Li-Fang Liu

A high performance liquid chromatography fingerprinting and ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry chemical profiling approach to rapidly find characteristic chemical markers for quality evaluation of dispensing granules, a case study on Chuanxiong Rhizoma

A high performance liquid chromatography-photodiode array detector (HPLC-PDA) fingerprinting and ultra high performance liquid chromatography-photodiode array detector coupled with quadrupole time-of-flight mass spectrometry (UHPLC-PDA-QTOF-MS/MS) based chemical profiling approach was developed to rapidly find characteristic chemical markers for quality control of dispensing granules, taking Chuanxiong Rhizoma (CR) as a model herb. Firstly, CR crude drugs, their traditional decoctions and CR dispensing granules were analyzed by HPLC-PDA to rapidly establish the fingerprints and thereby generate the simulative median chromatograms of CR crude drugs, decoctions and dispensing granules, and by comparing the simulative median chromatograms, major characteristic peaks of CR decoctions and dispensing granules could be determined. Secondary, UHPLC-PDA-QTOF-MS/MS was used to identify the major characteristic peaks of CR decoctions and dispensing granules. The identities of three major peaks were elucidated and confirmed to be ferulic acid (1), senkyunolide I (2) and senkyunolide H (3) by comparing the mass/UV spectra and retention times with that of the reference compounds. Thirdly, an HPLC-PDA method was validated to quantify the three characteristic components in commercial CR dispensing granules. The average contents of ferulic acid and senkyunolide H were found to be less than 1.0mg/g, whereas that of senkyunolide I was 4.40mg/g in CR dispensing granules, which indicated that senkyunolide I might be chosen as a suitable quantitative marker, while ferulic acid and senkyunolide H as qualitative markers for the quality evaluation of CR dispensing granules. It is suggested that this newly established approach could be used to practically and rapidly find suitable marker compounds for quality control of dispensing granules derived from other medicinal herbs.

Inhibition of Mitochondrial Fission and NOX2 Expression Prevent NLRP3 Inflammasome Activation in the Endothelium: The Role of Corosolic Acid Action in the Amelioration of Endothelial Dysfunction

AIMS Corosolic acid (CRA) is a natural triterpenoid with antioxidative activity. This study was designed to elucidate the mechanism through which CRA protected vessel endothelial homeostasis by combating oxidative stress. RESULTS In endothelial cells, CRA induced dynamin-related protein 1 (Drp1) phosphorylation at Ser637 and thus inhibited mitochondrial fission in response to oxidative stress. It promoted AMP-activated protein kinase (AMPK) activity in an LKB1-dependent manner, and silencing AMPK abrogated its inhibitory effect on Drp1 activation and mitochondrial fission. CRA inhibited the translocation of p47(phox) and p67(phox) and the overexpression of gp91(phox) induced by palmitate (PA), demonstrating its action in suppression of NOX2 activation. Drp1 knockdown reduced PA-induced gp91(phox) expression, while Drp1 induction was also diminished by gp91(phox) knockdown, suggesting the reciprocal relationship between NOX2 and Drp1. Knockdown Drp1 or gp91(phox) attenuated PA-induced NLRP3 induction and enhanced inhibitory effects of CRA. Oral administration of CRA in high-fat diet mice reproduced similar regulation in the aorta endothelium, further confirming its protection on endothelial homeostasis in vivo. INNOVATION This study demonstrated that the defect in mitochondrial morphology is associated with the oxidative stress and NLRP3 inflammasome activation in the endothelium. Drp1 and NOX2 regulated each other and worked together to induce NLRP3 inflammasome activation, suggesting that modulation of Drp1 phosphorylation (Ser637) might be a potential therapeutic target for combating oxidative stress in vessel diseases. CONCLUSION CRA prevented mitochondrial fission by regulation of Drp1 phosphorylation (Ser637) in an AMPK-dependent manner, and this action contributed to blocking NOX2 oxidase signaling and suppressing NLRP3 inflammasome activation in the endothelium. Antioxid. Redox Signal. 24, 893-908.

Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis

Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-β (TGF-β1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-β1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-β1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1β further increased TGF-β1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-β1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis.

Sulfur dioxide residue in sulfur-fumigated edible herbs: The fewer, the safer?

