Identification of the absorptive constituents and their metabolites in vivo of Ziziphi Spinosae Semen by UPLC-ESI-Q-TOF-MS/MS.

In this research, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) was used for detection and identification of the absorptive constituents and their metabolites in rat plasma, urine and feces following oral administration of Ziziphi Spinosae Semen alcohol extract. After structure elucidation, a total of 12 compounds in rat plasma, comprising seven prototypes and five metabolites, 28 compounds in urine, comprising 17 prototypes and 11 metabolites, and 23 compounds in feces, comrpising 17 prototypes and six metabolites, have been tentatively identified by comparison with standard compounds and reference literature information. To the best of our knowledge, this is the first comprehensive and systematical metabolic study on the seed. Mostly importantly, we propose that gastric acid could convert jujubosides into an absorbable form of ebelin lactone oligosaccharides, which may be responsible for the low bioavailability and specific bioactivities of these compounds. Additionally, we deduced that the absorption site of ebelin lactone oligosaccharides is located in the stomach, and that the ebelin lactone form of jujubosides may be more suitable for absorption than its hydrolysis product. Our investigation will be helpful to narrow the scope for potentially active ingredients of the seed, and pave the way for determination of the pharmacological mechanism of the seed.

UPLC-ESI-Q-TOF-MS/MS analysis of anticancer fractions from Ophiocordyceps xuefengensis and Ophiocordyceps sinensis.

Ophiocordyceps xuefengensis (O. xuefengensis), a new species of caterpillar fungus, has been identified as the sister taxon of Ophiocordyceps sinensis (O. sinensis). The aims of the present study are to evaluate the anticancer activity and to qualitatively analyze the potential bioactive chemical constituents of O. xuefengensis and O. sinensis, comparatively. An MTT assay was used to evaluate the in vitro anticancer activities of different fractions from O. xuefengensis and O. sinensis. The results show that ethyl acetate fractions of O. xuefengensis and O. sinensis have significant in vitro anticancer activity. These two bioactive fractions were analyzed by ultra-performance liquid chromatography-electrospray ionization with quadrupole-time of flight tandem mass spectrometry technology. A total of 82 compounds and 101 compounds were identified or tentatively characterized in the bioactive fractions of O. xuefengensis and O. sinensis, respectively. Among these compounds, 68 existed in both O. xuefengensis and O. sinensis. A total of 67 compounds were reported in O. xuefengensis and 8 compounds were reported in caterpillar fungus for the first time. This is the first detailed comparative analysis of the in vitro anticancer activity and chemical ingredients between O. xuefengensis and O. sinensis. The application of this work will provide reliable fundamental pharmacological substances for the use of O. xuefengensis by Yao people.

[Rapid identification of constituents of Urtica hyperborea using UPLC-ESI-Q-TOF-MS/MS method].

This paper deals with the application of ultra-performance liquid chromatography tandem quadrupole time of flight mass spectrometry(UPLC-ESI-Q-TOF-MS/MS) method to rapidly determine and analyze the chemical constituents of methanol extract of Urtica hyperborea. We employed UPLC YMC-Triart C18(2. 1 mm×100 mm,1. 9 µm) column to UPLC analysis with acetonitrile-water(containing 0. 4% formic acid) in gradient as mobile phase. The flow rate was 0. 3 m L·min-1 gradient elution and column temperature was 30℃; the injection volume was 4 µL. ESI ion source was used to ensure the data collected in anegative ion mode. The chemical components of U. hyperborea were identified through retention time,exact relative molecular mass,cleavage fragments of MS/MS and reported data.The results indicated that a total of 31 compounds were identified,including 8 flavonoids,14 phenolic compounds,8 phenylpropanoids(4 coumarins and 4 lignans),and 1 steroidal compound,13 of which were confirmed by comparison. The UPLC-ESI-Q-TOF-MS/MS method could rapid identify the chemical components of U. hyperborea. The above compounds were discovered in U. hyperborea for the first time,which could provide theoretical foundation for further research on the basis of the pharmacodynamics of U. hyperborea.

