolet spectral fingerprint (UVFP) index, information index, fluctuation index, information fluctuation index combined with the quantified UV fingerprint method (QUFM) had been established and put into practice in the Ginkgo Tablets (GT) quality evaluation. The flowing injection analysis (FIA) coupled with a diode array detector was applied as a novel method to obtain the UVFP in the region of 190-400 nm at which the absorption can reflect all the information of the chemical constituents contained π→π*, n→π* and n→σ* transition. The result showed that all batches were qualified (Grade ≤3) except S8 for its too high contents. It was proved that this method made the expression of superposed information in UVFP of traditional Chinese medicine (TCM) digitized and simple. What's more, an approach which can test the total chemical content with the chromophoric characteristics in the complex system of TCM rapidly, simply and accurately was achieved by the application of QUFM. In one word, it made the exploration of the general characteristic information of the molecular absorption complex TCM in the ultraviolet regions feasible and possible.

Kava components down-regulate expression of AR and AR splice variants and reduce growth in patient-derived prostate cancer xenografts in mice.

Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5'6'-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.

Rhodiola rosea extracts and salidroside decrease the growth of bladder cancer cell lines via inhibition of the mTOR pathway and induction of autophagy.

The incidence of human urinary bladder cancer increases markedly with age, suggesting a mechanistic connection between aging and bladder carcinogenesis and a potential use of anti-aging agents in bladder cancer chemoprevention. Rhodiola rosea, growing in high altitude or cold regions of the world, has been reported to have anti-aging effects in Drosophila. We demonstrated that a R. rosea extract and one of its bioactive components, salidroside, inhibited the growth of bladder cancer cell lines with a minimal effect on nonmalignant bladder epithelial cells TEU-2. Interestingly, the R. rosea extract and salidroside component exhibited a selective ability to inhibit the growth of p53 knockout primary mouse embryo fibroblasts (p53-/- MEFs) compared to their wild-type counterparts. The growth inhibitory effects of the R. rosea extract and salidroside were, however, attenuated in TSC2 and p53 double knock MEFs (TSC2-/-, p53-/- MEFs), suggesting that TSC2 protein is, at least in part, required for the growth inhibitory effects of the R. rosea extract and salidroside. The R. rosea extract and salidroside treatment of UMUC3 cells resulted in an increase of AMP-activated protein kinase (AMPK)-α phosphorylation and a decrease of 4E-BP1 phosphorylation, leading to increased binding of 4E-BP1 to m7 GTP. These results indicate that the R. rosea extract and salidroside inhibit translation initiation. Furthermore, both the R. rosea extract and salidroside treatment of UMUC3 cells caused a significant percentage of cells undergoing autophagy. Therefore, the R. rosea extract and salidroside deserve further study as novel agents for chemoprevention of bladder carcinogenesis.

Antihyperlipidemic effect of protodioscin, an active ingredient isolated from the rhizomes of Dioscorea nipponica.

Developing drugs against metabolic-related disorders, including obesity and hyperlipidemia, is of importance for human health. Here, a bioactive phytochemical, protodioscin, isolated from the rhizomes of Dioscorea nipponica (Rhizoma Dioscoreae Nipponicae), was identified for its anti-hyperlipidemic effect. In hyperlipidemic rats, the post-administration of protodioscin significantly reduced the time of blood coagulation. In addition, the blood levels of triglyceride, cholesterol, low-density and high-density lipoproteins were also changed accordingly. Taken together, this was the first report of an antihyperlipidemic effect of protodioscin. Its great availability in various vegetables and medicinal plants will be useful in developing health food supplement(s) and/or drug(s) against hyperlipidemia.

A milk-based wolfberry preparation prevents prenatal stress-induced cognitive impairment of offspring rats, and inhibits oxidative damage and mitochondrial dysfunction in vitro.

Lycium barbarum (Fructus Lycii, Wolfberry, or Gouqi) belongs to the Solanaceae. The red-colored fruits of L. barbarum have been used for a long time as an ingredient in Chinese cuisine and brewing, and also in traditional Chinese herbal medicine for improving health. However, its effects on cognitive function have not been well studied. In the present study, prevention of a milk-based wolfberry preparation (WP) on cognitive dysfunction was tested in a prenatal stress model with rats and the antioxidant mechanism was tested by in vitro experiments. We found that prenatal stress caused a significant decrease in cognitive function (Morris water maze test) in female offspring. Pretreatment of the mother rats with WP significantly prevented the prenatal stress-induced cognitive dysfunction. In vitro studies showed that WP dose-dependently scavenged hydroxyl and superoxide radicals (determined by an electron spin resonance spectrometric assay), and inhibited FeCl(2)/ascorbic acid-induced dysfunction in brain tissue and tissue mitochondria, including increases in reactive oxygen species and lipid peroxidation and decreases in the activities of complex I, complex II, and glutamate cysteine ligase. These results suggest that dietary supplementation with WP may be an effective strategy for preventing the brain oxidative mitochondrial damage and cognitive dysfunction associated with prenatal stress.

Hydroxytyrosol protects against oxidative damage by simultaneous activation of mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells.

Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress.

Anti-convulsant effect and mechanism of Astragalus mongholicus extract in vitro and in vivo: protection against oxidative damage and mitochondrial dysfunction.

Astragalus mongholicus (AM) is a traditional medicinal herb used as a neuroprotective agent for its anxiolytic, antidepressant, antiamnestic, and antiaggresive effects. However, the mechanisms underlying its anti-convulsant properties are not well studied. In the present study, we examined the anticonvulsant effects on pentylenetetrazol (PTZ)-induced seizures in mice and the possible mechanisms of protection against oxidative damage and mitochondrial dysfunction in vitro. The behavioral studies showed that the root extract of AM had powerful anticonvulsant effects against seizures induced by PTZ and the biochemical studies showed that root extract of AM inhibited PTZ-induced increase in lipid peroxidation, protein oxidation and reactive oxygen species, and enhanced mitochondrial function. Electron spin resonance spectroscopy studies demonstrated that the extracts from the root and aerial parts of AM possess potent effects on scavenging hydroxyl and lipid free radicals. We found that AM extract significantly protected malondialdehyde-induced oxidative damage by ameliorating activities of the mitochondrial complexes I, II, malate dehydrogenase and mitochondrial membrane potential. These data suggest that the anti-convulsant effects of AM extract may be mediated by its protective actions against oxidative damage and amelioration of mitochondrial dysfunction.

Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry.

Protodioscin (3-O-[alpha-L-rhamnopyranosyl-(1-->2)-{alpha-L-rhamnopyranosyl-(1-->4)}-beta-D-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-(25 R)-furost-5-ene-3 beta,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20-125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.