The residual content of sulfur dioxide is frequently regarded as the exclusive indicator in the safety evaluation of sulfur-fumigated edible herbs. To examine the feasibility of such assessment criteria, here the variations in residual sulfur dioxide content during sulfur-fumigation and the potential mechanisms involved were investigated, using Angelicae Sinensis Radix (ASR) as a model herb. The residual sulfur dioxide content and ten major bioactive components in sulfur-fumigated ASR samples were dynamically examined at 13 successive time points within 72 h sulfur-fumigation. The relationship between the content variation tendency of sulfur dioxide and the ten chemicals was discussed. The results suggested that sulfur dioxide-involved chemical transformation of the original components in ASR might cause large consumption of residual sulfur dioxide during sulfur-fumigation. It implies that without considering the induced chemical transformation of bioactive components, the residual sulfur dioxide content alone might be inadequate to comprehensively evaluate the safety of sulfur-fumigated herbs.

Effect of different drying methods on the quality of Angelicae Sinensis Radix evaluated through simultaneously determining four types of major bioactive components by high performance liquid chromatography photodiode array detector and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry.

In the present study, the effect of drying methods on the quality of Angelicae Sinensis Radix (DG), was evaluated by newly developed high performance liquid chromatography photodiode array detector (HPLC-DAD) and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS). Ten major bioactive components including two phenolic acids, two hydroxyl phthalides, four alkyl phthalides and two phthalide dimers were selected as evaluation chemical markers and the newly-established method was qualitatively and quantitatively validated. DG slices and whole roots dried in shade, sun light, hot air, vacuum, microwave, far infrared ray and combination of microwave and far infrared ray as well as the fresh DG samples were determined by the established methods. DG slices dried in hot air kept the similar chemical composition to that of fresh DG, while DG whole roots dried in vacuum retained highest contents of the major components. Coniferyl ferulate and ligustilide degraded significantly in DG slices dried by microwave, far infrared ray and their combination. The influence of such chemical changes induced by different drying methods on the bioactivities of DG warrants further investigation, so that the optimal drying method can be obtained for the standardization of DG herb.

A strategy to identify and quantify closely related adulterant herbal materials by mass spectrometry-based partial least squares regression

In this study, a new strategy combining mass spectrometric (MS) techniques with partial least squares regression (PLSR) was proposed to identify and quantify closely related adulterant herbal materials. This strategy involved preparation of adulterated samples, data acquisition and establishment of PLSR model. The approach was accurate, sensitive, durable and universal, and validation of the model was done by detecting the presence of Fritillaria Ussuriensis Bulbus in the adulteration of the bulbs of Fritillaria unibracteata. Herein, three different MS techniques, namely wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI/MS), ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and UPLC-triple quadrupole tandem mass spectrometry (UPLC-TQ/MS), were applied to obtain MS profiles for establishing PLSR models. All three models afforded good linearity and good accuracy of prediction, with correlation coefficient of prediction (rp2) of 0.9072, 0.9922 and 0.9904, respectively, and root mean square error of prediction (RMSEP) of 0.1004, 0.0290 and 0.0323, respectively. Thus, this strategy is very promising in tracking the supply chain of herb-based pharmaceutical industry, especially for identifying adulteration of medicinal materials from their closely related herbal species.

UPLC/ESI-QTOF-MS-based metabolomics survey on the toxicity of triptolide and detoxication of licorice

Triptolide (TP) from Tripterygium wilfordii has been demonstrated to possess anti-inflammatory, immunosuppressive, and anticancer activities. TP is specially used for the treatment of awkward rheumatoid arthritis, but its clinical application is confined by intense side effects. It is reported that licorice can obviously reduce the toxicity of TP, but the detailed mechanisms involved have not been comprehensively investigated. The current study aimed to explore metabolomics characteristics of the toxic reaction induced by TP and the intervention effect of licorice water extraction (LWE) against such toxicity. Obtained urine samples from control, TP and TP + LWE treated rats were analyzed by UPLC/ESI-QTOF-MS. The metabolic profiles of the control and the TP group were well differentiated by the principal component analysis and orthogonal partial least squares-discriminant analysis. The toxicity of TP was demonstrated to be evolving along with the exposure time of TP. Eight potential biomarkers related to TP toxicity were successfully identified in urine samples. Furthermore, LWE treatment could attenuate the change in six of the eight identified biomarkers. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan, pantothenic acid, and porphyrin metabolism. Therefore, it was concluded that LWE demonstrated interventional effects on TP toxicity through regulation of tryptophan, pantothenic acid, and porphyrin metabolism pathways, which provided novel insights into the possible mechanisms of TP toxicity as well as the potential therapeutic effects of LWE against such toxicity.