Exploring on the bioactive markers of Codonopsis Radix by correlation analysis between chemical constituents and pharmacological effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Codonopsis Radix is a commonly used traditional Chinese medicine, and has the effect of strengthening spleen and tonifying lung, nourishing blood and engendering liquid. In addition, it is also used as important food materials. AIM OF THE STUDY: The aim of the study was to explain the underlying correlations between chemical constituents and pharmacological effects and explore the bioactive markers of Codonopsis Radix. MATERIALS AND METHODS: Codonopsis Radix samples from Min county, Gansu province processed with different methods were taken as the materials, UPLC-ESI-Q-TOF-MS/MS analysis was conducted to identify the compounds and establish UPLC fingerprint. Meanwhile, hematopoietic and immunologic functions of Codonopsis Radix were investigated to obtain relevant pharmacological index. Then, the correlation analysis between chemical constituents in UPLC fingerprints and pharmacological effects was carried out. The plant name was confirmed to the database "The Plant List" (www.theplantlist.org). RESULTS: According to the results of canonical correlation analysis, tryptophan, syringin, tangshenoside I, codonopyrrolidium A, lobetyolin and two unknown compounds might be the potential bioactive markers related to the hematopoietic and immunologic functions of Codonopsis Radix, which could be recommended as the index compounds. CONCLUSION: This study illustrated the underlying correlations between chemical constituents and pharmacological effects, explored the pharmacological material basis, and could lay a foundation for the improvement of quality standard of Codonopsis Radix.

Impact of ultrasonication techniques on the preparation of novel Amiloride-nanoemulsion used for intranasal delivery in the treatment of epilepsy.

AIM: To develop a nanoemulsion-nanoformulation in order to enhance brain bioavailability for Amiloride (Amilo) via intranasal (i.n.) drug delivery in the brain. MATERIAL AND METHODS: Oleic Acid, Tween-20 and Carbitol were selected as oil, surfactant and co-surfactant, respectively. For nanoemulsion preparation, an aqueous micro titration method followed by a high energy ultra-sonication method was used whereas three-factor three-level central composite design was employed to get the best formulation. The independent variables selected for the optimization were %oil, % Surfactant and co-surfactant (Smix) and sonication time (seconds). RESULTS: Based on the constraints applied for independent and dependent variables, the optimized formulation was selected with 2.5% oil, 10% Smix and a sonication time of 45 s. The experimental values observed for dependent variables such as hydrodynamic diameter (nm), % transmittance and % cumulative drug release were found to be 89.36 ± 11.18 nm, 99.23 ± 0.84% and 80.36 ± 5.48%, respectively. Results showed; a spherical shape (transmission electron microscopy and scanning electron microscopy - assisted morphological characterization), polydispersity index (0.231 ± 0.018), zeta potential (-9.83 ± 0.12 mV), refractive index (1.38 ± 0.042), viscosity (41 ± 5 cp), pH (6.4 ± 0.18) and drug content of 98.28 ± 0.29%, for optimized Amiloride-loaded-Nanoemulsion (Amilo-NE). For bioavailability evaluation, ultra-performance liquid chromatography-mass spectroscopy based bioanalytical method was developed and validated for pharmacokinetics, biodistribution, brain-targeting efficiency (1992.67 ± 45.63%) and nose-to-brain transport (586.18 ± 11.63%) whereby an enhanced Amilo-brain bioavailability was observed as compared to intravenous administration (i.v.). Furthermore, Amilo-NE enhanced the treatment in seizure threshold i.e. both rodent models of epilepsy (increasing current electroshock and pentylenetetrazole) induced seizures in mice. CONCLUSION: A significant role of Amilo-NE as observed after high targeting potential and efficiency of the formulation supports the easy brain targeting for Amilo-NE.

Identification of a novel ACE-inhibitory peptide from casein and evaluation of the inhibitory mechanisms.

