Qibin Chen

A LC/QTOF-MS/MS Application to Investigate Chemical Compositions in a Fraction with Protein Tyrosine Phosphatase 1B Inhibitory Activity from Rosa Rugosa Flowers

INTRODUCTION Rosa rugosa flowers used as herbal medicine possess many activities. A fraction extracted by ethyl acetate exhibited strong inhibitive activity against protein tyrosine phosphatase 1B (PTP1B) in vitro. OBJECTIVE Establish an efficient method of LC coupled to quadrupole time-of-flight (QTOF) with tandem MS/MS to investigate the compositions in the active fraction. METHODS Chemical compositions were separated and investigated by LC/QTOF-MS/MS in negative electrospray ionisation (ESI) mode at different collision energy (CE) values. The maximal structural information was obtained for the identification of components. RESULTS A total of 75 compounds including tannins, their related compounds and flavonoids were identified or partially characterised according to accurate mass and the characteristic fragments at low and high CE. Meanwhile, the fragmentation pathways of gallotannins and ellagitannins (hexahydroxydiphenoyl group and lactonised valoneoyl group) were studied and proposed and were used to trace tannins in crude extracts. CONCLUSION The results suggest that this fraction is a source of PTP1B inhibitory activity with a potential for treating diabetes.

Highly Conjugated Norditerpenoid and Pyrroloquinoline Alkaloids with Potent PTP1B Inhibitory Activity from Nigella glandulifera

Three norditerpenoid alkaloids, nigelladines A-C (1-3), and one pyrroloquinoline alkaloid, nigellaquinomine (4), all possessing new skeletons with highly conjugated systems, were isolated from Nigella glandulifera. The 8aS-configuration for 1 and 2 was determined by comparison of the experimental and calculated electronic circular dichroism spectra. These alkaloids exhibited potent protein tyrosine phosphatase 1B (PTP1B) inhibitory activity but are devoid of cytotoxicity against the A431 cell line at 100 μM.

Separation and purification of two new and two known alkaloids from leaves of Nitraria sibirica by pH-zone-refining counter-current chromatography.

The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica. The separation was performed with a solvent system of MtBE-n-BuOH-H2O (2:2:5, v/v) at a flow rate of 2.0mL/min. And 15mM triethylamine (TEA) was added to the upper organic phase, while 10mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6mg), and a known alkaloid, nitraramine (5.0mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica. The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n-hexane-n-BuOH-H2O (1.5:3.5:5, v/v) and (2:3:5, v/v), respectively. TEA (10mM) was added to the upper phase, and 10mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0mg), and a new alkaloid, schoberimine (3.0mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC-ELSD, and their structures were identified by ESI-MS, 1D and 2D NMR.

Anti-diabetic effect of three new norditerpenoid alkaloids in vitro and potential mechanism via PI3K/Akt signaling pathway.

Diabetes is a metabolic disease with the characteristic of high blood glucose (hyperglycemia). In our previous study, we found that nigelladines A-C (compounds A-C), three norditerpenoid alkaloids from the seeds of Nigella glandulifera Freyn (Ranunculaceae) exhibited protein of tyrosine phosphatase 1B (PTP1B) inhibitory activity in vitro. In the present study, we further investigated their anti-diabetes activities in L6 moytubes and illuminated the mechanisms of action of compounds A-C. Several parameters of glucose metabolism such as glucose consumption, glycogen content and hexokinase activity were increased by compounds A-C. The results suggested that compounds A-C improved glucose metabolism through promoting synthesis of glycogen. Expression of PTP1B protein was inhibited by compounds A-C in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by compounds A-C and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the insulin-mediated induction of insulin receptor substrate-1 (IRS-1) and glycogen synthase kinase-3β (GSK-3β) were also suppressed by wortmannin. Western blot results indicated that compounds A-C-induced IRS-1/Akt activation was likely a consequence of PTP1B inhibition. Compounds A-C promoted glycogen synthesis through Akt-mediated GSK3 phosphorylation. Therefore, activation of PI3K/Akt insulin signaling pathway and suppression of PTP1B is the molecular mechanism that contributes to the anti-diabetic effect of compounds A-C in cellular models. The three alkaloids potentially serve as lead compounds for the development of antidiabetic drugs.

A sample pretreatment strategy for pH-zone-refining counter-current chromatography.