Global detection and semi‐quantification of Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus by a non‐targeted multiple reaction monitoring approach

Methods based on triple quadrupole tandem mass spectrometry have been widely used and reported as highly selective and sensitive methods for quantifying substances of herbal medicines. However, most of them were limited to targeted components, due to the difficulties to optimize the multiple reaction monitoring transitions without authentic standards. This study proposed a novel strategy for non-targeted optimization of multiple reaction monitoring method based on the diagnostic ion guided family classifications, tandem mass spectrometry database establishment, and transitions and collision energy screening. Applying this strategy, 59 Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus have been classified, and 51 of these Fritillaria alkaloids were successfully detected by the optimal multiple reaction monitoring method. For semi-quantification, the easy-to-obtain Fritillaria alkaloids of each type, such as verticinone for cevanine type and peimisine for jervine type, were used as the reference standards to calibrate the other Fritillaria alkaloids in the same type. The method was demonstrated a good linearity (R(2) > 0.998) with satisfactory accuracy and precision, and the lower limits of quantification of verticinone and peimisine were estimated to be 0.076 and 0.216 pg, respectively. In addition, the results suggested that the proposed strategy might obtained high quality metabolomics data in discrimination of Fritillaria unibracteata and Fritillaria ussuriensis.

Discovery of characteristic chemical markers for inspecting sulfur-fumigated Radix Angelicae Sinensis by ultra-high performance liquid chromatography–quadrupole/time-of-flight mass spectrometry based metabolomics and chemical profiling approach

An UHPLC-QTOF-MS/MS based metabolomics and chemical profiling approach was developed to identify the characteristic chemical markers for inspecting sulfur-fumigated Radix Angelicae Sinensis (S-RAS). Two sulfur-containing compounds in S-RAS were discovered and deduced to be (3Z)-6-sulfite-ligustilide and (3E)-6-sulfite-ligustilide. Furthermore, utilizing extraction ion method with the typical ion (m/z 271.06) of (3Z)-6-sulfite-ligustilide/(3E)-6-sulfite-ligustilide as the diagnostic ion, another six sulfur-containing components were determined in S-RAS, and identified as 6, 8-disulfite-ligustilide and 8-sulfite-ligustilide or their isomers. The generation mechanisms of these compounds were proposed as the addition reaction of sulfurous acid with (Z)-ligustilide and/or (E)-ligustilide, two major naturally occurring components in RAS, at the double bonds of C6C7 and C3C8 positions. Using (3Z)-6-sulfite-ligustilide and (3E)-6-sulfite-ligustilide as the chemical markers, fourteen of sixteen batches of commercial RAS samples were inspected to be S-RAS, suggesting that the newly generated sulfur-containing compounds could be used as characteristic chemical markers for inspecting S-RAS among commercial RAS samples.

Dose-response characteristics of Clematis triterpenoid saponins and clematichinenoside AR in rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine metabolomics.

&NA; Clematidis Radix et Rhizoma is a traditional Chinese medicine widely used for treating arthritic disease. Clematis triterpenoid saponins (TS) and clematichinenoside AR (C‐AR) have been considered to be responsible for its antiarthritic effects. However, the underling mechanism is still unclear because of their low bioavailability. To address of this issue, metabolomics tools were performed to determine metabolic variations associated with rheumatoid arthritis (RA) and responses to Clematis TS, C‐AR and positive drug (Triptolide, TP) treatments. This metabolomics investigation of RA was conducted in collagen‐induced arthritis (CIA) rats. Liquid chromatography/mass spectrometry and multivariate statistical tools were used to identify the alteration of serum and urine metabolites associated with RA and responses to drug treatment. As a result, 45 potential metabolites associated with RA were identified. After treatment, a total of 24 biomarkers were regulated to normal like levels. Among these, PC(18:0/20:4), 9,11‐octadecadienoic acid, arachidonic acid, 1‐methyladenosine, valine, hippuric acid and pantothenic acid etc, were reversed in Clematis TS and C‐AR groups. Tetrahydrocortisol was regulated to normal levels in Clematis TS and TP groups, while 3,7,12‐trihydroxycholan‐24‐oic acid was regulated in C‐AR and TP groups. Biomarkers like citric acid, p‐cresol glucuronide, creatinine, cortolone were reversed in TP group. Graphical abstract Figure. No caption available. HighlightsMetabolomics study of Clematis triterpenoid saponins (TS) and clematichinenoside AR (C‐AR) on collagen‐induced arthritis rat was investigated.The Clematis TS and C‐AR treatment groups presented a dose‐dependent reversal effect on metabolic trajectory and corresponding biomarkers.45 potential biomarkers were identified in response to RA.Glycerophospholipid catabolism, sphingolipid metabolism, arachidonic acid metabolism were filtered out as the potential target pathways.