Various bioactive peptides are continuously being identified from casein hydrolysates. In this work, a novel angiotensin I-converting enzyme (ACE)-inhibitory (ACEI) peptide, NMAINPSKENLCSTFCK, derived from the αs2-casein fragment residues 25-41, was screened and identified by UPLC-ESI-Q-TOF-MS/MS from tryptic casein hydrolysate. The IC50 value of the peptide, determined by an HPLC method, was 129.07 µM. The Lineweaver-Burk plot showed that this peptide acted as a mixed-type inhibitor against ACE, which might be attributed to the peptide being susceptible to degradation by ACE, indicating that the mixed-type inhibition could partly be a result of newly generated peptide fragments. The physicochemical characteristics and the secondary structure were evaluated by circular dichroism analysis and online prediction software (Expasy, PepDraw, and ProtParam) to identify the basic characteristics of this peptide. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACEI activity of the peptides.

Study on Chemical Constituents in Polygoni Cuspidati Folium and its Preparation by UPLC-ESI-Q-TOF-MS/MS.

A rapid and credible analytical method was developed using online UPLC-ESI-Q-TOF-MS/MS to identify chemical constituents in Polygoni cuspidati folium and its preparation. By accurate mass measurements within 6.5 ppm error for [M-H]- ion in routine analysis, 26 chemical constituents, including tannin, derivatives of phenylpropionic acid, stilbene, flavonoid, anthraquinone, torachryson and its derivatives, were identified or tentatively characterized. Among them, five constituents (compounds 19-23) were firstly reported in Polygoni cuspidati folium, other 17 constituents were coexisting in both Polygoni cuspidati folium and its preparation. Fragmentation behaviors of different categories of constituents were also investigated to confirm the results. This established UPLC-ESI-Q-TOF-MS/MS method, with reliance and efficiency for the identification the major constituents, would be the basis for quality control of Polygoni cuspidati folium and its preparation.

Rapid characterization of metabolites in soybean using ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) and screening for α-glucosidase inhibitory and antioxidant properties through different solvent systems.

This work was the first to investigate on the simultaneous characterization of metabolite profiles in soybean using UPLC-ESI-Q-TOF-MS/MS. Twenty two compositions were observed within 14 min from the methanol extract and confirmed as twelve isoflavones of three types and ten soyasaponins (Ab, Af, I-III, αg, ßg, ßa, γg, and γa). Moreover, the patterns of two chemicals showed considerable differences in seven solvent systems by HPLC analysis and their optimal extraction was achieved by 70% methanol (isoflavone: 4102.69 µg/g; soyasaponin: ten peaks). The second abundant isoflavones were detected in 50% methanol (4054.39 µg/g), followed by 30% methanol, 100% methanol, 10% methanol, CH2Cl2, and acetone extracts with 3134.03, 2979.49, 1681.33, 366.19, and 119.00 µg/g, respectively. Soyasaponins exhibited similar tendencies as those of isoflavones. The highest total phenolic was found as 2.10 ± 0.05 mg GAE/g in 70% methanol with remarkable differences by comparing other extracts. Specifically, this extract showed potent α-glucosidase inhibitory (81%) and antioxidant capacities (DPPH: 93% and ABTS: 95%) at a concentration of 1.0 mg/mL. Our results may be contributed to enhancing the value to functional foods and evaluating the secondary metabolites concern to antioxidant properties using solvent system in soybean.

Intranasal delivery of quercetin-loaded mucoadhesive nanoemulsion for treatment of cerebral ischaemia.

