Xiaodong Cong

Identification of metabolites of crude and processed Fructus Corni in rats by microdialysis sampling coupled with electrospray ionization linear quadrupole ion trap mass spectrometry

A microdialysis (MD) sampling coupled with electrospray ionization linear quadrupole ion trap mass spectrometry (LTQ-MS(n)) method has been developed for rapid and sensitive analysis of rat microdialysate metabolite profile of Fructus Corni, a well-known Traditional Chinese Medicine (TCM). The purified samples were separated by a reversed-phase HPLC with C₁₈ column under a gradient elution. Parent compounds and metabolites of crude and processed Fructus Corni of Jiu Zheng Pin (JZP, JZP is produced after steaming the crude drug pre-steeped in wine) were detected by the on-line MS(n) detector in negative scan model. The identification of the metabolites and their structural elucidation were performed by comparing the changes in molecular mass and defining sites of biotransformation based on the accurate MS(n) spectral information of diagnostic fragment ions. In this work, we used such strategies for the identification of the parent compounds and metabolites of crude and processed Fructus Corni in rats, and seven parent compounds and three new metabolites of Fructus Corni were found in rats for the first time. This study provides important structural information regarding to the metabolism of crude Fructus Corni and its JZP. Furthermore, this work also demonstrated the possibilities of using microdialysis sampling coupled with LC-MS(n) approach for identification of bioactive compounds from TCM in vivo.

Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time-of-flight-mass spectrometry.

We herein present a chemical profiling method to efficiently process the information acquired by ultra fast liquid chromatography (UFLC)-electrospray ionization source in combination with hybrid ion trap and high-resolution time-of-flight mass spectrometry (UFLC-(ESI)-IT-TOF/MS), facilitating the structural determination of serial components contained in crude or processed traditional Chinese medicine (TCM). Under the optimized UFLC and IT-TOF-MS(n) conditions, over 39 compounds were separated and detected in crude or processed Fructus corni within 25 min. The components were identified by comparing the mass spectra and retention time with reference compounds, or tentatively assigned by elucidating low-energy collision-induced dissociation (CID) fragment ions and matching empirical molecular formula with that of the published compounds. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used to elucidate the processed mechanism of F. corni, which is regularly affected by the processing conditions. This study provides a novel approach and methodology to identify the complicated components from various complex mixtures such as crude TCM, processed TCM, and biological samples. It can be used as a valid analytical method for further understanding the processing mechanism of TCM, along with the intrinsic quality control of TCM and its processed product.

Simultaneous chemical fingerprinting and quantitative analysis of crude and processed Radix Scrophulariae from different locations in China by HPLC

A validated liquid chromatography method was first developed to evaluate the quality of crude and processed Radix Scrophulariae extracts through establishing chromatographic fingerprint and simultaneous determination of five bioactive compounds, namely 5-hydroxymethylfurfural (5-HMF), acteoside, angroside C, harpagoside and cinnamic acid. The chromatographic were separated on an Agilent Zorbax Extend C(18) column (250 mm × 4.6 mm, 5 μm) and detected by diode array detector (DAD). Mobile phase was composed of (A) aqueous phosphoric acid (0.03%, v/v) and (B) acetonitrile using a gradient elution. Analytes were performed at 30 °C with a flow rate of 1.0 mL/min and UV detection at 280 nm. All calibration curves showed good linear regression (r(2) ≥0.9996) within the tested ranges, and the recovery of the method was in the range of 98.12-103.38%, with RSD values ranging from 0.6 to 2.8%. In addition, the contents of those five bioactive compounds in crude and processed Radix Scrophulariae prepared by different locations of China were determined to establish the effectiveness of the method. The results demonstrate that the developed method is accurate and reproducible and could be readily utilized as a suitable quality control method for the quantification of Radix Scrophulariae.

Argirein alleviates corpus cavernosum dysfunction by suppressing pro-inflammatory factors p66Shc and ER stress chaperone Bip in diabetic rats

Objectives  The aim was to investigate whether argirein, which releases rhein and l‐arginine after medication, could improve erectile dysfunction (ED) in diabetic rats through normalising the abnormalities of nitric oxide synthase (NOS), p66Shc and immunoglobulin heavy‐chain binding protein (Bip), in the corpus cavernosum (CC).

Male hypogonadism induced by high fat diet and low dose streptozotocin is mediated by activated endoplasmic reticulum stress and IκBβ and attenuated by argirein and valsartan.

Male hypogonadism is frequently accompanied with type 2 diabetes due to testicular dysfunction, but the origin of the pathogenesis is not known. We measured whether pro-inflammatory factors including endoplasmic reticulum (ER) stress chaperones and inhibitory κBβ (IκBβ) contribute to testis damage in type 2 diabetic rats produced by a high-fat diet (HFD) and low dose streptozotocin (STZ). We determined whether these can be attenuated by the anti-inflammatory activity of argirein a derivative of rhein as compared to valsartan. Reduced testosterone and LH (luteinizing hormone) levels in serum were significant in association with a decrease in the levels of mRNA and steroidogenic acute regulatory protein (StAR), insulin receptor substrate (IRS-1), activated IκBβ and ER stress chaperone C/EBP homologous protein (CHOP) in the diabetic testis and sperm count, motility and sexual behaviors were reduced in vivo. Additionally, Leydig cells cultured with high glucose showed upregulated IκBβ, ER stress sensor PERK (PKR-like ER kinase) and p-Akt/Akt in vitro. These changes may be due to a component of inflammation linked to activated NADPH oxidase and were significantly alleviated by either argirein or valsartan. In conclusion, diabetic testopathy induced by a HFD and low STZ is characterized by an entity of inflammation and is alleviated by argirein and valsartan through normalizing activated IκBβ and ER stress.