A sample pretreatment method using acid-base extraction was established for pH-zone-refining counter-current chromatography (CCC) to increase the yield of the target compound. As the most important step, selection of two-phase solvent system for pH-zone-refining CCC should follow some rules. One of these rules is that when pH of two-phase solvent system is around 2 (for an acidic analyte) or 10 (for a basic analyte), partition coefficient values of K(acid) or K(base) should be much greater than 1. According to this rule, a new pretreatment method was developed that could substantially increase the yield of the target compound in pH-zone-refining CCC. The separation of the crude alkaloids extracted from the seeds of Sophora alopecuroides was selected as an example to show the advantage of this method. Before CCC separation, 20 g of crude sample was extracted 3 times with the selected two-phase solvent system composed of MtBE-CH₃CN-H₂O (2:2:3, v/v), where pH of the upper phase was adjusted with triethylamine to 10. A 10 g amount of the second basic extract thus obtained was subjected to pH-zone-refining CCC separation. As a result, 169 mg of sophoramin and 696 mg of sophoearpin were isolated from the second basic extract, where the yields have been doubled compared with those of the untreated sample (crude sample). Experimental results showed that the present method for pretreating the crude sample could produce substantially higher yields of target compounds in pH-zone-refining CCC.

Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti-influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well-known traditional Uighur medicine for the treatment of colds. In the present study, high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid.

Isolation of new polyacetylenes from the roots of Eurycoma longifolia via high-speed counter-current chromatography

Eurycoma longifolia is a tropical plant of diverse applications in folk medicine, which occurs in Southeast Asia. In this study, pre-purified fraction (0.86g) of the crude extracts from the roots of E. longifolia, was subjected to preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water (HEMWat) at a volume ratio of 5:2:5:2 (v/v). Longifolione A (1, 19mg, purity 96.0%) and longifolione C (3, 317mg, purity 96.2%), together with longifolione B (2, purity 77.6%) were isolated in one run. The whole mobile and stationary phase was then blown out, concentrated in vacuo, and subjected to second HSCCC purification. Using HEMWat at a volume ratio of 6:1:6:1.2 (v/v), this fraction yielded two more new polyacetylenenes, longifolione D (4, 5mg purity 94.5%) and longifolione E (5, 33mg purity 96.3%). All of these five compounds are new natural products and isolated from E. longifolia for the first time. The established protocol for large-scale isolation of these polyacetylenes from E. longifolia was simple, efficient, and economical.

Biological activity and LC-MS profiling of ethyl acetate extracts from Nitraria sibirica (Pall.) fruits

Abstract Nitraria sibirica is a traditional Uighur medicine. This study was undertaken to investigate the bioactivity of N. sibirica fruit extract and to evaluate their chemical compositions. The ethyl acetate extract from N. sibirica fruits exhibited the potential antioxidant activity (SC50 = 30.17 ± 0.06 μg/mL) and protein tyrosine phosphatase 1B inhibitory activity (IC50 = 7.15 ± 0.03 μg/mL) in vitro. In order to investigate the active constituents in this extract, a LC-QTOF-MS/MS method was developed and established. A total of 28 compounds including seven cinnamic acids, nine benzoic acids and 12 flavonoids were identified or partially characterised according to the accurate mass and the characteristic fragment ions at low and high collision energy. Most of them were reported for the first time in this plant. Phytochemical profiles of the active extract will help the development and utilisation of N. sibirica in food and medicine.

Piperidine Alkaloids with Diverse Skeletons from Anacyclus pyrethrum

Fifteen new piperidine derivatives, pyracyclumines A-J (1-10), including five pairs of enantiomers, (+)-1/(-)-1 to (+)-5/(-)-5, together with three known compounds, agrocybenine (11), 4,6,6-trimethyl-5,6-dihydro-2(1 H)-pyridone (12), and 3,5,5-trimethyl-1,5-dihydro-2 H-pyrrol-2-one (13), were isolated from the roots of Anacyclus pyrethrum. Pyracyclumines A, B, and H (1, 2, and 8) possess a novel 6/5/6/6 dimeric piperidine skeleton, a unique 6/5/6 dimeric piperidine skeleton, and a 1,4,6-triazaindan skeleton, respectively. Pyracyclumine C (3) is based on a rare cyclopentane-piperidine framework. The structures of the isolated compounds were established by analysis of their NMR and HRESIMS data. The racemic pyracyclumines A-E (1-5) were further separated by chiral HPLC to give the enantiomers (+)-1/(-)-1 to (+)-5/(-)-5, for which the absolute configurations were determined by comparison of their experimental and calculated ECD spectra. The plausible biogenetic pathways of these piperidine alkaloids were proposed starting from the basic units of compounds 12 and 13. All of the isolated compounds were tested for their inhibitory effects on menin-mixed lineage leukemia 1 protein-protein interaction.

Alkaloid constituents from Viola tianschanica

Eighteen previously undescribed alkaloids, tishaviolamines A-J, including eight pairs of enantiomers, together with two known benzylisoquinoline alkaloids, (-)-bicuculline and (-)-corlumine, were isolated from Viola tianschanica. Among them, tishaviolamine A-E were demonstrated to possess three types of unpresented skeletons. The structures of these alkaloids were established by comprehensive analyses of the 1D, 2D-NMR and (+)HRESIMS data. The absolute configurations of enantiomers were determined by comparing their calculated ECD spectra with the experimental ones. The menin-mixed lineage leukemia 1 protein-protein interaction inhibitory effect of the isolated compounds were also measured.