BACKGROUND: Quercetin (QUR), as an antioxidant flavonoid, exhibits potential role in the amelioration of cerebral ischaemia; however, poor solubility as well as oral absorption results low serum and tissue levels for this drug. PURPOSE OF THE STUDY: To enhance bioavailability, this study aims to prepare QUR nanoemulsions and administer via non-invasive nasal route in order to evaluate the drug targeting in brain. METHODS: Quercetin mucoadhesive nanoemulsion (QMNE) was prepared (ionic gelation method) and optimized using various parameters, that is, particle size, entrapment efficiency, zeta potential and ex vivo permeation study. RESULTS: The results observed for optimized QMNE were as follows: mean globule size (91.63 ± 4.36 nm), zeta potential (-17.26 ± 1.04 mV), drug content (99.84 ± 0.34%) and viscosity (121 ± 13 cp). To evaluate the extent of bioavailability for QMNE via post-intranasal (i.n.) administration, Ultra performance liquid chromatography-mass spectroscopy (UPLC-ESI-Q-TOF-MS/MS)-based bioanalytical method was developed and validated for pharmacokinetics, biodistribution, brain-targeting efficiency (9333.33 ± 39.39%) and brain drug-targeting potential (2181.83 ± 5.69%) which revealed enhanced QUR brain bioavailability as compared to intravenous administration (i.v.). Furthermore, improved neurobehavioral activity (locomotor and grip strength), histopathology and reduced infarction volume effects were observed in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic rats model after i.n. administration of QMNE. CONCLUSION: This study supports a significant role for QMNE in terms of high brain-targeting potential and formulation efficiency due to ease of access and effective targeting in brain.

Characterization of metabolite profiles from the leaves of green perilla (Perilla frutescens) by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry and screening for their antioxidant properties.

The objective of this research was to access the determination of metabolite profiles and antioxidant properties in the leaves of green perilla (Perilla frutescens), where these are considered functional and nutraceutical substances in Korea. A total of 25 compositions were confirmed as six phenolic acids, two triterpenoids, eight flavonoids, seven fatty acids, and two glucosides using an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) technique from the methanol extract of this species. The individual and total compositions exhibited significant differences, especially rosmarinic acid (10), and linolenic acids (22 and 23) were detected as the predominant metabolites. Interestingly, rosmarinic acid (10) was observed to have considerable differences with various concentrations in three samples (Doryong, 6.38 µg/g; Sinseong, 317.60 µg/g; Bongmyeong, 903.53 µg/g) by UPLC analysis at 330 nm. The scavenging properties against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals also showed potent effects with remarkable differences at a concentration of 100 µg/mL, and their abilities were as follows: Sinseong (DPPH, 86%; ABTS, 90%) > Bongmyeong (71% and 84%, respectively) > Doryong (63% and 73%, respectively). Our results suggest that the antioxidant activities of green perilla leaves are correlated with metabolite contents, especially the five major compositions 10 and 22-25. Moreover, this study may be useful in evaluating the relationship between metabolite composition and antioxidant activity.

Mass spectrometry analysis and in silico prediction of allergenicity of peptides in tryptic hydrolysates of the proteins from Ruditapes philippinarum.

BACKGROUND: Ruditapes philippinarum is nutrient-rich and widely-distributed, but little attention has been paid to the identification and characterization of the bioactive peptides in the bivalve. In the present study, we evaluated the peptides of the R. philippinarum that were enzymolysised by trypsin using a combination of ultra-performance liquid chromatography separation and electrospray ionization quadrupole time-of-flight tandem mass spectrometry, followed by data processing and sequence-similarity database searching. The potential allergenicity of the peptides was assessed in silico. RESULTS: The enzymolysis was performed under the conditions: E:S 3:100 (w/w), pH 9.0, 45 °C for 4 h. After separation and detection, the Swiss-Prot database and a Ruditapes philippinarum sequence database were used: 966 unique peptides were identified by non-error tolerant database searching; 173 peptides matching 55 precursor proteins comprised highly conserved cytoskeleton proteins. The remaining 793 peptides were identified from the R. philippinarum sequence database. The results showed that 510 peptides were labeled as allergens and 31 peptides were potential allergens; 425 peptides were predicted to be nonallergenic. CONCLUSION: The abundant peptide information contributes to further investigations of the structure and potential function of R. philippinarum. Additional in vitro studies are required to demonstrate and ensure the correct production of the hydrolysates for use in the food industry with respect to R. philippinarum. © 2017 Society of Chemical Industry.

Rapid screening and identification of lycodine-type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Lycodine-type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS/MS) for comprehensive characterization of lycodine-type alkaloids for the first time. The lycodine-type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine-type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine-type alkaloids and demonstrates the superiority of UPLC with a high-resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.