Activation of AQP4, p66Shc and endoplasmic reticulum stress is involved in inflammation by carrageenan and is suppressed by argirein, a derivative of rhein.

Objectives  We investigated the effect of argirein on acute inflammation edema and examined that aquaporin 4 (AQP4), p66Shc and activating transcription factor (ATF‐6) might be involved in carrageenan‐induced rat paw inflammation and be reversed by argirein, rhein and indometacin, but not l‐arginine.

Isoproterenol-induced FKBP12.6/12 downregulation is modulated by ETA and ETB receptors and reversed by argirhein, a derivative of rhein.

Aim:To investigate which endothelin receptors mediated isoproterenol (ISO)-induced downregulation of FKBP12.6/12 in cardiomyocytes and study whether argirhein, a novel compound containing rhein and L-arginine that has anti-inflammatory activity, could reverse the downregulation of FKBP12.6/12 induced by ISO.Methods:Neonatal rat cardiomyocytes were incubated with ISO to downregulate FKBP12.6/12. Then the cells were treated with a selective ETA blocker (PD156707) and a ETB blocker (IRL1038), a dual ETA/ETB antagonist (CPU0213), and argirhein, respectively. FKBP12.6/12 expression was assayed by RT-PCR, Western blot, and immunocytochemistry.Results:The expression of FKBP12.6 mRNA was reduced by 37.7% (P<0.01) and 28.9% (P<0.05) relative to the control by ISO 1 and 0.1 μmol/L, respectively, but no response to ISO 0.01 μmol/L was observed in vitro. FKBP12.6/12 protein expression was reduced by 47.2% (P<0.01) and 37.8% (P<0.05) by ISO 1 and 0.1 μmol/L, respectively. This decrease was reversed significantly by PD156707, or IRL1038, and CPU0213. CPU0213 was more potent than either PD156707 or IRL-1038. Argirhein 10 μmol/L blunted the downregulation of FKBP12.6/12 by ISO, as demonstrated by the rising mRNA and protein levels and by the fluorescent density of the ISO-incubated cardiomyocytes.Conclusion:In cardiomyocytes, the ISO induced downregulation of FKBP12.6/12 is modulated by both ETA and ETB. A new compound, argirein, reversed the down-regulation of FKBP12.6/12 expression in myocardial cells stimulated with ISO.

Simultaneous determination of four bioactive compounds in Verbena officinalis L. by using high-performance liquid chromatography

Background: Verbena officinalis L., called mabiancao in Chinese, is derived from the aerial part of Herba Verbanae. It is a traditional Chinese medicine commonly used in China and northern Europe, which is widely used for clearing away heat and detoxicating, promoting blood circulation, and removing blood stasis. This paper describes a sensitive and specific assay for the determination of four bioactive compounds in V. officinalis L. Materials and Methods: In this paper, the four components were separated on an Agilent Zorbax Extend C18 column (250 mm × 4.6 mm × 5 μm) and detected by a diode array detector. The mobile phase was composed of (a) aqueous phosphoric acid (0.1%, v/v) and (b) acetonitrile using a gradient elution. Analytes were performed at 30°C with a flow rate of 1.0 ml/min and UV detection at 203, 238, and 331 nm. Results: All calibration curves showed good linear regression (r2 ≥ 0.9999) within tested ranges. Overall intra- and interday variations were less than 1.84%, and the average recoveries were 97.32–102.81% for analytes. Discussion and Conclusion: The proposed method would be sensitive enough and reliable for comprehensive quality control for clinical use and modernization of V. officinalis L.

Screening and identification of potential active components in crude Fructus Corni using solid-phase extraction and LC–LTQ-linear ion trap mass spectrometry

Context: Fructus Corni is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc. (Cornaceae). It has attracted increasingly much attention as one of the most popular and valuable herbal medicine in clinic. This paper applied a rapid and validated method to the intrinsic quality control of Fructus Corni. Materials and methods: The components of crude Fructus Corni were investigated by means of solid-phase extraction (SPE) and LTQ-linear ion trap mass spectrometry (MS) technique in the negative ion mode. Results: The 29 detected compounds were identified by comparing the retention time and mass spectrometry data and retrieving the reference literatures. Discussion and conclusion: It was concluded that a rapid and validated method was successfully applied based on SPE-LC-DAD–LTQ-linear-MSn which showed high sensitivity and resolution that was more suitable for identifying main components in Traditional Chinese medicines (TCMs) and their prescriptions, which would be helpful to their quality control.

Novel software-based method to classify structurally similar compounds combined with high performance liquid chromatography–quadrupole time of flight mass spectrometry to identify complex components of herbal medicines

The components of herbal medicines (HMs) are usually extremely complex, belonging to hundreds of compound classes with diverse chemical and physical properties. Full characterization of HMs is hugely important in order to identify the individual chemical constituents and provide a first step toward determining which components are responsible for the therapeutic effects of a particular medical plant. In this study, a novel software-based approach was developed to classify structurally similar compounds, and this was combined with high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) to identify the individual components in an extract of Mentha haplocalyx. A total of 33 compounds were tentatively identified in samples of M. haplocalyx extract, including 9 new minor constituents reported for the first time. Semi-quantitative analysis of the extract sample was also carried out. Software validation and robustness tests were performed. The results of this study demonstrate the enormous potential of this strategy, using classification based on structural similarity together with HPLC-QTOF-MS, for the identification and quantification of complex components in HMs and related products.